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Plant Hormones
Table of Content
1
...
Auxin Hormone
3
...
Cytokinins Hormone
5
...
Ethylene Plant Hormone

What are Plant Hormones?
Plants need sunlight, water, oxygen, minerals for their growth and development
...
Apart from these, there are some intrinsic factors that regulate the growth and
development of plants
...


•

Plant hormones are chemical compounds present in very low concentration in plants
...


•

These hormones are produced in almost all parts of the plant and are transmitted to
various parts of the plant
...
Roles of different hormones can be
complementary or antagonistic
...
along with extrinsic factors
...


Charles Darwin first observed the phototropism in the coleoptiles of canary grass and F
...

Went first isolated auxin from the coleoptiles of oat seedlings
...

•Auxins
...


Inhibitors and promoters

•Abscisic acid
...
They are widely used in agricultural and horticultural practices
...

Auxins are very widely used in plant tissue culture and usually form an integral part of nutrient
media
...
The word auxin has
a Greek origin: auxein means to enlarge or to grow
...

Since they are capable of initiating cell division they are involved in the formation of meristems
giving rise to either unorganised tissue, or defined organs
...

The choice of auxins and the concentration administered depends on:

• the type of growth and/or development required;
• the rate of uptake and of transport of the applied auxin to the target tissue;
• the inactivation (oxidation and/or conjugation) of auxin in the medium and within the
explant;
• the natural levels and the endogenous synthesis within the explant;
• the sensitivity of the plant tissue to auxin (and other hormones as well);
the interaction, if any, between applied auxins and the natural endogenous substances
...
But endogenous
occurrence of 4-chloro-IAA (2) (Engvild, 1985) and of indole-3-butyric acid (IBA) (3) (LudwigMüller and Epstein, 1991) have also been demonstrated
...
, 1967) and there are precursors and metabolites of IAA present in plant tissues, like indole3-pyruvic acid, tryptamine (Cooney and Nonhebel, 1991) or tryptophol (Rayle and Purves, 1967;
Percival et al
...

In addition, the intermediate of agrobacterial IAA Biosynthesis, indole-3-acetamide (5), has been
detected in plant tissues (Saotome et al
...
Most of the IAA produced within plants is
conjugated to other compounds to form esters, amides or glycosyl esters
...
Conjugation seems to be a mechanism for storing IAA in cells,
stabilising the level of free auxin in the plant, and metabolising its excess (Ljung et al
...
, 2004)
...

IAA may be used as an auxin in plant tissue culture media, but it tends to be oxidised in culture
media and is rapidly metabolised within plant tissues
...

IAA has also been used with other regulants to induce direct morphogenesis (including the
rooting of microcuttings), and for meristem and shoot cultures (e
...
, of Bougainvillea,
Chaturvedi et al
...
, 1981; Citrullus, Barnes, 1979; and Sinningia, Haramaki,
1971; Grunewaldt, 1977)
...
, 1997)
...

These analogues have different structures but similar biological properties and are also called
auxins
...

The synthetic auxins (including selenienylated IAA and 2,4-D) are most commonly used in
tissue cultures
...
g
...
) may serve as an efficient source of IAA in tissue cultures (Magnus et al
...


PHYSICAL AND CHEMICAL PROPERTIES OF AUXINS

Natural and synthetic auxins are low-molecularweight, organic substances, containing either an
Indole or an aromatic ring
...
With the exception of IAA they are
stable and persist in the media for tissue cultures
...
Both naturally occurring indolic auxins and their synthetic counterparts, in spite of their
different structures, have similar physiological effects
...
55 Å from a carboxyl group
...
The relative degree of activity of individual auxins in different growth processes is very
variable
...

Probably due to its high instability, IAA is usually less effective than synthetic auxins like 2,4-D
or NAA
...
Auxin biosynthesis is usually
thought to be more intensive in meristematic regions and young growing organs such as
rapidly growing leaves, apical buds, root tips, and developing inflorescences
...
The above is especially true for monocotyledons, whereas in dicotyledons the
highest concentration of IAA occurs in the subapical zone, which also grows most rapidly (Law
and Davies,1990)
...
IAA
concentrations are high in young, fast growing organs and decline with age and are affected by
external and Krekule, 1984)
...
g
...
The level is not constant during
the day: daily oscillations in IAA concentration in Chenopodium rubrum plants have been
described
...
Various diurnal fluctuations were observed in different
photoperiodic regimes
...
The age of the motherplant, the conditions
under which it has been growing and the season of the year at which explants are taken, can all
be influential (Cassells, 1979)
...
The presence of an associated
organised meristem can cause callus to grow more vigorously, or to be capable of
organogenesis (Fakhrai et al
...
This suggests that meristematic cells are particularly active
sites for the biosynthesis and/or the release of natural growth factors favouring cell growth
(Clare and Collin, 1974)
...
However, as described below, Trp-independent
pathways have also been considered
...

The most common IAA-biosynthetic pathway in plants appears to be the indolepyruvate one; it
begins by the transamination of Trp catalysed by the enzyme tryptophan transaminase (or
tryptophan amino transferase) (Forest and Wightman,1972)
...
, 1972; Purves and Brown, 1978)
...
(NAD-dependent Indoleacetaldehyde
dehydrogenase) or by oxidation (indoleacetaldehyde oxidase) (Wightman and Cohen, 1968;
Miyata et al
...

The indole-3-acetaldoxime pathway starts by formation of indole-3-acetaldoxime followed by
it’s conversion to Indole-3-acetonitrile and final hydrolysis (nitrilase) to IAA (Thimann and
Mahadevan, 1964; Normanly et al
...

Pathway is typical for, e
...
, Brassicaceae and Resedaceae
...
At this point this pathway
joins with the indolepyruvate pathway
...
e
...

There is one more IAA biosynthetic pathway starting from Trp: it takes place in some plant
pathogenic bacteria and consequently also in plants transformed by Agrobacterium
...
, 1987 for review)
...
, 1999)
...
, 1993)
...

Hence, all the above mentioned pathways exist in plants, some of them may function in parallel
or in some proportion and/or sequence depending on the existing physiological conditions
...
Conjugates are
compounds in which hormone molecules are bound in a covalent manner with other lowmolecular substances, and therefore lose their physiological activity
...

Auxin is known to form two main classes of conjugates: amides (peptides) and glycosyl esters
...
Glycosyl esters of

IAA involve compounds such as various isomers of o-indole-3acetyl-ß-D-glucopyranose and
indole-3-acetyl-myoinositol, indole-3-acetylglucosyl-rhamnose, etc
...
, 1999, Ljung et al
...
, 2004)
...

In contrast to conjugation reactions, auxin degradation is an irreversible process
...
The oxidative decarboxylation may be catalysed by a non-specific activity
traditionally called “IAAoxidase/peroxidase” and leads to the subsequent formation of
degradation products such as indole-3methanol, indole-3-aldehyde, methylene oxindole and
indole-3-carboxylic acid (Hinman and Lang, 1965; reviewed by Reinecke and Bandurski, 1987;
Bandurski et al
...
, 1999)
...
Similar pathways are functional also for degradation of some conjugates,
e
...
, indole3-aspartate (see Slovin et al
...
The knowledge on the whole complex
of auxin

Metabolism (including biosynthesis) has been recently summarised in Ljung et al
...
, (2004)
...
, 1987, Klems et al
...
This reversible
Conjugation may regulate level of free active substances

EFFECT OF SYNTHETIC AUXINS ON IAA LEVELS

The synthetic auxins, which are which are added exogenously to control the growth and
organisation of cultured tissues, may affect endogenous IAA levels
...
Callus cultures of Arabidopsis thaliana can be initiated and
maintained on a medium containing 2,4-D, but progressively lose their morphogenic capacity
the longer they are maintained on it, until after 6-8 months there is no regeneration at all
...
, (1979) to be closely paralleled by a
progressive decline in the activity of peroxidase enzymes through successive transfers of the
callus
...
Reduction in peroxidase levels would be expected to result
in higher endogenous IAA Concentrations, but the decrease in peroxidase enzymes appears in

this case to have been associated with a decrease in natural auxin biosynthesis
...
,
(1998) found a correlation between decreased rhizogenic activity and the activity of a rootspecific peroxidase in tobacco callus
...
, 1992)
...
This suggests that 2,4-D interfered directly with IAA synthesis or
hastened IAA conjugation/degradation
...
, 1978)
...
, 1995)
...

In carrot hypocotyl explants, neither 2,4-D nor NAA, both of which induced callus formation,
had any effect on endogenous IAA concentration
...
, 1996)
...
This was probably due to the high intracellular
content of 2,4-D in Arabidopsis cultures created during preceding cultivation in 2,4D-containing
medium and its release following transfer to growth-regulator-free medium (Meijer et al
...


STABILITY IN CULTURE MEDIA

IAA and to some extent also IBA are heat labile and decompose during autoclaving
...
In the dark, there can be more than a ten-fold
decrease in concentration over a four-week period in the absence of inocula (Campbell and
Sutter, 1986; Nissen and Sutter, 1988)
...
, 1986)
...
75 mg/1) IAA was reduced to less
than the limit of detection (0
...

Because, for oxidative degradation, oxygen is required, in solid media the degradation of IAA is
likely to be significantly less
...
Other auxins such as NAA and 2,4D are not oxidized (Dunlap et al
...


TRANSPORT OF AUXINS

Auxins seem to be the only group of plant hormones exhibiting on the level of the whole plant
or its parts active transport in a polar manner in addition to long-distance movement via
vascular tissues (Hopkins, 1995)
...
The free IAA present in the
phloem sap is probably synthesised and exported from the mature leaves
...

The phenomenon of polar auxin transport was demonstrated first in the late 1920s (Went, 1928)
...
The polarity of auxin transport was explained (Rubery and Sheldrake, 1974; Raven, 1975)
by the different permeability of opposite parts of cells for dissociated and undissociated
molecules of indole-3-acetic acid (IAA and IAAH, respectively), and by an asymmetric
localisation (basal in the stem cells) of a so-called auxin efflux carrier (translocating the IAA
anion outside the cell)
...
Now, it is believed – on the basis of biochemical,
physiological and molecular biological data that at the level of the individual cell, both passive
diffusion and an auxin-uptake (influx) carrier drive auxin translocation into the cell while an
auxin efflux carrier drives auxin transport out of the cell
...
g
...
, 1995; Bennett et al
...
Now several genes are known, coding for both putative carriers and for the
NPA-binding site
...
,
1998; Morris, 2000; Friml and Palme, 2002; Morris et al
...
The effects of potential auxin
transport inhibitors on tissue and organ culture are described below
...
, (1996)
have studied auxin accumulation at the cell level in detail
...

On the basis of different behaviour of both auxin uptake and efflux carriers towards NAA and
2,4-D, Delbarre et al
...
This approach has been used for identification of new auxin
inhibitors of aryl and transport aryloxyalkylcarboxylic acid type (Imhoff et al
...
Based on
studies of auxin transport inhibitors, the hypothesis arose that, unlike the auxin uptake carrier,
the auxin efflux carrier has a rather complex structure
...


As no nervous system is present, the main signalling systems are hormone-dependent
(Libbenga and Mennes, 1995)
...
Auxins and cytokinins
impact at several levels in many different processes of plant development
...
e
...
The auxin:cytokinin ratio
represents an important signal in the formation of cell phenotype and also in the onset and
maintenance of the process of cell division (Stickens et al
...

The ability of auxins (together with cytokinins) to manage key events in plant morphogenesis
was documented, among others, by Skoog and Miller's (1957) discovery of the regulation of
organogenesis in vitro by means of the auxin:cytokinin ratio in culture media
...
g
...
, 1994; Leyser et al
...
, 1996)
...
Nevertheless, the main steps in auxin- (as well as other hormone-) signalling
can be generally described as:
1
...
the signal transduction cascade,
3
...
Receptor-like auxin-binding proteins have been identified and
characterised by various techniques (traditional ligand-binding studies, photoaffinity labelling
and genetic approaches) as recently reviewed by Napier et al
...
, (2004)
...
e
...
This protein exists in the form of a dimer of 22-kDa subunits
...

Additionally, several other genes encoding this auxin-binding protein (ABP1) have been
sequenced from other plants (Arabidopsis thaliana and other dicots including tobacco
(reviewed in Napier et al
...
All homologues share a common primary amino acid sequence
containing an N-terminal signal peptide for transit into the endoplasmic reticulum,
glycosylation site and the C-terminal KDEL (LysAsp-Glu-Leu) sequence for retention in the

lumen of endoplasmic reticulum
...
, (2002)
...
, 1981 and references therein, Zazimalova et al
...
Nevertheless, one
Jones's (1994) statement: "There is not enough information to label any single ABP as the auxin
receptor
...


AUXIN SIGNAL TRANSDUCTION PATHWAY(S)

There are several perception/transduction mechanisms known in animal and plant cells
(Libbenga and Mennes, 1995; Walden and Lubenow, 1996)
...
,2001;
Scherer, 2002)
...


Recent findings suggested the involvement of
Targeted protein degradation in auxin signalling
...
Ubiquitin is a small and highlyconserved protein which facilitates protein degradation
...

On the other hand, if auxin controls the ubiquitin-mediated degradation of those proteins,
which are unique for particular phases of the developmental programme (Fig 5
...
, 2000;Leyser, 2001; Kepinski and Leyser, 2002; Hellman and Estelle, 2002;
Dharmasiri and Estelle, 2004)
...
Numerous
auxin-responsive genes have been identified (Takahashi et al
...
Several
families of genes have been identified in a variety of different plants and organs that were
rapidly induced after auxin treatment, namely Aux/IAA, GST (glutathione-S-transferase),
SAUR (small-auxinupregulated RNAs), ACC (aminocyclopropane carboxylic acid) synthase,
GH3 genes and many others (summarised in Guilfoyle, 1999)
...
The main players in the control of
transcription by auxin are two families of transcription factors: ARFS (auxin response factors),
which can bind to the auxinresponse elements within auxin-responsive genes, and Aux/IAA
proteins, repressors, the expression of which are auxin-regulated
...
At higher
auxin concentrations, the ARF-Aux/IAA complexes dissociate and Aux/IAAs are
ubiquitinated; ARFS then activate transcription (Ward and Estelle, 2001; Guilfoyle and Hagen,
2001; Kepinski and Leyser, 2002; Tiwari et al
...

Auxin controls ubiquitin-of hypothetical proteins (E1-E5, i
...
enzymes, repressors, transcription
factors, etc
...
Degradation of the
proteins necessary for one stage results in the commencement of the next one
...
( Buchanan et al
...


PHYSIOLOGICAL EFFECTS OF AUXINS
In most auxin effects, a bell-shaped concentration/activity curve can be observed
...
1 10 μM) the effect usually increases with concentration, but concentrations
higher than 10 µM are often inhibitory
...


AT THE CELLULAR LEVEL

A stimulatory effect on cell elongation can be demonstrated in segments of coleoptiles or stems
at physiologically significant IAA concentrations
...
1-10 µM
...
, 1971) and/or changes
in gene expression (see above)
...
This hypothesis can explain the time-course of a typical biphasic
auxin-induced elongation curve consisting of short-term (acid growth) and long-term (gene
expression) responses (see also Cleland, 2004 for recent review)
...
, 1991, Funada et al
...


Auxin, together with cytokinins, is also involved in bud initiation and growth
...
Auxins exert an effect on DNA

replication, while cytokinins seem to exert some control over the events leading to mitosis
(Jouanneau, 1971,cf Chapter 6)
...
Auxin starvation resulted in G2-arrest in tobacco cell suspension (Koens et al
...

Activation of cell division is also coupled with activation of cdc 2, the main cell cycle regulating
kinase (John et al
...
Cells are thought not to enter mitosis unless cytokinin is present
...
Auxin is gradually canalised by a positive
feedback mechanism where increasing conductivity of auxin conducting cells leads to canals of
cells efficiently transporting auxin (Sachs, 2000)
...

Inhibition of polar auxin transport leads to many abnormalities and in embryos it can lead to
death (Liu et al
...

Auxins are known for their ability to promote adventitious root formation
...
, 1993)
...
, 2001)
...
It has been
shown that IBA is readily converted to IAA, but it probably also has an effect on its own (van
der Krieken et al
...
Removal of the
tip, the main auxin source, or inhibition of auxin transport leads to the outgrowth of axillary
buds
...
Uneven distribution of auxin is
considered to cause differential growth rates in different sides (upper/lower or
irradiated/shaded) of coleoptile or root and their bending in gravitropic or phototropic
reactions (Friml, 2003)
...
g
...
In this respect, each tissue culture system is unique, and the effects of
different

Concentrations of auxins and other hormones must be tested for each case individually and
only to some extent can the results can be transferred to other cultures
...
Applied auxins seem to
be capable of fundamentally altering the genetically programmed physiology of whole plant
tissues, which had previously determined their differentiated state
...
How auxin brings about this
reprogramming is understood only to a very limited extent
...
, (1989) found that
auxins cause DNA to become more methylated than usual and suggested that this might be
necessary for the re-programming of differentiated cells
...
A high rate
of DNA methylation was found in the early somatic embryo stage in cultures of Cucurbita pepo
L (Leljak-Levanic et al
...


Irvine et al
...
From the effective compounds, 96% had structures known
to be associated with auxin activity
...
However, since cultures maintained on 2,4-D may become genetically variable,
some investigators prefer NAA or IAA, or a transfer of callus to a medium containing one of
these alternative compounds once it has been initiated by 2,4-D
...

To induce callus growth from explants of dicotyledonous plants, a cytokinin is usually added to
the medium in addition to an auxin
...
The combined use of
auxin and cytokinin in tissue cultures is considered separately in a later section
...
The relatively high levels of auxin added to liquid media to obtain dispersion will
prevent morphogenesis, but might induce embryogenesis if the cells are still competent
...


Some reduction of chlorophyll formation in the presence of 2,4-D was noted in callus cultures
of pea, tomato and potato by Hildebrandt et al
...
e
...
5 µM, or NAA from 50
to 5 µM)
...
Other workers have made similar observations
...
, 1973), but in other tests IAA or NAA have been reckoned to
be less inhibitory to chlorophyll formation than 2,4-D (Davey et al
...


ORGAN CULTURES

An auxin is almost invariably required to promote the initial growth of meristem and shoot tip
explants
...
It is important to choose an auxin at a concentration that will promote
growth without inducing callus formation
...
Rhizogenesis is usually achieved by treatment with auxin alone
...
, 2003)
...
Auxin-induced root formation is thought to require, or
induce, the promotion of polyamine synthesis (Friedman et al
...


Sometimes tissues, organs or strains of cells arise that are able to grow without the addition of
any auxin to the medium
...
Sharp et al
...
It was suggested that
division of the pro-embryogenic cells and their development into embryos is only resumed at
lower auxin concentrations
...
It is possible that in
these instances, embryogenesis has been induced by endogenous auxin, the concentration of
which has then been subsequently reduced by metabolism to permit embryo formation
...
, 2002)
...
, 2002)
...
Embryo
formation coincides with the withdrawal of auxin and a rise in cellular pH
...
, 2002)
...
, 1989), subjecting tissues to high
osmotic potential (0
...
6 M mannitol in Ishikawa et al
...
3 M NaCl in
Kiyosue et al
...
5-1
...
, 1990)
...
In the induction of somatic embryogenesis from immature cotyledons of Glycine
max, Lazzeri et al
...
The number of embryos obtained was reduced if the
concentration ratio of auxin: sucrose was high, or vice versa
...
, 2001)
...
, 2002)
...
NAA and IBA are favoured auxins for shoot culture
...
1) is used only rarely in tissue cultures, and then almost exclusively for the
induction of callus and indirect embryogenesis in monocotyledons such as Avena, Oryza, and
Panicum
...
, (1983) found that in Triticum aestivum, some varieties produced
embryogenic callus with 2,4-D, while others responded only to 2,4,5-T
...
1) is often effective in inducing the formation of embryogenic callus in
monocotyledons
...
5
µM) (Jarret et al
...
5-18
...
The use of 9
...
6-4
...
6 µM) (Carman et al
...
This is probably because dicamba is metabolised quickly in
wheat (Chang and Van den Born, 1971), possibly more quickly than 2,4D
...
1) is sometimes used to induce and/or maintain callus
or suspension cultures of broadleaved trees, or to induce the formation of embryogenic callus,
where it may be more effective than 2,4-D (Beyl and Sharma, 1983)
...
g
...
06-4
...
Mok and Mok (1977) found that the growth rate and yield of callus from different
species and varieties of Phaseolus, were greater in the presence of picloram than with 2,4-D
...
In only a very
few instances has this auxin been used for meristem or single node culture and then at very low
concentrations (e
...
, 0
...
4 µM) in combination with a cytokinin
...
, 2003)
...
e
...
1) (Tadino et al
...
1) induce
somatic embryogenesis in Panax ginseng (Kevers et al
...


