In this article we will discuss about:- 1. Meaning of Gibberellins Acid 2. Occurrence of Gibberellins Acid 3. Biosynthesis and Metabolism 4. Mechanism of Action 5. Cell Division and Cell Elongation 6. Miscellaneous Functions.
Meaning of Gibberellins Acid:
In East Asia, rice plants infested with the fungus Gibberella fujikuroi displayed remarkable vigorous longitudinal growth. Immediately after the second world war, the active principle was isolated from this fungus which was called gibberellin. Chemically its structure is like a gibbone skeleton and is a diterpene. The gibberellic acid is commonly detected in higher plants and is designated as GA3.
Every GA has a carboxyl group at carbon-7 and is essentially needed for biological activity, and C19—GAs are more biologically active than GA20 GAs. Additionally GAs with 3-B hydroxylation, 3- B, 13- dihydroxylation, or 1, 2-unsaturation are usually more active than those with both 3-B-OH and 1, 2-unsaturation exhibit the highest activity.
Introduction of a -OH at the 2 position inactivates the molecule and may be regarded as a mechanism for removing hormones from the active pool.
The presence of GA is detected on the basis of the biological assay in dwarf mutants of rice, maize or bean. In these mutants synthesis of GA is genetically blocked and exogenous application with GA induces normal growth.
So far more than 110 individual gibberellins are known and additional members are added every year. Of these 25 are isolated from G. fujikuroi. All of these have the same ring structure derived from the isoprenoid synthetic pathway (Figs. 20-7, 20-8). However, their distribution is specific and some tissues may have one or more gibberellins. Different tissues react differently to various gibberellins.
There is also a view that several of the gibberellins are intermediate forms of several other compounds or some of the gibberellins. They are synthesized in different parts of the plant (Fig. 20-8) but actively growing tissues produce maximum amount of them. These are embryos or developing tissues. They are readily transported in the plant and pass through phloem or xylem.
Large amount of the plant gibberellin remains in a bound or inactive form. During seed germination much of the gibberellin is released and this is due to its release from the bound state. Further its synthesis is also dependent upon feedback control.
Several compounds are described in the literature which prevent gibberellin action. These are growth retardants (Cycocel, CCC; phoshon-D or AMO-1618 (Fig. 20-11)) which inhibit its synthesis primarily. However, in nature how its inactivation is brought about is not clear. Perhaps several of the growth hormones may be antagonizing their action and these may include ABA or even IAA.
Methods of Isolation:
Recently combined gas chromatography-mass spectrometry (GC-MS) are used for the definitive identifications of gibberellins and their metabolites. However, this technique is not applicable to the analyses of non-volatile GA-conjugates since a minimum level of purity as 5% is required.
Because of the sophisticated instruments involved the method cannot be used by ordinary workers. Mass fragmentation (mass fragmentography) is normally employed where the amount of sample is extremely low, e.g., quantitative analysis of GA’s in wheat chloroplasts or suspensor cells of embryo.
Some workers have also recommended the use of high performance liquid chromotography (HPCL) in GA analysis. GA’s which differ by the presence or absence of a double bound (GA 4, 7; 5, 20; 1, 3) could be separated in the form of their p-nitrobenzyl esters by argentation HPLC or thin layer chromotography.
Some workers have used bromophenacyl esters in HPLC analysis of GA’s. There are reports available where as small a sample as 2 nanograms of GA was sampled through electron capture detection.
All said and discussed it may be inferred that bioassays are far too convenient to perform in an ordinary plant physiology laboratory to identify GA’s but their exact biological potencies are difficult to quantify. Some workers have refined different bioassays to identify as many as six new GA’s using as many bioassays.
In barley aleurone layer bioassay total protein release in response to GA was measured. However, this bioassay is less sensitive than the classical a-amylase assay.
Occurrence of Gibberellins Acid:
Currently as many as 110 gibberellins have been positively spotted from Gibberella fujikitroi and or higher plants. Bearder and Sponsel (1977) have given their complete structures.
From 50 kg of Cucurbita pepo seeds following amounts of GA’s were sampled:
GA39 = 50 mg
GA49 = 3 mg
GA48 = 27 mg
A new non-polar GA was extracted from needles of Picea sp. and tentatively identified as an isomer of GA9. Further GA51 is an endogenous constituent of pea seeds. It is interesting to mention that simple gibberellins have been synthesized but the total synthesis of GA3 has resisted several attempts.
Slightly more than one-third of the GAs have retained the full complements of 20 carbon atoms and are called C20-gibberellins. The others have lost carbon atom number 20 and are thus known as C19-gibberellins. All gibberellins which are chemically characterized are assigned an ‘A’ number and this number does not imply chemical relationships, it roughly represents the order of discovery.
