AUXIN TRANSPORT

Synthesis of IAA is not always site of action, so must be moved to other locations:

IAA lipid soluble in undissociated form, weakly acidic.

^{ATP-proton pump pumps out H+, keep pH gradient.}

^{IAA intercellular transport:}

• ^{diffuses apoplastically. this may be one means of transport between adjacent cells in a given organ}
• ^{translocated in xylem (tracheary elements) and phloem (sieve elements) along with flow of water.}
• ^{polar transport. in shoots, transport is basipetal (away from apex).}
• ^{in roots, depends on developmental stage. In most roots, transport towards root tip through vascular parenchema is acropetal (towards apex). In rapidly growing roots near tip, transported in epidermis and subepidermis}
• ^{Transport relies on specific proteins.}

^{Efflux located at one end of cell. Requires energy. 5-20 mm/h transport (slower than non-polar transport, but more IAA moves this way (at least basipetally down shoot), i.e. larger transport capacity)}

^{Polarity (direction) of auxin transport is independent of orientation of plant with respect to gravity. (you can’t fool the plant by turning it upside down)}

^{NAA also travels through vasculature (tracheids and sieve elements), so this background value must be subtracted.}

^{IAA made in shoot moves up epidermal cells}

^{(gravitropism?)}

^{IN ROOTS: IAA moves in phloem parenchyma from shoot to root (lateral root development, root elongation) and also transported from root tip.}

^{Historical side note of interest on regulation of auxin transport by ethylene:}

^{1932 Van der Loon -Ethylene modifies seed growth, changes endogenous auxin distribution}

^{1966-7 Ethylene treatment of intact cotton plants and etiolated pea seedlings caused inhibition of basipetal auxin transport.}

^{1969 Osborne and Mullins-auxin and ethylene bind to carrier protein at two different sites. When ethylene binds, either auxin binding or function of the protein in transporting auxin is inhibited. (allosteric effect?)}

^{ETHYLENE INHIBITS AUXIN POLAR TRANSPORT AT SITE OTHER THAN IAA BINDING SITE}

^{1988 Jerry Suttle- reduction of IAA movement in ethylene-treated tissues is the result of reduced transport capacity rather than reduced velocity. (at low auxin concentrations, auxin transported/time similar with or without ethylene treatment.}

^{ethylene may cause decrease in concentration or activity of efflux carrier.}

^{TDZ (thidiazuron; a cotton defoliant, used to get cotton plants to drop green leaves prematurely for easier harvest of cotton bolls) increases ethylene synthesis and therefore inhibits auxin transport. Maybe when ethylene is synthesized during senescence, ripening or leaf abscission, this ethylene also inhibits auxin transport.}

^{Many proteins needed for auxin transport}

^{Efflux carrier inhibited by set of compounds called PHYTOTROPINS}

^{Phytotropins include:}

^{NPA N-1-napthylphthalamic acid}

^CPD

^CMF

^{Phytotropins inhibit efflux carrier protein, thus stimulate IAA uptake (more IAA remains in tissue since it isn't transported out).}

^NPA:

^{inhibits lateral root formation (0.05 uM)}

^{inhibits gravitropic response (>0.05 uM)}

^{inhibits root elongation (1 uM)}

^{NPA binding protein (called NBP=NPA Binding Protein) in cell membrane-}

^{Screen Arabidopsis for transport mutants that interact with NPB. Selected for mutants that appear normal even when in presence of phytotropin (ie, roots ELONGATE EVEN IN THE PRESENCE OF NPA). These mutants may be mutants of the genes for NPA-binding protein or some protein that interacts with NPA-binding protein, i.e. they are IAA transport mutants. TIR3 protein up-regulates the level of active NBP in membrane. (Ruegger et al. 1997 Plant Cell 9: 745-757)}

^{Labile cytosolic component-??}

^{Efflux carrier- PIN family-of transport mutants. Encode proteins with 12 membrane spanning domains.}

· PIN1 mutants- shoot inflorescence looks like a pin. Little lateral branching. Decreased auxin transport in inflorescences. PIN1 expression is located throughout globular embryo, but eventually attains polar distribution. In basipetal end of parenchymal cells in vascular tissue. In seedlings, phytotropin phenocopies PIN1 mutant. May be transmembrane component of auxin efflux carrier.