At high concentrations most synthetic auxins are phytotoxic to field-grown broad-leafed plants
...
This detrimental effect was reported also for high levels of IAA and is probably due
to a dramatic increase in ethylene production
...


An unusual auxin 2-chloro-3(2,3-dichlorophenyl) propionitrile (CDPPN) (9), which is related to
orthonil (PRB-8) (10), was found by Nemeth (1981) to produce up to 90% more shoots in apple
shoot cultures than the same concentration of IBA (5µM)
...
In
these cases it may stimulate growth or induce morphogenesis (e
...
, callus growth of Nicotiana
glauca x N
...


MIXTURES OF AUXINS

Some investigators have employed mixtures of many different auxins (e
...
, Blackmon et al
...
However, a mixture of more
than one auxin can be particularly effective for root induction and a mixture of a synthetic auxin
and IAA has been found by many workers to be more effective than the synthetic compound on
its own
...
,
1983)
...
, (1988), who found that adding 0
...
62 µM 2,4-D, consistently enhanced the formation of somatic embryos from
scutellum callus of wheat genotypes
...
As mentioned above, mixtures of auxins are also more
effective in inducing regeneration of wheat, barley and triticale (Przetakiewicz et al
...


AUXIN UPTAKE AND METABOLISM IN TISSUE CULTURES

It is not quite correct to talk about uptake, because what we can measure, is in fact
accumulation, i
...
, the amount of a regulator in a tissue, which was taken up from the medium
and not yet metabolized
...
The compounds are subsequently
absorbed into cells as whole molecules (via uptake carrier or diffusion, see above), but
dissociation then causes them to be retained within the cell, because the plasmalemma is
impermeable to auxin anions (Norris and Bukovak, 1972; Raven, 1979; Edwards and Goldsmith,
1980; Minocha and Nissen, 1985; Minocha, 1987)
...
Besides uptake
through the tissue surface, in cultures using segments, diffusion through the cut surface must
be taken into account
...
The rate of uptake of NAA
into tobacco pedicel explants was proportional to the concentration in the medium and its
presence is necessary for 4 d only (Smulders et al
...


Total 2,4-D uptake (¹4C-labelled) was found to be higher in easy-to-root juvenile clones of
Sequoiadendron giganteum than in difficult-to-root mature stem cuttings (Berthon et al
...

An unequal distribution of free 2,4-D between apical and basal ends of cuttings was found in
both types of shoots; the accumulation was higher in the basal parts
...
2,4-D uptake and
metabolism have also been studied in embryogenic and non-embryogenic maize lines
...
The embryogenic line also metabolised 2,4-D more actively than the
nonembryogenic one (Bronsema et al
...
Another comparison of 2,4-D uptake, distribution
and metabolism was performed with explants of cucumber (Cucumis sativus L
...
Cotyledon explants take up more 2,4-D, have a more pronounced basipetal
gradient of 2,4-D level and conjugate 2,4-D more actively than hypocotyl explants (Fig
...
4;
Klems et al
...


EFFECT OF AUXIN TRANSPORT INHIBITORS ON SHOOT CULTURES

Polar transport of auxin can be inhibited by NPA (8) or 2,3,5-triiodobenzoic acid (TIBA) (11)
...
2,4,6Trichlorophenoxyacetic acid (2,4,6-T) (12) and p-chlorophenoxyisobutyric acid (PCIB) (13) are
probably genuine anti-auxins
...


Application of two drops of a 2µM solution of TIBA to the apices of cultured rose shoots had the
same effect as manually tipping the shoots, and increased the number of axillary shoots
subsequently produced
...

TIBA or B-NAA improved adventitious root generation in sugar beets as well
...
NPA prevented
the growth of tobacco callus when incorporated into the medium at 200 µM, but 2 20 µM
promoted growth in conjunction with IAA
...
5 µM kinetin initiated buds only when NPA was present (Feng and Linck, 1970)
...

PCIB (13) counteracted the inhibitory effect of 2,4-D on adventitious bud formation on sections
of Chondrilla juncea roots (Kefford and Caso, 1972) and, together with the cytokinin
benzyladenine, it promoted the formation of adventitious shoots from Solanum melongena
callus, when combinations of IAA and BAP were ineffective
...
, (1978) thought this
might be because PCIB overcame excessive endogenous IAA in the tissue
...
,on rooting of apple microcuttings (Fig
...
5)
...
, (1982) similarly suggested that explants of some species may have a too high
level of endogenous auxin for shoot buds to be produced either directly or from associated
callus
...
5 – 5 µM TIBA but no auxin, caused a marked increase in shoot bud formation, probably
because the TIBA prevented endogenous auxin moving to sites of potential shoot bud
formation
...
These could be induced to develop into shoot buds
when TIBA was added to the medium
...


Callus of Colocasia esculenta produced normal shoots when 50 µM TIBA was added to the
medium whereas shoots produced with combinations of auxin and cytokinin had an abnormal
morphology (Nyman and Arditti, 1984)
...
, 1984)
...
Regeneration of roots on tomato hypocotyl cuttings is suppressed by TIBA (Tyburski
and Tretyn, 2004)
...
, 2003)
...
In anther cultures of Hordeum (Clapham, 1973) and Zea mays
(Genovesi and Collins, 1982), TIBA was more effective than normal auxins in inducing callus
formation and embryogenesis
...
They took particular care to wash residual auxin from cell
clusters and thought that the stimulatory effects of anti-auxins noted by others, were due to
their failure to take this precaution
...
, 2002),
Whereas in culture of Elutherococcus senticosus, TIBA suppressed embryo formation while cell
division was not affected (Choi et al
...


Auxin polar transport was reported to be essential for bilateral symmetry during early plant
embryogenesis
...
2) and
9hydroxyfluorene-9-carboxylic acid (16) induced formation of fused cotyledons in in vitro
culture of early globular embryos of Indian mustard (Liu et al
...
Also the symmetry of
zygotic embryos is affected by auxin polar transport
...
e
...
The relative
position of the shoot apical meristem was anomalous and no root meristem differentiated
(Fischer and Neuhaus, 1996)
...
BNAA, phenylpropionic acid, and 2-(o-chlorophenoxy)-2methylpropionic acid, together with
TIBA, prevented the re-callusing of indirectly formed Sapindus trifoliatus embryos, but did not
permit embryo germination
...
The compound seemed to act as an anti-auxin (Desai et al
...


GROWTH REGULATORY EFFECTS OF PHENOLS

Compounds that carry one or more hydroxyl groups on an aromatic ring are termed phenolic
compounds (for examples see Table 5
...
There are many papers on the growth regulating
properties of phenolic compounds in tissue cultures
...
Most plant responses have involved a synergism with auxins, particularly IAA,
so that a mode of action that is dependent on the regulation of internal IAA levels has seemed
probable
...
This has led to two hypotheses as to their growth regulatory activity:

1
...
,
1970; James and Thurbon, 1981b
...
Some monophenolics increase the rate, while some 3-substituted phenols
depress it
...
Relatively large amounts of
natural inhibitors of IAA oxidase have been reported to be present in meristematic and
juvenile tissues, but not in normal mature differentiated cells until they are wounded
(Stonier and Yoneda, 1967; Stonier, 1969)
...
Growing tobacco cells on media that favour the
induction of auxin habituation (see Syono and Furuya, 1974) caused an increase in an
inhibitor of auxin destruction (Syono, 1979)
...
g
...
, 1969;
Hammerschlag, 1982) have questioned whether the stimulatory effect of phenols in
promoting rooting is not due to some other function than that of preventing IAA
destruction
...
,
2000)
...
On the other hand, enhancement of quercetin (Table
5
...
, 2001)
...
Morphogenic activity is induced by the products formed when compounds such as
phloroglucinol (18) and phloridzin (19) (see below) are oxidised
...
, (1988)
advanced this hypothesis when they found that phloroglucinol only promoted rooting
in apple clones with sufficient polyphenol oxidase activity to cause significant oxidation
of the compound
...
It should be noted however that phenols may have other, unexpected roles
...
2) is now emerging as an important component of plant responses to
DIOTIC stress

CORRELATIONS WITH ENDOGENOUS LEVELS

The natural rate of formation of some phenolic compounds has been observed to depend on the
rate of growth of cultured tissues (Barz, 1977) and on auxin/cytokinin levels in the medium
(Sargent and Skoog, 1960; Skoog and Montaldi, 1961)
...
A rise in the content of endogenous phenolics, including monoferuloyl,
monocaffeoyl and mono-p-cumaroyl tartaric acids, was observed during root formation in Vitis
vinifera in vitro (Mato et al
...
Correlation between flavonoid accumulation and root
formation was also described in Eucalyptus gunii (Curir et al
...
Additionally, a correlation
between level of endogenous derivatives of cinnamic and benzoic acids with embryogenesis
was reported in Medicago falcata (Cvikrová et al
...


Rawal and Mehta (1982) found that shoot formation from haploid tobacco callus occurred as the
content of natural phenolic substances declined, while cellular differentiation occurred with
increasing phenolic accumulation
...
Also, the level of aromatic amines, like tyramine or phenetylamine, seems
to be correlated to morphogenetic events (Martin-Tanguy and Carre, 1993, Cvikrová et al
...
On the other hand, a decreased content of phenolic acids (mainly derivatives of cinnamic
acid (Table 5
...
) cell suspension culture after treatment
with an inhibitor of phenylalanine ammonia lyase (PAL), 2-aminoindan-2-phosphonic acid
(AIP) (20), was connected with a decreased level of IAA, lower IAA-oxidase activity in later
stages of the culture and with slower growth of the culture (Hrubcová et al
...
In
embryonic cultures of sessile oak, inhibiton of phenylpropanoid synthesis by AIP led to
increased number of well developed somatic embryos (Cvikrová et al
...


ROOT FORMATION

Some observations on natural levels of auxin protectors might suggest that their low levels are
coupled with root initiation, but high levels with root growth
...
, 1982)
...
The activity of peroxidases in the induction medium increased during 7-11
days and then decreased, roots appearing as phenols were decreasing (Monteuuis et al
...

In each of these plants, peroxidase activity was inversely correlated with phenol content
...
The best time to

explant shoot tips from adult chestnut material to a rootinducing medium, was during one of
the first two peaks of growth of shoots, which coincided with the occurrence of maximum
quantitites of natural
...
4-chlororesorcinol (21), a
polyphenol oxidase inhibitor (i
...
inhibiting the conversion of monophenols and dihydric
phenols to polyphenols) has been found to improve the rooting and subsequent growth of
cuttings (Gad et al
...
In shoot cuttings of Cedrus deodara adventitious rooting was
stimulated both by IBA and by coumarin (Table 5
...
, 2002)
...

James and Wakerell (1982) found that the compound was without effect on the apple variety
M26’, but promotory on ‘M9’, while Whiteley and Abbott (1977) reported that the growth of
shoot cultures of Malus ‘Golden Delicious’, ‘Egremont Russet’ and ‘Bramley’ was completely
inhibited by 0
...
Hutchinson (1985) found that 1 mM phloroglucinol
more than doubled the number of shoots produced by the apple cultivar ‘Northern Spy’ during
the first two subcultures: there was no increase in shoot number by the fourth subculture
...
For
example, added to the medium at 500 µM, phloroglucinol (and catechol- see below) increased
adventitious shoot formation from Rubus callus (Compton and Preece, 1988)
...
g
...
, 1982)
and Ficus carica (Moraceae) (Pontikis and Melas, 1986)
...
Adding phloridzin (19) and phloroglucinol to the
medium, increased the number of somatic embryos produced from embryogenic callus of oil
palm (Hanower and Hanower, 1984)
...
This effect has been especially noted in several apple cultivars (Jones et al
...
Welander and Huntrieser (1981) found that IBA plus 0
...
9 mM IBA plus 1 mM phloroglucinol induced the best
rooting of juvenile shoots
...
, 1980), strawberry and Rubus genotypes in
conjunction with IBA (but not NAA) (James, 1979; Sobczykiewicz, 1987)
...
, 2003), in micro-shoots of Decalepis hamiltonii (Reddy et al
...
Interestingly, phloroglucinol is able to
suppress accumulation of phenolic substances in the callus of water chestnut, enabling plant
regeneration from the callus (Hoque and Arima, 2002)
...
, 1982)
...
Phloretic acid was also found by Jones and Hatfield
(1976) to increase the proportion of apple shoots that could be rooted, but it was less active than
phloroglucinol
...
g
...
, 1999)
...
The stimulatory effect of the
compound is, however, now thought to be largely independent of this effect (Jones and
Hopgood, 1979; Jones and James, 1979)
...


THE EFFECT OF CATECHOL

Catechol is another strong reducing agent that has been reported to regulate the rate of IAA
oxidation in plant tissues
...
m²
...


However, 60 µM IAA together with 50 µM catechol resulted in a rooting response that was
almost equal to that produced by the optimum NAA level, suggesting that catechol protected
IAA from light-induced degradation At low irradiance (7 umol
...
s¹), IAA alone produced
almost as many roots as IAA + catechol in high lighting
...


Catechol (and phloroglucinol, 500 µM), increased the number of adventitious shoots formed
from leaf callus of tobacco (Compton and Preece, 1988)
...
This last test was not conducted under sterile conditions so that

catechol could have acted as an antiseptic
...


OTHER PHENOLIC COMPOUNDS

Like catechol, chlorogenic acid (Table 5
...
It is a strong
reducing agent, which has been noted to stimulate callus growth of Prunus avium stem
segments (Feucht and Johal, 1977; Feucht and Schmid, 1980) and to promote the growth of olive
callus (Lavee and Avidan, 1982)
...
2),
chlorogenic acid has been added routinely to media containing NAA and IBA to promote
rooting of Beta vulgaris (Margara, 1977) and Brassica (Margara and Leydecker, 1978) shoots in
vitro
...
Shoots kept in darkness all rooted in response to IAA alone
...
Compounds of this kind have been found to affect a wide variety of processes,
low levels sometimes exerting a stimulatory role, but higher levels are often inhibitory
...

Scopoletin (Table 5
...


Coumarin and related compounds, have been found to both stimulate and decrease protein
synthesis, respiration and photophosphorylation and decrease carbohydrate metabolism
(Brown, 1981), but increased growth in conjunction with IAA has been reported (Neumann,
1960)
...


Tobacco pith callus grown on the medium of Linsmaier and Skoog (1965) with 12 µM IAA and
12 µM kinetin is wholly unorganised, but if grown on which is auxin-habituated (i
...
, auxin
autotrophic) the same medium supplemented with 600 µM Ltyrosine (23), 80 µM adenine
sulphate and 2
...
Tyrosine is a substrate for the enzyme phenylalanine ammonia
lyase which converts it to pcoumaric acid
...
2) than callus, (Zador et al 1985)
Other compounds, which have been suspected to be naturally-occurring inhibitors of IAA
oxidase, may increase callus growth in certain circumstances
...
, 1962;
Thimann, 1963; Feucht and Nachit, 1978), catechin and flavandiols (Feucht and Nachit, 1977;
Feucht and Schmid, 1980) and chemicals of the B-inhibitor complex’ (BennettClark and Kefford,
1953)
...
, 1967), and various phenolic acids such as caffeic acid, chlorogenic acid, and
sinapic acid (Thimann, 1963)
...
It was
shown to participate in the mechanism of colonization of alfalfa roots by Sinorhizobium meliloti
(Yang et al
...
,
1996) and to protect auxin from oxidation (Brennan, 1996)
...
Ethylene accumulated in the tissue culture vessels may then inhibit the growth
and

Development of many tissue culture grown plants
...


GIBBERELLINS
There are more than 100 gibberellins (GA1, GA2, GA3…
...
They are acidic in
nature
...

Gibberellins (Gas), a class of diterpenoid phytohormones, produced by plants and some fungi
play an important role in modulating diverse processes throughout plant growth and
development
...
Recent studies on GA biosynthesis,
metabolism, transport, and signaling, as well as cross talk between GA and other plant

hormones and environmental cues have achieved great progress along with the advancement of
molecular genetics and functional genomics
...

Bioactive Gas promote plant growth and development by promoting the degradation of the
DELLA proteins, a family of nuclear growth repressors
...

Evidence also shows that Gas act as mobile molecules that can pass through the plasma
membrane for cell-to-cell transport
...


FUNCTIONS OF GIBBERELLINS

Gibberellins (Gas) are a large family of tetracyclic diterpenoid plant hormones that regulate
many different aspects of plant growth and development through the entire life cycle of the
plant, including promotion of cell division and elongation, seed germination, stem and
hypocotyl elongation, root growth, and flowering induction (Daviere et al
...
, 2016)
...
, 2008, 2012)
...
The rice plants infected by the pathogenic fungus Gibberella fujikuroi
exhibited excessive stem elongation such that they fell over easily (Silverstone and Sun, 2000)
...

There are at least 136 fully characterized Gas, named from GA1 to GA136, which have been
identified from various bacteria, fungi, and plants (Hedden and Thomas, 2012; Silverstone and
Sun, 2000)
...
The genetic evidence has revealed that
GA1 and GA4 are major active Gas in most plant species although GA3 has been identified in
plants
...
, 1998; Magome et al
...
, 2013; Ueguchi-Tanaka et al
...

GA homeostasis is tightly feedback controlled by GA metabolism and signaling (Hedden and
Kamiya, 1997)
...
The rice
SD1 (SEMI-DWARF1) gene encodes a key GA biosynthetic enzyme, OsGA20ox2, and the wheat
Rht1 gene encodes the GA repressor DELLA (Peng et al
...
, 2002; Spielmeyer et
al
...
Therefore, GA biosynthesis and signaling are critical to plant growth and
development, and also of vital importance in agriculture
...


GIBBERELLIN BIOSYNTHESIS, INACTIVATION, TRANASPORT AND
REGULATION
GIBBERELLIN BIOSYNTHESIS PATHWAY

More than 136 Gas have been identified so far
...
, 2007; Yamaguchi, 2008;
Yamaguchi and Kamiya, 2000; Zi et al
...

Only a few Gas, including GA1, GA3, GA4, and GA7, exhibit biological activity toward various
aspects of plant growth and development in the whole life cycle, such as seed germination, stem
elongation, leaf expansion, flowering, and fruit and seed development (Hedden and Sponsel,
2015; Plackett et al
...
, 2004)
...


The GA biosynthesis pathway has been well established through extensive biochemical and
genetic studies in higher plants over a long period, which requires a set of enzymes, including
terpene synthases (TPSs), cytochrome P450 monooxygenases (P450s), and 2-oxoglutaratedependent dioxygenases (2ODDs)
...
These enzymatic reactions, as well as the production of bioactive Gas, are strictly
and precisely controlled in both their timing and subcellular localization at the tissue and organ

levels (Hedden and Thomas, 2012; Itoh et al
...
Accordingly, GA
biosynthesis can be generally divided into three stages in three subcellular compartments:
plastids, the endomembrane system, and the cytosol (Fig
...
1) (Han and Zhu, 2011; Hedden and
Thomas, 2012; Yamaguchi, 2008)
...
, 1997; Helliwell et al
...
, 1998; Sun and Kamiya, 1994)
...
4
...
Loss-of-function
mutations of CPS or KS in Arabidopsis and rice cause severe GA-deficient dwarf phenotypes,
whereas overexpression of both CPS and KS greatly increases levels of the early intermediates
ent-kaurene and ent-kaurenoic acid, with only slight increases in later metabolites (Sakamoto et
al
...
, 1992; Yamaguchi et al
...
As a result, these overexpression lines have
wild-type levels of bioactive Gas and do not exhibit any GA overdose phenotypes (Fleet et al
...
, 2004)
...
This GA homeostasis is likely
attributed to the strict control of gene expression
...
, 2003; Olszewski et al
...
, 1997a)
...
, 2003; Koornneef
and Van der Veen, 1980; Silverstone et al
...
The AtCPS (also called GA1) gene was first
isolated and identified from the ga1 mutants, also known as “non-germinating GA-dwarfs”
(Aach et al
...
, 2001b; Sun and Kamiya, 1994; Sun et al
...
The
Arabidopsis ga1 mutants are male-sterile dwarfs whose phenotypes can be converted to the
wild-type by repeated application of exogenous GA (Koornneef and Van der Veen, 1980)
...
, 2001)
...
,
2004)
...
, 2014)
...
The OsCPS1 protein is essential for GA biosynthesis (Toyomasu et
al
...
The rice loss-of-function mutation in OsCPS1 exhibits GA-deficient dwarfism,
indicating its critical role in early GA biosynthesis in rice
...
However, OsCPS2 can complement the severe dwarf phenotype of the

Oscps1-1 mutant with a transgene driven by the OsCPS1 promoter (Sakamoto et al
...
, 2015)
...
Similarly, two pumpkin
CPS genes, CmCPS1 and CmCPS2,, display different expression patterns
...
These CPS genes may fulfill
their functions in GA biosynthesis at different developmental stages (Smith et al
...