GA3-a C20 gibberellin is called gibberellic acid was the first one isolated and characterized, now commercially available form. Contrarily G1and G20– GA19, are most active and hence most important GAs in higher plants.
Though large number of gibberellins are reported, most of them have slight or no biological activity and many are intermediates in the synthesis of active forms or represent metabolic products that have retained basic structure. Some workers have suggested that GA1 may be the main active GA regulating stem elongation in higher plants.
Biosynthesis and Metabolism of Gibberellins Acid:
The main pathway of GA biosynthesis has been worked out in Gibberella fujikuroi. GA’s have also been synthesized from cell-free systems from higher plants especially immature cucurbitaceous seeds and some legume seeds.
In general ent-Kaur-ene and GA12 aldehyde are the pivotal intermediates in the fungal and higher plant systems. Kaurene synthetase is the first enzyme which catalyzes the first step in GA biosynthesis and has been purified from the endosperm of some seeds (fig. 20-10). No resolution of activity A and activity B was made out, and the two activities differed in regard to several properties.
Presently it is not possible to infer whether the two reactions are catalyzed at the same site, or at different sites of the same protein or even separate proteins. Molecular weight of kaurene synthetase was nearly 45,000 daltons. The activity of the enzyme is inhibited by Phosphon-D or AMO 1618. One view is that only the natural intermediates ent-Kaur-16 ene, but not other isomers, are efficiently incorporated into GA3.
Even though demonstrated it is also doubted whether Phosphon-D or AMO 1618 acted exclusively via inhibition of synthetase enzyme. One view is that AMO 1618 retards growth through deprivation of sterols needed for membrane synthesis.
Likewise phosphon-D is shown to uncouple photophosphorylation and inhibits photosynthetic electron transport at higher concentrations. Similarly CCC has been shown to affect photosynthetic membranes.
Another inhibitor, ancymidol, which inhibits the oxidation of kaurene to kaurenol was also an active inhibitor of the cytochrome P-450- dependent oxidation of kaurene, kaurenol and kaurenal by the microsomal fraction of the endosperm.
Beyond GA12-aldehyde the pathways forming multitude of GA’s vary in Gibberella fujikuroi and higher plants. The chief difference lies in the divergent capacities to hydroxvlate gibberellins skeleton at C-3 and C-13 positions. The biological activity of GA is also linked with the extent and position of hydroxylation and their subsequent metabolism. Series of C19 GA’s are obtained after C-20 is removed.
In vivo studies in Gibberella sp. indicate that C-20 of GA13-7-aldehyde is lost as CO2 during conversion of the latter to GA3. Currently it is not clear which GA metabolite is the immediate substrate for the reaction leading to the removal of C-20.
During the sequence of the metabolic steps leading from GA12-aldehyde to several GAs many biologically active GA’s are formed which are subsequently deactivated by substitution reactions and or conjugations.
In brief it may be mentioned that GA metabolism is being studied at very high sophisticated levels. Most of the studies are based on immature seeds. The precise relationship between metabolism of GA and the effects of applied GA in excised lettuce elongation is accompanied by its conversion to glucose either and its ester. The latter are polar metabolites.
The amount of these polar metabolites is proportional to the exogenous GA1concentration. Once GA1 is removed the growth is sustained and in this way the induced growth differs from the auxin-induced growth. GA3 is quickly converted to GA20.
A casual relationship between GA9-induced growth and its metabolism has been suggested. Some workers have also suggested a relation between the GAq metabolism and its effect on apical growth and senescence in peas.
On the other hand no correlation was observed between conversion of GA1 to GA8 and its effect on growth in dwarf rice seedlings. Similarly effect of GA1 metabolism on the induction of α-amylase activity has also been worked out.
There are some reports which do not support the contention that ABA inhibition of pea stem growth is enacted through GA inactivation. GA, however, can be inactivated by 2β-hydroxylation and formation of conjugates especially glucosylated derivatives. The glucose conjugates are either O- β-D- glucopyranosides or O-β-D-glucopyranosyleters.
Most of the available data do not support the once accepted contention that GA’s may be stored as glucose esters from which biologically active GA’s are released. The general opinion is that plants have capacity to release GA’s from the bound forms enzymatically and through their glucosylation they are converted into esters. During seed germination these esters are hydrolysed.
Some work on the GA synthesis and metabolism, compartmentation in chloroplasts has also received some attention. There is also a report showing that vacuoles isolated from barley protoplasts were capable of converting GA1 to GA8through 2 β-hydroxylation.
