Biosynthesis of gibberellins.pptx

Biosynthesis, metabolism, physiological role and mechanism of gibberellins

1. BIOSYNTHESIS, TRANSLOCATION, PHYSIOLOGICAL ROLE AND MECHANISM OF GIBBERELLINS BY B. PAVAN KUMAR NAIK 1 st YEAR Ph.D DEPARTMENT OF HORTICULTURE FACULTY OF AGRICULTURE ANNAMALAI UNIVERSITY

2. 1 INTRODUCTION OF GIBBERELLINS 2 BIOSYNTHESIS AND METABOLISM OF GIBBERELLIN 3 ENDOGENOUS GA 1 LEVELS ARE CORRELATED WITH TALLNESS 4 GIBBERELLINS TRANSPORT IN PLANTS 5 ENVIRONMENTAL CONDITIONS CAN ALTER THE TRANSCRIPTION OF GIBBERELLIN BIOSYNTHESIS GENES 6 MECHANISM OF GIBBERELLINS 7 PHYSIOLOGICAL ROLE CONTENTS

3. The discovery of gibberellins is quite fascinating and dates back to about the same period when auxins were discovered, but it was only after 1950s they came into prominence. A young Japanese scientist Kurosawa - rice seedlings - fungus Gibberella fujikuroi . Cause – Excess shoot elongation, yellowish green leaves taller plants – poorly developed grains. These are the symptoms of ‘ Backanae disease’ (meaning foolish) which is known to Japanese for over a century. In 1926 - filtered extract of this fungus - Backanae disease in healthy rice seedlings. In 1935, Yabuta isolated the active substance which was quite heat stable and gave it the name gibberellin. INTRODUCTION OF GA

4. In the 1930s Japanese scientists succeeded in obtaining impure crystals of two fungal growth-active compounds, which they termed gibberellin A and B. Conti…

5. Structure of Gibberellic acid Tokyo University isolated 3 gibberellins- gibberellin A gibberellin A1, gibberellin A2, and gibberellin A3 Gibberellin A3 and gibberellic acid proved to be identical.

6. The structural feature that all gibberellins have in common, and that defines them as a family of molecules, is that they are derived from the ent-kaurene ring structure : Conti…

7. In 1958 a gibberellin (gibberellin A1) was conclusively identified from a higher plant (runner bean seeds, Phaseolus coccineus ): As more and more gibberellins from fungal and plant sources were characterized, they were numbered as gibberellin AX (or GAX), where X is a number, in the order of their discovery. Conti…

8. C 20-Gas., e.g , GA12, GA27, GA53 etc. Others have only 19 (C19-GAs), having lost one carbon to metabolism . C 19 –Gas., e.g , GA1, GA3, GA20 etc. Conti…

9. BIOSYNTHESIS AND METABOLISM OF GIBBERELLIN

10. Gibberellins constitute a large family of diterpene acids and are synthesized by a branch of the terpenoid pathway . The elucidation of the gibberellin biosynthetic pathway would not have been possible without the development of sensitive methods of detection . BIOSYNTHESIS AND METABOLISM OF GIBBERELLIN

11. Systems of measurement using a biological response, called bioassays , were originally important for detecting gibberellin-like activity in partly purified extracts and for assessing the biological activity of known gibberellins. The use of bioassays, however, has declined with the development of highly sensitive physical techniques that allow precise identification and quantification of specific gibberellins from small amounts of tissue Gibberellins Are Measured via Highly Sensitive Physical Techniques

12. Conti…

13. Gibberellins are tetracyclic diterpenoids made up of 4 - isoprenoid units. Terpenoids are compounds made up of five-carbon (isoprene) building blocks Gibberellins Are Synthesized via the Terpenoid Pathway in Three Stages In plant GAs are biosynthesized in apical tissues and 3 main sites of biosynthesis Developing seeds and fruits , The young leaves of developing apical buds and elongating shoots , and The apical regions of roots .