Therefore, proper tissue or cell-specific expression of CPS genes is critical for their biological
roles in the biosynthesis of Gas and other terpenoids; this also suggests that Gas act in specific
cells
...
, 1996, 1998b)
...
By contrast, the rice
genome contains a family of KS-like (OsKSL) genes, including a pseudogene OsKSL3, which are
responsible for the biosynthesis of various diterpenoids (Tezuka et al
...
, 2007)
...
, 2005; Sakamoto et al
...
, 2007)
...

The ZmKS1 locus contains a tandem array of three TPS genes, ZmTPS1, ZmKSL3, and ZmKSL5
...
, 2016)
...


In the second stage of the GA biosynthesis pathway, the conversion of entkaurene to GA12 via
stepwise oxidation is catalyzed by ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase
(KAO)
...
, 1998, 1999, 2001a; Ko et al
...
, 2010)
...
, 1998, 1999)
...
The loss-of-function mutant of GA3 is also GA-deficient dwarf
...
, 1998), consistent with high levels of bioactive Gas during plant flowering
...
In the rice genome, genes for five CYP701A subfamily members, OsKO1 to

OsKO5, are tandemly arrayed on chromosome 6 as a result of gene duplication (Sakamoto et al
...


Notably, only OsKO2 (D35) exhibits KO activity and is required for GA biosynthesis (Itoh et al
...
, 2012)
...
The pea (Pisum sativum) homologous gene PsKO1 (also known as LH) of the
CYP701A subfamily was isolated and confirmed to have KO activity in GA biosynthesis
...
, 2004)
...
Moreover, expression of the KO gene is not under feedback regulation by Gas
(Hedden and Phillips, 2000; Olszewski et al
...


The next three steps of the GA biosynthesi busys pathway, from ent-kaurenoic acid, ent-7hydroxykaurenoic acid, and GA12-aldehyde to GA12, are catalyzed by the action of KAO
(Helliwell et al
...
These reactions occur on the outer membranes of the plastid and
require the transport of ent-kaurene from the organelles by an unknown mechanism
...
Two KAO genes in Arabidopsis,
AtKAO1 and AtKAO2, that encode CYP88A proteins are expressed in all tissues from
germinating seeds to developing organs (Helliwell et al
...
, 2014)
...
In comparison
with wild-type plants, the Atkao1 Atkao2 double mutant exhibits the typical GA-deficient
phenotype, similar to that observed in the severely GA-deficient ga1-3 mutants
...
Moreover, AtKAO1 and AtKAO2 exhibit
equivalent enzymatic activities in GA catabolism in a yeast expression system (Helliwell et al
...
, 2014)
...
, 2014)
...
The Oskao mutant also has a severe dwarf phenotype without flower or seed
development with extremely low levels of intermediates and bioactive GA1 (Sakamoto et al
...
Similar to Arabidopsis, pea has two KAO genes, PsKAO1 (also known as NA) and
PsKAO2
...
In these organs, GA
levels are greatly reduced in the na mutant
...
, 2003)
...
By contrast, in the third stage of GA biosynthesis, GA12 and GA53 are
converted to various GA intermediates and bioactive Gas by GA 20-oxidase (GA20ox) and GA
3-oxidase (GA3ox) through two parallel pathways
...
,
1995; Plackett et al
...
, 2013; Sponsel et al
...
, 1995)
...
The non-13-hydroxylation branch has been
extensively studied, and all key enzymes have been identified
...
GA12 is also a substrate for the 13-hydroxylation branch in the production of
GA53 (13-OH GA12), which is a precursor for GA1 (Phillips, 1998; Yamaguchi, 2008)
...
Recently,
two rice cytochrome P450 genes, CYP714B1 and CYP714B2, were found to encode GA13ox
(Magome et al
...
Recombinant CYP714B1 and CYP714B2 can convert GA12 into GA53
...
GA4 is more
active than GA1 in stem elongation in rice (Zhu et al
...
Interestingly, transgenic
Arabidopsis plants overexpressing CYP714B1 or CYP714B2 are dwarfs due to the decreased
levels of GA4 in spite of the increased levels of GA1, supporting the notion that GA4 is more
active than GA1 in promoting plant growth (Magome et al
...
Therefore, CYP714B1 and
CYP714B2 play important roles in plant growth and development through catalyzing GA 13hydroxylation that finetunes GA homeostasis
...
GA9 and GA20 are precursors of bioactive Gas, which
are converted by GA20ox via oxidation of C-20 to an aldehyde followed by the removal of this
C atom and the formation of a lactone (Coles et al
...
, 1994; Yamaguchi, 2008)
...
A
loss-of-function mutant of AtGA20ox1 has a reduced stem height but otherwise appears to be
developmentally normal
...
, 1999; Hedden and Phillips, 2000; Huang et al
...
,
1995; Plackett et al
...
Loss-of-function mutant studies of AtGA20ox1 and AtGA20ox2
identified partial functional redundancy between the two paralogs, which have partially
overlapping expression patterns throughout development (Rieu et al
...
Through
systematic mutant analysis, AtGA20ox1, AtGA20ox2, and AtGA20ox3 were shown to have

significant effects on floral organ and anther development, while AtGA20ox4 and AtGA20ox5
were shown to have very minor roles (Plackett et al
...
The OsGA20ox2 (SD1) gene is wellknown as the “Green Revolution” gene and is the most important gene employed in modern
rice breeding (Ashikari et al
...
, 2002; Qian et al
...
, 2002)
...
Another
gene, OsGA20ox1, has also been identified and shown to affect plant stature (Oikawa et al
...
, 1997)
...
, 2012; Yano et al
...
OsGA20ox1 and
OsGA20ox2 control plant stature through coordinative regulation of bioactive GA levels
(Ashikari et al
...
, 2004)
...

GA3ox enzymes are also encoded by multiple genes in all plant species
...
, 1995; Hedden and
Phillips, 2000; Itoh et al
...
, 2011; Yamaguchi et al
...
The AtGA3ox1
(GA4) gene was genetically identified by T-DNA tagging and expression in Escherichia coli
(Chiang et al
...
, 1998)
...

While Atga3ox2 single mutants do not have any phenotype, the Atga3ox1 Atga3ox2 double
mutant has a smaller leaf diameter, a shorter phenotype, and a severe seed germination defect
compared with the Atga3ox1 single mutant (Mitchum et al
...
In rice, OsGA3ox1 and
OsGA3ox2 (D18) have been functionally identified (Itoh et al
...
Different expression
patterns of the two OsGA3ox genes were found in vegetative and reproductive organs:
OsGA3ox2 was broadly functional in vegetative and reproductive organs, whereas OsGA3ox1
preferentially functioned in the reproductive organs (Itoh et al
...
, 2003;
Sakamoto et al
...
ZmGA3ox2 is the causative gene for the maize dwarf1 (d1) phenotype
(Chen and Tan, 2015)
...
Therefore, different paralogous ODD enzymes have redundant
functions in GA biosynthesis
...
, 1999; Huang et al
...
, 2004; Plackett et al
...
Whether GA 3oxidation is a rate-limiting step in GA biosynthesis remains controversial
...
, 2004)
...
, 2008)
...
, 2006)
...
, 2013)
...
2
...
In general, the majority of Gas are inactive, providing a means to
rapidly regulate their bioactive levels
...


Deactivation mechanisms and pathways to effectively regulate bioactive hormone levels are
critical for proper plant growth and development
...
The most studied
deactivation pathway is 2β-hydroxylation, which is catalyzed by a class of GA 2-oxidase
(GA2ox) enzymes that are soluble 2- oxoglutarate-dependent dioxygenases (2ODDs) (Huang et
al
...
, 2008; Olszewski et al
...
, 2014; Thomas et al
...
The GA2ox family was characterized through genetic, transgenic, and
biochemical approaches
...
,
2010; Lee and Zeevaart, 2005; Lo et al
...
, 2001b; Schomburg et al
...
, 2014)
...
,
2001b; Schomburg et al
...
, 2014; Thomas et al
...
Different GA2ox enzymes
convert bioactive Gas and their precursors as C20-Gas and C19-GA substrates to limit bioactive
GA levels and regulate many stages of plant development
...
The C20GA2ox
subgroup only acts on C20-GA precursors, such as GA12 and GA53, to form GA110 and GA97,
but not on C19-Gas
...
, 2008)
...
The GA2ox family
can be divided into three classes on the basis of the phylogenetic relationships (Lee and
Zeevaart, 2005)
...
Arabidopsis has five C19GA2ox genes AtGA2ox1, AtGA2ox2, AtGA2ox3,

AtGA2ox4, and AtGA2ox6 (AtGA2ox5, is a pseudo gene) (Olszewski et al
...
,
1999; Yamaguchi and Kamiya, 2000) and two C20GA2ox genes (AtGA2ox7 and AtGA2ox8)
(Schomburg et al
...
C19-GA2-oxidation limits bioactive GA content and regulates plant
development at various stages during the plant life cycle: C19GA2ox enzymes prevent seed
germination, delay vegetative and floral phase transitions, limit the number of flowers
produced per inflorescence, and suppress elongation of the pistil prior to fertilization (Rieu et
al
...
Genetic analysis revealed that C19GA2ox enzymes participate in a major GA
inactivation pathway that limits bioactive GA content and regulates plant growth and
development (Lo et al
...
, 2008a)
...
Double Atga2ox7 Atga2ox8 mutants had higher levels of active Gas
and displayed GA overdose phenotypes (Schomburg et al
...
In the rice genome, a total of
10 putative GA2oxs were identified, including seven C19GA2ox genes (OsGA2ox1-4,
OsGA2ox7-8,, and OsGA2ox10) and three C20GA2ox genes (OsGA2ox5, OsGA2ox6, and
OsGA2ox9), which are differentially regulated and act in concert or individually to control the
homeostasis of GA levels (Lee and Zeevaart, 2005; Lo et al
...
, 2001b, 2004;
Sasaki et al
...
, 2014)
...
, 2011)
...
4
...
The CYP714D1 gene
(well-known as ELONGATED UPPERMOST INTERNODE, EUI) encodes the GA 16α,17epoxidase in rice (Zhu et al
...
Then 16α,17- epoxides are converted to 16α,17-[OH]2-Gas (GA 16,17-dihydrodiols) by
EUI-related enzymes
...
,
2006); while the ectopic expression of EUI under the control of the 35S promoter or the
promoters of the rice GA biosynthesis genes OsGA3ox2 and OsGA20ox2 dramatically reduced
GA levels, as well as plant morphology (Zhang et al
...
, 2006)
...
GA 16,17-dihydrodiols have been found in diverse plants, and
the EUImediated GA deactivation is a conserved mechanism in plants
...
, 2006), which should be further catalyzed
into (OH)2-Gas by another unrecognized enzyme(s) that can hydrolyze epoxy-Gas
...
,
2011a)
...
, 2013)
...
, 2013)
...
, 2013)
...
, 2013)
...
The overexpression of PtCYP714A3 in rice led to a semidwarf phenotype and
reduced endogenous bioactive GA levels
...
, 2016)
...
4
...
, 2007)
...
In contrast,
overexpression of AtGAMT1 reduces the level of the major bioactive GA4, resulting in typical
GA deficiency semidwarf phenotypes and increased tolerance to drought stress in transgenic
plants (Nir et al
...
, 2007)
...
However, whether the methylation
of Gas is a common deactivation reaction in higher plant species remains to be determined
...
The uncertainties regarding the mechanisms of the GA metabolism
pathway require further study
...
GA homeostasis must be fine-tuned with both GA
biosynthesis and catabolism
...
, 2014; Hirose et al
...
Therefore, the levels of bioactive Gas are maintained via feedback and
feedforward regulation of GA metabolism (Hedden and Phillips, 2000), including regulation of
the transcription of core GA signaling components, such as the GA receptor GID1 and repressor
DELLA (Griffiths et al
...


Unlike later GA biosynthesis genes, CPS genes appear not to be regulated by a feedback
mechanism (Silverstone et al
...
GA dynamics are mainly targeted to 2ODDs in the GA
metabolism pathway to establish homeostasis
...
, 1999)
...
, 1995; Zhang et
al
...
, 2006)
...
, 1998a)
...
,
1998)
...
,
2013)
...
, 2007)
...
, 2014)
...
, 2008; Zhu et al
...
In
contrast, the expression of the GA deactivation genes AtGA2ox1, AtGA2ox2, OsGA2ox1, and
OsGA2ox3 is upregulated upon GA3 application or high endogenous bioactive GA levels
(Sasaki et al
...
, 1999; Zhang et al
...
, 2006)
...
, 2010)
...
, 2014; Dill and Sun, 2001; Fukazawa et al
...
, 2003; UeguchiTanaka et al
...
, 2007)
...
, 2007)
...
, 2007)
...
,
2003; Ueguchi-Tanaka et al
...
Moreover, the DELLA binding transcription factor GAF1
(GAI-ASSOCIATED FACTOR1) regulates AtGA20ox2 and AtGID1b and is thereby involved in
feedback regulation of GA biosynthesis (Fukazawa et al
...
YAB1, a member of the YABBY
family of C2C2 zinc finger transcription factors, as a mediator of feedback inhibition of GA
biosynthesis, regulates OsGA3ox2 and OsGA2ox3 gene expression in rice (Dai et al
...
The
precise sites and timing of GA biosynthesis and responses must be controlled for proper plant
growth and development
...
1–100 ng/g fresh

weight) in most vegetative and floral tissues, consistent with activities of metabolic enzymes
(Hedden and Phillips, 2000)
...
, 2008;
Silverstone et al
...
, 2011a, 2008; Zhu et al
...
Therefore, different GA
metabolism and signaling genes are expressed with different temporal and spatial patterns
...
The CPS gene is expressed with low levels in rapidly growing
tissues and vascular elements of expanded leaves, which might act as a source of Gas or their
precursors (Fleet et al
...
, 1997a)
...
, 1998; Yamaguchi et al
...
Moreover, AtCPS is expressed in the provasculature of the embryonic axis in
germinating seeds and AtKO and AtGA3ox genes are expressed in the cortex and endodermis
(Yamaguchi et al
...
The intermediate ent-kaurene is relatively volatile and has been
found to be released into the external environment, which might contribute to reducing the flux
through the GA biosynthesis pathway in the AtCPS overexpression line (Fleet et al
...
, 2004)
...
, 1993)
...
The different paralogous 2ODD genes show distinct but
overlapping expression patterns, and as such contribute unequally to different developmental
processes
...
, 2008; Mitchum et al
...


Differential expression of AtGA2ox genes during the plant life cycle is also associated with seed
germination and development of seedlings, flowers, and secondary shoots
...
,
1997; Silverstone et al
...
, 1999; Zhu et al
...
Several AtGA2ox enzymes
were downregulated in correlation with rapid seed germination when increased GA levels are
necessary for seed germination (Lo et al
...
In rice, OsGA20ox2 and OsGA3ox2 are
predominantly expressed in the flowers (Kaneko et al
...
, 2010), probably establishing a feedforward regulatory loop of bioactive
GA abundance during the procreative period
...
, 2008; Zhu et al
...


ENVIRONMENTAL REGULATION OF GA METABOLISM

Many environmental responses are regulated through GA abundance, and GA metabolism is
regulated by environmental signals, such as light, temperature, water, and nutrient status, as
well as by other abiotic and biotic stresses
...
GA metabolism is
sensitive to changes in light quantity, quality, or duration, which may result in increased or
decreased GA content
...
, 1998) and
Arabidopsis seeds (Yamaguchi et al
...
Expression of LsGA3ox1 was dramatically
increased and LsGA20ox2 expression was decreased by red light ® treatment, while LsGA3ox2
and LsGA20ox2 expression were unaffected in seeds under any light conditions
...
, 1998)
...
, 1998a)
...
Cryptochromes can also regulate GA20ox and GA2ox expression
in Arabidopsis seedlings (Achard et al
...
, 2007) and rice seedlings (Hirose et al
...
High levels of AtGA20ox1 and low levels of AtGA2ox genes expression were found
in Arabidopsis seedling hypocotyls grown in the dark
...
These studies indicate that the biosynthesis and accumulation of bioactive Gas
are critical to regulate the length of the seedling hypocotyls (Achard et al
...

Blue light affects bioactive GA level through regulating the expression of AtGA2ox1,
AtGA20ox1, and AtGA3ox1, which requires cryptochromes (Zhao et al
...
In rice seedlings,
phytochromes mediate the repression of GA biosynthesis capacity, including the repression of
two GA20ox genes, OsGA20ox2 and OsGA20ox4, and a GA3ox gene, OsGA3ox2, and the
induction of four GA2ox genes, OsGA2ox4 to OsGA2ox7 (Hirose et al
...
A further study
indicated that independent photoreceptors in rice separately but cooperatively mediate GA
metabolism, for example, blue light sensed by cryptochrome 1 (cry1a and cry1b) induced the
expression of the four GA2ox genes (OsGA2ox4–OsGA2ox7) (Hirose et al
...
Phytochrome
B, together with auxiliary action of phytochrome A, mediates the repression of GA20ox genes
(OsGA20ox2 and OsGA20ox4)
...


The circadian clock directly influences GA biosynthesis and signaling
...
, 1997)
...
, 1996)
...
In Arabidopsis, PIL5 is a negative regulator of
phytochrome-dependent seed germination
...
, 2006, 2007)
...
Moreover, ELF3 negatively regulates AtGA20ox1 and AtGA20ox2 expression, which
depends strongly on the redundant activities of PIF4 and PIF5 (Filo et al
...


Endogenous GA levels are also regulated by cold, salt, and high temperature stimuli through
GA biosynthesis pathway enzymes and GA deactivation enzymes (Colebrook et al
...
, 2008)
...
The AtGA3ox1 gene, but not other GA3ox genes
(AtGA3ox2, AtGA3ox3, and AtGA3ox4), is induced by cold temperature in dark-imbibed seeds,
and Atga3ox1 mutant seeds are defective in both cold-stimulated GA4 accumulation and
germination, compared to wild-type seeds (Yamauchi et al
...
The embryonic regulators
LEAFY COTYLEDON2 (LEC2) and FUSCA3 (FUS3) are involved in multiple aspects of
Arabidopsis seed development, including negative regulation of GA biosynthesis or action
...
, 2004)
...
, 2004)
...
The MADS
domain transcriptional regulator AGAMOUS-Like15 (AGL15) promotes somatic embryogenesis
by binding DNA and regulating gene expression
...
, 2004)
...
, 2002,
2004)
...
, 2009), to negatively regulate bioactive GA levels (Curaba et al
...
,
2004)
...


Cold treatment also induced a transient increase in AtGA2ox1 transcript levels (Achard et al
...
In Arabidopsis, six AtGA2ox genes, including AtGA2ox1, AtGA2ox2, AtGA2ox4,

AtGA2ox6, AtGA2ox7, and AtGA2ox8, were upregulated under high-salinity stress; thus,
endogenous GA levels could be reduced as a result of the induction of GA2ox leading to
repression of growth as an adaptation to stress (Magome et al
...
The salinity-responsive
DWARF AND DELAYED FLOWERING 1 (DDF1) gene encodes an AP2 (Apetala2)
transcription factor of the DREB1 (dehydration-responsive element binding protein)/CBF (Crepeat binding factor) subfamily
...
, 2004)
...
, 2008)
...
,
2014)
...
Therefore, the change in abiotic stress tolerance in CmBBX24 RNAi lines may be related
to the influence of increased levels of bioactive Gas through expression of GA biosynthesis
genes (Yang et al
...
Gas also influence drought tolerance in plants (Colebrook et al
...

Overexpression lines of AtGA20ox1 or Atga2ox quintuple mutants with high GA content
presented decreased drought tolerance, while Atga20ox1 Atga20ox2, Atga3ox1 Atga3ox2
double mutants, or Atga20ox1 Atga20ox2 Atga20ox3 triple mutant with low GA levels or GA
deficiency presented dramatically increased drought tolerance compared to the wild-type
Arabidopsis (Colebrook et al
...


The GA-deactivating enzyme EUI is also involved in response to biotic and abiotic stresses
through the regulation of active Gas in rice and Arabidopsis (Yang et al
...
The eui
mutants accumulate exceptionally large amounts of biologically active Gas, while the level of
GA4 is dramatically reduced in EUI overexpression transgenic plants (Zhu et al
...

Interestingly, eui mutants or EUI overexpression compromises or increases, respectively,
resistance to bacterial blight and rice blast
...

Similarly, GA application also decreased the disease resistance
...
, 2008)
...
Therefore,
Gas play a negative role in rice basal disease resistance (Yang et al
...


