In this article we will discuss about Gibberellins. After reading this article you will learn about: 1. Biosynthesis of Gibberellins 2. Responses of Plants to Gibberellins 3. Mode of Action 4. Bioassay Methods.
Contents
Biosynthesis of Gibberellins:
Gibberellins are cyclic di-terpenoids or tetraisoprenoids. Biosynthesis of gibberellins follows the basic pathway of isoprenoid biosynthesis. All the carbon atoms of isoprene (C5H8) are derived from acetate. 5-Carbon isoprene monomer contains 2 carbons derived from carboxyl (COOH) group of acetate and 3 carbons derived from methyl (CH3) carbon of acetate.
First step is the formation of active acetic acid, i.e., acetyl CoA in which acetic acid is esterified with CoA. ATP drives the reaction.
Three molecules of acetyl CoA are produced which take part in the following 2 successive condensing reactions:
First condensing reaction:
Second condensing reaction:
Next step is the transformation of BOG – CoA to mevalonic acid (MVA). This consists of reduction by which BOG – CoA is reduced in 2 successive NADPH-requiring steps to primary alcohol level, i.e., mevalonic acid (MVA).
Then mevalonic acid is phosphorylated by MVA kinase with ATP as phosphate donor.
In second step, MVA phosphate is again phosphorylated by phospho-MVA kinase to produce MVA pyrophosphate (MVA – PP).
In the next step, carboxyl group of MVA – PP (MVA pyrophosphate) is eliminated as CO2, which occurs simultaneously with elimination of water to produce isopentenyl pyrophosphate (IpPP).
Here ATP provides the necessary energy to drive the reaction.
Then IpPP is converted into dimethylallyl pyrophosphate (DMAPP), which is an isomer of IpPP by enzyme IpPP isomerase.
One molecule of dimethylallyl pyrophosphate (DMAPP) then serves as an acceptor of one IpPP molecule with elimination of pyrophosphate (PP) and formation of one molecule of diisoprenoid alcohol pyrophosphate or geraniol pyrophosphate (GPP) which is a monoterpene.
Further growth of polyisoprene chain involves the addition of further IpPP units. Thus, sesquiterpene alcohol pyrophosphate, i.e., farnesol pyrophosphate (polymer of 3 isoprenes) is produced from geraniol pyrophosphate (GPP). Then farnesol pyrophosphate accepts another IpPP to form geranyl geraniol pyrophosphate (GGPP).
Then geranyl geraniol pyrophosphate (diterpenoid or tetraisoprenoid) is folded in various ways and then converted into a partially cyclized compound, copalyl pyrophosphate (CPP). This CPP is finally transformed into a fully cyclic compound kaurene. CPP is the last non-cyclic compound in the pathway and kaurene is the first cyclic compound in the pathway.
Kaurene is successively converted into kaurenol, then kaurenal and then kaurenoic acid. At C-7 position of ring 2 of kaurenoic acid, a hydroxyl (OH) group is attached forming 7-hydroxykaurenoic acid. Then ring contraction occurs in kaurene by extrusion of C-7 in the form of CHO and gibberellin is first formed which is an aldehyde form of GA12.
In gibberellic acid (GA3) formation, there is lactone bridge formation between C19 and C20. In this molecule, there are two unsaturated double bonds, one between C-1 and C-2 and the other between C-16 and C-17.
There are 2 OH groups — one at C-3 and the other at C-13. Compounds like GA3 where a lactone formation takes place are 19-C compounds, while there is a large group of GAs, which are 20-C compounds. Reactions up to the formation of kaurene are catalyzed by enzymes present in soluble fraction and occur in the presence of ATP and Mg2+, while those beyond kaurene are present in particulate fraction.
The biogenetic scheme assumes that the first GAs formed are aldehydic gibberellane intermediaries which occur both in the fungi and the higher plants. The major difference between a fungal GA and a higher plant GA lies in the presence or absence of OH groups in positions 3 and 13.
It is possible that two pathways exist in higher plants while one is operative in fungi. One of the pathways in higher plants is identical with the fungal pathway that is characterized by initial hydroxylation in position 3. In the other pathway which is unique in higher plants, the initial hydroxylation is in the position 13.