14. The gibberellin biosynthetic pathway can be divided into three stages, each residing in a different cellular com partment. ( Hedden and Phillips 2000 ) Conti…

15. Stage – 1 Production of terpenoid precursors and ent-kaurene in plastids Geranyl Diphosphate (GPP)-10 C Farnesyl Diphosphate (FPP) F PP-15 C GGPP-20 C Copalyl Diphosphate Synthase (CPP) Kaurene Synthase PLASTID

16. Stage – 2 Oxidation reactions on the ER form GA 12 and GA 53 Kaurene Oxidase (KO) Kaurene acid Oxidase (KAO) ROUGH ENDOPLASMIC RETICULUM (RER)

17. Stage – 3 Formation in the cytosol of all other gibberellins from GA 12 or GA 53 GA 12 GA 12 13 – OH PATHWAY NON 13 – OH PATHWAY GA 13ox GA 20ox GA 20ox GA 20ox GA 3ox GA 2ox GA 20ox GA 20ox GA 20ox GA 3ox GA 2ox CYTOSOL

18. The gibberellins of tall pea plants containing the homozygous Le allele (wild type) were compared with dwarf plants having the same genetic makeup, except containing the le allele (mutant). Le and le are the two alleles of the gene that regulates tallness in peas, the genetic trait first investigated by Gregor Mendel in his pioneering study in 1866. We now know that tall peas contain much more bioactive GA 1 than dwarf peas have. ( Ingram et al. 1983) GA 1 Is the Biologically Active Gibberellin Controlling Stem Growth

19. Conti…

20. Endogenous GA 1 Levels Are Correlated with Tallness

21. S hoots of gibberellin-deficient le dwarf peas are much shorter than those of normal plants (internodes of 3 cm in mature dwarf plants versus 15 cm in mature normal plants), the mutation is “leaky” (i.e., the mutated gene produces a partially active enzyme) and some endogenous GA1 remains to cause growth. Different le alleles give rise to peas differing in their height, and the height of the plant has been correlated with the amount of endogenous GA1. Endogenous GA1 Levels Are Correlated with Tallness Stem elongation corresponds closely to the level of GA1. Here the GA1 content in peas with three different alleles at the Le locus is plotted against the internode elongation in plants with those alleles. The allele le-2 is a more intense dwarfing allele of Le than is the regular le-1 allele. There is a close correlation between the GA level and internode elongation. (After Ross et al. 1989.)

22. There is also an extreme dwarf mutant of pea that has even fewer gibberellins. This dwarf has the allele na (the wild-type allele is Na), which completely blocks gibberellin biosynthesis between ent-kaurene and GA12-aldehyde. As a result, homozygous (nana) mutants, which are almost completely free of gibberellins, achieve a stature of only about 1 cm at maturity ( Reid and Howell 1995 ) Endogenous GA1 Levels Are Correlated with Tallness The phenotype of 21-d-old seedlings of wild-type  NA  (WL1769) and two independent mutants,  na-1  (WL1766) and  na-2  (L81).

23. Endogenous GA1 Levels Are Correlated with Tallness Phenotypes and genotypes of peas that differ in the gibberellin content of their vegetative tissue. (All alleles are homozygous). Davies, 1995

24. Conti…

25. Conti… GA 1 is the biologically active gibberellin that regulates tallness in peas Ingram et al. 1986; Davies 1995 n ana shoot tip can convert the GA20 from the dwarf into GA1.

26. GIBBERELLINS TRANSPORT IN PLANTS

27. Gibberelins have been found from both phloem and xylem exudates from a variety of plants. Unlike auxins , the transport of gibberellins in plants is non-polar. It is believe that gibberellins are translocated through phloem according to a flow pattern which is similar to those of carbohydrates and other organic solutes. Gibberellins are not translocated in plant as free molecules but probably in their bound form as gibberellin-glycosides. The movement of gibberellins from scutellum to the cells of the aleurone layer in the germinating cereal seeds is well established. GIBBERELLINS TRANSPORT IN PLANTS

28. Seed germination may require gibberellins for one of several possible steps: The activation of vegetative growth of the embryo, the weakening of a growth-constraining endosperm layer surrounding the embryo, and the mobilization of stored food reserves of the endosperm. Conti… Gibberellin application also stimulates the production of numerous hydrolases, notably α-amylase, by the aleurone layers of germinating cereal grains.

