gibberellic acis-biosynthesis and mechanism of action

gibberellic acid , its biosynthesis and signalling

1. BIC 501 – PLANT BIOCHEMISTRY (2+1)

2. GIBBERELLIC ACID – BIOSYNTHESIS AND ITS MECHANISM OF ACTION SUBMITTED BY GOWSALYA R 2023502106

3. GIBBERLIC ACID – DEFINITION Gibberellic acids are diterpene plant hormones that are biosynthesized from geranylgeranyl diphosphate, a common C20 precursor for diterpenoids, which control diverse aspects of growth and development including seed germination, stem elongation, flowering, fruit development, and the regulation of gene expression in the cereal aleurone layer.

4. HISTORY First discovered by – Japanese plant pathologist kurosowa in 1928. Found in – Rice plant He observed – the plant with excessive stem elongation (devoid of seed nature) Due to – the plants infected by the fungus called Gibberella fujikuroi ( G.monoformis ) and caused the Bakane disease (meaning – Foolish seedling) The substance was extracted and purified from the culture filtrate of Gibberella fujikuroi and the chemical was named as gibberlic acid.

5. STRUCTURE - C 19 H 22 O 6 Basic structure – Gibbane ring Gibberellic acid

6. Biochemical characteristics : Reproductive parts contain much higher concentrations of gibberellins than the vegetative parts. Gibberellins are responsible for cell wall loosening and enlargement. Immature seeds are especially rich in gibberellins (10-100 mg per g fresh weight) and are most favourite plant parts for isolation of gibberellins and their study. Gibberellins in two different forms – free gibberellins and bound gibberellins. Bound gibberellins usually occurs as gibberelines -glycosides. Two forms of GA – C-19 (active form) and C-20 (inactive form). C-19 bioactive PGR - GA 1 GA 3 GA 4 GA 7.

7. LOCATION OF GIBBERELIC ACID SYNTHESIS Apical tissue Young leaves Immature and developing fruit Root regions

8. FUNCTIONS Growth of meristem or buds via in-vitro Adventitious root as well as shoot formation Induce maleness and promotes growth of dwarf plants Seed germination and used for breaking of dormancy Possess pollenicide effect (pollen tube growth) Intensifies transcription, photosynthesis and respiration Replaces chilling and light requirement of plants Delays senescence of fruits Enhances seedless fruits (Parthenocarpy) Stimulate α -amylase production in germinating seeds Elongation of internodes

9. BIOSYNTHESIS OF GIBBERELLIC ACID The gibberellins which are chemically related to Terpenoids are thought to be formed by the condensation of a 5C precursor – an isoprenoid unit called as isopentenyl pyrophosphate (IPP) through a number of intermediates to give rise to gibberellins. Stages : 1.Formation of terpenoid precursors and ent -kaurene in plastids 2.Oxidations to form GA 12 and GA 53 on ER through GA 12 aldehyde 3.Formation of all other GAs from GA 12 or GA 53 in cytosol

10. Formation of terpenoid precursors and ent -kaurene in plastids GA is biosynthesized from a 5-C precursor IPP. The IPP may be synthesized either in plastids or cytosol. From IPP, 10-C (GPP), 15-C (FPP) and 20-C (GGPP) precursors of terpenoids are formed by condensation of 5-C units (IPP). After the formation of GGPP, the pathway become specific for GAs. GGPP is converted by two cyclization reactions through copalyl pyrophosphate into entkaurene . These reactions are catalyzed by the enzymes cyclases which are located in proplastids and not in mature chloroplasts and infact constitute the first step that is specific for GAs.

11. Oxidations to form GA 12 and GA 53 on ER through GA 12 aldehyde The ent -kaurene is transported from plastids to ER (endoplasmic reticulum). Now a methyl group on ent -kaurene at 19 th carbon position is oxidized to carboxylic group which is followed by contraction of ring B from 6-C to 5-C ring structure to form GA 12 which is precursor to all other GAs in plants. Hydroxylation of GA 12 at C-13 results in the formation of GA 53. The enzymes catalyzing the above oxidation reactions are mono- oxygenases which are located on ER and utilize cytochrome P450 in these reactions.

12. Formation of all other GAs from GA 12 or GA 53 in cytosol All other steps in the biosynthesis of GAs from GA 12 of GA 53 are carried out in cytosol by soluble enzymes called dioxygenases. These enzyme require molecular O 2 and 2-oxoglutarate as cosubstrates and use ferrous ion and ascorbic acid as cofactors. Environmental factors such as temperature and photoperiod are known to affect biosynthesis of gibberellins.