This would account for the fact that if in a fungal GA, there is a single OH group, it is always present in position 3, whereas in higher plant GAs the single OH group is always borne on position 13. This would mean that there are a large number of gibberellins in higher plants which are lacking in Gibberella.
(a) Bound Gibberellins:
Several GA-like substances may occur in bound form which are specially found in seeds and fruits. In contrast to typical GAs, these substances cannot be extracted from acidified aqueous solution with ethyl acetate but can be done with n-butanol.
Treatment of these substances with acid, alkali or enzyme like ficin, emulsin results in the release of typical less polar GAs. These substances are called water-soluble, butanol-soluble or bound GAs. Some authors have suggested the existence of protein-bound GAs.
Other polar forms of GA have been chemically identified as conjugates of glucose from higher plants whereas acetyl GA has been isolated from Gibberella. Such GA-glucosides, glucosyl esters and other conjugated GAs are termed conjugated GAs which are inactive in growth regulation but become active on hydrolysis.
Storage function can be attributed to the bound forms as shown by the inter-conversion between free and bound GAs during seed development and germination.
During seed development, part of the free GA is converted to bound form, while during early germination, part of the bound GA is reconverted to free GA. Another function may be associated with GA transport. Conjugated GAs may represent storage GAs which are transported to tissues requiring the hormone.
(b) Gibberellins Transport:
Gibberellins are transported in the entire conducting system, i.e., both in the phloem and in the xylem. GA transport occurs passively along with the flow of assimilates or of water, salt and other organic compounds. In general, GA movement is non-polar in contrast to polar transport of auxin.
In a few instances, basipetal polar transport of GA has been reported, but such polarity may be really due to movement from a source to a growth centre and is not a true polar movement.
Responses of Plants to Gibberellins:
(a) Stem Growth:
Gibberellins are well known for their remarkable effects on intact plants. GAs applied to intact plants induce appreciable elongation of stem tissue, the effect being more pronounced in rosette and dwarf species, producing tall plants. The effect of GA on stem extension in general is based on pronounced cell elongation and not on cell division.
It should, however, be noted that GA-induced stem growth can also be accompanied by an increase in cell division. In the sub-apical meristem of rosette and caulescent plants, GA has been shown to increase both the cell number and cell size.
(b) Bolting and Flower Formation in Long-Day Plants:
Of all the plant hormones, only GAs have been shown to effectively cause flower formation in a wide variety of plants. In general, long-day plants (LDP) and cold-requiring plants are responsive to exogenous GAs when kept under non-inductive condition, while short-day plants (SDP) and day- neutral plants (DNP) are not.
GA-sensitive LDP and cold-requiring plants usually grow as rosettes in non-inductive conditions but either transfer to inductive LD condition or GA treatment in non-inductive SD condition results first in stem elongation (bolting) and then flower formation can be induced.
In such rosette LD plants, the flower inducing effect of GA is thought to be mediated through its effect on stem elongation (i.e., bolting) and not on the synthesis of flowering hormone. Anton Lang reviewed the situation and presented evidences against the direct effect of GA on flowering.
These are:
(i) Flower formation can be induced by GA in many rosetted LDP whereas SD plants in general do not respond to GA.
(ii) GA-sensitive LDP and cold-requiring plants usually grow as rosettes in non-inductive conditions. Flower-forming effect of GA on LD plants is generally restricted to rosettes only, whereas GA is almost without effect on caulescent LD plants,
(iii) GA has been shown to be most effective when applied to the stem apex suggesting its effect on stem growth, whereas the processes related to photo-induction occur in leaves,
(iv) LD plants treated with GA under SD (non-inductive) condition respond first by stem elongation and then by flower formation,
(v) Correlations between endogenous GA level are more close with stem growth rather than with flowering,
(vi) Treatment with inhibitors of GA biosynthesis can completely suppress LD-induced stem elongation (bolting) in LDP but has no effect on flower formation during photo-induction.
Even though GAs are not directly involved in the transition to flowering in many LDP and cold-requiring plants, some grafting experiments with LDP Silene and cold-requiring Chrysanthemum point to the possibility that exogenous GA acts indirectly through the production of the floral stimulus.
