LS3121 Plant Physiology Lecture 14 2001 T. Y. Lin
Plant
Hormones and Growth Regulators (Part I)
1. Characteristics
of a plant hormone
(1). An organic compound synthesized in plant.
(2). It can be translocated.
(3). It causes a physiological response in very low
concentrations.
2. Plant
hormones and growth regulators
3. The classes of plant hormones
(1). Auxins
(2). Gibberellins
(3). Cytokinins
(4). Ethylene
(5). Abscisic acid
(6). Salicylic acid
(7). Jasmonic acid (JA) and methyl jasmomate (MeJA)
Auxins
I. Background experiments
(1).
(2). Went, 1926, auxin was present in the tip of oat
coleoptiles.
II.
Biosynthesis, transport and
metabolism of auxin
1. Natural and synthetic auxins
(1). The principal auxin in higher plants is
indole-3-acetic acid.
(2). The synthetic auxins: PAA, 2,4-D, 2,4,5-T
2. The definition of an auxin
(1). Induce cell elongation
in isolated coleoptiles or stem sections.
(2). Induce cell division
in callus tissues in the presence of a cytokinin.
(3). Promote lateral root
formation at the cut surfaces of stems.
(4). Induce pathenocarpic
tomato fruit growth.
(5). Induce ethylene
formation.
3. The detection and analysis of
auxin
(1). Bioassays (sensitivity 0.02-0.2
mg)
a. The Avena
(oat) coleoptile curvature assay
(a). Germinate seeds in darkness and
decapitate to deplete hormone reserves.
(b). A block of agar containing the growth
substance to be assayed is placed asymmetrically on the decapitated stump. Two hours later, the curvature resulting
from greater growth under the block is measured.
b. The straight
growth test
(a). Decapitated coleoptiles are incubated in the
solution to be assayed.
(b). Changes in length during a 12 h period are
measured.
(c). Growth is proportional
to log [IAA].
(2). HPLC-GC-MS methods (sensitivity
1 pg)
a. The deuterium in the 4, 5, 6
and 7 positions of IAA indole ring is not exchanged in alkaline solution so d4-IAA
is used as an internal standard.
c. A known amount of d4-IAA
and sufficient 14C-IAA to permit monitoring.
d. IAA is purified by
DEAE-Sephadex and reverse phase HPLC.
The sample is then methylated with diazomethane and chromatographed on a
GLC column of intermediate polarity.
The IAA coming from the plant may be monitored at its molecular ion,
189, and at its major fragment ion, 130 (d4-IAA at 193 and 134).
(3). Immunoassays:
antibodies are prepared by coupling IAA (ng) to
serum albumin with formaldehyde.
4. Multiple pathways exist for
the biosynthesis of IAA
(1). IAA synthesis begin with the shikimic pathway
or occur from tryptophan.
The pathway from tryptophan to IAA
(2). IAA is also synthesized
from indole or from indole-3-glycerol phosphate.
5. Auxin transport
(1). Auxin movement (parenchyma cells) is slow
(1 cm h-1) and preferentially in a basipetal
direction in stems. Transport in roots
is preferentially acropetal.
(2). Polar transport
of auxin requires metabolic energy since IAA is not transported under
anaerobic conditions or in the presence of ATP synthesis inhibitors.
(3). Polar transport-a
chemiosmotic model
a. Auxin enters cells by passive diffusion or active transport (H+ symporter).
b.
Cells use plasma
membrane ATPases to pump H+ from the
cytosol to walls.
b. The lower
pH of walls keeps the carboxyl group of an auxin
less dissociated than in the cytosol.
c. Noncharged
auxins cotransport with H+ from the cell wall into cytosol.
d. IAA-
builds up and moves out from the basal end (carrier
proteins).
(4). Phytotropins (inhibitor auxin transport)
bind to specific sites on the plasma membrane.
(5). Auxin is transported nonpolarly in the
phloem.
(6). Most IAA in plant is in a covalently
bound form.
a. IAA is found to be
conjugated to glucose, myo-inositol, amide, glucan and glycoprotein. The bound auxins do not stimulate plant
cell growth.
b. Possible roles of bound IAA:
storage and transport.
(7). Multiple pathways degrade IAA.
(8). There are two subcellular pools of IAA:
the cytosol and the chloroplasts.
III.
Physiological effects of auxin
1. Cell elongation
(1). Auxins induce cell
elongation in stems and coleoptiles (10-6 to 10-5 M).
(2). Auxin induces the synthesis of ethylene, a root growth inhibit.
(3). Low concentration
of auxin (10-10 to 10-9 M) promotes the growth of intact
root, but higher concentration of auxin (10-6 M) inhibits growth.
(4). The minimum
lag time for auxin-induced growth is 10 minutes.
a.
The stimulation of growth by auxin requires
energy.
b.
Proteins with high turnover rates are involved.
c.
Inhibitors of RNA synthesis also inhibit
auxin-induced growth.
(5). Auxin induced
proton extrusion acidifies cell wall, increasing extensibility.
a.