13. Mevalonate pathway

14. BIOSYNTHESIS OF GIBBERLIC ACID

15. GGPP -geranylgeranyl diohosphate Gaxx - Gibberellic acid CPS - ent-copalyl diphosphate synthase KS - ent -kaurene synthase KO - ent -kaurene oxidase KAO - ent -kaurenoic acid oxidase GA 13ox - gibberellin 13-oxidase GA 20ox - gibberellin 20-oxidase GA 3ox - gibberellin 3-oxidase GA 2ox - gibberellin 2-oxidase

16. SIGNALLING PATHWAY GA 1 from the embryo first binds to a cell surface receptor. The cell surface GA receptor complex interacts with a heterotrimeric G-protein, initiating two separate signal transduction chains. A calcium independent pathway involving cGMP, results in the activation of signalling intermediates. The activated signalling intermediate binds to DELLA repressor proteins in the nucleus. The DELLA repressors are degraded when bound to the GA signal. The inactivation of the DELLA repressors allows the expression of the MYS gene, as well as other genes, to proceed through transcription, processing and translations.

17. SIGNALLING PATHWAY The newly synthesized MYB protein then enters the nucleus and binds to promoter genes for α-amylase and other hydrolytic enzymes. Transcription of α-amylase and other hydrolytic genes is activated. α-amylase and other hydrolases are synthesized on the rough ER. Proteins are secreted via the golgi . The secretory pathway requires GA stimulation via calcium-calmodulin dependent signal transduction pathway.

18. GIBBERELIC ACID SIGNALLING PATHWAY GA ABSENT

19. GA PRESENT

20. 1.Elongation of intact stems Many plants respond to application of GA by a marked increase in stem length ; the effect is primarily of internode elongation. Applied gibberellin promotes internodal elongation in a wide range of species Mechanism of action Illustration of internodal elongation in dwarf varieties after treatment with GA

21. 2.Role of gibberellic acid in germination

22. 3.Role of gibberellic acid under abiotic stress

23. 4.Cell Elongation and Cell Division Gibberellin increases both cell elongation and cell division , as evidenced by increases in cell length and cell number in response to applications of gibberellin. For example, internodes of tall peas have more cells than those of dwarf peas , and the cells are longer. Mitosis increases markedly in the subapical region of the meristem of rosette long-day plants after treatment with gibberellin. (After Sachs 1965)

24. Continuous recording of the growth of the upper internode of deep-water rice in the presence or absence of exogenous GA3. The control internode elongates at a constant rate after an initial growth burst during the first 2 hours after excision of the section. Addition of GA after 3 hours induced a sharp increase in the growth rate after a 40-minute lag period (upper curve). The inset shows the internode section of the rice stem used in the experiment. The intercalary meristem just above the node responds to GA. (After Sauter and Kende 1992.)

25. However, the most dramatic stimulations are seen in dwarf and rosette species, as well as members of the grass family. Exogenous GA3 causes such extreme stem elongation in dwarf plants that they resemble the tallest varieties of the same species. Accompanying this effect are a decrease in stem thickness, a decrease in leaf size, and a pale green color of the leaves. It appears that dwarfness of such varieties is due to internal deficiency of gibberellins. Dwarf pea plants without (left) or with GA (right) treatment (5 μg ) for one week.

26. 5.Gibberellins Influence Floral Initiation Gibberellin can substitute for the long day or cold requirement for flowering in many plants. In plants where flowers are unisexual rather than hermaphroditic, floral sex determination is genetically regulated. However, it is also influenced by environmental factors, such as photoperiod and nutritional status, and these environmental effects may be mediated by gibberellin. In maize, for example, the staminate flowers (male) are restricted to the tassel, and the pistillate flowers (female) are contained in the ear. Exposure to short days and cool nights increases the endogenous gibberellin levels in the tassels 100-fold and simultaneously causes feminization of the tassel flowers. Application of exogenous gibberellic acid to the tassels can also induce pistillate flowers.

27. Benefits of GA 3 applications (Research evidences) Species Benefits References Barley (Hordeum vulgarae ) Increase the hydrolytic enzyme activity MacLeod and Millar (1962) Maize ( Zea mays L .) Overcome of salt stress Tuna et al., (2008) Tomato ( Solanum lycopesicum ) Decrease of seed germination time Balaguera -Lopez et al., (2009) Soyabean ( Glycine max ) Amelioration of salt stress effects Hamayun et al., (2010) Sugarcane ( Saccharum officinarum ) Salt tolerance Shomeili et al., (2011) Sunflower ( Helianthus annus ) GA 3 and pressmud application allowed the growth of sunflower plants in Cr- contaminated soil Saleem et al., (2015) Seedless grapes ( Vitis vinifera L.) Increase of grape bud size Casanova et al., (2009)

28. Future thrust The acquired knowledge on GA3 regulation and biosynthesis has opened new opportunities for improving regulatory circuits by genetic engineering strategies. Moreover, current methods have enabled directed genetic modifications in the metabolic pathways that result in higher improvements than those obtained by random mutagenesis. Since GA3 biosynthesis is an overly complex process, the actual research has explored a global regulatory approach for a multiple-level enhancement that involves both genetic regulation and metabolic engineering with promissory results. Therefore, further studies must be directed toward evaluating the combined effects from the simultaneous manipulation of positive global regulators overexpression, metabolic precursors, and bioprocess conditions .