Role of inhibitors of GA biosynthesis on SDP Pharbitis nil is rather opposite to the effect in LDP and is quite complex where both stem elongation and flower formation are shown to be prevented.
Thus in Pharbitis, GAs are required for floral initiation by increasing the ability of the apex to respond to the floral stimulus. In long-short-day plants (LSDP) Bryophyllum, on the other hand, GAs can play essential role in the production of the floral stimulus but not simply initiation.
(c) Induction of Seed Germination and Enzyme Production During Germination:
Of all the growth hormones, only gibberellins are known to consistently enhance germination and are positively implicated in many seed processes. GAs can cause germination in seeds that normally require cold temperature (stratification) or light to induce germination. Exogenous application of GAs can replace at least part of the chilling or light requirement in such seeds.
During germination of a cereal grain, starch and other complex food reserves present in the storage part, i.e., endosperm of grain undergo conversion into simpler compounds. In the 1960s, Paleg in Australia and Varner in U.S.A. established that gibberellins can stimulate these conversions.
A cereal seed contains two parts, the embryo which is the living part and the endosperm which is the metabolically inactive non-living part. The endosperm is surrounded by one or two layers of cells called the aleurone layer. During germination of a cereal seed, hydrolytic enzymes, specially α-amylase, are formed in the aleurone layer and then secreted into the endosperm where starch is hydrolysed.
When the embryo is dissected out of the seed, the aleurone cells are not able to produce a-amylase which indicates that the embryo is the site for the production of gibberellins which are transported to the aleurone cells where the hydrolytic enzymes are synthesized and secreted into the starchy endosperm.
The incubation of isolated aleurone layers in media containing specified concentrations of GA has been used extensively as a model for understanding hormone action in plants.
(d) Breaking of Dormancy and Growth of Dormant Buds:
Buds of evergreen deciduous trees usually become dormant in autumn, enter a period of rest in winter and growth is arrested till the onset of spring.
Bud dormancy is overcome by extended cold period and by long day and red light. Under these conditions, an increase in gibberellins takes place. Exogenous GA application is able to overcome both seed and bud dormancy by acting as a substitute for low temperature, long day or red light.
(e) Fruit Setting and Growth:
A number of deciduous fruit trees such as apple, pear and some citrus species can be induced to set fruit with GA or a combination of GA with auxin. GA is also used extensively on seedless grape varieties to increase the size and quality of the fruit. It has been suggested that the application of GA increases the mobilization of carbohydrates to the developing fruit.
(f) Malting of Barley:
GA is used to increase the yield of barley malt extract. Application of GA to germinating barley supplements the endogenous GA content and accelerates the production and release of hydrolytic enzymes that degrade storage proteins and carbohydrates of the endosperm into sugars and amino acids that comprise the malt extract.
(g) Change of Sex Expression:
In Cucumis, perfect flowers are initiated but one sex organ fails to develop which leads to the development of monoecious plant with imperfect flowers. Treatment with exogenous GA results in male flower formation.
It has been observed that staminate plants contain more GA-like substances than their pistillate counterparts. In Spinacia sp. and Cannabis sativa, which are dioecious plants with male and female flowers in separate plants, application of exogenous GA increases the tendency for the formation of male flowers.
Mode of Action in Gibberellins:
Experiments on the mechanism of GA action in higher plants have been made along two distinct lines. One aspect is concerned with the role of GA on stem elongation while the other deals with the effects of GA to promote RNA and protein synthesis in germinated seeds and seedlings.
(a) GA and Stem Elongation:
Gibberellins produce spectacular effects in intact plants leading to the elongation of stem tissue, this effect being more pronounced in rosettes and dwarf species. A striking example of internode elongation is found in deep water rice (Oryza sativa).
In order to enable the upper foliage of the plant to remain above water, the internodes elongate as the water level rises. GAs play a critical role in this enormous inter nodal growth of deep-water rice.
Such dramatic stimulation of internode elongation of deep-water rice is due to increased cell division activity in intercalary meristem. The increase in length of a shoot results from increase in length of existing as well as newly divided cells. Simple cell division cannot lead to increase in volume unless the cells expand.