The AtGA3ox1 gene plays an essential role during seed imbibition and is induced by cold
temperatures in dark-imbibed seeds, suggesting that the GA biosynthesis pathway and the
cellular distribution of bioactive Gas are altered during seed imbibition under low temperature
conditions (Yamauchi et al
...
The zeaxanthin epoxidase gene ZEP (also known ABA1) and
three 9-cis-epoxycarotenoid dioxygenase genes NCED2, NCED5, and NCED9, which are the
enzymes in ABA (abscisic acid) biosynthesis, are all upregulated and ABA levels are elevated
under high temperatures in Arabidopsis seeds
...
, 2008)
...
Simultaneously, the negative GA regulators SPINDLY (SPY) and RGALIKE 2
(RGL2) are enhanced to suppress GA signaling pathway (Toh et al
...
High temperatures
stimulate ABA synthesis and repress GA synthesis and signaling through the action of ABA in
Arabidopsis seeds
...
, 2008)
...
In this section, we focus on the regulation of GA metabolism by ABA, auxin,
ethylene, brassinosteroid (BR), and cytokinin (CK)
...
, 2006; Weiss and Ori, 2007)
...
, 2007), whereas GA biosynthesis is increased in ABA-deficient aba2 mutant seeds via
activation of AtGA3ox1 and AtGA3ox2 expression (Seo et al
...
Furthermore, activation of
GA biosynthesis genes is also observed in the aba2-2 mutant during seed development (Seo et
al
...
ABA reduces bioactive GA levels by decreasing AtGA20ox1 and increasing
AtGA2ox6 transcript levels (Zentella et al
...
In addition, aba2-2 mutants show significant
expression of GA biosynthesis genes and repression of SPY expression even at high
temperatures
...
, 2008)
...
The rice eui mutant with a higher level of
bioactive GA was less sensitive to ABA, while the EUI overexpression lines of Arabidopsis and
rice, with a GA phenotype, were hypersensitive to ABA (Yang et al
...
This evidence
suggests that Gas play a role in the abiotic stress response probably through modulating the
ABA signaling pathway
...
, 2010)
...
The
expression of both EUI and the ABA biosynthesis gene OsNCED-1 is directly controlled by
OsAP2-39, illustrating a mechanism that leads to homeostasis and balance of these hormones
(Yaish et al
...


The regulation of GA biosynthesis by auxin has been attributed to changes in the expression
levels of the genes encoding GA biosynthesis or deactivation enzymes
...
, 2000)
...
In Arabidopsis, the
AUXIN/indole-3-acetic acid (Aux/IAA) and auxin response factor proteins (see auxin chapter
in this book for details) directly regulate the expression of GA metabolism genes through
upregulating the expression of AtGA20ox and AtGA2ox (Frigerio et al
...
The auxin
regulation of PsGA2ox1 is partly dependent on DELLA, while PsGA2ox2 was upregulated by
auxin in a DELLA-independent manner (O’Neill et al
...
This finding suggests that auxin
differentially regulates the expression of GA biosynthesis genes involved in pea and
Arabidopsis plants
...
, 2006)
...
These observations suggest that developmental regulation
and organ-, tissue-, and cell- specific regulation override both auxin and GA metabolic
regulation, ensuring the appropriate temporal and spatial levels of Gas
...
BR regulates the
biosynthesis of GA to promote plant growth (Unterholzner et al
...
Exogenous BR
treatment and overexpression of BR biosynthesis gene DWARF4 (DWF4) increase expression of
several GA20ox genes (AtGA20ox1, AtGA20ox2, and AtGA20ox5) in Arabidopsis (Lilley et al
...
It has also been found that BR regulates cell elongation by modulating GA metabolism in
rice (Tong et al
...
Under physiological conditions, BR promotes GA accumulation by
regulating the expression of GA metabolism genes to stimulate cell elongation
...
, 2014)
...
Moreover, AtGA20ox1 can rescue many of the developmental
defects of BR mutants, and the transcription factors BES1 and BZR1 functioning in BR signaling
likely mediate this GA-mediated response through binding the promoters of AtGA20ox1 and
other GA biosynthesis genes (Unterholzner et al
...
Therefore, cross talk between BR and
GA occurs not only at the level of signals but also at the level of hormone biosynthesis
(Hofmann, 2015; Tong and Chu, 2016; Unterholzner et al
...


The role of ethylene in GA metabolism is less clear
...
In the ctr1-1 mutant,
which exhibits constitutive ethylene responses, the levels of GA1 and GA4 are substantially
reduced and the expression levels of AtGA3ox1 and AtGA20ox1 genes are elevated through a

negative feedback mechanism, suggesting that the ethylene signal is initially targeted to the GA
metabolism pathway (Achard et al
...
Ethylene also regulates GA metabolism during fruit
set in tomato (Shinozaki et al
...
The ethylene-insensitive Sletr1-1 mutant fruits have
elevated levels of bioactive Gas, most likely through increasing transcription of SlGA20ox3, and
repressing transcription of SlGA2ox4 and SlGA2ox5
...
, 2015)
...
Overexpression of AtERF11 resulted in elevated bioactive
GA levels by upregulating expression of AtGA3ox1 and AtGA20ox genes
...
, 2016)
...


Antagonistic effects are exerted by GA and CK in many developmental processes (GreenboimWainberg et al
...
A balance of CK and GA signals are required for normal SAM (shoot
apical meristem) function (Jasinski et al
...
, 2001a)
...
CK can also regulate GA
levels by directly inhibiting GA biosynthesis and indirectly promoting GA deactivation (Weiss
and Ori, 2007)
...
KNOX activity is mediated by both GA and CK (Jasinski et al
...
CK and KNOX both
induce the expression of GA2ox at the SAM probably to block bioactive Gas (Jasinski et al
...
The potato StBEL5 (BEL1-like transcription factor) and POTH1 (potato homeobox1)
mediate developmental processes by regulating CK and GA levels
...
, 2004)
...

The fine-tuning of gene expression in GA biosynthesis and metabolism pathways co-ordinately
control the levels of Gas (Hedden and Thomas, 2012)
...
, 2012; Eriksson et al
...
, 1983; Proebsting et al
...
, 2011; Regnault et al
...
,
2013; Tal et al
...
Biochemical and micrografting experiments have demonstrated the
translocation of Gas from synthetic sites to the tissues and organs that require Gas for growth
and development (Regnault et al
...


Studies in pea provided the first evidence for the transport of Gas from root to shoot, by
employing grafting experiments in a GA biosynthesis mutant na mutant that blocks ent-7αhydroxykaurenoic acid conversion to GA12-aldehyde, resulting in reduced levels of active Gas
within the shoot (Ingram and Reid, 1987)
...
, 1992)
...
, 1992)
...
, 2001)
...
,
2006)
...
, 2008)
...
Thus, the mobility of the shoot-derived
Gas contributes to regulate hypocotyl xylem expansion (Ragni et al
...

Similarly, leaf-derived GA1 and GA20 are mobile signals that induce GA-promoting internode
elongation, cambial activity, and fiber differentiation in tobacco stems (Dayan et al
...
The
GA precursor GA12 is also a long-distance mobile GA signal through the vascular system
...
, 2015)
...
, 2016)
...
,
2013), which is consistent with previous studies that the endodermis is the major GA-responsive
tissue in the roots (Heo et al
...
, 2013; Ubeda-Tomas et al
...
, 2011b)
...
Surprisingly, GA3-Fl
accumulation in the mesophyll of rosette leaves in the Arabidopsis tem1-1 tem2-2 mutants was
increased and distributed throughout a much larger leaf area in comparison with wild-type
plants (Matías-Hernández et al
...
Indeed, TEM is known to inhibit GA biosynthesis, and
also represses the expression of several GA transporters, including NITRATE
TRANSPORTER1/PEPTIDE TRANSPORTER FAMILY (NPF) NPF2
...
10, and NPF3
...
Therefore, TEM is essential for GA distribution
...
The NPF family proteins were initially identified as nitrate or peptide
transporters (Léran et al
...
, 2007), and were later found to also transport auxin,
ABA, GA, and/or JA (jasmonic acid) hormones (Chiba et al
...
, 2012; Krouk et
al
...
, 2015)
...
1 has been proven to be a unique GA transporter in plants (Tal
et al
...
The AtNPF3
...
Interestingly, expression of AtNPF3
...
Another nitrate/peptide transporter GTR1
(glucosinolate transporter1, also known as NPF2
...
, 2012)
...
Consistent with this observation, the gtr1 mutants exhibit severely impaired
filament elongation and anther dehiscence (Saito et al
...
Therefore, levels of bioactive Gas
in special tissues or cells are determined not only by local GA biosynthesis and catabolism but
also by GA translocation through a GA transporter
...
However, a breakthrough was
achieved with the discovery of the GID1 protein as a GA receptor in rice (Ueguchi-Tanaka et al
...


In order to investigate the GA signaling molecular mechanism, the rice gid1 mutant was
identified, showing the severe dwarf phenotype with wide, dark green leaf blades
...
The gid1 mutant must be maintained as a heterozygote because of absence of fertile
flowers, and it appears to be completely insensitive to GA (Sasaki et al
...
, 2005)
...

Additionally, the endogenous accumulation of GA1 in 1-month-old gid1-1 shoots was up to
100-fold higher than that in wild-type plants
...
The GA-binding

kinetic analysis revealed that the half-time for both association and dissociation between GSTGID1 and 16,17-dihydro-GA4 was within 5 min
...
The GA-binding kinetics may be critical for soluble
receptors, because the sensitivity of the system to subtle intracellular GA concentrations
alterations results in profound and compounding effects on gene regulation (Ueguchi-Tanaka et
al
...
The GID1-GFP fusion protein was primarily localized in nuclei, with a fainter
cytosolic signal, and uniconazole or GA3 treatment did not change the subcellular localization
...
, 2005)
...
, 2007)
...
, 2006)
...
Expression of
Arabidopsis AtGID1a, AtGID1b, or AtGID1c genes could rescue the rice gid1-1 dwarf
phenotype (Nakajima et al
...
Similar to rice, the AtGID1a-GFP fusion protein was
predominantly localized to the nucleus, and to the cytoplasm, and this localization was not
altered by treatments with GA or with the GA biosynthesis inhibitor PAC (Fleck and Harberd,
2002; Silverstone et al
...
, 2005; Willige et al
...
Cytosolic
AtGID1a also plays an important role in initiating GA signaling and responses
...
A possible explanation is
that activated cytosolic AtGID1a interacts with DELLA proteins before they enter the nucleus
(Livne and Weiss, 2014)
...
, 2006), whereas the DELLA domain of
GAI alone can mediate GA-dependent AtGID1a-GAI interactions and that the presence of the
adjacent VHYNP domain does not contribute to enhance this interaction
...
, 2006; Hirano et al
...

Thus the DELLA domain acts as a receiver domain for GA-GID1 (Willige et al
...


In Arabidopsis, AtGID1a, AtGID1b, and AtGID1c genes are expressed in all tissues and have
both overlapping and specific roles in growth and development (Griffiths et al
...
, 2007)
...
These genetic evidences
suggest that AtGID1 isoforms are the GA receptors in Arabidopsis and have some degree of
functional redundancy (Griffiths et al
...
Indeed, the Atgid1a Atgid1b Atgid1c triple
mutant did not germinate readily and only started to grow when the seed coat was removed
after imbibition
...
By comparing Atgid1 multiple mutants with sly1 Atgid1
double mutants, roles of AtGID1a, AtGID1b, and AtGID1c in proteolytic and non-proteolytic
GA signaling were demonstrated
...
, 2014)
...
, 2007)
...
The GA-binding site of GID1 protein corresponds to the
active site of the HSL domain, and four helices at the N terminus and the central part of GID1
form a lid closure (Fig
...
3)
...
DELLA interacts with the GID1-GA complex at its N-terminal region,
and as a result of DELLA binding, the GID1-GA complex is stabilized (Fig
...
3) (UeguchiTanaka et al
...
In 2008, two research groups independently demonstrated crystal
structures of rice GID1 (OsGID1)-GA complex and the Arabidopsis GID1 (AtGID1a)-GADELLA complex (Murase et al
...
, 2008)
...
4
...
The AtGID1a protein is monomeric and is
composed of one α/β core domain with an N-terminal extension that extends up the core
surface toward the DELLA domain
...
, 2008)
...
, 2008)
...
, 2008)
...
, 2007)
...
, 2010; Murase et al
...


THE EVOLUTIONARY CONSERVATION OF GA-GID1-DELLA MODULE

Bioactive Gas promote growth and development throughout the life cycle of the flowering
plant
...
, 2007; Sun, 2011;
Yasumura et al
...
The proteins encoded by the Selaginella moellendorffii homologs of
GID1, DELLA, and GID2/SLY1 genes in vascular plants functioned well, in a manner similar to
that in flowering plants, whereas the homologous proteins in Physcomitrella patens did not
interact or functionally substitute for their flowering plant homologs (Hirano et al
...
, 2007)
...
moellendorffii
shows 47% similarity to that of flowering plants, and includes an Arg265 residue whichh is
important for GA binding (Vandenbussche et al
...
However, SmGID1 proteins showed
lower binding affinity for bioactive GA4 and low specificity for various other Gas when
compared with GID1 of flowering plants
...
Taken together,
these results indicate that GID1 evolved from a member of the HSLs and was further modified
to have higher affinity and more strict selectivity for bioactive Gas by adapting the amino acids
involved in GA binding in the course of step-by-step evolution (Shimada et al
...


DELLA PROTEINS: THE KEY MEDIATORS OF GA SIGNALLING

Through genetic analysis and biochemical studies, several key components in GA signaling
have been identified
...
They function genetically as growth repressors,
and include examples in wheat (Rht1), maize (d8), barley (SLN1), rice (SLR1), grape (VvGAI),
and tomato (PROCERA) (Boss and Thomas, 2002; Jasinski et al
...
, 2001)
...
The first identified DELLA protein was GAI, which was isolated from the Arabidopsis
gai-1 mutant (Peng and Harberd, 1993)
...
, 1997)
...
, 1997b, 1998)
...
, 2001)
...
, 2002)
...
The DELLA proteins have a conserved C-terminal region and a more
divergent N-terminal GA perception region (Fig
...
4)
...
, 1997)
...
, 2009;
Dill et al
...
, 2002; Peng et al
...
, 2007), and polymeric Ser/
Thr/Val motifs (poly S/T/V), which could be targets of phosphorylation or glycosylation
...
, 2001; Silverstone et al
...
, 2010)
...
The different Arabidopsis DELLA proteins have both overlapping and
specific roles in the regulation of plant growth and development
...
, 2001)
...
Both SLY1 and GID2 encode homologous F-box proteins and
function as subunits of the SCF E3 ligase complex, which is required for GA-mediated
degradation of DELLA proteins (Dill et al
...
The sly1 null mutant fails to degrade DELLA
proteins and exhibits GA-insensitive dwarf phenotypes (McGinnis et al
...
However, the
sly1-10 dwarf phenotype is suppressed in the gai-t6 rga-24 double mutant (Dill et al
...
, 2004)
...
, 2004; Fu et al
...
, 2003)
...
, 2004)
...
, 2004)
...

Thus, the affinity among components of DELLA-SCFSLY1/GID2 proteins complex is important
in the regulation of the accumulation of DELLA proteins (Fu et al
...


Previous studies have shown that protein kinase inhibitors strongly inhibited GA-mediated
degradation of barley SLN1 protein, but phosphorylation of DELLA proteins enhanced their
interaction with the F-box component of SCFSLY1/GID2 in Arabidopsis, barley, and rice (Fu et
al
...
, 2004)
...
, 2004; Itoh et al
...
Thus, phosphorylation
of DELLA proteins may be essential for their biological functions, but might not be necessary
for GA-mediated proteasome-dependent protein degradation
...
, 2011)
...
, 2015)
...
In the Atsiz1-2 mutant, SLY1 was less abundant than that in wild-type plants,
but the RGA protein was more abundant in the Atsiz1-2 mutant than in wild-type plants
...
These
findings reveal that sumoylation of F-box protein SLY1 is critical for GA-mediated degradation
of DELLA proteins (Kim et al
...


Arabidopsis has a homolog of SLY1 known as SNEEZY (SNE) or SLEEPY2 (SLY2), and the fulllength SLY1 and SNE genes have 55% DNA and 33% amino acid sequence homology (Ariizumi
et al
...
No apparent phenotypic difference was observed in sne mutants when compared
with wild-type plant in the vegetative phase, but loss- of-function mutations in SNE caused
increased ABA sensitivity in seed germination
...
Furthermore, overexpression of SNE (SLY2) under the
control of cauliflower mosaic virus 35S promoter could partially rescue the sly1 dwarf
phenotype, suggesting that SNE can functionally replace SLY1 (Fu et al
...
,
2004)
...
, 2011)
...
, 2015)
...

GA perception is mediated by its receptor GID1, and the binding of bioactive GA to GID1
induces the formation of a GA-GID1-DELLA protein complex, which enhances their binding to
the E3 ubiquitin-ligase SCFSLY1/GID2 complex (Griffiths et al
...
This then results in the
degradation of DELLA proteins by the 26S proteasome (Daviere et al
...
, 2011;
Hartweck, 2008; Jiang and Fu, 2007; Shimada et al
...
, 2014), thereby relieving the
growth suppression caused by DELLAs and resulting in GA-promoted growth and other GA
responses (Fig
...
5)
...
Thus, a proteolysis-independent model was proposed to explain
these phenomena, and the inactivation may result directly from protein–protein interactions or
indirectly through posttranslational modification (Ariizumi et al
...
,
2008)
...
, 2008)
...
, 2013)
...
For example, GAF1 interacts with both DELLA
proteins and co-repressor TOPLESS RELATED (TPR)
...
, 2015)
...
, 2001; Fu and Harberd, 2003)
...
,
2004; Gomi et al
...
, 2005, 2007; Itoh et al
...
, 2003)
...
Studies showed that dephosphorylation of serine/threonine was
probably necessary for GA-mediated degradation of RGL2 in tobacco BY2 cells, and six
mutations in conserved serine/threonine residues

(RGL2S441D, RGL2S542D, RGL2T271E, RGL2T319E, RGL2T411E, and RGL2T535E) that
mimicked a state of constitutive phosphorylation caused a resistance to GA-induced
degradation (Hussain et al
...
Further experiments demonstrated that tyrosine
phosphorylation might be a prerequisite for GA-induced degradation of RGL2 (Hussain et al
...
However, exogenous GA treatment induced accumulation of both the phosphorylated
and non-phosphorylated SLR1 proteins with similar kinetics in callus of the gid2 mutant, and
GA-induced degradation of both forms of SLR1 proteins with similar half-life in rice wild-type
callus cells, suggested that the phosphorylation of DELLA proteins is not essential for their GAmediated degradation (Itoh et al
...
The rice SLR1 protein can be phosphorylated by
EARLY FLOWERING1 (EL1) in vitro
...


Several studies have shown that microcystin-LR (inhibitor of PP1/PP2A-type phosphatase)
markedly inhibited the degradation of maltose binding protein (MBP)-tagged RGA fusion
protein in a cell-free proteasome assay, suggesting that the degradation of the DELLA proteins
requires serine/threonine dephosphorylation activity (Wang et al
...
A study has shown
that phosphatase TOPP4, which is a member of the protein phosphatase 1 (PP1) family in
Arabidopsis, acts as a positive regulator in GA signaling and promotes the GA-mediated
degradation of DELLA proteins through directly dephosphorylating DELLA proteins (Qin et
al
...
To investigate the serine/threonine sites of DELLA proteins, six conserved
serine/threonine sites of Arabidopsis RGA were substituted with alanine (RGA6A) or aspartic
acid (RGA6D) to mimic the states of constitutive dephosphorylation and phosphorylation,
respectively
...
, 2014)
...
The Arabidopsis SPY gene encodes an O-GlcNAc transferase that acts as a negative
regulator of GA signaling via O-GlcNAcylation of DELLA proteins (Filardo et al
...
, 1998; Shimada et al
...
, 2001)
...
, 2006; Silverstone et al
...

Studies have shown that SWI3C, the core component of Switch (SWI)/Sucrose Non-fermenting
(SNF)-type chromatin-remodeling complexes (CRCs), could interact with both SPY and DELLA
proteins, suggesting a potential role for DELLA and SPY in the modulation of GA responses
through the regulation of chromatin architecture (Sarnowska et al
...


Although Arabidopsis O-GlcNAc transferase SECRET AGENT (SEC) could modify the DELLA
proteins, genetic analysis revealed that SEC and SPY play distinct roles in the regulation of GA
signaling
...
The
transient co-expression experiments in tobacco cells showed that RGA is O-GlcNAcylated by
SEC but not by SPY
...
These findings reveal that O-GlcNAcylation of
DELLA proteins by SEC provides a fine-tuning mechanism for integration of developmental
and environmental signals into GA responses (Zentella et al
...