Further conversion of GA to its glucoside and other products was also seen with protoplasts. Different solvents have also been employed for the extraction of chloroplasts GA’s. For instance Triton X100 could extract GA4 and GA9 from chloroplast membranes whereas methanol was not suitable for GA extraction in wheat chloroplasts.
Figure 20-9A shows schematic representation of gibberellin biosynthesis in three stages.
Stage I:
Mavaionic acid pathway leading to formation of terpenoid 5-carbon (C5) isoprenoid unit isopentenylpyrophosphate (IPP) is synthesized from acetyl-coenzyme A and used to form C20 geranylgeranyl-pyrophosphate (GGPP).
Stage II:
Synthesis of GAs via ent-kaurene from GGPP and its conversion to GA12 – 7 -aldehyde. This is the first compound in the pathway with the true gibberellin ring system and hence precursor of gibberellins. The pathway to die synthesis of this aldehyde is same in fungus and higher plants.
Stage III:
This stage comprises the biosynthesis of all gibberellins from GA12 – 7-aldehyde.
It may be stated that the first two steps involve the cyclization of GGPP and are inhibited by the antigibberellin dwarfing genes or agents like AMO-1618, CCC, Phosphon-D. In this way deficiency of gibberellins is created.
Figure 20.10 β is the tentative pathways for the gibberellin biosynthesis in pea. The chief pathways are shown with bold arrows and occur in shoots and seeds.
Mechanism of Action of Gibberellins Acid:
Immunohistochemical analysis has associated a-amylase in GA-treated cells with the perinuclear region rather than the aleurone grain membrane, a-amylase synthesis is believed to be due to the formation of new ER from the nuclear membrane or envelope. It has also been suggested that α-amylase was released without the participation of secretory organelles.
Available information also supports the viewpoint that the mode of release of α-amylase was particulate rather than soluble. However, soluble mode of α-amylase secretion cannot be ruled out. Very recently Okita(1979) have shown that a possible precursor of α-amylase in wheat aleurone layers which was Ca. 1500 dalton was larger than the exsecreted form of the enzyme.
Available reports have also shown that Ca2+ was necessary for specific binding and this explains the essentiality of Ca for GA-induced synthesis and secretion of α-amylase in aleurone layer. Further ABA eliminates the binding of GA1, When wheat aleurone layers were incubated in GA3 acid lipase and phospholipase D activated initially associated with this layer disappeared.
The initiation of this process seems essential for the binding of GA to aleurone layer and the enzyme thus released possibly catalyzed lipid mobilization and turnover. However, it remains to be verified whether aleurone layer was the primary site of action of GA.
Studies have also proved that a correlation between the rate of in vivo production of α-amylase and the amount of translatable mRNA for the enzyme and also the fact that the transcription inhibition prevented formation of α-amylase only during the initial 12 hour of GA treatment gives evidence that α-amylase is translated from st-mRNa formed during the initial 12 hour of GA treatment.
These data may be taken to mean that GA stimulates a-amylase mRNA synthesis but does show that it interacts directly or in association with a receptor protein with the transcription process. There are also some studies where 5-FU treatment of aleurone layer synthesized α-amylase, having decreased thermal stability. Apparently 5-FU led to the production of α-amylase with altered physical properties.
Subsequent reports did not support these studies. It may be added that cells of the aleurone layer are encircled with thick cells made up of arabinoxylan. Once GA is applied endoxylanase is formed, releasing xylose and arabinose in the medium. Aleurone layer has also been shown to release carboxypeptidase but GA does not seem to affect its synthesis or release.
Gibberellin-tannin antagonism:
Some workers have suggested GA-tannin atnagonism for GA- induced a-amylase and phosphatase in barley.
GA3 could overcome the inhibitory effect of tannins and the latter have been suggested as native plant growth inhibitors. No precise information exists on the GA-induced DNA turnover in aleurone layer cells.
In castorbean endosperm GA application led to the assembly of a special class of glyoxysomes having high specific activity of isocitrate lyase. Obviously application of GA stimulated the processes that occur also in the absence of exogenous GA. This system appears to be less suitable for GA-action studies as compared with barley endosperm.
GA-induced growth has been investigated in several other systems including growth of excised lettuce hypocotyl sections; elongation inhibition caused by light could also be overcome by GA; K and Na which have no effect on the growth of the sections in the absence of GA, synergestically stimulated the growth response.
In fact the increase in growth was due to cellular extensibility. The temperature dependence of GA responses is attributed to changes in membrane fluidity. Direct interaction of hormone with membranes has also been shown in dwarf maize coleoptile cells. However, no distinction between protein and liquid components in the membrane was made out.