29. Conti ….

30. The highest levels of gibberellins are found in immature seeds and developing fruits . However, because the gibberellin level normally decreases to ZERO in mature seeds, there is no evidence that seedlings obtain any active gibberellins from their seeds. Gibberellins Are Biosynthesized in Apical Tissues

31. Work with pea seedlings indicates that the gibberellin biosynthetic enzymes and GA3ox are specifically localized in young, actively growing buds, leaves, and upper internodes . In Arabidopsis, GA20ox is expressed primarily in the apical bud and young leaves , which thus appear to be the principal sites of gibberellin synthesis. Elliott et al. 2001 Conti…

32. The gibberellins that are synthesized in the shoot can be transported to the rest of the plant via the phloem. Intermediates of gibberellin biosynthesis may also be translocated in the phloem. Indeed , the initial steps of gibberellin biosynthesis may occur in one tissue, and metabolism to active gibberellins in another. Gibberellins also have been identified in root exudates and root extracts , suggesting that roots can also synthesize gibberellins and transport them to the shoot via the xylem. Conti…

33. FIGURE Gibberellin is synthesized mainly in the shoot apex and in young developing leaves. This false color image shows light emitted by transgenic Arabidopsis plants expressing the firefly luciferase coding sequence coupled to the GA20ox gene promoter. The emitted light was recorded by a CCD camera after the rosette was sprayed with the substrate luciferin . The image was then color-coded for intensity and superimposed on a photograph of the same plant. The red and yellow regions correspond to the highest light intensity. ( Courtesy of Jeremy P. Coles, Andrew L. Phillips, and Peter Hedden , IACR-Long Ashton Research Station.) Conti…

34. Case Study

35. The time courses of the changes in radioactive content of hypocotyl and epicotyl. The radioactivity in the hypocotyl was highest at 50 min, and was higher under CL-DT and DT-DT than under CL-CL condition for both GA1, and GA20 applications. When GA20 was applied to the cotyledon of seedlings grown under CL-CL, radioactivty was observed in the epicotyl from 50 min to 10 h after application. However , little radioactivity was observed in the epicotyl when GA1, was applied to cotyledon and undetectable at 50 min. The translocation of both GA1, and GA20 from the cotyledon to the hypocotyl was greater under DT condition than under CL after GAs application. In contrast, the translocation of GA20 from the cotyledon to the epicotyl was promoted by CL conditions after GA application . (Young-Yell Yang et al., 1996) Translocation patterns of labeled GAs from the cotyledon to hypocotyl and epicotyl

36. Table: Distribution of radioactivity in hypocotyl and epicotyl at 50 min, 3, 10, 24 and 48hr after GA application to the cotyledon

37. The height of ‘w106’ was significant shorter than that of ‘CG ’ (Fig. a). Therefore , we conducted a microscopic observation of shoots of the ‘CG’ and ‘w106’ plants using paraffin sectioning. The cell sizes in transverse sections were not obviously different between the ‘CG’ and ‘w106’ plants (Fig.  b ). However , the cell lengths in longitudinal sections were obviously shorter in ‘w106’ than in ‘CG’ plants (Fig.  c ). Thus , the defective cell elongation appears to be the main cause for the reduced shoots and dwarf architecture in watermelon . Phenotypic and genetic analyses Yuyan Sun et al., 2020 Case Study