The posttranslational sumoylation of DELLA proteins suggests an additional mechanism for
controlling their stability and activity
...
The SUMOconjugated DELLA proteins interact with a SUMO-interacting motif in GID1 in a GAindependent manner
...
, 2014)
...
The DELLA proteins
lack a DNA-binding domain and there is no evidence of direct DNA binding
...
The first one is to
interact with DNA-binding domains of transcription factors and inhibit their DNA-binding

activity, and consequently repress target gene expression
...
4
...

Accumulating evidence suggests that DELLA proteins are able to integrate multiple hormone
and environmental signals to control plant growth and development by modulating stability
and/or function of DELLA proteins (Fig
...
7)
...
Bioactive GA promotes seed germination, while ABA is involved in the
establishment and maintenance of seed dormancy (Koornneef et al
...
Genetic analysis
revealed that ABSCISIC ACID INSENSITIVE 3 (ABI3) and ABI5 are negative regulators of seed
germination (Piskurewicz et al
...
Previous studies have shown that RGL2 represses
Arabidopsis seed germination via the regulation of the transcriptional level of XERICO, a gene
encoding a RING-H2 zinc finger protein, which promotes endogenous ABA biosynthesis
...
, 2013)
...
In Arabidopsis, the transcription fac-

Tors BZR1 and BRASSINOSTEROID-INSENSITIVE 1 EMS-SUPPRESSOR1 (BES1) are positive
regulators of BR signaling
...
Conversely, the GA-induced
degradation of DELLA proteins releases transcription factors (Bai et al
...
, 2012; Li et al
...
Both DELLA and BZR1 proteins interact with PIF4
(Bernardo-Garcia et al
...
, 2007) to form a DELLA-BZR1-PIF4 complex which
enables plant growth to be regulated by GA, BR, and environmental factors acting through a
core transcription network (Bai et al
...


Additionally, DELLA proteins interact with auxin response factor ARF6, which together with
BZR1 and PIF4 can create a BZR1-ARF6-PIF-DELLA module, which provides a means to
regulate Arabidopsis hypocotyl cell elongation (Oh et al
...


There is cross talk between GA and ethylene signaling in the regulation of Arabidopsis apical
hook development
...
Ethylene responses are regulated by DELLA
proteins through interaction with the DNA-binding domains of EIN3 and EIN3-LIKE1 (EIL1)
proteins, which in turn repress EIN3/EIL1-regulated HOOKLESS1 (HLS1) expression and
apical hook formation (An et al
...
A DELLA-interacting protein RELATED TO
APETALA2
...
3) was found to be involved in the regulation of apical hook development
...
3 protein belongs to the group VII ETHYLENE RESPONSE FACTOR of the
APETALA2/ethylene responsive element binding protein superfamily, and the interaction with
GAI impairs the DNA-binding activity of RAP2
...
, 2014)
...


Cytokinins and Gas are known to exert antagonistic regulation of multiple developmental
processes in plants (Weiss and Ori, 2007)
...
, 2015;
Moubayidin et al
...
In addition, GA represses the expression of ARR1 at early stages of
meristem development, and DELLA proteins are recruited by ARR1 to the promoters of
cytokinin-regulated genes, where DELLA proteins act as transcriptional co-activators, thereby
enhancing their expression (Marín-de la Rosa et al
...


Studies have shown that plant-specific transcriptional factor TCP (TEOSINTE
BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR) is involved in strigolactone
(SL) signal transduction pathways (Braun et al
...
, 2015; Guan et al
...
, 2015), which inhibit shoot branching and function during rhizospheric
communication with symbiotic fungi and parasitic weeds
...
, 2014; Resentini et al
...
The observation that
DWARF14 (D14, a SL receptor) can physically interact in yeast with rice DELLA protein SLR1
suggests that DELLA proteins might mediate cross talk between SL and GA signaling
(Nakamura et al
...
However, experimental evidence identifying the function of a D14DELLA interaction has not yet been forthcoming
...
The interaction between DELLA and SPL proteins interferes with SPL
transcriptional activity, decreases the expression of miR172 in leaves and of MADS-box genes at
the shoot apex under longday conditions, which in turn delays floral induction (Yu et al
...

Flowering is promoted by GA in Arabidopsis by activating floral meristem identity genes such
as LEAFY (LFY) and SUPPRESSOR OF OVEREXPRESSION CONSTANS1 (SOC1)
...
, 2004, 2007a; Moon et al
...
A new study showed that DELLA proteins physically interact
with the critical flowering activator CONSTANS (CO), and the regulation of GA-promoting
flowering in leaves under long-day conditions is mediated in part through DELLA-mediated
repression of CO (Xu et al
...
Other DELLA-interacting proteins include BOTRYTIS
SUSCEPTIBLE1 INTERACTOR (BOI), BOI-RELATED GENE1 (BRG1), BRG2, and BRG3 which
are collectively referred to as BOIs, and belong to the RING domain protein family
...
, 2013)
...
, 2010)
...
, 2008; Feng et al
...
The PIF homolog SPATULA
(SPT) regulates seed dormancy and restrains cotyledon expansion and fruit growth (Fuentes et
al
...
, 2011)
...
, 2010)
...
, 2012)
...


In addition to functioning as transcriptional repressors, DELLA proteins can function as
transcriptional co-activators
...
Expression of SCL3 is induced by
DELLA proteins and repressed by GA treatment
...
,
2011; Zhang et al
...
The Arabidopsis genome contains 16 INDETERMINATE DOMAIN
(IDD) proteins, which are characterized by a distinct arrangement of zinc (Zn) finger motifs
(Colasanti et al
...
Several IDD proteins such as IDD1, IDD3, IDD5, IDD9, IDD10, and a
homologous protein GAF1 interact directly with DELLA proteins, and regulate GA signaling in
Arabidopsis (Feurtado et al
...
, 2014; Yoshida et al
...
The interaction
between DELLA and IDD1 regulates seed dormancy (Feurtado et al
...
Indeed, IDD3,
IDD5, IDD9, and IDD10 not only interact with DELLA but also with SCL3 protein
...
, 2014; Yoshida and UeguchiTanaka, 2014)
...
Furthermore, the accumulation of SCL3
protein also acts as a co-repressor; an increased level of the SCL3-IDD protein complex can
suppress its own expression (Yoshida et al
...


Studies have shown that cellulose synthesis is also regulated by the GA-DELLA regulatory
system in rice
...
In contrast, SLR1
interacts directly with NACs, which in turn inhibits an NACMYB-CESA signaling cascade
...
, 2015)
...
, 2009)
...
, 2013)
...
However, DELLA proteins physically interact with PKL and block the PKL-PIF3
interaction, resulting in deactivation of these genes (Zhang et al
...
More interestingly, a
study has revealed that GA regulates microtubule organization via an interaction between the
DELLA proteins and the prefoldin complex (PFD), a co-chaperone required for tubulin folding
(Locascio et al
...
Without GA, the DELLA-PFD interaction retained the localization of the
complex in the nucleus, thus compromising α/β-tubulin heterodimer availability in the
cytoplasm
...
, 2013)
...
Both light
and GA regulate many important plant developmental processes
...
Interaction of DELLA proteins with the bHLH
transcription factors PIF3 and PIF4 blocks PIF transcriptional activity by binding to the DNArecognition domain of these factors, resulting in inhibition of PIFmediated expression of target
genes and hypocotyl elongation (de Lucas et al
...
, 2008)
...
The
interaction between DELLAs and PIFs integrates both GA and light signals to modulate
photomorphogenesis
...
Studies have
demonstrated antagonistic roles of Gas in JA-mediated plant development and defense against
biotic or abiotic stress
...
, 2010; Wild et al
...
, 2012)
...
, 2012)
...


In contrast to their antagonistic roles in modulating growth and defense, GA and JA signaling
also synergistically regulate several developmental processes
...
, 2011, 2014)
...
, 2012)
...
, 2009)
...


•

GA inactivation pathways involving covalent modification and conjugation have been
revealed, which are important for controlling endogenous GA levels
...


•

The key components of GA signaling have been identified, and evolutionary conserved
“Relief of Repression” regulatory model is demonstrated
...


•

DELLA proteins integrated with other hormonal and environmental signaling molecules
control plant growth and development
...

Studies have begun to uncover how GA promotes plant growth and development at the
molecular level through GID1-mediated degradation of DELLA proteins
...

However, several questions still remain to be resolved
...
Recent biochemical and micrografting studies have suggested that the
movement of various Gas from production sites to the tissues and organs that require
Gas contributes to the coordinated regulation of a diversity of processes associated with
plant growth and stress adaptation
...
However,
the molecular basis of GA mobility and long-distance coordination is little understood
...

Although soluble GA receptor GID1 protein is present in the cytoplasm, studies
revealed that GA-GID1-DELLA protein complex is localized to the nucleus
...


•

The de-repression regulatory model suggests that GA promotes plant growth via the
GA-mediated degradation of the DELLA proteins
...
For
example, Arabidopsis fruit growth is induced by GA in both global (lacking GAI, RGA,
RGL1, RGL2 and RGL3) and ga1 global mutants, whereas exogenous GA treatment is
unable to restore fruit growth in a Atgid1a Atgid1b Atgid1c triple mutant (Fuentes et al
...
The implication is that a DELLA-independent pathway is involved in the control
of GA responses, although the components in DELLA-independent GA signaling have
yet to be identified
...

Previous studies showed that both sly1 mutants accumulate more DELLA proteins but
display less severe dwarf phenotypes than that of ga1-3 or Atgid1a Atgid1b Atgid1c
triple mutant, and the upregulation of GID1 could rescue the sly1 or gid2 dwarf
phenotypes without decreasing the accumulation levels of DELLA proteins (Ariizumi et
al
...
, 2008)
...


•

It is known that DELLA proteins physically interact with various transcription factors
that integrate multiple developmental and environmental signals into cell proliferation
and differentiation through GA-mediated degradation of DELLA proteins
...


•

Future studies should use a combined biochemical and systems biology approach to
identify new components involved in GA responses and generate a robust framework
for GA signaling that integrates multiple developmental and environmental signals to
ensure normal plant growth and adaptation in a fluctuating environment
...
Cytokinins are naturally synthesised
in the plants where rapid cell division occurs e
...
root apices, shoot buds, young fruits, etc
...


In the 1940s and 1950s, a wide variety of substances ranging from yeast extract to tomato juice
were identified that that could initiate and sustain the proliferation of normal plant stem tissues
in culture
...
In the 1950s, Skoog and Miller found that autoclaved herring sperm
DNA was a potent activator of the proliferation of cultured tobacco pith cells (Miller et al
...
, 1956)
...
In the presence of auxin (but not in its absence) kinetin
stimulated tobacco pith parenchyma tissue to proliferate in culture
...


Since their discovery, cytokinins have been implicated to play a role in almost all aspects of
plant growth and development, including cell division, shoot initiation and growth, leaf
senescence, and photomorphogenic development (Mok and Mok, 1994)
...
The most common class of cytokinins have isoprenoid side chains,
including the most abundant cytokinin in Arabidopsis, trans-zeatin
...
While most of the effects of zeatin have been attributed to
the trans form, recent results suggest that the cis form may also be biologically active (Martin et
al
...


In addition to the free base forms, cytokinins can also be present in the plant as a riboside (in
which a ribose sugar is attached to the 9 nitrogen of the purine ring) or a ribotide (in which the
ribose moiety contains a phosphate group)
...
, 1994;
Mok and Mok, 2001)
...


BIOSYNTHESIS

Mature tRNAs from most organisms, including plants, contain cis-zeatin as a modified base
...
, 1972) as the released cis-zeatin could subsequently be converted to
active trans-zeatin by zeatin isomerase (Mok and Mok, 2001)
...


An enzymatic activity that converts AMP and dimethylallyl pyrophosphate (DMAPP) to the
active cytokinin iPMP (isopentenyladenosine-5’-monophosphate) was firstly identified in
Dictyostelium discoideum (Taya et al
...
Subsequently, the ipt gene from Agrobacterium
tumefaciens was shown to encode an enzyme with similar activity (Akiyoshi et al
...
Ipt
genes have also been identified in several other bacteria, and IPT activity was detected in crude
extracts from a variety of plant tissues, but the plant enzymes were not purified and the
corresponding genes were not cloned
...
Phylogenetic analysis suggested that
AtIPT2 and AtIPT9 encode a putative tRNA-ipt while the other seven AtIPTs formed a distinct
clade more closely related to the bacterial ipt gene
...
coli resulted in the secretion of the cytokinins iP (isopentenyladenine) and zeatin,
confirming that they encode cytokinin biosynthetic enzymes (Takei et al
...
Additionally,
calli overexpressing AtIPT4 under the control of the CaMV 35S promoter regenerated shoots
even in the absence of cytokinin, while CaMV 35S::AtIPT2 calli were still dependent on
exogenous cytokinin (Kakimoto, 2001)
...
The
product of the plant enzyme is likely to be iPTP (isopentenyladenosine-5’-triphosphate) and
iPDP (isopentenyladenosine-5’-diphosphate), which can be subsequently converted to zeatin
(Figure 2)
...


Another study in Arabidopsis indicates that an alternative cytokinin biosynthetic pathway
exists in plants (Åstot et al
...
The authors compared the biosynthetic rate of zeatinriboside5’-monophosphate (ZRMP; zeatine ribotide) and iPMP in wild-type and transgenic plants

Designed to inducibly overexpress the Agrobacterial ipt gene
...
In vivo deuterium labeling revealed a 66-fold higher biosynthetic rate of
ZRMP as compared to that of iPMP, the proposed direct product of IPT
...
The authors suggested the presence of an iPMPindependent pathway, in which ZRMP is directly synthesized by IPT from AMP and an as yet
unidentified side chain precursor (Figure 2)
...


METABOLISM

Cytokinins can be conjugated to form a glycoside in which a sugar molecule, generally
glucose, is attached to the 3, 7, or 9 nitrogen of the purine ring, or to the oxygen of the
zeatin or dihydrozeatin side chain
...
It is assumed that the N7 and N9

modifications irreversibly inactivate cytokinins, but the precise in vivo role of these Nglucosyl-conjugates is unknown (Mok and Mok, 2001)
...

These forms can easily be converted into active cytokinins by b-glucosidases
(Brzobohaty et al
...
Thus, it is believed that O-glycosylated cytokinins are inactive,
stable storage forms that play an important role in the regulation of cytokinin
homeostasis
...


Many plant tissues contain cytokinin oxidases, enzymes that cleave the N6-side chains
from a subset of cytokinins (Figure 3; Jones and Schreiber, 1997)
...
Substitution of other functional groups on the
purine ring, as well as O-glycosylation, also prevents cytokinin oxidase cleavage
(reviewed in Mok and Mok, 2001)
...
Cytokinin oxidase irreversibly
inactivates cytokinins, and could be important in regulating or limiting cytokinin
effects
...
Kieber,
unpublished data)
...
, 1999; Morris et al
...
The enzymes catalyzing each reaction are shown
in red
...
P
(C) Phylogenic tree of cytokinin oxidase predicted proteins, including all the
Arabidopsis enzymes
...
In each case, the enzyme was purified sufficiently to obtain peptide sequence and
the gene was subsequently cloned using degenerate PCR
...
This and other data
suggest that these enzymes are FAD-dependent amine oxidases
...


The Arabidopsis genome encodes seven cytokinin oxidase genes (Table 2; Figure 3;
Bilyeu et al
...
Like their
maize counterparts, the predicted Arabidopsis proteins contain a signal sequence for
the secretory pathway, and they may be therefore extracellular proteins, but no in vivo
localization studies have yet been reported
...
A component possibly involved in this transport, a
purine transporter called AtPUP1, has been isolated from Arabidopsis by the functional
complementation of a yeast mutant deficient in adenine uptake (Gillissen et al
...

The ability of AtPUP1 to transport adenine was competitively inhibited by free
cytokinin bases, suggesting that AtPUP1 may be a cytokinin/purine transporter
...
, 2001b)
...
The level of cytokinin observed in the xylem sap was sufficient to
elevate the expression of a cytokinin primary response gene in detached leaves
...
Thus, cytokinins may represent a longdistance signal for nitrogen/nutrient-availability from the root to the shoot, presumably
to coordinate shoot and root development
...
, 1997)
...

As seen in other systems, elevated cytokinin levels affect the morphology of the shoot
...
Thus, it was concluded that cytokinins may act as paracrine
signal, at least with respect to apical dominance and leaf senescence
...

The ratio of cytokinin to auxin determines the type of organs regenerated from
undifferentiated callus tissue in vitro: callus placed on media with a high cytokinin to
auxin ratio usually produces many shoots and few roots while callus placed on media
with a low cytokinin to auxin ratio usually produces few shoots and many roots; equal
concentrations result in the proliferation of undifferentiated callus (Skoog and Miller,
1957)
...
Application of cytokinin to whole plants or detached leaves tends to
delay senescence (Gan and Amasino, 1996)
...
, 1993; Crowell and Amasino, 1994), and
etiolated seedlings grown in the presence of cytokinin adopt a morphology similar to
light-grown seedlings (Chory et al
...
Cytokinins have been shown to influence
sink/source relationships, germination, the formation of vascular tissue, and cotyledon
expansion in many different plant species (Mok and Mok, 1994)
...

These studies have used many different plant species and experimental designs,
sometimes making comparisons difficult
...
To avoid these problems, endogenous levels of cytokinin have been
altered by creating transgenic plants expressing the Agrobacterium ipt (Akiyoshi et al
...
, 1984), under the control of various promoters (Smigocki and Owens,
1988; Medford et al
...
, 1989; Estruch et al
...
, 1991;
Smigocki, 1991; Li et al
...
These studies have

largely supported the roles established by exogenous application of cytokinins, though
the precise role of cytokinins in plant development is still unclear
...
These transgenic tobacco plants developed normally except that
senescence was greatly delayed, resulting in dry mass and seed yields 50% greater than
wild-type plants
...
, 1999)
...
In contrast, the 9-glucoside conjugate of zeatin was elevated after 8 hrs and
remained elevated at 72 hrs after a heat shock in these transgenics
...
Overall, the biomass of the heat-shocked transgenics increased
...
The leaves of the
heat- shocked transgenics were serrated at the margins, which is similar to the
phenotype of Arabidopsis plants that overexpress the KNAT1 and STM genes
...


CYTOKININ AND THE SHOOT APICAL MERISTEM

The shoot apical meristem is a highly specialized group of cells from which the majority
of the aerial portion of the plant is derived by reiterative development (Kerstetter and
Hake, 1997)
...


One mechanism by which cytokinin may influence shoot apical meristem development
is by regulating gene expression
...
, 1994; Kerstetter et al
...

Transgenic plants that have an elevated level of cytokinin as a result of over-expression
of the ipt gene have some phenotypes reminiscent of transgenic plants over-expressing
kn1, such as a delay in senescence, reduced apical dominance, and ectopic shoot

formation (Kerstetter and Hake, 1997)
...
, 1999)
...


A similar relationship between cytokinin levels and expression of the maize kn1 gene
was observed when kn1 was over-expressed in tobacco
...
, 1999)
...
Remarkably, older SAG:kn1
leaves had cytokinin levels 15 times higher than wild-type plants, suggesting that kn1
may inhibit senescence by increasing cytokinin levels
...
Alternatively, the elevation of cytokinin in connection with ectopic expression
of kn1 may not accurately reflect the endogenous relationship between cytokinin and
kn1 homologs
...
, 2001)
...
In contrast, the
growth of the root system was enhanced, indicating that cytokinins may have opposing
roles in shoot and root development
...
Leaves were formed from a
significantly decreased number of cells, which was partly compensated for by an
increased cell size
...
The reduced cell proliferation in the shoot apical
meristems as a result of lowered cytokinin levels is consistent with an in vivo role for
this hormone in the regulation of cell division
...
A brief outline of the relevant
details of two-component signaling in prokaryotic and fungal systems is presented in
the following section
...


Two-component regulators are the major routes by which bacteria sense and respond to
various environmental cues (Hoch and Silhavy, 1995; Perraud et al
...
The E
...
The two
components generally consist of a sensor kinase that perceives environmental stimuli
and a response regulator

That propagates the signal, often by directly regulating transcription of target genes
(Figure 4)
...
Active sensor kinases are dimers
that trans-phosphorylate on a conserved histidine residue in the transmitter domain
...
Most response regulators also contain output
domains that typically act as transcription factors (Stock et al
...
However, there are
also instances in which the receiver domain regulates the activity of a separate protein,
as is the case with CheY, which is an E
...
CheY is comprised solely of a receiver domain and regulates the
activity of the flagellar motor via an interaction with the FliM protein (Welch et al
...


