2. Gibberellins Plant Physiology II BS Botany 7th semester
Habib-ur-Rehman Athar
Institute of Pure & Applied Biology
Bahauddin Zakariya University, Multan

3. Discovery of Gibberellins
1926 E. Kurosawa discovered that a substance produced by a fungal pathogen Gibberella fujikuroi, caused the ‘foolish seedling’ disease in rice.
This substance caused the plant to grow spindly, tall and pale colored.
1935 T Yabuta and Y Sumiki isolated this substance from the fungal pathogen Gibberella and named it Gibberellin
1938 isolated crystals of GA-A and GA-B
1955 Stodola from USDA and Brian from ICI London isolated Gibberellic Acid
1956 West and Phinney UCLA isolated GA from plants
1958 MacMillan in Bristol England identified GA1 from beans
1956 J. MacMillan isolated a similar compound from a plant (immature bean seeds)
More than 125 compounds belonging to the class of Gibberellins have been isolated from a wide variety of plants (24 occur in Gibberella fujikuroi and 101 in other higher plants)
They are found in all parts of the plant body
Highest concentrations of gibberellins are found in developing seeds
Different gibberellins differ in structure and biological activity

4. “Foolish seedling disease” in rice

5. Plant Hormones Gibberellins Types of gibberellins used in horticulture
Several different gibberellins (GA) produced by plants
Large, complicated molecules not synthesized
Commercial gibberellins produced by fungus
Site of gibberellin production in plants:
Shoot and root tips (apical meristems)
Young, expanding leaves
Embryos
Fruits
Tubers

6. Plant Hormones Gibberellins Action of gibberellins in plants:
Stimulate cell elongation
Dwarf plants treated with gibberellins produce normal growth
Applied to grapes to elongate the peduncle (stem of flower cluster) and pedicels (stem of single flower), making looser cluster
Promotes cell division in vascular cambium
Promotes seed germination
causes production of enzymes that break down starch into energy needed for growth
Used by beer brewers to stimulate sugar production in barley malt (sugar is converted into alcohol during fermentation)
Influences flower and fruit development

7. Bioassay of Gibberellins
Three bioassays for gibberellins
i) Lettuce hypocotyl elongation bioassay
ii) The dwarf rice leaf sheath bioassay
iii) Barley aleurone layer a-amylase bioassay
Assays i and ii depend on growth responses
Assay iii is based on induction of enzyme production and secretion by GA

9. Biosynthesis of Gibberellins
Gibberellins are tetracyclic diterpenoids made up of 4 isoprenoid units. There are two pathways for biosynthesis of gibberellins
Mevalonic acid-dependent pathway
Mevalonic acid-independent pathway
First pathway operated in cytosol and is involved in sterol biosynthesis
Second pathway operated in chloroplast and is involved in carotenoids synthesis

10. Mevalonic acid-dependent pathway

11. Mevalonic acid-independent pathway [Stage 1]
To Stage 2

12. Mevalonic acid-independent pathway [Stage 2]

13. Mevalonic acid-independent pathway [Stage 3]

14. Physiological Roles of Gibberellins
Seed Germination-Barley de novo amylase synthesis (Varner 1964)
When applied externally, they can reverse the effect of certain dwarfing mutations
Studies with such mutants have shown that although the application of mall gibberellins can cause this effect, they have to be converted to a particular form (gibberellin A1) before they can have any biological effect.
Gibberellins can substitute for the dormancy breaking treatments (cold or light) in certain seeds like lettuce tobacco and wild oats.
Gibberellins promotes in germination and radicle growth in malting barley seeds.
External application of gibberellins can promote development of seedless fruits (parthenocarpy) in some species (e.g. Currants, apples cucumbers
Flower induction
Control of sex expression
Delays Senescence

15. Gibberellins promote stem elongation and leaf expansion
They cause these effects by promoting both cell division and cell elongation
The mechanism by which GA regulates cell elongation is different from that of Auxins which involve cell wall acidification but the effect of GA on wall loosing takes place without acidification
GA regulates xyloglucan endotrans glycosylase (XET). XET facilitates the penetration of expansins (cell wall proteins that cause loosening in acid conditions). Thus both XET and expansins are required for GA – stimulated cell elongation

16. Dwarf pea plant treated with gibberellin

17. Result: shorter stems
Result: longer stems

18. Gibberellins promote stem elongation and leaf expansion

20. GA regulates the transcription cell cycle kinases – cell division
GA activates the cell division cycle first at the change from G1 to S phase, leading to an increase in mitotic activity. To do this, GA induces the expression of the genes for Kinases (CDKs) which are involved in regulation of cell cycle

24. After imbibition, certain grass seed embryos secrete gibberellins, which promote synthesis of hydrolytic enzymes in aleurone (eg. Amylase etc. which help mobilize the storage starches for germination).

26. Starch partially digested
(48 hr. Water)
Starch undigested
(48 hr. 1 ppb Gibberellin)
Starch partially digested
(48 hr. 100 ppb Gibberellin)
Starch well digested

27. External application of gibberellins can also enlarge fruit size in grapes
GA can induce parthenocartpy and fruit enlargement

28. Gibberellins can cause bolting in Brassicacea members even in the absence of inducing photoperiod or cold treatment

29. Wheat embryo (X4) After soaking in distilled water
Embryonic leaves
Coleoptile
Following imbibition, separation of scutellar epithelium
Shoot apex
Radicle
Scutellum
Endosperm

30. GA3 150mg/L
GA3 100mg/L
GA3 200 mg/L
15 dS m-1 NaCl M.H.-97

31. Physiological mechanism Gibberellin-Induced Stem Elongation
GA-induced growth begins after 40 min in deep water rice, 2-3 hours in peas
Recently, a close correlation between GA-stimulated growth and the activity of the enzyme xyloglucan endotransglycosylase (XET) has been observed for many tissues (Potter and Fry 1994; Smith et al. 1996).
XET has the potential to cause molecular rearrangements in the cell wall matrix that could promote wall extension. Auxin-induced growth is not associated with an increase in XET activity. Thus the effect is specific for gibberellins.
One possibility is that XET facilitates the penetration of expansins into the cell wall. According to this view, GA and auxins may work together to promote cell wall loosening: Auxin induces proton extrusion, while GA stimulates XET activity, which allows expansin proteins to penetrate into the wall, where they become activated by the acidic pH.