The effect of GA on stem extension is mainly based on cell elongation. In sub-apical meristems, however, both the rate of cell division and the size of the meristem are increased by GA which indicates that GA-induced stem growth can also be accompanied by an increase in cell number.
It has been proposed that GA has an effect on the timing of the mitotic cycle. GA shortens the interphase by promoting DNA synthesis in cells which are arrested in the G, phase of the cell cycle (jones and McMillan, 1984). Early studies on the biochemical basis of GA-induced elongation were made in terms of acid-growth hypothesis proposed to explain auxin-induced growth.
This theory is based on acid-stimulated growth and indicates that auxin stimulates the activity of a plasma-membrane bound proton pump which acidifies the cell wall causing cell-wall loosening and ultimately growth. Recent experimental evidence shows that GA induces growth by mechanisms other than acidification of the cell wall.
It has been observed that the lag time before GA-stimulated growth begins is longer than that for auxin. For instance, it is about 40 minutes in deep-water rice, whereas in peas, it is of the order of 2 to 3 hours. These longer lag times point to the possibility that cell-wall acidification as mediated by auxin is not the mechanism of GA action.
Many physiological processes in plants have been proposed to be controlled by calcium. Based on the observation that CaCl2 solutions block GA-induced elongation, Moll and Jones (1981) proposed that the removal of Ca2+ ions bound to the cell wall and the movement of Ca2+ ions into the cytosol are associated with the stimulation of growth by GA.
Wall extensibility depends on the removal of Ca2+ ions bound to the cell wall whereas an increase in the cytoplasmic calcium concentration stimulates vesicle flow from the Golgi bodies to the cell wall which increase the rate of cell-wall synthesis and turnover which in turn increase the rate of elongation.
The rate of incorporation of 14C- labelled glucose in the cell wall increases in Avena internodes after exposure to GA.
In addition to increasing cell-wall extensibility, GA also prevents reactions which cause cell-wall stiffening. Phenolic cell-wall components like ferulic acid are converted into diferuloyl crosslinks by a cell-wall peroxidase which reduce the flexibility of cell wall and consequently limits its expansion.
GA has been proposed to regulate cell expansion by inhibiting peroxidase activity. Peroxidase also catalyses the conversion of water-soluble phenolics into hydrophobic quinones. GA inhibits peroxidase activity which results in a decrease in hydrophobicity of the cell wall, thus increasing its plasticity and loosening action of hydrolytic enzymes.
Peroxidase also controls the formation of a network of cell-wall protein extensin. Extensin molecules are cross-linked by isodityrosine residues and a peroxidase is involved in their synthesis. Inhibition of peroxidase by GA results in the loosening of extensin network.
Another possible mechanism by which GA controls elongation is through the regulation of cell-wall lignification. The enzyme phenylalanine ammonia lyase (PAL) controls the production of phenylpropanoid precursors of ferulic acid and lignin. When growth is inhibited in light, PAL activity is high. In contrast, GA reduces PAL activity which results in high rates of elongation.
In a study on the effect of GA on stem elongation, a close correlation between GA-stimulated growth and activity of the enzyme xyloglucan endotransglycosylase (XET) has been observed in many tissues. XET is an enzyme that hydrolyzes xyloglucan internally and transfers one of the cut ends to the free end of an acceptor xyloglucan molecule.
Thus, XET has the capacity to cause molecular rearrangement in the cell-wall matrix that promote cell extension. It has been further observed that auxin does not increase XET activity. So, this effect is specific for gibberellins.
One possibility is that XET facilitates the penetration of expansions into cell walls. Expansins are cell-wall proteins that cause cell-wall loosening at acid pH by weakening hydrogen bonds between cell-wall polysaccharides. Thus, both expansins and XET may be required for GA-stimulated growth.
In Arabidopsis, three main classes of mutations affecting plant height have been selected:
(a) Gibberellin-insensitive dwarfs.
(b) Gibberellin-deficient mutants in which the GA deficiency has been overcome by a second suppressor mutation, so that the plants look like the normal counterpart.