38. Phenotypic and genetic analyses

39. Conti…

40. ENVIRONMENTAL CONDITIONS CAN ALTER THE TRANSCRIPTION OF GIBBERELLIN BIOSYNTHESIS GENES

41. Gibberellins play an important role in mediating the effects of environmental stimuli on plant development. Environmental factors such as photoperiod and temperature can alter the levels of active gibberellins by affecting gene transcription for specific steps in the biosynthetic pathway. Yamaguchi and Kamiya 2000 ENVIRONMENTAL CONDITIONS CAN ALTER THE TRANSCRIPTION OF GIBBERELLIN BIOSYNTHESIS GENES

42. The presence of light has many profound effects. Some seeds germinate only in the light, and in such cases gibberellin application can stimulate germination in darkness. The promotion of germination by light has been shown to be due to increases in GA1 levels resulting from a light-induced increase in the transcription of the gene for GA3ox, which converts GA20 to GA1 ( Toyomasu et al. 1998). This effect shows red/far-red photoreversibility and is mediated by phytochrome Light regulation of GA 1 biosynthesis

43. Conti… When a plant grows in the light, the rate of extension slows down through regulation by changes in hormone levels and sensitivity. (A) When dark-grown pea seedlings are transferred to light, GA1 level drops rapidly because of metabolism of GA1 , but then increases to a higher level, similar to that of light-grown plants, over the next 4 days.

44. (B) To investigate the GA1 response in various light regimes, 10 mg of GA1 was applied to the internode of GA-deficient na plants in darkness, 1 day after the start of the light, or 6 days of continuous light, and growth in the next 24 hours was measured. The results show that the gibberellin sensitivity of pea seedlings falls rapidly upon transfer from darkness to light, so the elongation rate of plants in the light is lower than in the dark, even though their total GA1 content is higher. Conti… After O’Neill et al. 2000 .

45. Potato tuberization is another process regulated by photoperiod ( Figure A). Tubers form on wild potatoes only in short days (although the requirement for short days has been bred out of many cultivated varieties), and this tuberization can be blocked by applications of gibberellin . The transcription of GA20ox was found to fluctuate during the light–dark cycle, leading to lower levels of GA1 in short days . Potato plants overexpressing the GA20ox gene showed delayed tuberization , whereas transformation with the antisense gene for GA20ox promoted tuberization , demonstrating the importance of the transcription of this gene in the regulation of potato tuberization . Carrera et al . 2000 Photoperiod control of tuber formation Figure A: Tuberization of potatoes is promoted by short days. Potato ( Solanum tuberosum spp. Andigena ) plants were grown under either long days or short days. The formation of tubers in short days is associated with a decline in GA1 levels. Courtesy of S. Jackson .

46. MECHANISM OF GIBBERELLINS

47. 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, A. Elongation of intact stems Illustration of internodal elongation in dwarf varieties after treatment with GA

48. 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 Cell Elongation and Cell Division

49. Conti…

50. 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 difference in the initial growth rates of the two treatments is not significant here, but reflects slight variation in experimental materials . 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.) Conti…

51. 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. Conti… Dwarf pea plants without (left) or with GA (right) treatment (5 μg ) for one week.

52. GAI (Gibberellin insenstive dwarfs mutants) RGA (Gibberellin deficiency reversion mutants) SPY (Spindle or slender mutant) GAI + RGA – amino terminal (protein) = DELLA (DELLA Repressors) Repressors

53. Functional DELLA repressors have been found in several crop plants that have dwarfing mutations , analogous to gai-1 , in the genes encoding these proteins. Most notable are the rht (reduced height) mutations of wheat that have been in use in agriculture for 30 years. These alleles encode gibberellin response modulators that lack gibberellin responsiveness , leading to dwarfness ( Peng et al. 1999; Silverstone and Sun 2000). Cereal dwarfs such as these are very important as the foundations of the green revolution that enabled large increases in yield to be obtained. Normal cereals grow too tall when close together in a field, especially with high levels of fertilizer. The result is that plants fall down (lodge), and the yield decreases concomitantly. The use of these stiff- strawed dwarf varieties that resist lodging enables high yields. DELLA Repressors Have Been Identified in Crop Plants

54. The Negative Regulator SPINDLY Is an Enzyme That Alters Protein Activity “Slender mutants” resemble wild-type plants that have been treated with gibberellin repeatedly. They exhibit elongated internodes, parthenocarpic (seed-free) fruit growth (in dicots), and poor pollen production. Slender mutants are rare compared to dwarf mutants .