Extended versions of the basic two-component system of histidyl- aspartyl
phosphorelay signaling have been discovered in both prokaryotes and eukaryotes
(Perraud et al
...
The common thread linking multi-step phosphorelays is
that they involve four sequential phosphorylation events that alternate between
histidine and aspartate residues, although the number of proteins harboring these
phosphorylation sites varies
...
This pathway consists of three components: a
hybrid histidine kinase called SLN1 that contains a fused receiver domain, a response
regulator (SSK1) that mediates the output of the pathway, and a third component,

YPD1, a histidine phosphotransfer protein that mediates phosphotransfer between the
receiver domain of the sensor histidine kinase and the receiver domain of the response
regulator
...
The
nonphosphorylated form of the SSK1 response regulator activates SSK2;
phosphorylation of SSK1 by SLN1 via YPD renders it inactive
...
These are all found as gene families, and include
histidine kinases, histidine phosphotransfer proteins (AHPs), and two classes of
response regulators (ARRs)
...


THE HISTIDINE KINASE GENE amily

The Arabidopsis genome encodes a number of genes that share significant sequence
similarity to bacterial histidine kinases, including the ethylene receptors, the
phytochromes, and cytokinin receptors (Table 3; Figure 5)
...
, 2000)
...
Similarly, the histidine kinase-like domains
present in phytochromes are highly divergent and are unlikely to possess histidine
kinase activity
...


Several of the Arabidopsis sensor histidine kinases contain a C-terminal receiver
domain, a feature shared by other eukaryotic histidine kinase homologs
...
This second receiver
domain lacks some of the highly conserved residues found in other receivers, and in
AHK3 and AHK4 the putative phosphoaccepting aspartate is replaced by a glutamate
residue
...
, 2001)
...
AHK2, AHK3 and AHK4 share a region of
sequence similarity between the predicted transmembrane regions (Ueguchi et al
...
, 2001), which has been shown to contain the cytokinin binding domain
(see below)
...
It has been named the CHASE (cyclases/histidine kinases
associated sensing extracellular) domain and is present in the extracellular or
periplasmic regions of receptors containing an intracellular histidine kinase or
nucleotide cyclase domains
...


CYTOKIN RECEPTORS ARE SIMILAR TO HISTIDINE KINASES

The first evidence linking cytokinin perception to a histidine kinase came from a screen
for Arabidopsis genes whose overexpression resulted in callus proliferation in the
absence of exogenously supplied cytokinin (Kakimoto, 1996)
...
This analysis identified a gene called CKI1,
which encodes a protein with sequence similarity to histidine kinases
...
Recently, a loss-of-function allele of CKI1 has been
identified and shown to result in a female gametophytic lethal phenotype, consistent
with the expression pattern of the gene (T
...
While
this indicates an important function in female gametophyte development, it does not
address the role of CKI1 in cytokinin responsiveness
...
coli
resulted in a constitutive rescue of a histidine kinase mutation (Yamada et al
...
coli mutant (see below)
...
Thus, the
role of CKI1 in cytokinin signaling remains unclear
...
, 2001; Suzuki et al
...
The cytokinin
response 1 (cre1) mutant was identified in a screen for mutants impaired in the
responses of callus tissue to cytokinin, namely greening and shoot formation (Inoue et
al
...
The cre1 mutants are also less sensitive to cytokinin inhibition of root growth
in intact seedlings
...


Further evidence that CRE1 is a cytokinin receptor came from experiments based on the
ability of CRE1 to complement histidine kinase deficient yeast and E
...
In one report, CRE1 was found to rescue the growth
defect of a yeast strain lacking the SLN1 histidine kinase only when cytokinins were
present in the media (Inoue et al
...
This indicates that CRE1 function was activated
in response to cytokinins
...
The complementation of sln1D by
CRE1 required YPD1, indicating that CRE1 was acting through the yeast
phosphotransfer pathway
...
coli multistep phosphorelay system (Suzuki et al
...
,
2001; Yamada et al
...
As
with the complementation of sln1D by CRE1, the complementation in these systems
occurred in a cytokinin-dependent manner
...
pombe cells designed to express
CRE1 has confirmed that CRE1 binds the radiolabeled cytokinin isopentyl adenine with
high affinity (Yamada et al
...
Competition assays with non-labeled compounds
showed that other N6 substituted aminopurines, such as trans-zeatin and aromatic
cytokinins such as benzyl adenine could also bind CRE1, but various adenine
derivatives that were inactive in cytokinin response assays did not
...
Isopentenyl-adenosine (a cytokinin riboside)
failed to compete for binding to CRE1, consistent with the notion that only the free base
forms of cytokinins are active
...
, 1995; Mähönen et al
...
, 2001)
...
e
...
Indeed, the wol
mutation disrupts cytokinin binding in vitro
...
, 2001)
...
,
2000; Inoue et al
...
, 2001)
...
,
2000)
...
, 2000)
...
, 1995), resulted in an increased number of cell layers in the
vasculature compared to the wol single mutant and the detection of phloem-specific
markers in the vascular cylinder
...


No shoot phenotype has been described for cre1/wol mutants (Mähönen et al
...
, 2001; Ueguchi et al
...
g
...
Lossoffunction mutations of these closely related AHK genes may reveal a role for these genes
in the growth and development of the shoot
...
Similarly, the
Arabidopsis response regulators (ARRs) and histidine phosphotransfer proteins (AHPs)
appear to act downstream of the CRE1/AHK receptors in cytokinin signaling
...
, 1998)
...
The type-A ARRs
contain a receiver domain, but lack a classic output domain
...
In addition, most of the type-A ARRs are induced by exogenous
cytokinins, while the steady-state level of the type-B ARRs is unaffected by cytokinin
(Brandstatter and Kieber, 1998; Taniguchi et al
...
, 1999; Kiba et al
...
, 2000)
...


TYPE-A ARRs ARE CYTOKININ PRIMARY RESPONSE GENES

The Arabidopsis genome encodes ten type-A ARRs that fall into five pairs with highly
similar amino acid sequences (Figure 6), which may reflect an evolutionarily recent
duplication of the Arabidopsis genome (Vision et al
...
The amino acid sequences
of the receiver domains of the typeA ARRs are very similar, but the sequences of the
small Cterminal extensions (< 100 amino acids) are more divergent (Imamura et al
...
, 2000)
...
Thus, these are
cytokinin primary response genes
...
, 2000), which implies that a transcription factor(s) is activated in
response to cytokinin (see below)
...
, 1998; Kiba et

al
...
, 2000)
...
, 1998; Urao et al
...
, 1999), which may reflect an
alteration of endogenous cytokinin levels in response to these stimuli
...
,
2000)
...
The expression of ARR5 mRNA in shoot and
root apical meristems was confirmed by whole mount in situ analysis of seedlings and
is consistent with likely sites of cytokinin action
...
, 2001)
...
Furthermore, the level of ARR4 protein was highly
elevated in response to light, but the steady-state level of ARR4 mRNA is not altered by
light (Brandstatter and Kieber, 1998)
...


TYPE-B ARRs ACTS AS TRANSPORTATION FACTORS

There are eleven type-B ARRs in the Arabidopsis genome that are characterized by the
presence of a receiver domain and a large C-terminal extension (Schaller et al
...

Unlike type-A ARRs, the steady-state level of typeB ARRs is not affected by application
of cytokinins (Imamura et al
...
, 1999; Lohrmann et al
...
The Cterminal domains of the type-B ARRs contain potential nuclear localization signals
(Sakai et al
...
, 1999), and several type-B ARRs have been
demonstrated to localize to the nucleus based on observations of GUS or GFP fusion
proteins expressed in onion epidermal, parsley, or Arabidopsis cells (Lohrmann et al
...
, 2000; Hwang and Sheen, 2001; Lohrmann, 2001)
...
, 1999; Lohrmann, 2001)
...
, 2000)
...
, 2001)
...
, 2000)
...


In summary, the C-terminal domains of the type-B ARRs act as classic two-component
output domains; they bind DNA in a sequence-specific manner and are capable of
activating transcription
...


HISTIDINE PHOSPHOTRANSFER PROTEINS IN ARABIDOPSIS

Histidine phosphotransfer proteins mediate transfer of a phosphoryl group from the
receiver domain of an activated hybrid sensor histidine kinase to the receiver domain of
a response regulator in a phosphorelay signal transduction pathway (Figure 4)
...
, 2000)
...
There is also a single pseudohistidine phosphotransmitter in Arabidopsis in which the conserved histidine is
replaced with an asparagine residue
...
, 1998; Suzuki et al
...
, 2000)
...
A number of the Arabidopsis phosphotransfer proteins were able to
complement a deletion of YPD1, the histidine phosphotransfer protein component of
the budding yeast osmosensing phosphorelay pathway described above

(Miyata et al
...
, 1998)
...

coli assay system (YojN-RcsB) employed to examine CRE1 function (Suzuki et al
...
, 2001b; see above) suppressed the cytokininresponsive phosphotransfer,
indicating that AHPs were competing with the endogenous E
...
, 2001b)
...
coli membrane preparations can
phosphorylate the AHP1 and AHP2 at the conserved histidine residue (Suzuki et al
...
, 1998; Imamura et al
...
,
2001b)
...
, 1998; Suzuki et al
...
, 2000; I
...
Kieber, unpublished)
...
How specificity is achieved in this system is
question that has yet to be addressed
...
Furthermore, yeast two-hybrid and in vitro analyses indicate that the
AHPs mediate phosphotransfer reactions to the receiver domains of type-A and type-B
ARRs
...
, 2001)
...
A loss-offunction ARR1 mutation results in reduced sensitivity to cytokinin in both root
elongation assays in intact plants and shoot regeneration assays in tissue culture, while
overexpression of ARR1 increases the sensitivity to cytokinin in these assays
...
Transgenic plants engineered to overexpression ARR2 displayed
phenotypes consistent with an activation of the cytokinin response pathway, including

excess cell proliferation and shoot and leaf formation in cultured cells as well as delayed
leaf senescence in excised leaves (Hwang and Sheen, 2001)
...
This is consistent with previous results that
suggested that the receiver domain of type-B ARRs inhibits the activity of the Cterminal output domain (Sakai et al
...


Further evidence linking the type-B ARRs to the cytokinin signaling came from studies
employing a transient expression system in Arabidopsis protoplasts (Hwang and
Sheen, 2001)
...
Arabidopsis mesophyll protoplast transfected with this
construct displayed a strong increase in luciferase activity specifically in response to
cytokinin, similar to the induction of the endogenous gene
...
This
indicates that type-B function is likely to be a rate limiting factor in the activation of the
ARR6 gene
...


Taken together, these results suggest that the type-B ARRs act as positive regulators of
cytokinin responsiveness, including the induction of type-A ARR gene expression
...
How type-B function is regulated, and in particular the role
of phosphorylation in its activation remains unresolved
...
coli (Inoue et al
...
, 2001b; Ueguchi et al
...
CKI1 was localized to the plasma membrane in this
system
...
Expression of mutant forms of CRE1 in which the conserved
histidine and aspartate residues in the transmitter and receiver domains were mutated
failed to elicit an elevated ARR6LUC response to cytokinin, supporting the model that
phosphotransfer within CRE1 is required for its signaling function
...


Overexpression of several type-A ARRs in the protoplast system suppressed the
cytokinin induction of ARR6-LUC, indicating that type-A ARRs negatively regulate
their own expression
...


Several lines of evidence also implicate the AHPs as mediators of cytokinin signaling,
linking the activation of CRE1 by cytokinin binding to the activation of the type-B
ARRs
...
Secondly,
overexpression of AHP2 from a CaMv 35S promoter in transgenic Arabidopsis resulted
in modest hypersensitivity to exogenous cytokinin in root and hypocotyl elongation
assays (Suzuki et al
...
These results, coupled with the two-hybrid and in vitro
phosphorylation experiments described above, indicate that these histidine
phosphotransfer proteins are likely to mediate signaling between CRE1 and the type-B
ARRs
...


The data described above are generally consistent with the simple model for
phosphorelay signal transduction in cytokinin signaling presented in Figure 7
...
This induces autophosphorylation on a histidine
residue within the transmitter domain and subsequent transfer of the phosphate to an
aspartate residue within the fused receiver domain
...
The activated type-B ARRs bind to elements within

the promoter of the type-A ARRs to increase their rate of transcription
...
Analogy to bacterial systems, in vitro studies and conservation of the sites of
phosphorylation all suggest that phosphorylation of the AHPs and ARRs play a role in
regulating their function
...
At least in the cases of ARR2 and ARR6,
phosphorylation at the conserved aspartate residue predicted to be the
phosphoaccepting residue may not be required for regulation of ARR6 transcription
(Hwang and Sheen, 2001)
...
Cytokinin
signaling beyond the phosphorelay

While it is becoming clear that the signal transduction chain mediating the perception of
cytokinin and the induction of the type-A ARRs is mediated by a classic phosphorelay
system, how this translates into alteration in cellular function is unknown
...
These targets
and potential novel elements may be represented in some of the cytokinin action
mutants, the cytokinin responsive genes, and/or the yeast two-hybrid interactors that
have been identified
...


Proteins that interact with two-component elements

ARR4 was found to interact with the red light photoreceptor PHYB in a yeast twohybrid screen (Sweere et al
...
Overlaps between the action of light and cytokinin
on plant development and induction of gene expression have long been observed
...
, 1994)
...
, 2001) provides
an interesting and direct link between light and cytokinin signaling
...
Yeast two-hybrid analysis showed that
the interaction of ARR4 with PhyB was mediated by the extreme N-terminus of PhyB
...
Consistent with this, overexpression of ARR4 resulted in

hypersensitivity to red light as assayed by measuring the lengths of hypocotyls in
seedlings grown continuously in varying doses of red light and by comparison of the
root length and the number of leaves at flowering between transgenic and wild-type
plants
...


Additional elements that interact with AHPs and the type-A ARRs have been identified
in yeast two-hybrid screens
...
, 1989; Yamada et al
...

A novel nuclear localized protein, TCP10, was found to interact with several AHPs
...
, 1999; Suzuki et al
...


CYTOKININ ACTION MUTANTS

A number of Arabidopsis mutants have been reported that may affect cytokinin action,
in addition to the mutations in two-component elements described above
...
Cyr1 mutants have pale
green leaves, abbreviated shoot development and incomplete cotyledon and leaf
expansion (Deikman and Ulrich, 1995)
...
However, cytokinin-primary response genes are induced normally in
cyr1 and cytokinin induces ethylene biosynthesis to wild-type levels in cyr1 mutants (J
...


Two CYTOKININ-HYPERSENSITIVE mutants, ckh1 and ckh2, were identified as
showing increased sensitivity to cytokinin in tissue culture (Kubo and Kakimoto, 2000)
...
Adult ckh1 and ckh2 plants
are short, but do not have any other striking morphological alterations
...
,
1998)
...
The pas1 mutants also have short primary roots and
lack secondary roots, a phenotype reminiscent of wol mutants (Scheres et al
...
, 2000; Inoue et al
...
Finally, the rosette of pas1-1 mutant is small
and abnormal and pas1 plants rarely produce flowers

(Vittorioso et al
...


Mutations in STUNTED PLANT 1(STP1) affect cell division rate and growth rate, but
not overall plant morphology
...
However, root growth in the stp1
mutant does show reduced sensitivity to cytokinins
...


Several Arabidopsis tissue culture lines were identified that were able to grow in vitro
on media lacking added auxin and cytokinin (Frank et al
...
These lines grew as
shoot-like callus, root-like callus or as undifferentiated tissue
...
The steady-state level of transcript of the CKI1 and STM genes were
elevated in all six shooty lines, and the level of STM transcript was elevated in three,
which, coupled with the observation that they have WT levels of cytokinins, suggests
that they may be affected in cytokinin signaling
...
, 1998a; Vogel et al
...
This
analysis revealed that cytokinin elevated ethylene production primarily via a posttranslational activation of ACS5, which encodes one member of the Arabidopsis ACC
synthase gene family
...
This mutation does not affect the
morphology of the adult plant
...
, 1993)
...
The AMP1 gene has recently been cloned and
found to encode a putative glutamate carboxypeptidase (Helliwell et al
...
This
homology makes it unlikely that this gene directly affects cytokinin biosynthesis
...
Cytokinins and gene expression

Many changes in gene expression, in addition to the induction of type-A ARRs, have
been detected in response to application of exogenous cytokinins (reviewed in
Schmülling et al
...
Genes showing cytokinin-responsive expression are good
candidates for components of cytokinin signaling and effector pathways to various
cellular and developmental responses
...


Additional genes responsive to cytokinin have been identified by cytokinin treatment of
soybean or tobacco tissue culture cells that were starved for cytokinin
...
, 1990)
...
Sequence analysis revealed that two of these genes were homologous to
ribosomal proteins and one was homologous to a pollen allergen protein (Crowell et al
...
Several studies have also examined cytokinin-regulated gene
expression during tissue differentiation, including nodulation, senescence, floral
development, lateral bud induction, and various aspects of light development
...
However, most of these genes are also
induced by other stimuli, most notably light and auxin, and none of these genes are
induced with kinetics suggestive of an immediate early response gene
...
Additional

cytokinin responsive genes have been identified using microarrays and these may
reveal additional elements involved in cytokinin action (T
...
Roshette and J
...


CYTOKININS AND THE CELL CYCLE

Cytokinins are required, in concert with auxin, for cell division in culture in a wide
variety of plant cells
...
Immunocytochemistry and direct measurements of cytokinin
revealed high cytokinin levels in mitotically active areas, such as the root and shoot
meristems, and very low levels are found in tissues where the cell cycle is arrested (Mok
and Mok, 1994; Dewitte et al
...
Application of exogenous cytokinin to some organs
that normally lack this hormone has been shown to induce cell division
...


Several observations suggest that cytokinins may play a role in the G2/M transition
(reviewed in Hare and Staden, 1997), though a decisive link is lacking
...
, 1993), and cytokinins were
demonstrated to influence the activity, via the phosphorylation state, of a cdc2-like
kinase in tobacco protoplasts (Zhang et al
...


Compelling evidence that cytokinin regulates the G1/S transition in the cell cycle has
been obtained by Murray and co-workers (Riou-Khamlichi et al
...
This group
previously identified three different Arabidopsis genes encoding D-type cyclins by
complementation of a yeast strain deficient in G1 cyclins, and found that one, CycD3,
was induced in cultured cells by exogenous cytokinin application (Soni et al
...
In
Arabidopsis, cytokinin rapidly upregulates the expression of CycD3, a cyclin that plays
a key role in the regulation of plant cell division both in vitro and in vivo (Soni et al
...
, 1999)
...
Riou-Khamlichi and co-workers found, using in

situ hybridization, that in Arabidopsis CycD3 was expressed in the shoot meristem, leaf
primordia and axillary meristems, and its induction by cytokinin was also specific to
those tissues (Riou-Khamlichi et al
...
Thus, this gene is expressed primarily in
proliferating tissues, as expected if it is an important element regulating cell division
...
Normally, when explanted into culture, cells require both auxin
and cytokinin in the media in order for cell division and callus formation to occur
...
To
demonstrate a role for CycD3 in cell division, the levels of S-phase associated histone
H4 mRNA were examined in the leaf explants
...

Finally, the expression of CycD3 and histone H4 mRNA was observed in parallel with
DNA synthesis during synchronous activation of quiescent Arabidopsis cells and S
phase was found to occur significantly after the induction of CycD3, which implies that
CycD3 may be involved in the G1/S transition
...


SUMMARY AND PERSPECTIVES

Arabidopsis genes encoding key enzymes of cytokinin biosynthesis and metabolism
have been identified, and the near future should reveal much about the mechanism and
regulation of cytokinin biosynthesis and metabolism
...
This pathway is, at least in outline, very similar to the
bacterial two-component phosphorelay paradigm
...