• ^{PIN2 mutants- agravitropic. Expressed at anticlinal basal end of epidermal cells in root tip.}
• ^{GNOM- localizes PIN1 within embryo. Membrane guanine-nucleotide exchange factor on ADP-ribosylation factor G protein, important for vesicle trafficking. PIN1 distribution may thus be dependant on vesicle trafficking.}

^{Influx carrier- purified. 44 kD polypeptide}

• ^{aux1- encodes permease located in root tips. Important for development of lateral roots, vascular development, gravitropism.}

• ^{AGR gene family}
• ^{AGR1- mutant agravitropic, reduced sensitivity to ethylene and phytotropins. Homologous to bacterial transporters. If expressed in yeast, causes increase efflux of IAA from cells.}

^{Auxin polar transport is important in itself for developmental responses.}

^vasculature

• ^{Buds induce vascular formation in graft.}
• ^{IAA can replace buds, get same effect.}
• ^{Polar movement by auxin -> long continuous vascular strands in vascular tissues.}
• ^{Ath-8 – homeobox gene needed for vascular development. IAA induces Ath8 gene expression specifically in provascular cells}
• ^{Lop1-auxin transport mutant - also needed for vascular development}
• ^{RCN1- needed for NPA activity, but doesn’t affect NPA binding. Phosphatase regulatory subunit. Indicates phosphorylation/dephosphorylation may be important for IAA transport.}
• ^{High concentration of auxin may be required for establishment of root formation.}

^{Auxin transport may be important to establish embryo axis}

^{Somatic globular embryo:}

^{Block auxin action, w/antagonist (PCII) --no growth}

^{Block auxin transport w/phytotropin—no establishment of axis. Globular embryo keeps growing}

^{Add IAA or 2,4-D ---growth, no morphogenesis}

^{Gnom is critical for formation of embryo axis}

SIGNAL TRANSDUCTION

Receptors-molecules a hormone interacts with directly to initiate its response pathway(s). Each hormone may have multiple types of receptors.

Cellular regulation of a hormonally induced response can occur because of changes in

· level of the hormone

· levels of the receptor

· levels of post-receptor factors (i.e., the receptor is there, but a signal transduction component is missing)

WHAT molecules might bind to auxin??

· IAA receptors involved in signal transduction (could be any type signal transduction molecule)

· metabolic enzymes

· transport proteins

· acceptor or sequestering proteins

· non-protein molecules just “accidentally”

EXPECTED CHARACTERISTICS OF A PLANT HORMONE RECEPTOR:

· high, specific binding constant, saturable binding kinetics, reversible binding

· binding of real hormone shows competition from hormone analogues as expected by in vivo responses to such analogues

· low temperature dependence

· correlations of level of receptor and sensitivity to hormone

· If the hormone receptor is eliminated, response of the plant to the hormone should be eliminated

Difficulties:

· Hormone receptor may loose ability to bind during isolation

· Receptors present at low concentrations, hard to isolate.

· Receptors may not function when over-expressed in heterologous system.

SCATCHARD PLOT- plots data on binding of hormone to receptor to determine the dissociation constant for the hormone to the binding molecule. Particularly useful to determine whether one or more binding molecules are present in the solution being tested. If two molecules bind hormone are present, the plot is not linear. This shows the importance of obtaining binding data to a number of concentrations of added hormone. To eliminate complications due to molecules that do not bind specifically, but rather bind irreversibly, at each concentration of radiolabeled hormone added, free cold hormone is added, and the bound-radiolabeled hormone is again quantified. The amount of hormone still bound after this chase is the amount of irreversible (“non-specific”) binding.