55. B. Mobilization of Endosperm food reserve

56. PHYSIOLOGICAL ROLE

57. Certain light sensitive seeds e.g., lettuce and tobacco show poor germination in dark. Germination starts vigorously if these seeds are exposed to light or red light. This requirement of light is overcome if the seeds are treated with gibberellic acid in dark. Seed Germination & Dormancy

58. Case Study

59. Case Study

60. Bolting is a survival mechanism in a plant. If the environmental conditions necessary for the growth of the plant are fluctuating or are becoming stressful for the survival of the plant, it will try to produce the next generation (seeds) as quickly as possible. Essentially , it is the premature production of a flowering stem (or stems) on a plant before it can be harvested. Bolting GA induced bolting in Arabidopsis

61. Case Study

62. Conti…

63. Conti…

64. 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. Gibberellins Influence Floral Initiation

65. Case Study

66. Conti…

67. Applications of gibberellins can cause fruit set (the initiation of fruit growth following pollination) and growth of some fruits, in cases where auxin may have no effect. For example, stimulation of fruit set by gibberellin has been observed in apple ( Malus sylvestris ). Gibberellins Promote Fruit Set

68. Case Study Manabu Watanabe et al., 2008

69. Effects of single and combined treatment of plant growth regulators on the fruit set and shape of parthenocarpic apple fruits The fruit retention rate in ‘ Ohrin ’ decreased from 8 DAT in the control, 2,4-DP-alone, and CPPU-alone treatments, and from 14 DAT in the GA3-alone and GA3-combined treatments (Fig. 2A). The wave pattern of fruit drop in ‘ Ohrin ’ was bimodal in control, 2,4-D Palone , CPPU-alone and GA3 + 2,4-DP treatments, and was uni -modal in the GA3-alone and GA3 + 2,4-DP + CPPU treatments. Regarding ‘Fuji’, the control, 2,4-DPalone, and CPPU-alone treatment showed a fruit retention rate decrease from 6 DAT (Fig. 2B). The fruit retention rate of GA3-alone and GA3+2,4-DP treatments decreased from 21 DAT; that of CPPU-combined treatments decreased from 28 DAT. ‘Fuji’ showed one peak period of fruit drop, except for the control.

70. Conti…

71. Conti...

72. Conti…

73. A major use of gibberellins is to increase the stalk length of seedless grapes. Because of the shortness of the individual fruit stalks, bunches of seedless grapes are too compact and the growth of the berries is restricted . Gibberellin stimulates the stalks to grow longer, thereby allowing the grapes to grow larger by alleviating compaction, and it promotes elongation of the fruit. Conti... Gibberellin induces growth in Thompson’s seedless grapes. The bunch on the left is an untreated control . The bunch on the right was sprayed with gibberellin during fruit development. (Sylvan Wittwer )

74. GA application for increasing fruit size Variety: Thomson’s Seedless Every 1 – 1.5 weeks application of GA. GA – 3 times for increasing fruit size @ 3, 2, 2 gm Cuttings @ Oct Harvesting @ March Farmer: Naveen Verma (2022)