32. CDK's and Gibberellin-Induced Cell Division
Transitions between the different phases of the cell cycle are regulated by cyclin-dependent protein kinases (CDKs). Sauter and colleagues (1995) measured the transcript levels of two genes (CDC2) encoding cyclin-dependent protein kinases in deepwater rice in the presence or absence of GA. The expression of one of the CDC2 genes was increased after 1 hour of GA treatment, as was the expression of two corresponding mitotic cyclin genes.
These results supported the hypothesis that GA promotes cell division by increasing the level of a specific Cdc2 protein kinase along with the M cyclins required for the entry into mitosis. Thus, GA may stimulate cell division in the intercalary meristem of deepwater rice by several mechanisms

33. Gibberellin-Induced α-amylase mRNA
Total mRNA was extracted from GA3-treated and control tissue and translated in vitro in the presence of the radioactively labeled amino acid methionine. Upon precipitation of the reaction products with α-amylase antibody, the radioactivity in the α-amylase was quantified. On the basis of the amount of immunoprecipitated protein, more α-amylase was synthesized from mRNA extracted from GA-treated tissue than from mRNA of the controls, and the increase in the presumptive α-amylase mRNA preceded the appearance of α-amylase in the medium (Higgins et al. 1976).
The method just described measures only α-amylase mRNA. To obtain a direct measurement of the total amount of α-amylase mRNA, researchers made α-amylase cDNA clones from isolated α-amylase mRNA, which is the principal mRNA made by aleurone cells and thus is relatively easy to obtain, and they used this mRNA to make [32P]cDNA probes. By hybridizing the radioactively labeled cDNA probes to Northern (RNA) blots, researchers showed that the level of α-amylase mRNA is strongly enhanced by gibberellic acid (Chandler et al. 1984).

34. Gibberellin effects on enzyme synthesis and mRNA synthesis
Gibberellin effects on enzyme synthesis and mRNA synthesis. (A) Synthesis of α-amylase by isolated barley aleurone layers is evident after 6–8 hours of treatment with GA3 (10–6 M). (B) Messenger RNA extracted from aleurone cells and translated in vitro showed an increase in presence of α-amylase mRNA, and the appearance of the mRNA preceded the release of the α-amylase from the aleurone cells by 12 hours. The α-amylase mRNA in this case was measured by the in vitro production of α-amylase as a percentage of the protein produced by the translation of the bulk mRNA

35. Gibberellin Signal Transduction

36. Phytohormones – Signal Transduction Tolerance
Plant – ON / OFF SWITCH
Growth & Development
Phytohormones – Signal Transduction Tolerance
Production of Natural Products

38. Composite model for the induction of α-amylase synthesis in barley aleurone layers by gibberellin. A calcium-independent pathway induces α-amylase gene transcription; a calcium-dependent pathway is involved in α-amylase secretion. (The SPY negative regulator was omitted for clarity.)

44. The proteins GAI and SPY act as repressors of GA responses
The proteins GAI and SPY act as repressors of GA responses. Gibberellin acts by deactivating these repressors.
Calcium ions act as second messengers for many hormonal and environmental responses in various plants. GA increases a slow rise in cytosolic Ca2+ within 1 to 4 hours after exposure to the hormone, and thus precedes the onset of α-amylase synthesis.
Removal of the extracellular calcium inhibited both the secretion of α-amylase and the increase in calcium, indicating that calcium is being taken up from the external medium. Consistent with a role for calcium in α-amylase production, GA, in the presence of calcium, increased the level of calmodulin in barley aleurone layers by twofold, and the effect begins as early as 2 hours after the start of incubation. Recall that calmodulin binds to calcium ions, and the resulting calcium–calmodulin complex is capable of activating specific enzymes, such as Ca2+–calmodulin-dependent protein kinases.

45. Recent studies by Gilroy (1996) have suggested that GA stimulates the secretion of α-amylase and other hydrolases via a calcium-dependent pathway, whereas GA appears to stimulate expression of the α-amylase gene via a calcium-independent pathway. The operation of different signal transduction pathways for enzyme secretion and gene expression is consistent with the observation that GA stimulates both secretion and the synthesis of some enzymes (e.g. α-amylase), but only the secretion of other enzymes (e.g., β-1,3-glucanase and ribonuclease).
Cyclic GMP is a possible candidate for a calcium-independent signaling intermediate involved in GA-induced gene expression. cGMP has been implicated as a second messenger in phytochrome-regulated gene expression. GA causes a transient rise in cGMP levels in barley aleurone layers after a lag period of only 1 hour (Pensen et al. 1996). An inhibitor of guanylyl cyclase, the enzyme that synthesizes cGMP from GTP, blocks GA-induced α-amylase production, and the inhibition can be overcome by membrane-permeant analogs of cGMP (Pensen et al. 1996). These findings suggest that cGMP is one of the components of the signal transduction pathway involved in the GA response.
In conclusion, GA signal transduction seems to involve calcium ions as well as cyclic GMP, but the detailed signaling pathways have not been worked out. α-Amylase secretion is regulated by a calcium-dependent pathway, whereas α-amylase gene expression is regulated by a calcium-independent pathway.

46. Thanks