(c) Mutants with a constitutive GA response called slender mutants.
In these mutants genes responsible for the synthesis of same signal transduction component have been identified even in morphologically different plants. This has been possible due to mutations either in regulatory or in functional (repression) domains of the protein. GA controls growth by signal transduction through this repressor protein.
The regulatory and repression domains of the repressor protein are encoded by GAI and RGA genes respectively. Those genes belong to a large multi-gene family in Arabidopsis. The repressor domain is active in the absence of gibberellin. A GA-induced signaling intermediate binds to the regulatory domain, targeting it for destruction and thus facilitating growth.
The repressors have highly conserved regions with nuclear localization signals. The gai (gibberellin insensitive) mutant is dwarf, while rga is tall, indicating that the mutations are in different domains of the protein. The gai mutation erases the sensitivity of the repressor to GA due to mutation in regulatory domain. The rga mutation, on the other hand, prevents the function of the repressor resulting in growth.
The effects of GA treatment on different mutants of Arabidopsis have been shown in the following Fig. 13.15.
(b) GA and Synthesis of RNA and Protein:
Plant hormones in general affect RNA and protein synthesis.
Study on the response of cereal aleurone to GA, particularly with reference to α-amylase synthesis has made a significant contribution to our understanding of GA action in plant cells. The incubation of isolated aleurone layers in media containing specified concentrations of GA has been extensively used as a model system for the study of the molecular basis of hormone action in plants.
The main observation is that GA, either coming from the embryo in intact seeds or added to the media in case of isolated layers, can stimulate the aleurone cell to synthesize and secrete a number of hydrolytic enzymes, particularly α-amylase. These enzymes are responsible for mobilization of stored endosperm reserves which provide the growing seedling with a supply of simple products.
As early as 1960, Paleg in Australia and Yomo in Japan discovered that GA addition to embryo less endosperm could substitute for the embryo in initiating substrate breakdown. Subsequently, workers in Varner’s laboratory could remove the aleurone layer from starchy endosperm.
When isolated aleurone layers were incubated with GA containing Ca2+, a range of hydrolytic enzymes are produced de novo of which 60-70 per cent made up by a-amylase. Following synthesis, most enzymes except those involved in fat metabolism are secreted into the medium.
Vigorous research was initiated to reveal the molecular mechanism that lead to de novo protein synthesis and the structural events that permit the secretion of these proteins.
GA has been shown to influence two enzymes of the CDP-choline pathway of lecithin biosynthesis which signifies its role in membrane synthesis. Choline kinase, the first enzyme of this pathway does not increase, but phosphorylcholine-cytidyl transferase and phosphorylcholine-glyceride transferase increase following GA treatment.
The endoplasmic reticulum (ER) is the site of α-amylase synthesis and synthesis of RNA is necessary for GA-induced enzyme synthesis and it is also involved in its secretion. It was later, however, shown that probably no additional ER is synthesized but there is reorganization of pre-existing ER in response to GA treatment.
GA causes de-repression of genes for α-amylase synthesis and synthesis of new RNA is necessary for GA-induced enzyme synthesis. Inhibitors of RNA synthesis inhibit α-amylase synthesis and poly-(A)-rich RNA corresponding to mRNA specific for a-amylase enzyme is synthesized in GA-treated tissue.
Thus, it has been argued that GA may not cause a general increase in translatable RNA, rather it stimulates the activation of some RNAs, like a-amylase mRNA.
Evins and Varner observed that there is an increase in the synthesis of ribosomes (rRNA) following GA treatment together with ribosome aggregation leading to polyribosome formation and these polyribosomes are attached to ER where new protein is synthesized.
The observation that inhibitors of protein synthesis like cycloheximide inhibited GA-induction of hydrolase formation signifies that the enzyme production is due to protein synthesis de novo. It is now clear that GA brings about enzyme-specific selective mRNA synthesis which is associated with polyribosome formation and de novo synthesis of a particular hydrolase.
Thus, GA operates directly at the transcriptional level during mRNA synthesis whereas its effect on the translation of new mRNAs into proteins may be indirect.
