Plant Hormones-Gibberellins

 

Gibberillic acid was first discovered in Japan under unusual circumstances.  Farmers in Japan, before the World War, found that their paddy crop plants were afflicted with a strange disease called Bakane.  The diseased plants showed unusual growth where plants were very tall, weak and sterile.  This disease affected 20-30% of the crop plants and the farmers were subjected to great loss.

Investigation into the cause, Hori a Japanese plant pathologist discovered that this disease was due to a fungus called Gibberella fujikoroi, but it is now identified as Fusarium moniliforme.  Later Sawada and Kurosowa found that the extracts of this fungus simulated what the fungus could cause on infection.  Then a group of Japanese workers led by Yabuta and Sumuki obtained an active principle from the extracts of the causative fungus, and later Yabuta and Hayashi identified the active principle as Gibberellic acid, which is new popularly called as Gibberellins (GA).  Western scientists did not know anything about these discoveries before the First World War.  After the war, they came to know about GA.  Then they searched for this substance in higher plants and found this substance in all known higher plants.  In fact they succeeded in obtaining GA in pure crystalline form and also they established its chemical structure.

Maryland mammoth

Distribution

 

Almost every kind of plant, including algae, ferns, fungi, bacteria, is known to contain GA.  Quantitative analysis of various parts of the higher plants shows that young leaves, which are just unfurling, are rich in GA.  Even germinating seedlings contain significant amount of GA.  Further studies revealed that proplastids found in young and developing leaves act as the site of GA synthesis and it is of great interest to know that their synthesis and release from plastids is under the subtle control of phytochromes.  Though GAs is synthesized in plastids, they are translocated to different regions of the plant body through sieve tubes by active translocation mechanism.

The diagram shows the concentrations and spatial distribution of different phyhormones in a plant body under normal conditions.

 

Structure and Classification of GA

 

Gibberellins are diterpenoid acids derived from tetra cyclic diterpenoid known as Kaurene.  The basic carbon skeleton of GA is known as Gibbane ring.  But today the term Gibbane is in use.  However, the systematic nomenclature of GAs is based on Kaurene and Gibbane structures.

 

 

 

Modified solvent extraction methods and the use of sophisticated analytical tools like HPLC etc. have greatly helped plant biochemists in identifying different forms of GAs.  So far, 52 to 57 different kinds of Gas have been identified.  Some of the gibberellins are known to be conjugated with glucose as glucose esters.

 

Chemical analysis of different parts of the plant body for GAs shows that different kinds of GAS are located in different parts of the plant body; their content and kinds vary at different stages of development of the plant body.  Two or more GAs are found in the same plant but in different organs.  In addition, Gibberellins exist in a dynamic state, in the sense; they undergo rapid interconversions from one kind to another, which however depends upon the intrinsic or extrinsic factors.  It is also known that some of the gibberellins which are active in one organ are found to be inactive in another organ and vice versa.  The dynamic equilibrium between different kinds of GAs, their synthesis and their multifaceted effects in plants is really fascinating.

Biosynthesis:

Biosynthesis of GA takes place in young plastids.  Acetate is the precursor for the synthesis of all kinds of gibberellins. 

 

In a series of multi step reactions acetate is used to produce a diterpene compound called Kaurene.  Specific enzymes responsible for each creates have been more or less identified.  The site of synthesis is proplastids or plastids or both.  Plastids are also the site of fatty acid synthesis in plants.

 

 

 

 

 

 

 

Kaurene thus synthesized is converted to Gibberellic acid, which is then subjected to hydroxylation, methylation or glycosylation to produce different forms of Gibberellins.  The remarkable feature of the biosynthetic pathway of GAs is that the same pathway is also used by plastids to synthesize certain plant pigments, Abscisic acid and some sterols.  The needed enzymes for the biosynthesis of the above said compounds are regulated by phytochromes and other factors.  The plastogenome and nuclear genome play a significant role in regulating the biosynthesis of Gibberellins and other mentioned compounds, how plastogenome contributes to the synthesis and its regulation is not known.