The wall-loosing
factor (WLF), the acid growth theory,
H+ is the WLF.
(a). Auxin causes cells to extrude protons actively into the cell wall.
(b). The apolplastic
pH activates wall-loosening enzymes.
b.
Specific proteins
mediate acid-induced loosening of the cell wall.
(a). Hydrolases are not cell wall-loosening
enzymes.
(b). Expansin causes
wall loosening in response to acid pH.
2. Phototropism and
gravitropism
(1). Phototropism
a.
Cholodny-Went model:
phototropism may be mediated by an unequal lateral distribution of auxin at the
tip (on the shaded side).
b.
A falavoprotein,
NPH1 (116 kDa, an autophosphorylating protein kinase) is a blue light
photoreceptor for phytochrome.
(2). Gravitropism
a.
In soybean hypocotyls, gravitropism leads to a
rapid asymmetry in the accumulation of small auxin
up-regulated RNAs (SAURs).
b.
Arabidopsis AUX1 code for a
protein similar to permease.
c.
Gravity perception involves amyloplasts in cells
of starch sheath and root cap.
(a). Statoliths are amyloplasts that function
as gravity sensors. Statocytes are the
specialized gravity-sensing cells (the starch-statolith
hypothesis).
(b). Gravity can be detected in the absence of
statoliths (the plasmalemma central control
model).
(3). The root cap
may play a role in the lateral redistribution of auxin.
3.
Auxin regulates apical
dominance (the direct-inhibition
model)
(1). Auxin level in the stem inhibits the growth of lateral buds.
(2). [Auxin] in auxiliary bud increased
following decapitation of shoot apex.
(3). Auxin makes the shoot apex a sink for
cytokinin from the root.
(4). Apical dominance of Elytrigia repens
is associated with
4.
Auxin promotes the
formation of lateral roots and adventitious roots.
5.
Auxin delays the
onset of leaf abscission.
6.
Auxin regulates
floral bud development.
7.
Auxin promotes fruit
development (parthenocarpy).
8.
Auxin induces
vascular differentiation.
III. The Mechanism
of Auxin Action
1.
A possible auxin receptor
(1). Auxin-binding sites: ER, plasmalemma, and
tonoplast.
(2). Auxin-binding protein 1 (ABP1) (22 kDa) forms a
dimmer in ER.
(3). ABP1 participates in the rapid auxin-induced increase
in membrane voltage.
2.
The MAP kinase cascade may mediate auxin
effects.
3.
Auxin may induce the ubiquitination of nuclear
proteins.
4.
Auxin-induced genes
(1). Early genes
a.
Aux/IAA (5 to 60 min): 19 to 36 kDa, a
hydrophilic polypeptide, a nuclear localization signal, and a DNA-binding
motif.
b.
SAUR (2 to 5 min): insensitive to cycloheximide,
a cluster of 5 similar genes, 10 kDa
c.
GH3 (5 min), 70 kDa
(2). Late genes: GST-like genes, ACC synthase
(3). Each auxin response
domain is a composite structure of two AuxREs,
a variable constitutive element adjacent to a
conserved TGTCTC element.
(4). Early auxin genes may
be under negative control of a short-lived repressor.
a.
The expression of early
genes is stimulated by cycloheximide.
b.
Cycloheximide rapidly inhibits auxin-induced
proton extrusion and growth.
c.
Auxin may promote the ubiquitination
of the repressor protein.
(5). Fusicoccin
activates preexisting H+-ATPases.
a.
Fusicoccin does not stimulate the expression of
auxin-induced genes.
b.
Fusicoccin may displace the autoinhibitory
C-terminal domain of the enzyme from the catalytic site.
c.
Fusicoccin binds to protein complexes on the
plasma membrane.
Gibberellins
I. Biosynthesis,
metabolism, and transport of gibberellin
1.
Gibberellin-like compounds
were first measured by bioassays.
(1). Gibberellins are
related compounds defined by chemical structure.
(2). Rice plants were induced to be tall by a
chemical secreted by the fungus Gibberella fujikuroi.
(3). The bioassays
a. Lettuce
hypocotyl elongation: sensitivity 1 to 100 ng/0.2 ml water.
b. Dwarf rice/microdrop: sensitivity 0.1 to 100 ng of
GA per plant.
c. a-Amylase
production in germinating cereal grain: 0.1 to 100 ng ml-1.
(4). Gas
chromatography-mass spectrometry provides definitive analysis
a. Purification: an internal
control to plant extract.
b. HPLC for separation.
c. Detection and analysis
Chemical
modification: put a methyl group on all carboxyl groups and a trimethylsilyl
group on all hydroxyl groups. GAs
and other compounds partition between the helium and a silica column surface.
2.
Gibberellins biosynthesis
(1). Gibberellins are tetracyclic diterpenoids made
up of four isoprenoid units.
(2). Cyclization geranylgeranyl
pyrophosphate to ent-kaurene (cyclases).