(c) GA Binding: Possible Location of Gibberellin Receptor:
The newly received idea about the mode of action of hormones is the existence of a receptor protein either on the cell surface or within the cytoplasm, migration and association of the receptor-hormone complex to the nucleus and the formation of mRNA, which is translated into protein in the cytoplasm.
Recent evidence indicates that the GA receptor is localized on outer face of the plasma membrane. Some experiments have shown that the entry of GA into the cell is not required for activity, and membrane proteins may be involved in the early GA signaling events in aleurone cells.
Recent evidences indicate that the GA receptor is localized on the surface of the plasma membrane. Some GA analogues have been synthesized which being membrane impermeable, are not able to cross the membrane. However, these are found to be active when added to aleurone protoplasts indicating that entry into the cell is not necessary for activity.
In another experiment, Gilroy and Jones injected GA3 into barley aleurone protoplasts, but it produced no effect. On the contrary, when the protoplasts are immersed in GA3, the hormone produces a-amylase. So, GA is perceived only on the outer face of the plasma membrane. Thus, it may be concluded that membrane proteins may be involved in the early GA signaling events in aleurone cells.
(d) GA Regulates Cell Cycle in Intercalary Meristems:
The internodes of deep-water rice dramatically increase their growth rate in response to submergence. The growth response occurs mainly in the intercalary meristem of the youngest internode just above the node.
Transitions between different phases of cell cycle are regulated by cyclin-dependent protein kinases (CDKs). Sauter and his colleagues (1995) measured the transcript levels of two genes (CDC 2) or cell division cycle 2 genes encoding cyclin-dependent protein kinases in deep-water rice in presence or absence of GA.
Expression of one of the CDC 2 genes was increased after 1 h of GA treatment along with the expression of two corresponding mitotic cyclin genes. Thus, GA promotes cell division by increasing one CDC 2 protein kinase and also M cyclins required for the entry into mitosis.
(e) Regulation of α-Amylase Gene Expression:
It is now well established that GA induces the synthesis of the enzyme α-amylase in aleurone cells of germinating cereal grains. The GA from the embryo induces the transcription of the genes for α-amylase mRNA. In a study on GA responsiveness to the α-amylase gene, the map of the promoter region of the α-amylase gene has been examined.
This study has revealed that several promoter elements of the gene confer GA responsiveness. One particular sequence TAACAAA box has been named gibberellin response element (GARE). The second sequence involved in GA-induced gene expression is TATCCAC box. In addition, a third sequence (C/TCTTTTC/T) known as pyrimidine box may be required for full GA response.
These three sequences together have been known as gibberellin response complex (GARC). Specific transcription factors associated with GARC then interact with general transcription factors at the TATA box (so named because of abundance of two nucleotides). These regions are the sites of binding of specific transcription factors for α-amylase gene expression.
It is now believed that GA either increases the amount or promotes the activity of a protein that switches on the production of α-amylase mRNA by binding to an upstream regulatory sequence of the α-amylase gene. It has been suggested that MYB protein is a transcription factor that binds with the gibberellin response complex of the a-amylase promoter.
There is evidence that MYBs are transcription factors that regulate growth and development. The synthesis of MYB protein is itself induced by GA (GA-MYB).
This regulatory protein (GA-MYB) after binding the GA-response element TAACAAA in the α-amylase gene promoter, stimulates the synthesis of α-amylase mRNA, which is subsequently translated to the α- amylase enzyme during the germination of cereal grains. Thus, GA stimulates the expression of GA-MYB gene, and the MYB protein serves as a regulator of transcription of the α-amylase gene.
In a time-course study for the induction of GA-MYB mRNA and a-amylase mRNA by GA, it has been observed that the production of GA-MYB mRNA precedes a-amylase mRNA. This finding enables us to infer that GA-MYB plays the role of an early GA-response gene that regulates the transcription of the a-amylase gene, which is a later event.
Gibberellins transduce signal leading to a-amylase synthesis and its secretion in the following way:
1. GA produced in the embryonal axis is transported to and binds to a aleurone cell surface receptor.
2. The GA-receptor complex interacts with a heterotrimeric G-protein, switching on two separate signal transduction cascades.