 

Effect of GA on of Plants

 

1.   Effect on General Metabolism:

 

 Effect of various factors on cellular activities.

 

Gibberellins, as growth promoting hormones, accelerate the rate of cellular metabolic pathways such as respiration, protein synthesis etc.  During the germination of cereal grains, GA is known to promote mobilizing the food materials for the growing embryo.  It plays a significant role in metabolizing lipids to glucose and then to sucrose through gluconeogenesis process.  The promotive effect of GA on nitrogen metabolism and HMP pathway is very significant.  GA is also known for its effect on membrane transformation through rapid turnover of lipids.  Interestingly, GA also favor C3 pathway in C3 plants, but the same hormone adversely affects Hatch and Slack pathway in C4 plants.  Thus GA exerts a wider influence on general metabolic pathways.

 

 

 

 

2.   Effect on Transcription and Translation:

 

The metabolic effects exerted by Gibberellins show variations and it depends upon the structure, species and developmental stage of the plant body that receives it.  The presence GA binding proteins have been identified from various sources like pisum epicotyls, phase his etc.  Mol. Wt. of such proteins has been determined as 6.0 x 104 to 5.0 x 105 Daltons.  GAs are also known to elicit specific responses through such receptor proteins.

 

In cucumber hypocotyls, GA3 promotes the replication of DNA in plastids; thereby it exhibits its specific effect on biogenesis of chloroplasts.   Gibberellins are also known to stimulate transcriptional activity of DNA templates through the binding of GA to AMP rich regions of DNA.  In some cases, GA’ effect on transcriptional activity has been attributed to the activation of RNA polymerase through their receptor proteins, where the synthesis of all species of RNAs is enhanced.

 

The most specific effect of GA on transcription has been observed in the germinating barley grains.  GA3 induces the mRNA synthesis for alfa amylase enzymes in aleurone cells and the endospermal do not produce mRNA for alfa amylase.  Probably this effect of GA is one of the best exemplified actions of phytohormones on differential gone expression.  In spite of having voluminous data about GA’s effect on different plant species, nothing is clear how different GAs bring about different transcriptional activities leading to morphological expressions.

 

Gibberellins are also known to activate dormant mRNPs and the translating machinery.  This view is supported by the fact that GA enhances polysome formation even in the absence of concomitant synthesis of mRNA.  Synthesis of alfa amylase enzyme in barley aleurone layers, appearance of some new proteins during bolting and flowering are few examples to demonstrate the effect of Gibberellins on protein synthesis.

 

Effect on Genetic Dwarfism:

 

Genetic dwarfism, in many plants including garden peas and some species of French beans, is controlled by single genes.  Such genetic dwarfs respond very favorably to GA treatment and in response to the hormone they grow as tall as normal tall varieties. 

 

 

However the phenotypic transformation of genetic dwarfs into tall plants is not heritable.  Quantitative analysis of tall and dwarf peas and bean plants reveal the presence of high concentrations of GA in tall plants than is dwarf varieties, which suggests that dwarfism is due to a single gene mutation affecting one of the steps in GA biosynthesis.  Cross breeding experiment do support this view.

 

The fascinating aspect of Gibberellin’s effect on genetic dwarfs in its specific action is on internodal growth.  The internodes respond favorably than any other parts.  The elongation of internodes is possible by two simultaneous processes i.e. one by meristematic divisions of intercalary meristems and the second is by the elongation of meristematic cells.  The exact mechanism by which cell elongation is achieved is not clear, nonetheless, GA induced early growth of internodes can be inhibited by colchicine, but not by the inhibitors of transcription and translation.  This indicates that the cytoskeleton structures like microtubules are involved in GA mediated growth similar to that of auxin induced cell elongation.