(3). Oxidations to form GA12-aldehyde:
a methyl group is oxidized to a carboxylic acid. The B ring contracts from a six to a
five-carbon ring to give GA12-aldehyde
(P450 monooxygenases).
(4). Formation of all other gibberellins by GA12-aldehyde
(GA12)
a. Hydroxylation at carbon 13 or
carbon 3 or both.
b. A successive
oxidation at carbon 20 (CH2 ® CH2OH ®
CHO), followed by loss of carbon 20 as CO2.
c. GA20 is converted
to the biological active form, GA1, by 3b-hydroxylase.
3. GA1
may be the only GA controlling stem growth. (Le:
GA20 to GA1).
(1). Allele Na
completely blocks biosynthesis of ent-kaurene to GA12-aldehyde.
(2). GA1 (in tall plants) is rapidly metabolized to GA8 and GA8-catabolite.
(3). Stem elongation corresponds to the amount
of GA20 metabolized.
4. Gibberellin intermediates can
be transported.
5. GA biosynthesis is regulated.
(1). Photoperiod
a.
Shoots of spinach begin to elongate after increasing
day lengt ~ 14 days.
b.
The rise in GA1
is the crucial factor in regulating spinach stem growth.
c.
GA20-oxidase mRNA was in the highest amount in shoot tips and elongating stems,
and the level was higher under long-day conditions.
(2). Temperature
a.
Stratification and vernalization.
b. Return to high temperatures after
cold treatment, ent-kaurenoic acid is converted to GA9.
(3). Feedback control.
5. Gibberellins may be
conjugated to sugars.
II. Effects of gibberellins on growth
and development
1.
Gibberellin stimulates stem growth
in dwarf and rosette plants.
2.
GAs regulate the transition from
Juvenile to adult phases
English ivy
(GA3) and conifers (GA4 + GA7)
3.
GAs influence floral initiation and sex
determination.
(1). Primary role of GA in sex determination:
to suppress stamen development.
a. Exposure to short days
and cool nights increases endogenous GA levels in the tassel flowers 100-fold
and causes feminization of the tassel flowers.
b. Application of GA to the tassels can also induce
pistillate flowers.
(2). In dicots
such as cucumber and spinach, GA seems to have
opposite effect.
4.
GAs promote fruit set.
5.
GAs promote seed
germination.
6.
GAs have commercial applications.
(1). Fruit production: GAs increase the size
of seedless grapes and the distance between branches and delay senescence in
citrus plants, Gas.
(2). Malting of barley and increase sugarcane
growth and sugar yield.
(3). Uses in plant breeding
a.
GA4 and GA7 mixture is
used to enhance seed production in Pinaceae.
b. GA3
is used for Taxodiaceae and Cupressaceae.
b.
The promotion of male flowers in cucumber.
c.
The stimulation of bolting in biennial
vegetables such as beet and cabbage.
III. Mechanisms
of GA action
1.
GA stimulates cell elongation and
cell division.
2. GAs increase cell wall
extensibility.
(1). The lag
before growth stimulated by GA is often somewhat longer.
(2). GA stimulates
growth and the activity of xyloglucan endotransglycosylase (XET), which allows
expansin proteins to penetrate into the wall.
3.
GA regulates the cell cycle in
intercalary meristems.
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.
4.
GA response mutants may be
blocked in signal transduction.
(1). Dwarf
GA-insensitive mutants.
a.
Arabidopsis GAI gene encodes a
protein with nuclear localization signal and
leucine zipper DNA-binding motifs (a TF).
b.
The GAI repressor
may bind to a GA-induced signaling intermediate (or to
GA directly) and becomes inactivated.
(2).
a.
The spy mutant
can grow in the absence of GA biosynthesis.
c.
SPY is an N-acetyl glucosamine
transferase.
5.
Mobilizing endosperm reserves
(2). The
gibberellin receptor may be located on the plasma membrane and heterotrimeric
G protein may be involved.
(3). GA3
enhances the transcription of a-amylase
mRNA
mRNA: stimulating transcription or decreasing mRNA turnover?
a. Nuclear run-off
experiment to discriminate mRNA alternatives.
b. GA promotes the transcription
of a-amylase
mRNA.
(4). Several promoter elements confer GA
responsiveness.
a.
GA response element (GARE):
TAACAAA
b.
GA response complex (GARC):
TAACAAA, TATCCAC, C/TCTTTC/T.
(5). Transcription factors regulate a-amylase gene
expression.
a. The regulatory proteins and
DNA-binding.
b. Hypothesis: GA stimulates the
expression of the GA-MYB gene (TFs: GA induced MYB proteins), and the MYB
protein serves as a transcriptional regulator of the gene for a-amylase.
c.
To cause the MYB gene to be expressed, GA may
bring about the activation of one or more preexisting transcription factors.
(6). a-Amylase synthesis and secretion use two signal pathways.
a.
GA signal transduction seems to involve calcium ions
and cyclic GMP.
b.
a-A calcium-dependent pathway regulates amylase secretion,
whereas aa
calcium-independent pathway regulates -amylase gene expression.