3. A Ca2+ -independent pathway, involving cGMP to activate a signaling intermediate, which then binds to DELLA repressor protein in the nucleus.
4. When bound to the GA signal the repressor protein is degraded.
5. This inactivation of DELLA repressor allows the expression of the MYB gene, as well as other genes, to proceed through transcription, translation and processing.
6. The newly synthesized MYB protein then enters the nucleus and binds to the promoter gene for α-amylase and other hydrolytic enzymes.
7. Transcription of α-amylase and other hydrolytic enzyme genes is activated.
8. α-amylase and other enzymes are synthesized on the rough ER.
9. The synthesized proteins are secreted via the Golgi apparatus.
10. The secretion requires GA stimulation via a Ca2+ -calmodulin dependent signal transduction pathway (Fig. 13.17).
Bioassay Methods in Gibberellins:
The characteristic physiological responses elicited by gibberellins are:
(i) Elongation of internodes or leaf sheaths of dwarf plants
(ii) Hypocotyl elongation
(iii) Induction of amylase and probably other hydrolases in germinating cereal seeds
(iv) Lowering in some long-day plants. The responses (i), (ii) and (iii) are used extensively as typical bioassay methods.
(a) Dwarf Maize Test:
While normal maize plants attain a height of 3 m, some of the dwarf mutants do not grow more than 15 cm in height. The dwarfing genes are non-allelic and some of the mutants grow to normal heights when treated with GA.
The test solution is applied as a drop with a micropipette to the first unfolding leaf of a single-gene dwarf mutant when it emerges from the coleoptile. After 3-5 days, the length of the leaf sheath from the ligule to the coleoptile node is measured.
(b) Dwarf Pea Test:
This test is used most extensively and is fairly specific for gibberellins. Pea seedlings of a suitable dwarf variety measuring about 3 cm from the cotyledonary node to the hook are used as the test plants. Test solution is then applied with a micro syringe to the apex of each plant and the length of the shoot from the cotyledonary node to the highest visible node is measured after five days.
(c) Hypocotyl Elongation Test:
The hypocotyls of epigeally germinating seedlings are quite sensitive to GAs. Cucumber, lettuce and mustard hypocotyls are extensively used as the test materials. The test solution is applied on the apical buds of seedlings with a micropipette and the lengths of hypocotyls are measured after 3-4 days. The stimulation of hypocotyl growth by added GA is proportional to the logarithm of the concentrations over a wide range.
(d) Potato Eye Test:
From freshly harvested potato tubers, eyes are punched off with a cork borer. These pieces in a petri dish are treated with droplets of test solution and the number of sprouted eyes as a percentage of total number is calculated. This test showing the promoting effect of GA on the sprouting of potato provides a good bioassay method in some cases.
(e) Rumex Leaf Senescence Test:
Although retardation of senescence of detached leaves is a property exhibited by cytokinins, leaves of Rumex obtusifolius are peculiar in that they respond only to GA treatment. In this test, leaf discs cut off from the leaves with a cork borer placed in petri dishes lined with filter papers kept moistened with test solutions and incubated in darkness for 4-5 days.
While senescence starts in control leaves, showing lesser green colour, treated leaf discs still remain green from which chlorophyll is extracted and estimated. Retention of chlorophyll content can be utilized to detect and measure GAs.
(f) Amylase Formation Test:
This is probably the most specific of all the methods which is based on observations that GAs increase the extractable a-amylase of barley grains during germination and a de novo synthesis of the enzyme takes place in response to added GA.
Barley or rice seeds are soaked with water for 30 min. Husks are then removed and the de-husked seeds after sterilizing with calcium hypochlorite solution and washing with distilled water are cut transversely into two halves and only the endosperm halves free from embryo are used for the bioassay.
Ten such embryo less endosperm halves (half seeds) are placed in small dishes containing suitable buffer with Streptomycin to prevent microbial growth in addition to test solutions of GAs.
The dishes are then incubated at 24°C for 20 hours following which the half-seeds together with the incubation medium are extracted and α-amylase activity in the extract is tested according to the standard procedure using soluble starch as the substrate.