 

Dwarf rice plants also respond very well to GA treatment.  GA induced internode elongation in rice plants is due to the activation of inter-calary meristems.  After few cell divisions, the cell derivatives elongated 80-1000 times the original size.  Elongation, in this case, is due to the excretion of protons and labializing the cell wall to be plastic.   Meanwhile, intracellular turgidity also builds up due to the action of GA on plasma membranes.  The combined effect of loosening the cell wall, the increased turgour pressure results in the cell elongation.

 

 

 

Effect on Aleurone Layers in Cereal Grains:

 

Cereal grains of Zea mays, sorghum, Hordium, Oryza etc have a distinct layer around the endosperm called aleurone layer, the cells of which are rich in protein granules.  During germination, aleurone cells become active and with time, they secrete enzymes into endospermous tissue, where the reserve starch gets degraded to glucose and the same is utilized by the growing embryos.

 

 

 

 

 

There is a definite relationship between the activity of aleurone cells and developing embryo.  If the embryo is separated from the rest of the grain and incubated separately, aleurone cells fail to secrete any enzymes.  On the other hand if the embryo is also incubated with the rest of the grain, after a period of time, aleurone cells start secreting alfa amylase.  Instead of incubating embryo with the rest of the grain, if GA3 is added to the incubating medium, aleurone cells produce alfa amylase enzymes.  Thus it is clear that during the early part of germination the young embryo produces Gibberellins which on reaching the aleurone layer activates the cells to produce the required enzymes like alfa amylase, protease, phosphoryl choline glyceride transferase and phosphoryl choline citidyl transferase.  The last two mentioned enzymes are required in lecithin biosynthesis which activates membranes. Recently it has been reported that GA besides inducing the expression of alfa amylase inducing enzyme, it also triggers the expression of ribosomal RNA synthesis to a greater extent.

The synthesis of GA induced enzymes can be inhibited by actinomycin D or cycloheximides, which are the inhibitors of transcription and translation respectively.  This suggests that GA preferentially activates the gene expression for the above said enzymes. 

GA binding protein

 

Studies on GA induced molecular events clearly suggest than GA at very early stages activates preexisting mRNPs for alfa amylase but later the hormone activates specific genes in aleurone cells.  However, the increase in the levels of transcription for amylase and protease reaches maximum at 6-7 hours after hormone treatment.  The above results further supported by the fact, that when GA induced mRNAs from aleurone layer are translated in an vitro system, among the many proteins produced, the levels of alfa amylase and protease is found to be high.  In actuality, the newly synthesized enzymes are loaded into membrane vesicles and the same is secreted out of the aleurone cells into endospermous tissue, where they bring about hydrolytic activity and thus mobilize the food materials for the developing embryos.

 

 

 

Endosperms cells are dead cells.  In recent investigations it has been shown that GA not only induces the synthesis of mRNAs for alfa amylase but also induces the synthesis of RNA.  But ABA suppresses the GA induced mRNA for alfa amylase.  However ABA does not prevent the synthesis of RNAs.

GA signaling takes through trimeric G receptor protein.  This leads to pathways, one-Ca+ dependent pathway and the other Ca+ independent pathway; not much is known about how GA induces signal transduction.

The Ca+ independent pathway activates phosphorylation of DELLA proteins, which are repressors of GAMB gene ( GA induced Amylase-beta).  Phosphorylation of F-box proteins (SLN1 and SLR1) leads to ubiquitinated protein degradation.  This facilitates the GA-GA receptor complex bind to GAMYB gene promoter and activates the transcription of the genes. Translated product GAMY beta protein, a transcriptional regulator protein, enter the nucleus and binds Amylase gene regulatory elements called GARE (GA response elements) and induce amylase gene expression.  Transcripts on translation produce amylase proteins which are transferred into ER and loaded into vesicle and stored in cytoplasm.  They are released in response to GA through what is called Ca+ dependent pathway.  Note MYB is a family of genes there cans any where 80-108 in number; they are the most abundant transcription factors.

