Transcript Gibberellin-Induced α

Gibberellins
Plant Physiology II
BS Botany 7th semester
Habib-ur-Rehman Athar
Institute of Pure & Applied Biology
Bahauddin Zakariya University, Multan
Discovery of Gibberellins
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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.
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1935 T Yabuta and Y Sumiki isolated this substance from the fungal pathogen
Gibberella and named it Gibberellin
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1938 isolated crystals of GA-A and GA-B
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1955 Stodola from USDA and Brian from ICI London isolated Gibberellic Acid
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1956 West and Phinney UCLA isolated GA from plants
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1958 MacMillan in Bristol England identified GA1 from beans
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1956 J. MacMillan isolated a similar compound from a plant (immature bean seeds)
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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
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“Foolish seedling disease” in rice
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:
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Shoot and root tips (apical meristems)
Young, expanding leaves
Embryos
Fruits
Tubers
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 cell division in vascular cambium
• Promotes
• 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
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
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
Mevalonic acid-dependent
pathway
Mevalonic acid-independent pathway [Stage 1]
To Stage 2
Mevalonic acid-independent pathway [Stage 2]
Mevalonic acid-independent pathway [Stage 3]
Physiological Roles of Gibberellins
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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
Gibberellins promote stem elongation and leaf expansion
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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
Dwarf pea plant treated with gibberellin
Result: shorter stems
Result: longer stems
Gibberellins promote stem elongation and leaf expansion
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
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).
(48 hr. 100 ppb Gibberellin)
Starch well digested
(48 hr. 1 ppb Gibberellin)
Starch partially digested
(48 hr. Water)
Starch undigested
External application of gibberellins can also enlarge fruit size in grapes
GA can induce parthenocartpy and fruit enlargement
Gibberellins can cause bolting in Brassicacea members even in
the absence of inducing photoperiod or cold treatment
Wheat embryo (X4)
After soaking in distilled water
Embryonic leaves
Coleoptile
Following imbibition, separation of
scutellar epithelium
Shoot apex
Radicle
Scutellum
Endosperm
GA3 200 mg/L
GA3 150mg/L
15 dS m-1 NaCl
M.H.-97
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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.
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 cyclindependent 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.
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• 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
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 αamylase 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).
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 αamylase 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
Gibberellin Signal Transduction
Plant – ON / OFF SWITCH
Growth & Development
Phytohormones – Signal Transduction
Tolerance
Production of Natural Products
Composite model for the
induction of α-amylase
synthesis
in
barley
aleurone
layers
by
gibberellin. A calciumindependent
pathway
induces α-amylase gene
transcription; a calciumdependent pathway is
involved in α-amylase
secretion.
(The
SPY
negative regulator was
omitted for clarity.)
• 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 αamylase 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.
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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).
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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 GAinduced 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.
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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.