75. Hypothesis Sharad seedless

76. Conti…

77. Amin HASSANKHAH , Majid RAHEMI , Mohamad Reza MOZAFARI , Kourosh VAHDATI. (2018). Flower Development in Walnut: Altering the Flowering Pattern by Gibberellic Acid Application. Not Bot Horti Agrobo , 2018, 46(2):700-706. DOI:10.15835/nbha46211183 Bethke , P. C., Schuurink , R., and Jones, R. L. (1997) Hormonal signalling in cereal aleurone . J. Exp. Bot. 48: 1337–1356. Carrera , E., Bou , J., Garcia-Martinez, J. L., and Prat , S. (2000) Changes in GA 20-oxidase gene expression strongly affect stem length, tuber induction and tuber yield of potato plants. Plant J. 22: 247–256 Davies , P. J. (1995) The plant hormones: Their nature, occurrence, and functions. In Plant Hormones: Physiology, Biochemistry and Molecular Biology, P. J. Davies, ed., Kluwer, Dordrecht, Netherlands , pp. 1–12 . Elliott, R. C., Ross, J. J., Smith, J. J., and Lester, D. R. (2001) Feed-forward regulation of gibberellin deactivation in pea. J. Plant Growth Regul . 20: 87–94 . G. A. Martínez ,  A. R. Chaves  &  M. C. Añón   (1996). Effect of exogenous application of gibberellic acid on color change and phenylalanine ammonia- lyase , chlorophyllase , and peroxidase activities during ripening of strawberry fruit ( Fragaria  x  ananassa   Duch .). Journal of Plant Growth Regulation   volume 15 , Article number:  139. Haemyeong Jung, Seung Hee Jo, Won Yong Jung, Hyun Ji Park , Areum Lee, Jae Sun Moon, So Yoon Seong , Ju-Kon Kim, Youn -Sung Kim,* and Hye Sun Cho. Gibberellin Promotes Bolting and Flowering via the Floral Integrators RsFT and RsSOC1-1 under Marginal Vernalization in Radish (2020). 9, 594; doi:10.3390/plants9050594. Hedden , P., and Kamiya , Y. (1997) Gibberellin biosynthesis: Enzymes, genes and their regulation. Annu . Rev. Plant Physiol. Plant Mol. Biol. 48: 431–460 . Ingram, T. J., Reid, J. B., Potts, W. C., and Murfet , I. C. (1983) Internode length in Pisum . IV The effect of the Le gene on gibberellin metabolism. Physiol. Plant. 59: 607–616 . REFERENCE

78. Manabu Watanabe1 *, Hideyuki Segawa1 , Masanobu Murakami1 , Satoru Sagawa1 and Sadao Komori2. (2008). Effects of Plant Growth Regulators on Fruit Set and Fruit Shape of Parthenocarpic Apple Fruits. J. Japan. Soc. Hort. Sci. 77 (4): 350–357. O’Neill, D. P., Ross, J. J., and Reid, J. B. (2000) Changes in gibberellin A1 levels and response during de- etiolation of pea seedlings. Plant Physiol. 124: 805–812. Reid, J. B., and Howell, S. H. (1995) Hormone mutants and plant development. In Plant Hormones: Physiology, Biochemistry and Molecular Biology, P. J. Davies, ed., Kluwer, Dordrecht, Netherlands, pp. 448–485 . Yamaguchi , S., and Kamiya , Y. (2000) Gibberellin biosynthesis: Its regulation by endogenous and environmental signals. Plant Cell Physiol. 41: 251–257. Sauter , M., and Kende , H. (1992) Gibberellin-induced growth and regulation of the cell division cycle in deepwater rice. Planta 188: 362–368. Sachs, R. M. (1965) Stem elongation. Annu . Rev. Plant. Physiol. 16: 73–96. Young-Yell Yang', Isomaro Yamaguchi and Noboru Murofushi . 1996. Metabolism and Translocation of Gibberellins in the Seedlings of Pharbitis nil (II). Photoperiodic Effects on Metabolism and Translocation of Gibberellins Applied to Cotyledons, Plant Cell Physiol . 37(1): 69-75. Zeevaart , J. A. D., Gage, D. A., and Talon, M. (1993) Gibberellin A1 is required for stem elongation in spinach. Proc. Natl. Acad. Sci. USA 90: 7401–7405.

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