 

 

Effect on Vernalization:

 

Not all seeds released from the fruits germinate immediately.  Some pass through a period of dormancy.  And those seeds which exhibit temporary suspension of growth activity are called dormant seeds.  In order to make dormant seeds germinate they have to be subjected to scarification or stratification or some of them have to be subjected cold treatment.  Plants or seeds acquiring the ability to accelerate flowering in response to cold treatment is called vernalization. 

 

 

 

GA is  known to overcome cold treatment for plants requiring vernalization, but it can also break the seed dormancy.  Quantitative estimation of GA in vernalized seeds do not show any increase in GA content in response to cold treatment, however, during germination at normal temperature, the concentration of GA increases significantly; these studies suggest that cold treatment per se does not increase GA content, but provides the ability to synthesize more GA during germination and growth.  In some instances, where dormant seeds requiring far-red treatment to break the dormancy GA acts as the substituent.

 

Effect on Parthenocarpy:


Where auxins fail, GAs are found to be very effective in inducing Parthenocarpy.  In addition to it, GA induced parthenocarpic fruits are larger in size and sweeter in content.  GA is used to increase the total yield of grape fruits.  It is also highly effective in increasing the yield and sugar content of sugar cane.  However, the application of GAs should not be more than what is required in grape cultivation; otherwise fruits will be damaged .

 

Gibberellins and Auxin Interactions:

 

Gibberellins and auxins are found to induce cell elongation, parthenocarpy and metabolic activities including RNA and protein synthesis.  But GA and auxins have their own specific effects on different tissues of the plant body.  While GA promotes internode elongation, overcomes genetic dwarfism, induces amylase synthesis in aleurone cells and in some cases it can substitute cold treatment or far red treatment.  IAA does not elicit any of these effects.  On the other hand, auxins impose apical dominance, induce adventitious roots and induce cellulase synthesis. On the other hand GAs don’t  elicit any of these responses.  However, in the case of cell elongation though GA and IAA have independent actions in promoting the growth of etiolated normal pea stem sections, if both the hormones are provided together, their total effect is just additive but not synergistic.  On the contrary, if internodes of dwarf pea stems are treated with either GA or IAA, the promotive effect on stem segments is very little, but if both are provided together the stimulation in terms of growth is highly pronounced and the total effect is synergistic.  The above observation indicates that gibberellins need auxins for synergistic activity.  This particular conclusion is further substantiated by the fact that a decapitated internodal segment does not respond to GA treatment alone but if the apical meristem or an agar block containing auxin is placed on the decapitated segment, elongation of the internode is stimulated in the presence of GA.  This is because apical meristems do synthesize auxins, which interact with gibberellins to bring about the combined effect.  Recent studies in in vivo and invitro system strongly suggest that GA has an important role in promoting the biosynthetic pathway of auxin, thus in the presence of GA, the levels of auxins increase.  Probably GAs enhances the rate of IAA synthesis.

 

 

Microarray of GA induced gene expression

 

 

 

 

Effects

IAA

GA

Stem Growth

+

+

Parthenocarpy

+

+

Root initiation

+

-

Callus formation

+

-

Induction of amylase

-

+

Bolting and flowering

-

+

 

 

 

 

 

 

 

 

 

 

 

 

GA and ABA:

Plant hormonal interactions are fascinating for the simple reason that many of them act antagonistically and some cooperatively and few synergistically.  Some of the interactions are concentration dependent. GA and Auxin induce parthenocarpy; Cytokinins and auxins act antagonistically in Auxin induced new root formation.  Induction of dormancy and breaking dormancy are two opposite development pathways in seeds.  ABA can induce dormancy and GA can break the dormancy.  Cold treatment induces vernalization in some plants in ABA dependent manner, but vernalization can be broken by GA.

 

The diagram illustrates how ABA and GA act in opposite ways in response to environmental inputs.