There are several synthetic chemicals that block the synthesis of GAs and these are growth retardants or antigibberellins. These include AMO-1618, cycocel (CCC), Phosphon-D, ancymidol, B-nine or Alar (Fig. 20-11). These chemicals mimic the dwarfing genes by blocking specific steps in gibberellin biosynthesis and hence reducing the endogenous levels of GAs and suppressing internode elongation.
Floriculturits have used these inhibitors to make the flowers compact and short without reducing flower size. In wheat some of these inhibitors have been used to raise short and stiff stems and thus preventing lodging. These retardants are also sprayed on trees and turf grasses to check growth and hence turf mowing is precluded. In cherries and apple these inhibitors enhance fruit colour and produce firm fruits.
In lettuce hypocotyls GA or fusicoccin-induced growth the acid growth hypothesis does not seem tenable.
Recent studies have suggested the involvement of microtubules in GA-induced growth. The available cumulative evidences from several other laboratories have provided evidences that GA enhances hemicellulose and cellulose synthesis, and further that GA-effect was mediated through IAA. Evidently the response of a given tissue to a given hormone depends upon its ‘competence.’
The role of GA in the formation of bulbous swellings in Eichornia leaves; changes in the phyllotaxis of Xanthium sp.; sex expression of Cannabis sativus, Phoenix dactylifera, etc., and inhibition of betacyanin in Amaranthns caudatus as determined by the presence of tyrosine—the precursor of pigment production, are well documented.
General Mechanism of Action of GA3:
One of the possible mechanisms of GA3 action appears to be the promotion of changes in RNA synthesis during cell elongation. Apparently, GA3 action in all target tissues may be via its effect on the transcriptional process. GA3 may be enhancing the rate of synthesis of all classes of RNA, or it may be inducing some of the specific enzymes as in cereal aleurone.
Cell Division and Cell Elongation of Gibberellins Acid:
GA causes cell division and expansion causing stem elongation and the effect is similar to that of IAA but the mode of action is different. In general it has been demonstrated that GA action proceeds that of IAA at the same site. During stimulation growth effects cell elongation.
There is a body of evidence available to show that GA enhances protein synthesis or even stimulates DNA synthesis. Some workers have also demonstrated that GA stimulates cell division at the stem apex as well as stem elongation. In other words GA speeds up RNA synthesis.
Flowering:
Chailakhyan has suggested that GA is one of the two main hormones that together referred to as florigen—the flowering stimulus. GA is shown to cause rapid growth of flower primordium. Work of Nanda and his associates at Chandigarh in Inpatients has given different interpretation to Chailakhyan’s hypothesis.
Enzymes Synthesis:
GA is shown to induce several hydrolases especially amylase as in germinating barley seeds (Fig. 20-9A). GA also stimulates new mRNA’s. It is as if GA uncovers or de-represses specific genes leading to enhanced enzyme synthesis. There is also a view that GA acts on the nucleus of the cell.
Several other enzymes like invertase, etc. are also stimulated by GA. In cereal aleurone several of the enzymes are induced by GA and these include protease, amylase, esterase, peroxidase, malate dehydrogenase, catalase, etc. In the casterbean seeds, GA affects the activity of several enzymes, e.g., catalase, acid lipase, cytochrome oxidase, fumarase, malate dehydrogenase, enolase, etc.
Substitution of Light Effects:
Gibberellins can replace light effect in including seed germination in lettuce or tobacco. Presumably it functions by producing higher osmotic pressure in the embryonic parts.
The Role of Osmotic Potential:
It is generally suggested that GA lowers the π i.e., it makes Ψs more negative by increasing the solute concentration of the cell sap.
Effects on germination of dormant and non-dormant dicot seeds
The effects of GA3 on germination of dormant and non-dormant dicot seeds and on the growth of the seedlings is well known. Most studies have been undertaken on fat-storing seeds. As many as 16 enzymes have been shown to be affected in the seeds of castor seeds.
Fruit Growth:
Gibberellins are known to induce parthenocarpy in several plant species, e.g., grapes, tomato. These are also involved in the production of seedless fruits.
Miscellaneous Functions of Gibberellins Acid:
GA prevents senescence in leaves and are also known to participate in cambium activity and differentiation of tissues. Studies by Galun (1978) and Mohan Ram and Jaiswal (1974) have shown that GA plays an important role in sex reversion in cucumber and Cannabis.
They are closely related to steroids and several of them have strong hormonal effects. Extracts of several insect cause similar effects and many GAs produce ecdyon-like effects. Like the steroids, gibberellins may be having very specific effects in de-repressing genes and this activates specific enzymes.
Occurrence of several varieties of gibberellins in plant body points towards several modes of reactions and also diversity of sites of action. In short GA may be directly affecting preformed system or indirectly affecting through synthesis of RNAs.