It is interesting that the proposed cytokinin signal transduction pathway is remarkably
similar in its overall design to the auxin response pathway (reviewed by (Leyser, 2001)
...
These are counteracted by a related family of
hormone primary response genes that encode proteins lacking the DNA binding
domain, which negatively regulate the response pathway (type-A ARR or IAA genes)
...
The ARF/IAAs have been shown to form homo- and heterodimers, but it
remains an open question whether this is also the case of the type-A and type-B ARRs
...
How does cytokinin
act to regulate cell proliferation, and what others roles does it play in Arabidopsis
growth and development? How is the biosynthesis of cytokinin regulated? What is the
role of histidine-to-aspartate phosphorylation in the cytokinin signal transduction
chain? What are the outputs of the two-component signaling chain? How is specificity
of interaction among the various members of the gene families encoding the twocomponent elements achieved? Is there crosstalk between ethylene and cytokinin
signaling at the level of AHP phosphorylation? The cloning of genes corresponding to
the receptors, signaling elements, biosynthetic and metabolic enzymes, as well as
isolation of gain- and loss-of-function mutants in these genes provides a powerful suite
of tools to address these questions
...
ABAs act as an antagonist to Gas
...
It is also called “stress hormone” as it
increases the tolerance of plants
...
Pioneering studies by Hemberg found a water and ether soluble growth‐inhibiting
substance that is critical for the maintenance of bud dormancy in potato and Fraxinus (Hemberg
1949a, 1949b)
...
During the same period, a
substance that controlled the abscission of cotton fruits was discovered by Frederick Addicott
and named abscisin II (Ohkuma et al
...
The Addicott lab found that abscisin II also
promotes leaf abscission in cotton seedlings and inhibits indoleacetic acid‐induced growth of
Avena coleoptiles
...
1965; Addicott et al
...
Although the abscission‐
promotion role of ABA was considered by many to be an indirect effect of the elevated level of
ethylene (Cracker and Abeles 1969), recent studies have demonstrated that ABA promotes leaf
senescence and abscission independent of ethylene (Ogawa et al
...
2016)
...
Genetic
screens for viviparous mutants in maize and Arabidopsis, and for mutants that are insensitive
to sugar, salt, and ABA during germination lead to the identification of numerous components
involved in ABA biosynthesis and signaling
...
1984; Giraudat et al
...
1994,1997; Meyer
et al
...
1998; Finkelstein and Lynch 2000; Laby et al
...
2002)
...
Due to its high functional redundancy,
the ABA receptor Pyrabactin resistance 1 (PYR1) and PYR1‐like (PYL) proteins (hereafter
referred to as PYLs) were not revealed until 2009 by Sean Cutler and co‐workers through
chemical genetic screens for mutants that are insensitive to the ABA analog pyrabactin (Park et
al
...
In the meantime, regulatory components of the ABA receptors (RCARs) were isolated
through yeast two‐hybrid screens in the Erwin Grill lab (Ma et al
...
The function of the
proteins of PYL/ RCAR family was also demonstrated by in vitro reconstitution of the core
ABA signaling pathway (Fujii et al
...
2009; Miyazono et al
...
2009; Yin et al
...
2009a, 2009b; Gonzalez‐Guzman et al
...
2015; Miao et al
...
2018)
...


METABOLIC CONTROL OF ABA LEVELS

Plants quickly accumulate ABA which in turn activates several stress responses when subjected
to abiotic stresses such as drought and salt
...
Modulation of ABA levels in tissues and cells is
critical for balancing defense and growth processes when plants experience non‐optimal
environments
...


ABSCISIC ACID BIOSYNTHESIS

Abscisic acid is a sesquiterpenoid containing 15 carbon atoms
...
Phytopathogenic fungi synthesize
ABA through the mevalonate pathway with intermediates containing no more than 15 carbon
atoms, which is also called the “direct pathway” (Hirai et al
...
2018;
Takino et al
...
Plants synthesize ABA using the carotenoid pathway, which is also known
as the “indirect pathway
...
2013)
...


Genetic screens of viviparous mutants in maize have identified several ABA auxotrophic
mutants, named vp2, vp5, vp7, and vp9, which are defective in zeaxanthin synthesis (Reid

1990)
...
The Arabidopsis loss‐of‐function mutant, aba1, is defective in the gene for zeaxanthin
epoxidase (ZEP) which catalyzes the conversion of zeaxanthin to all‐trans‐violaxanthin via
antheraxanthin (Audran et al
...
This pathway then bifurcates into two pathways
...
2007)
...
1975; Schwartz et al
...
In Arabidopsis, based on
analysis of the sequence and function of homologous genes of VP14, NCED2, NCED3, NCED5,
NCED6, and NCED9 have been identified as participants in a rate‐limiting step in ABA
biosynthesis, where NCED3 is the critical enzyme for ABA synthesis at this point (Iuchi et al
...
2003)
...
Processes
that convert C40 to xanthoxin (C15) all take place in plastids, and in the cytoplasm, ABA2 and
AAO3 convert xanthoxin into ABA
...
ABA
catabolism is controlled by both ABA conjugation and catalytic hydroxylation
...
ABA can be catalyzed to phaseic acid (PA) by CYP707As, which in turn is
catalyzed to dihydrophaseic acid (DPA) by PA reductase (PAR)
...
2001; Cheng et al
...
The first mutants identified as defective in ABA synthesis
were in tomato and called flacca and sitiens, which are impaired in the oxidation of ABA
aldehyde to ABA (Tal 1966; Taylor et al
...
These data not only revealed an intact
biosynthesis pathway of ABA, but also showed that ABA synthesis is via an indirect pathway in
plants rather than a direct one based on C15 isoprenoid synthesis as is in fungi (Nambara and
Marion‐Poll 2005)
...
The maintenance of a basal level of free ABA
consistent with different tissues in different environments is paramount to the appropriate

growth and development states of the whole plant
...
ABA can be
glucosylated by a UDP‐glucosyltransferase (UGT) encoded by UGT71C5
...
2015b)
...
2006; Xu et al
...
The conjugation cycle established by glucosyltransferase and β‐glucosidase allows plants
to phenotypically adapt to their environment through ABA‐mediated responses by activating
and inactivating ABA rapidly
...
2004)
...
2016)
...
2016)
...
Ikegami et al
...
Other studies have confirmed that ABA is
synthesized in leaves and then transported to other organs (Zhang et al
...
Thus, transport
of ABA among cells, tissues, and organs is an important part of ABA function in systemic stress
responses of the whole plant
...
ABAH can diffuse passively through the plasma membrane, and the diffusion of ABA
largely declines with alkalization of the cytoplasm which increases during osmotic stresses
(Wilkinson and Davies 1997; Karuppanapandian et al
...
The active transport of ABA relies
on: (i) ATP‐binding cassette (ABCG) transporters; (ii) NRT1/PTR (NPF); (iii) multidrug and
toxic compound extrusion (MATE)‐type/DTX transporters (DTX50); and (iv) AWPM‐19 family
proteins (OsPM1), which were originally identified in rice (Kuromori et al
...
2012; Zhang et al
...
2015; Yao et al
...


In eukaryotes, there are eight subfamilies of ABC transporters, namely ABCA to ABCH
...
2010;
Kang et al
...
2011; Pawela et al
...
Among these transporters,
AtABCG25 functions in exporting ABA from vascular tissues to multiple sites such as guard
cells
...
2010; Kang et al
...
Besides ABCG transporters, AtDTX50 also acts as an exporter of ABA, and both
AtNPF4
...
2012; Zhang et al
...
2018)
...
However, AtABCG22 may
not transport ABA directly, although it is possibly involved in ABA efflux (Kuromori et al
...
Functionally, ABA transporters have been increasingly shown to be involved in
transpiration, root morphology, seed germination and other processes important to stress
...
With the introduction of FRET
(fluorescence resonance energy transfer) marker/sensors, ABACUS and ABAleon, detection of
ABA at a cell level has become attainable (Jones et al
...
2014)
...
2014)
...
2018)
...
2018c)
...
2018)
...
3 (Tan et al
...
The
subsequent transcriptional changes lead to the rapid release and increased synthesis of ABA,
while reducing ABA catabolism under drought stress, allowing the initiation of several ABA‐
mediated responses that affect the growth and survival of plants
...

Some biotrophic pathogens such as wheat rust fungi can promote increases in ABA that lead to
elevated cytoplasmic sugar accumulation by enhancing TaSTP6 expression (Huai et al
...


Upon infection by the necrotrophic pathogen Botrytis cinereal, the transcription factor WRKY33
promotes ABA biosynthesis by upregulating the transcription of NCED3 and NCED5 in
Arabidopsis (Liu et al
...
The tomato NAC transcription factor LeJA2 (for jasmonic acid 2)
upregulates expression of LeNCED1 that also promotes the biosynthesis of ABA that can limit
pathogen entry through stomata (Du et al
...


Hormone crosstalk also participates in the homeostasis of ABA
...
2018)
...
2015)
...
Collectively, all these processes finetunes the level of ABA during different
developmental stages and in response to various environmental changes
...


CORE ABA SIGNALING

Abscisic acid functions in plants through cellular recognition by the intracellular receptor, PYLs
(Ma et al
...
2009)
...
2009; Ma et
al
...
2009; Rubio et al
...
SnRK2s are then activated through
autophosphorylation, or they can be activated by other protein kinases such as Raf‐like
MAPKKKs (Lee et al
...
2015; Nguyen et al
...
SnRK2s regulate multiple
physiological responses through phosphorylating target substrates including ion channels,
transcription factors and transporters among others (Umezawa et al
...
2013)
...


The discovery of PYL/PP2C co‐receptors has led to a substantial effort to unravel the complex
signaling system that controls plant responses to ABA
...
Regulation of these components is critical to managing excessive and detrimental
defense responses under abiotic stress conditions
...


ABSCISIC ACID RECEPTORS

In Arabidopsis, the PYL ABA receptor family consists of 13 ABA receptors, and one non‐
responsive PP2C regulator PYL13 (Fujii et al
...
2013; Zhao et al
...
PYLs have
differing binding properties with ABA, and selectively interact with PP2Cs (Szostkiewicz et al
...
2011; Antoni et al
...
2013; Zhao et al
...
2017)
...
Generally, monomeric
PYLs such as AtPYL46 and AtPYL8‐10 have higher ABA binding affinity and interact with
PP2Cs in an ABA‐enhanced manner; while dimeric PYLs such as AtPYR1 and AtPYL1‐2 have
lower ABA binding affinity and interact with PP2Cs in an ABA‐dependent manner (Hao et al
...
In contrast, AtPYL13 and OsPYL12 interact with and inhibit several PP2Cs in an ABA‐
independent manner (Li et al
...
2013; He et al
...
2018),
indicating that they are not ABA receptors
...
2018; Cheng et al
...
Emergence of PYL receptors
apparently occurred before terrestrial occupation
...
2019)
...
2015; Wang et al
...
2017; Jahan et al
...


Although the PYLs function redundantly in their regulation of ABA coreceptor PP2Cs, some of
them function separately in regulating distinct downstream factors
...
2016)
...
2014; Xing et al
...
PYLs are differentially expressed in multiple
organs, cells, and during different growth stages (Gonzalez‐Guzman et al
...

2013)
...
2013; Zhao et al
...
AtPYL9 is highly expressed in senescent leaves, and
promotes ABA‐induced leaf senescence
...
2019)
...
This is demonstrated by the traits of “stratospheric” order PYL
mutants, including the pyl quattuordecuple mutant in Arabidopsis, and the ospyl septuple
mutant in rice (Miao et al
...
2018)
...
Another high
order pyl duodecuple mutant, with all PYL ABA receptors mutated except AtPYL6, is extremely
insensitive to ABA with respect to several physiological processes including seed germination,
seedling growth, stomatal movement, leaf senescence and gene expression response (Zhao et al
...
The ospyl septuple mutant in rice, with all group I (OsPYL1‐6 and OsPYL12) PYLs
mutated, is insensitive to ABA during seed germination and stomatal movement, and shows a
strong preharvest sprouting phenotype in field conditions (Miao et al
...
In contrast to the
severe growth defects of PYL mutants in Arabidopsis, mutations of group I OsPYLs promote
rice growth in the field (Miao et al
...
2018)
...

In the presence of ABA, pyrabactin resistance 1‐like (PYLs) form complexes with the PP2Cs,
allowing the activation of SnRK2s and downstream responses
...
PYLs are phosphorylated by several
protein kinases including TOR, AEL, CEPR2, and CARK1
...
CARK1
phosphorylates PYLs and may enhances their activity, whereas NO inactivates PYLs by tyrosine
nitration
...
Activities of PP2Cs can be enhanced by EAR1 and the receptor‐
like kinase PR5K2, and can be reduced by RDK1
...
SnRK2s can be activated by several other kinases including RAF10
and BIN2
...
Although PYLs
are essential for ABA‐mediated activation of SnRK2s, they are also involved in an antagonistic
regulation of SnRK2 activation during osmotic stress
...
These decorations control the fine regulation of responses to
environmental changes (Figure 2)
...
The TOR
kinase phosphorylates PYLs at a conserved site corresponding to PYL4 Ser114 that inactivates
PYLs (Wang et al
...
The AEL casein kinases phosphorylate PYLs at partially conserved
sites corresponding to PYR1 Ser109 and PYR1 Ser152 and promote ubiquitination and
degradation of PYLs (Chen et al
...
The plasma membrane‐localized leucine‐rich receptor‐
like kinase CEPR2 phosphorylates PYLs at a conserved site corresponding to PYL4 Ser54 and
promotes degradation of PYLs (Yu et al
...
A putative receptor‐like cytoplasmic kinase
(RLCK) VIII subfamily kinase CARK1 phosphorylates PYR1 and PYL1/2/3/8 at a less
conserved site corresponding to PYR1 Thr78 which enhances ABA responses, and the cark1
mutant Is less sensitive to ABA (Zhang et al
...
2019b)
...
2018b)
...

2014; Irigoyen et al
...
2016; García‐León et al
...
Consistent with their known
functions, the triple knockout mutant of CEPR2 and its homologs Phloem intercalated with
xylem (PXY) and PXY‐Like 2 (PXL2) are hypersensitive to ABA
...
2016, 2019; Chen et al
...

2018b; García‐León et al
...
2019a)
...
2012), which could be important in the establishment of homeostatic ABA
responses
...
It is well known that TOR is activated by glucose and other components that participate
in energy homeostasis (Xiong and Sheen 2012)
...
2015)
...
2018b; Yu et al
...


Regulators of ABA co‐receptors Protein phosphorylation and dephosphorylation are crucial for
maintaining the appropriate balance of ABAmediated growth regulation depending on the
environmental status of the plant (Zhu 2016; Shi et al
...
Protein
phosphatases can interact with and inhibit SnRK2s, SnRK1s, SnRK3s, and even mammalian
AMPKs, which are also core components in abiotic stress, ABA and energy signaling (Sanders
et al
...
Among the 80 PP2Cs in Arabidopsis, nine clade A PP2Cs including
ABI1/2, HYPERSENSITIVE TO ABA (HAB) ½, ABAHYPERSENSITIVE GERMINATION1
(AHG1), AHG3/PP2CA, HIGHLY ABA‐INDUCED (HAI) 1/2/3, and 3 clade E PP2Cs E‐
Growth‐Regulating PP2C (EGR) 1/2/3 function as negative regulators of stress responses
...
2009; Rubio et al
...
2009; Komatsu et al
...
Under stressed conditions, the ABA‐bound PYLs
interact with the conserved C‐terminal catalytic domains of clade A PP2Cs, which in turn
releases the inhibition of SnRK2s by the PP2Cs and allows activation of stress responses (Ma et
al
...
2009)
...
2017)
...
2017; Ding et al
...
Ancient PYL
without ABA binding affinity has been reported in algae, while PP2Cs and SnRK2s from algae
have conserved functions compared with those from higher plants (Lind et al
...
2018; Cheng et al
...
2019)
...
Although ABA and abiotic stresses inactivate PP2Cs to induce stress responses, the
expression levels of PP2Cs are actually upregulated by abiotic stresses and ABA through ABRE‐
BINDING FACTORS (ABFs), creating a feedback control loop to maintain new homeostatic
levels (Bhaskara et al
...
2019)
...
Indeed, several studies have indicated that
activities of PP2Cs can be enhanced by several PP2C binding proteins
...
2018a)
...
2019)
...
2019)
...
2017)
...
2015; Wu et al
...
2019; Julian et
al
...


Regulators of core protein kinases Abiotic stresses and ABA induce the activation of several
protein kinases including SnRK2s, CPK3, SOS2/ CIPK24/SnRK3
...
2001; Boudsocq et al
...
2009; Lin et al
...

2010; Umezawa et al
...
2013; Ding et al
...
2018)
...
2/3/6 protein kinases are activated by osmotic, salt, cold, and ABA treatment
...
2/3/6 from inhibition
...
These kinases appear to be
critical for the activation of SnRK2s and subsequent responses to ABA and abiotic stresses in
Arabidopsis and Physcomitrella patens (Huang et al
...
2015; Saruhashi et al
...
2016; Hwang et al
...
2019; Shinozawa et al
...

BRASSINOSTEROID INSENSITIVE 2 (BIN2), the Glycogen synthase kinase 3s
(GSK3s)/Shaggy‐like kinases (ASKs) repress brassinosteroid (BR) signaling, whereas they
enhance ABA signaling through specifical phosphorylation of SnRK2
...
3,
but not SnRK2
...
2014)
...
6 at Cys137 that inactivates SnRK2s (Wang et al
...
Although PYLs
are essential for ABA‐mediated activation of SnRK2s, they are also involved in an antagonistic
regulation of activation of SnRK2s by osmotic stress (Zhao et al
...
Overall, SnRK2s can be
activated by abiotic stresses and repressed by growthpromoting signals such as NO and BR
...
2013; Assmann and Jegla 2016;
Martin‐StPaul et al
...
2017)
...
Stomatal closure is the major process controlling the
transpirational water loss of plant
...

ABA‐induced Ca2+ signal involves the induction of reactive oxygen species (ROS) and inositol‐
1‐4‐5‐triphosphate (IP3), and may be decoded by CPK3/4/6/10/11/21/23 through activation
of SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1) and SLAC1 HO-

MOLOG 3 (SLAH3) to promote efflux of Cl−, which is also regulated by GHR1
...
CIPK23, CPK8‐CAT3 and rectifying channel GOAK, while
CPK13 inactivates KAT1 and KAT2, two K in CPK11‐Di19‐PR1/2/5 modules also regulate
stomatal closure
...
6/OST1 is a key regulator of Ca2+‐independent
stomatal closure
...
OST1 can also phosphorylate and inhibit
AKS1 to reduce expression of KAT1
...


Abscisic acid can induce the opening of Ca2+ channels which allows calcium ions to mediate
the closure of stomata pores (Mcainsh et al
...
2000; Pei et al
...
The
increase in cytosolic Ca2+ in guard cells in response to ABA likely involves the induction of
reactive oxygen species (ROS) and inositol‐1‐45‐triphosphate (IP3) (Gilroy et al
...

1996; Pei et al
...
2001; Mustilli et al
...
Ca2+ mediated signals could be
decoded by several Ca2+ sensors, including calcium dependent protein kinase (CPK) 3/
4/6/10/11, which may phosphorylate and activate the Slowtype (S‐type) anion efflux channels
including SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1) and SLAC1 HOMOLOG 3
(SLAH3) that are involved in stomatal closure and reduction of leaf water loss (Mori et al
...
2007; Zou et al
...
2012)
...
2010, 2011; Franz et al
...
2012; Demir et al
...
CPK8 was also reported to regulate stomatal closure that is
induced by ABA, ROS, and Ca2+ through the direct phosphorylation of CATALASE3 (CAT3)
(Zou et al
...
The CPK11‐Di19‐PR1/2/5 pathway also contributes to

Drought tolerance probably by affecting the stomatal movement (Liu et al
...
Besides CPKs,
CBL‐interacting protein kinase 23 (CIPK23) has also been implicated through genetic analyses
of ABA‐induced stomatal closure (Cheong et al
...
In addition to the modulation of anion
channel efflux, other ion channels can be regulated in a Ca2+ dependent manner
...
2003; van
Kleeff et al
...
Moreover,

+ flux CPK13 also inactivates KAT1 and KAT2, two K

in

Channels, and affects stomatal behavior through specific phosphorylation events (Ronzier et al
...
The activity

+

Of the H ‐ATPase of guard cells of fava bean was shown to be inhibited by Ca2+ in isolated
microsomal membranes (Kinoshita et al
...


Ca2+‐independent pathway

The ABA activated SnRK2
...
2000)
...
2010; Imes et al
...
OST1 up‐regulates the activity of SLAC1 and
KUP6, a KUP/HAK/KT family potassium efflux transporter, and inhibits KAT1 through
phosphorylation, which also affects stomatal movement (Kwak et al
...
2009;
Sato et al
...
2013)
...
2013)
...

2014)
...
2012; Devireddy et al
...


Both Ca2+‐dependent and Ca2+‐independent pathways mediate the decline of turgor of the
guard cells through membrane depolarization during water deficits, leading to reductions in
stomatal aperture
...

For example, ABI1 can dephosphorylate and inactivate CPK6 and OST1 in guard cells (Geiger et
al
...
2012)
...
Recent
studies have also suggested that salicylic acid (SA) may induce stomatal closure independent of
OST1 but dependent on CPK3/6 (Prodhan et al
...
There is a possibility that osmotic
Stresses may also induce Ca2+ elevation and regulate stomatal movement through Ca2+
sensors directly
...
An increase in CO2 concentration is able to reduce the number and size of
stomata (Woodward 1987; Gray et al
...
Stomatal movement is also triggered by high
concentrations of CO2 and this is impaired in ost1 mutants but not in ABA biosynthesis
mutants such as nced3/nced5 and aba2‐1 and in the ABA signaling mutant pyl112458
...
2011; Hsu et al
...
However, other study has shown that the effects of
CO2 on stomatal closure are dependent on ABA signaling, and therefore, the relationship
between CO2 and ABA needs further clarification (Chater et al
...
Recently, Dittrich et al
...