 

 

 

 

 

 

 

 


GA and Flowering:

 

Plants which require longer photoperiodic conditions for flowering are called long day plants and those plants requiring shorter photoperiodic are called short day plants.  Long day plants do not flower under short day conditions and vice versa.  Correct photoperiod treatments stimulate the synthesis of flowering hormone called florigin which inturn induces flowering by differential gene expression.  However, there are a large number of plants which do not require such photoperiodic conditions for flowering.

FCA and the control of Arabidopsis flowering time. (A and B) Schematic representation of the principal genetic pathways controlling flowering time in winter annual and rapid cycling accessions of Arabidopsis. Promotive activities are denoted by arrowheads, repressive activities are denoted by T-bars. The photoperiod (PP) and gibberellin signal transduction (GA) pathways are shown activating genes with a floral meristem identity function (FMI), while FLC is shown to repress this. FRI, the FCA-containing autonomous pathway (AUT) and vernalization pathway (VRN) regulate FLC in an antagonistic manner. The most frequently found difference between winter annual (A) and rapid cycling (B) Arabidopsis accessions is allelic variation at FRI, with most rapid cycling accessions carrying inactive, loss-of-function fri alleles. (C) A comparison of the phenotype of wild-type Ler plants and late flowering fca-1 mutant plants. Although grown for the same time and under identical conditions, wild-type plants have already flowered while the fca-1 plants have remained in the vegetative state and continued to produce more leaves as opposed to floral organs. (D) Schematic representation of the alternative processing of Arabidopsis FCA pre-mRNA. Exons are represented as filled boxes and intron by lines.

Some of the long day plants under non inductive short day conditions exhibit rosette leaves because of extreme condensation of internodes.  If such plants under non inductive conditions are sprayed with Gibberellins, they get stimulated and the internodes elongate dramatically; such GA induced elongated stems also initiate flowering, such a phenomenon is known as Bolting and Flowering.  Though bolting and flowering are two different events, GA has been found to effect the elongation of internodes first during which process flowering substances are also produced as a result floral buds are initiated.

Furthermore, quantification of GA in untreated plants and plants which are in flowering show very low amount of GA in the former and higher content in the later.  It amounts to the fact that during proper photoconductive conditions synthesis of Gibberellins increases.  However, GA has no effect on majority of short day plants.  Strangely particular variety of short day rice plants responds very well to GA treatment and produce flowers. 

 

From the above discussions, it is clear that Gibberellins actually over come long day treatments are photo period requiring plants.  The interrelationship between long day photoperiodic treatments and GA effect has been explained on the basis of the following facts.  Plastids synthesize and store the same under long day photo periodic condition.  At the same time, the phytochrome pigments which are also present within plastids, absorb red light and transform into far red light absorbing pigment.  This form of pigments labelises the plastid membranes and thus Gibberellins are released from the plastids.  Thus the concentration of GA increases in photo period induced long day plants.  In spite of it, the exact mechanism by which GA brings about differential gene expression during flowering is not clear.  Probably, it may also require other factors for floral induction.

 

Effect on inflorescence and floral structure:

 

Though GA is known to induce bolting and flowering in long day plants, the effect of GA on a short day plants like sorghum bicolor is very interesting.  In these plants, GA3 stimulates flowering even under non inductive conditions.  But in combination with far red light treatment GA’s effect is synergistic.  The hormone also brings about marked increase in the number of spikelets and glumes.  Along with these promotive responses, GA also increases the number of floral structures like ovaries and promotes fertilization, but inhibits stamen development.

Effects of GAs on Flowering and Flowering Genes”

“By Valerie Sponsel, Biology Department, University of Texas, San Antonio, TX, USA, ‘with  acknowdgment

Flowering in Arabidopsis, a plant for which more tools exist to study the molecular genetics of the process than in any other species, is regulated by four separate pathways: (a) the photoperiodic (long day) pathway, which operates in the leaves; (b) the convergent autonomous (leaf number)/vernalization (low temperature) pathway; (c) the carbohydrate (sucrose) pathway; and (d) the gibberellin pathway. The latter three pathways all operate in the shoot apical meristem. The four pathways converge on a number of floral pathway integrators that together regulate floral initiation.

The recent identification of FLOWERING LOCUS T (FT) as the gene expressed in leaves that encodes a phloem-mobile mRNA or protein that fits the description of a universal flowering stimulus, florigen, has been an exciting development after the decades-long search for this elusive flowering signal (Huang et al. 2005; and see Zeevaart 2006, for a review). Additional work is seeking to further define the integrative networks that exist between the different pathways. Web Essay 25.2, from which Figure 1 is taken, provides a complete consideration of this subject. This topic briefly addresses the GA pathway, when it operates, and what is known about how it is integrated with the other pathways.

  

 

Figure 1   Multiple developmental pathways for flowering in Arabidopsis: photoperiodism, the autonomous (leaf number) and vernalization (low temperature) pathways, the energy (sucrose) pathway, and the gibberellin pathway. The photoperiodic pathway is located in the leaves and involves the production of a transmissible floral stimulus, FT protein. In LDPs such as Arabidopsis, FT protein is produced in the phloem in response to CO protein accumulation under long days. It is then translocated via sieve tubes to the apical meristem. In SDPs such as rice, the transmissible floral stimulus Hd3a protein accumulates when the repressor protein, Hd1, is not produced under short days, and the Hd3a protein is translocated via the phloem to the apical meristem. In Arabidopsis, FT binds to FD, and the FT/FD protein complex activates the AP1 and SOC1 genes, which trigger LFY gene expression. LFY and AP1 then trigger the expression of the floral homeotic genes. The autonomous (leaf number) and vernalization (low temperature) pathways act in the apical meristem to negatively regulate FLC, a negative regulator of SOC1. The sucrose and gibberellin pathways, also localized to the meristem, promote SOC1 expression. (After Blázquez 2005.) (Click image to enlarge.)

In Arabidopsis, flowering occurs in long days (LD) even in the GA-deficient ga1-3 mutant, although it is somewhat delayed compared to wild-type plants. However, this mutant will not flower in short days (SD) unless treated with GA. Double mutants of ga1-3 and co, (the wild-type CONSTANS is normally expressed in leaves and is upstream of FT in the LD pathway) will not normally flower, either in SD or LD. From this and other information it is proposed that the LD and GA pathways are independent, with the GA pathway being essential for flowering in noninductive conditions. Input from the LD pathway (transduced through FT) and from the GA pathway converge on SOC 1 (SUPPRESSOR OF OVEREXPRESSION OF CO 1), that is expressed in the apical meristem. Thus, GA treatment to ga1-3 mutants in SD causes both enhanced expression of SOC in the apical meristem, and flowering. Over expression of SOC1 in ga1-3 mutants allows them to flower in SD without GA treatment (Moon et al. 2003).

An additional floral pathway integrator is LFY, the product of a floral meristem identity gene that is expressed in apical meristems. LFY expression, which is high in LD-grown plants, is only observed in SD-grown ga1-3 mutants that have been treated with GA. Constitutive LFY expression will also allow SD-grown ga1-3 to flower without GA treatment (Blazquez et al. 1998). Using deletion analysis, Blazquez and Weigel (2000) identified an 8 bp motif in the LFY promoter that is necessary for GA responsiveness, but not for response to day length. This sequence is a potential target for MYB transcription factors, and candidate MYBs whose expression increases in the shoot apex in response to exogenous GA4, or to elevated levels of native GA4, have been identified in both Lolium temulentum and Arabidopsis (Gocal et al. 1999, 2001b). Interestingly, LDs—but not GA treatment—cause a rapid increase in expression of the L. temulentum ortholog of FT (LtFT), indicating in this plant too that the LD and GA pathways converge downstream of FT (King et al. 2006).