Plants also regulate stomatal behavior to counter the ability of some pathogens to control
stomatal functions (stomatal immunity)
...
2018)
...
syringae (flg22), a member of the
pathogen‐associated molecular pattern (PAMP), induces stomatal closure by stimulating
SLAC1/SLAH3 in an OST1‐dependent manner in guard cells (Guzel Deger et al
...
Su et al
...
Also, the phytotoxin produced
by P
...
2006)
...
After that, subsequent
division of embryo cells is arrested at the mature embryo stage and storage products
accumulate
...
Upon rehydration, the embryo radicle enlarges by cell
elongation to break through the seed coat (germination) and the embryo enters the next
generation (Mansfield and Briarty 1996; Raz et al
...
Abscisic acid is involved in many
phases of embryo development during alternation of generations (sporophyte to gametophyte
to sporophyte)
...


Central regulation in embryo development The continuous growth of the embryo is arrested
during the transition from the embryogenesis phase to the early maturation phase, which is
primarily regulated through control of cell division (Raz et al
...
Embryo growth arrest in
mature seeds is controlled by FUSCA3 (FUS3), Leafy cotyledon 1 (LEC1) and LEC2, which is
evidenced by the fact that fus3, lec1, and lec2 mutants all fail to fully suspend embryo growth
and exhibit premature germination
...
2/3/6 triple and abi3/vp1 double mutant also show premature germination in
Arabidopsis, rice and maize (Robichaud et al
...
2009; Miao et al
...
However, ABA biosynthesis and signaling mutants aba1 and abi3
display normal embryo growth, indicating that FUS3, LEC1, and LEC2 control embryo growth
arrest independent of ABA signaling (Raz et al
...


During seed maturation, the ABI3/FUS3/LEC2 (AFL) subfamily of B3 transcription factors,
together with LEC1 and LEC1‐LIKE (L1L), compose a transcription control network called
LAFL (Figure 4) (Kwong et al
...
2014)
...
2013)
...
2014)
...
2017)
...
2013, 2014; Zhou et al
...


LAST PHASE OF SEED MATURATION

After the suspension of cell division during embryogenesis, plant seeds begin to accumulate
storage components and begin to desiccate
...
Abscisic acid also
functions in this final developmental stage and affects several important traits of the dormant
seed
...
The initiation of reserve
accumulation is mediated by several processes such as gene expression, posttranslational
modulation, strengthening the activity of enzymes and ATP production (Bewley et al
...


Before storage product accumulation, there is often a period of de‐greening that is important for
seed maturation and some commercial traits such as storability and seed oil quality (Delmas et
al
...
SnRK2s that are activated by ABA and the downstream transcription factor ABI3 are
often required for This de‐greening process, which can be observed by the presence of a
greenish‐brown seed coat in snrk2
...
2009;
Delmas et al
...
ABI3 interacts with the SnRK2 activated transcription factor ABI5, and these
components may function together in transcriptional regulation of ABA‐responsive genes
(Nakamura et al
...
Indeed, two stay‐green genes, SGR1 and SGR2, are targets of ABI3 and
function redundantly in regulating the de‐greening of seeds (Delmas et al
...


Abscisic acid functions in the storage of lipids, proteins and carbohydrates in seeds
...
2/3/6 triple mutants often exhibit reduced
accumulation of seed products, whereas overexpression of SnRK2
...
2010; Gonzalez‐Guzman et al
...
2018)
...
6, which mediates ABA signaling, but not ABAnonresponsive SnRK2
...
2010)
...
2/3/6 triple mutant, confirming that ABA can promote seed
storage accumulation through transcriptional regulation (Nakashima et al
...
Indeed, two
ABF transcription factors ABI5 and bZIP67, together with ABI3, and the AP2/ERF transcription
factor ABI4, control expression of genes related to numerous events involved in seed storage
processes that are downstream of ABA (Nambara et al
...
1994; Soderman et al
...
2013; Zinsmeister et al
...
The transcription factor bZIP67 together with
two other LEC1 inducible transcription factors, L1L and NUCLEAR FACTOR‐YC2 (NFYC2),
activate FATTY ACID DESATURASE 3 (FAD3) to affect omega‐3 fatty acid accumulation in
seeds (Mendes et al
...
ZmbZIP22 has also been reported to regulate seed storage events
...
2018;
Dong et al
...
Abscisic acid induces highly the expression of the DELAY OF
GERMINATION 1 (DOG1)‐LIKE 4 (DOGL4), which encodes a major inducer of reserve
accumulation during seed maturation (Figure 4) (Sall et al
...
Ectopic expression of DOGL4
enables the expression of seed maturation‐specific genes even during the germination process,
including the major seed reserve proteins ALBUMINs, CRUCIFERINs, and OLEOSINs (Sall et
al
...
Altogether, the classic PYLs‐SnRK2s‐ABFs signal cascade plays an essential role in the
assimilation and deposition of storage nutrients in plant seeds
...
For example, Arabidopsis 2S storage
protein 3 (At2S3), a representative seed storage protein gene, is dependent on FUS3 and
partially dependent on both ABI3 and the AP2/EREBP type transcription factor WRINKLED1
(WRI1) (Kagaya et al
...
2008)
...
2005a)
...
This suggests that LEC2 harbors a synergistic activity with ABI3, FUS3, and LEC1 to
affect reserve product accumulation (Santos Mendoza et al
...
The AP2/EREBP domain
protein, WRI1 also functions in multiple processes involved in the accumulation of oil and
sugars in seeds
...
2008;
To et al
...
WRI1 is regulated by LEC1, LEC2 and GmDREBL at the transcriptional level
...

2007; Mu et al
...
2009; Sirichandra et al
...
2016; Zhang et al
...
2017; Kong and Ma 2018)
...
The role of ABA in
controlling the activity of transporters for nutrients such as sugars and nitrogen in seed storage
processes is very likely but remains obscure (Baud et al
...
2015)
...
2005)
...
However, we need
further research on the effects of ABA on several nutrientspecific transporters and the
importance of these transporters in both seed storage and maturation
...
2013b)
...
The attainment of this
tolerance is associated with the accumulation of a series of protectants such as antioxidants,
sugars, and late embryogenesis abundant (LEA) proteins (Koornneef et al
...
Interactions between LEAs and sugars contribute to the formation
of a glassy state that suspends metabolic activities and protect seed tissues and cells from many
survival threatening events such as membrane damage (Buitink and Leprince 2008)
...
2009; Maia et al
...
2018)
...
2008)
...
2016)
...
Abscisic
acid is a key regulator in this process and it is noteworthy that only the embryo but not any
maternal tissues produces ABA
...
1983; Frey et al
...
Genetic screening of mutants deficient in
seed dormancy has led to the identification of several regulators of the hormone metabolism
and action signaling network that controls seed maturation and dormancy
...
2007, 2011; Nakabayashi et al
...
In rice, SEED DORMANCY 4 (OsSDR4) is considered to be a regulator that is involved in
seed dormancy with an unknown function
...

2010; Cao et al
...


Many mutations that affect ABA biosynthesis, sensing and signaling such as aba1, aba2/3,
nced6/9, snrk2
...
1982; Leon‐Kloosterziel et al
...
2006;
Nakashima et al
...
2018)
...
2015; Nguyen et
al
...
DOG1 also modulates seed dormancy as the mutant dog1 is completely nondormant
...
2006)
...
2003; Bentsink et al
...
2012)
...
2017; Nishimura et al
...
Although DOG1
interacts with another PP2C protein REDUCED DORMANCY5 (RDO5), RDO5 seems to
function in seed dormancy independent of both ABA and DOG1 (Xiang et al
...


GA is essential for seed germination, which is evidenced by the defects in seed germination of
several mutants with disrupted GA synthesis such as ga1, ga2, and ga3 (Debeaujon and
Koornneef 2000; Ogawa et al
...
DELLA proteins, RGA‐LIKE 2 (RGL2) and RGL3, are
central repressors of seed germination, indicating the important role of GA signaling in seed
germination (Lee et al
...
The GA‐insensitive mutant, sleepy1 (sly1), exhibits attenuated

seed germination due to enhanced accumulation of RGL2 (Ariizumi and Steber 2007)
...


2007; Ariizumi et al
...


The opposing roles of ABA and GA in seed dormancy and germination result in a balanced
control mechanism
...
2008; Lee et al
...
NF‐YC, together with
RGL2, promotes ABI5 expression to enhance ABA‐mediated repression of seed germination
(Liu et al
...
The COP9 Signalosome 1 (CSN1), which is recognized as a modulator of
ubiquitin E3 ligase, facilitates the degradation of RGL2 and CSN5A, and may inhibit ABI5
possibly through a physical interaction, therefore promoting seed germination (Jin et al
...

Expression of both RGL2 and ABI5 are also activated by exogenous ABA (Piskurewicz et al
...
Taken together, the balancing roles of GA and ABA on germination are mainly achieved
through effects on RGL2
...


During cold stratification, expression of CYP707As that participate in ABA catabolism, and
AtGA3ox1 that is involved in GA synthesis is elevated, leading to a high GA/ABA ratio, which
promotes seed germination (Okamoto et al
...
2016; Chen et al
...
The same
expression control pattern was found with the atper1 mutant that has a dysfunctional
seedspecific peroxiredoxin (Chen et al
...
In imbibed seeds, DELLA protein is degraded by
the elevated GA to attenuate transcriptional activities of the DELLAABI3‐ABI5 module, which
accelerates germination
...
2013)
...
2009, 2019; Xiang et al
...
2016)
...
PGG arrest
protects embryos from stressful surroundings that may threaten seedling survival
...
Seedling
growth will cease under such conditions and re‐enter the quiescent state
...
2018)
...
Abscisic acid‐deficient and ABA‐insensitive mutants are not able to
normally convert to PGG arrest, suggesting that ABA plays an essential role in the
establishment of this important stress adaptive response (Barrero et al
...
There may also be
multiple other participants in this process
...
8 and hence release the suppression of ABI3 to promote PGG arrest
(Wu et al
...
Recently, it was reported that RAF22 in Arabidopsis acts as a negative
regulator of PGG arrest that is independent of the canonical ABI5‐mediated ABA cascade
(Hwang et al
...


The multiple roles of ABA in several stages of seed development continue to be revealed
...
2004)
...

Their successful entry into and emergence from dormancy is critical to species survival in a
constantly changing environment that can abruptly become hostile to several life processes
...


THE ROLE OF ABA IN PLANTS DURING SEVERAL DEVELOPMENTAL STAGES

As we have already outlined, the biological regulations that involve ABA are ubiquitous and
impactful during multiple developmental stages of plants, and many of them are implemented
through ABF‐mediated transcriptional processes
...

These factors function redundantly in transcriptional regulation mediated by ABA (Fujita et al
...
ABI3 and ABI4 are two other important transcription factors that regulate ABA
responses
...


Abscisic acid in seeds and seedlings Abscisic acid has important functions during several stages
of the alternation of generations through gamete generation, subsequent fertilization and
embryo development
...
First, gamete formation by meiosis is the basis of most genetic variation
through which natural selection works
...
During the dormant period, plant embryos can survive

extreme environmental changes
...
1979; Perkins et al
...
After germination,
high‐concentrations of ABA attenuate the growth of the primary root, and in contrast, low‐
levels of ABA maintain the growth through restricting ethylene production controlled by
expression of ACS2/5, under water stress (Figure 5) (Spollen et al
...
2010; Li et al
...
Exogenous ABA can also promote the elongation of the seedling primary root in the
pyl112458 and pyl duodecuple mutants (GonzalezGuzman et al
...
2018)
...
2019)
...
ABA promotes root stem cell
maintenance through ABFs together with WOX5, and maintains primary root growth by
restricting the ethylene production through ACS2/5
...
ABA may promote bud dormancy by coordinating leaf
senescence, starch degradation and source to sink carbon translocation through transcriptional
reprogramming
...
Together, ABA regulates plant growth, development, and stress
responses
...
2010;
Zhang et al
...
2018)
...
2018)
...


ABSCISIC ACID IN ADULT PLANTS

Abscisic acid has several effects on adult plants
...
Abscisic acid induces several LEA class genes including
AIL1, RD29B, RAB18, EM1, and EM6 which are the classic ABA‐responsive marker genes

thought to protect plants from dehydration damage (Wang et al
...
Mutants defective in
multiple ABFs display wilty phenotypes under limiting water conditions
...
2010, 2015)
...
2016; Zhao et al
...
The expression of waxsynthesis‐
related genes, KCS2, CER1, LTP3, and WSD1, are strongly decreased in snrk2
...
2016)
...
Abscisic acid accelerates
the degradation of starch to sugars during osmotic stress through transcriptionally controlling
the expressions of β‐AMYLASE1 (BAM1) and α‐AMYLASE3 (AMY3) through several ABFs
...
2016)
...
2011; Gao et al
...
2019)
...
Abscisic acid is involved in several developmental processes associated with
flowering and transition of generations
...
2013; Liao et al
...
2018)
...


ABSCISIC ACID IN SENESCENT PLANTS

An important aspect of the transition to flowering and the successful production of dormant
structures (seeds) crucially involves the senescence process
...
Abscisic acid plays critical roles in
accelerating leaf senescence through transcriptional regulation (Gao et al
...

2016)
...
2016; Zhao et al
...
ABFs, such as ABI5 and EEL, also participate in
dark‐induced senescence (Sakuraba et al
...
Abscisic acid systematically controls energy
flow from source tissues (senescent leaves) to sink tissues (dormant seeds and floral meristem)
(Zhao et al
...
The floral meristems, under appropriate conditions, develop into
dormant embryos
...


PERSPECTIVES

Abscisic acid has many roles in regulating plant growth, development and response to various
environmental stresses
...
Due to technology limitations on ABA visualization, the regulation of
ABA metabolism is still poorly understood
...


Our understanding of how plants modulate ABA accumulation in response to environmental
stresses would be greatly facilitated by the identification of stress sensors
...
2014;
Ma et al
...
2019)
...
Innovative
technologies for ABA visualization will be a major tool that can greatly help to understand how
stress is perceived and leads to induction of Ca2+ signaling and ABA accumulation
...

2014; Devireddy et al
...
Several aspects of the spatiotemporal regulation of ABA
accumulation and signaling need further study, including: (i) long‐distance transmission of
stress signals to induce ABA; (ii) ABA dynamics at the cellular, tissue or organ levels
...
2013; Jones et
al
...
2014; Wu et al
...
Beside the Ca2+ and ROS signals, the small peptide
CLE25 may also participate in the long‐distance delivery of stress signals to promote ABA
biosynthesis in leaves (Ren et al
...


The diverse functions of ABA in regulating physiological processes are mediated largely
through the multiple downstream substrates of SnRK2s
...

However, many biochemical and genetic studies have indicated that ABA also modulates
senescence, abscission, vegetative dormancy, plant growth, carbon allocation, stem cell
maintenance and differentiation and several other processes, probably also involving
phosphorylation regulation by SnRK2 protein kinases (Fujii and Zhu 2009; Fujita et al
...
2009; Yoshida et al
...
2012; Umezawa et al
...
2013; Miao et al
...
2018; Shinozawa et al
...
Future efforts
should also be directed to understand further the regulation mechanism of these other
downstream responses
...
2018)
...
Carbon re‐allocation is an important aspect of leaf senescence, bud
and seed dormancy, and is closely related to the harvest index of crops (Savage et al
...

How ABA functions in stem cell maintenance and differentiation is a major underappreciated
aspect of stress adaptation (Han et al
...
2010; Wu et al
...
As sessile
organisms, plants cannot “flight” as animals do during stresses, and must “fight” against
stresses
...
Finally, much effort is needed to address the signaling
dilemma affecting the so‐called carbon/water trade‐offs that plants face during stress
...


ETHYLENE
It acts as a growth promoter as well as an inhibitor
...
It is synthesised in
the ripening fruits and tissues undergoing senescence
...


Ethylene is the only plant growth regulator that occurs in the form of a gas
...
It is produced
by almost all parts of higher plants and usually present in minute quantity but causes marked
effects
...
Like abscisic acid,
ethylene is usually considered as inhibitory hormone
...

Being volatile in nature, ethylene gas is not used directly
...


DISCOVERY

Ethylene has been used since the ancient times
...
The ancient Chinese
would burn incense in closed rooms to enhance the ripening of pears
...
In 1874 it was discovered that smoke caused pineapple fields to bloom
...
When the plants were allowed to grow in fresh air, they
regained their normal morphology and rate of growth
...
In 1912, A
...

Sievers and R
...
True demonstrated that the combustion gases from the kerosene stove were
beneficial in the ripening of lemons
...
C
...
I
...
The first report
that plant materials evolve ethylene came from H
...
Cousins, who observed that when oranges
and banana were stored together during shipping, some gas was emanated from oranges which
caused ripening of banana
...
Farmers in Florida would
commonly get their crops to ripen in sheds by lighting kerosene lamps, which was originally
thought to induce ripening from the heat
...
Denny discovered that it was the
molecule ethylene emitted by the kerosene lamps that induced the ripening
...
H
...
R
...


Since then it has been shown that ethylene is produced from essentially all parts of higher
plants
...

Ethylene production can also be induced by a variety of external aspects such as mechanical
wounding, environmental stresses and certain chemicals including auxin and other plant
growth regulators
...
This reaction is catalyzed by
the enzyme SAM synthetase( ATP- Methionine S- Adenosyl transferase)
...
In many cases, solenomenthionine has been found to be the better substrate for
ethylene biosynthesis than methionine
...
The next step in the biosynthesis of ethylene is the
conversion of SAM to 1-amino cyclopropane carboxylic acid (ACC), which is catalyzed by the
enzyme ACC synthase (ACS)
...
The final step in the biosynthesis of ethylene is oxidative cleavage of 1-amino
cyclopropane carboxylic acid (ACC) to form ethylene, CO2 and HCN by the enzyme
ACCoxidase (ACO), formerly known as the ethylene forming enzyme (EFE)
...
This reaction requires O2 and is activated by light
...
Fruit Ripening: Acceleration of fruit ripening was the first discovered effect of ethylene
and due to this character it is also called ripening hormone
...
Fruit ripening can be climacteric
or non-climacteric and both types react differently with exogenous application of
ethylene but this process is well marked in climacteric fruits Climacteric fruits ( e
...

apple, apricot, avocado, banana, blueberry, fig, mango, kiwi , tomato and papaya)
generate large amount of ethylene and show a marked increase in respiration as
ripening proceeds
...
So, the increase in ripening (climacteric) initiates just
after an enormous increase in ethylene production
...
) ethylene treatment
does not cause respiration climacteric and additional ethylene production and the rate of
ripening process remains unaffected
...
Seed germination: Ethylene plays a significant role in breaking seed dormancy and
inducing seed germination in plants like ground nut, wheat, lettuce and cocklebur
...
The maximum germination however is obtained at about 40-50ppm
ethylene
...
Flower inhibition and sex expression: In most of the plants, flowering is inhibited by
ethylene, although it is known to promote flowering in mango and pineapple
...
Ethylene also
changes sex expression in unisexual plants
...


4
...
These roots create anaerobic conditions and forms aminocyclopropane-1carboxylic acid which is transported up by the xylem to the leaf where it is converted to
ethylene in the presence of oxygen and induce epinasty i
...
the upper side of the petiole
of the leaf grows faster than the lower side and the leaf curves downwards, which
perhaps help the plant to lose water
...


5
...

When a growing shoot hits an obstacle while underground, ethylene production greatly
increases, preventing cell elongation and causing the stem to swell
...
If the
shoot does not reach the surface and the ethylene stimulus becomes prolonged, it affects
the stem’s natural geotropic response, which is to grow upright, allowing it to grow
around an object
...

When stems of trees are subjected to wind, causing lateral stress, greater ethylene
production occurs, resulting in thicker and sturdy tree trunks and branches
...
Acceleration of senescence and abscission: Ethylene promotes/ accelerates senescence
and abscission of plant parts, both natural and induced
...
Ethylene also induces abscission of leaves (eg
...
Mulberry, etc
...
g
...
)
and fruits (eg
...
When applied exogenously
(0
...


7
...


In conclusion, one or the other plant growth regulator influences every phase of growth
or development in plants
...


Additionally, more than one plant growth regulator, along with extrinsic factors, plays critical
role in plant growth and development
...
g
...




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