ABP1 (auxin binding protein 1)

First isolated as a protein that binds auxins according to criteria above. ABP1 is located in ER; about 10% in plasma membrane (determined from immunohistological evidence, studies of fractionated membranes, presence of KDEL, an ER target sequence, in ABP1)

Antibody to purified ABP1 used to screen expression library, ABP1 cDNA isolated.

Two independent approaches have resulted in isolation of ABP-1 genes

Antibodies to ABP1 block auxin-induced proton efflux in protoplasts.

ABP1 added to protoplasts stimulates proton efflux.

ABP1 knockouts have an embryo-lethal phenotype (embryo doesn’t develop past early globular stage)

Additional receptors (such as intracellular receptors)-??

^{57kd rice protein-binds to auxin, and to plasma membrane proton pump, may initiate acid-induced acid-induced growth via cell wall expansion.}

Aux1

^{May be also involved in auxin perception.}

^{Auxin response- (reviewed in Leyser}. Current Opinion in Plant Biology 2001, 4:382–386)

The AUX/IAA genes code for transcription factors that inhibit or induce transcription of a battery of auxin-response genes. Some of the AUX/IAA genes also repress or induce transcription of other AUX/IAA genes or of themselves.

Auxin, by an unknown mechanism, causes the modification of a set of AUX/IAA proteins. This modification targets these proteins for ubiquitination. The proteins are degraded, and the transcription of the battery of auxin-induced genes repressed by them in the absence of auxin can proceed.

Cell elongation and auxin

One of the better studied specific responses to a plant growth regulator is the induction of cell elongation by auxin, particularly in oat coleptile

Background.

Boysen-Jensen 1936 characterizes elongation response with coleoptiles. the goal to investigate a response using homogeneous cell population with an easy to monitor physiological response.

Elongation is simple to detect, but even in the case of the coleoptile,

1) cell types heterogeneous

By removing particular layers of cells, one can get idea of which layers are limiting elongation; in the case of Avena, all cell layer are important.

2) elongation growth in response to auxin is not uniform down length of coleoptile

3) elongation is developmental, and response to auxin is moderated by developmental stage of coleoptile and by environmental factors

**************

Cell enlargement involves an interaction of factors:

dV/dt+ m Lp (psi a -pie-Y)/m +Lp

m=wall extensibility

CELL WALL ###

Carbohydrates including

cellulose---D-glucose polymer w beta 1-4 linkages. in cotton cellulose is about 3000 glucoses, 500-8000 in soybean. celluloe strands are arranged into micelles, which are formed into microfibrils, which are formed into fibrils,

hemicelluloses---branched polysacharides (xylose, arabinose,glucose,galactose) with beta 1-4 backbone, and various other linkages

Proteins

hydroxyproline-rich glycoproteins such as extensin (structural)-glu

enzymes--glucosidases, hydrolases, cell wall biosynthetic enzymes

P + turgor pressure (psi a = P-pie)

P= pressure and is usually positive in cell w turgor

Psia has a value which is essentially same after elongation so

pie = osmotic (or solute) potential ie tendency of water to leave cell due to solute in the cell, relative to pure water, thus is usually negative in cell (ie there’s solute in cell)

Lp is the Hydraulic conductivity which is an attribute of the plasma membrane This value tend to be large, and thus can be ignored.

Y is the wall yield threshhold; the amount of force required to start the wall expanding

the terms used for these vary

****picture of a cell+equations

cell enlargement could be due to

-increase in m, the extensibility of the wall,

-change in the water potential (typically this is just the solute potential) outside the cell

-increase in (psi) the osmotic potential within cell

characteristics of cell elongation induced by auxin

-10-30 min lag time (typically)

-growth rate =log (IAA) (for range of IAA concs til saturated)

-requires energy probably as ATPase

inhibited by ATP synthesis inhibitors (KCN,DNP, azide)

or ATPase activity inhibitors (vanadate, DCCD, DES)

-requires cell turgor , i.e. there must be a force on the cell wall to induce its extension

-requires protein synthesis and RNA synthesis

Goes on for 24 h + therefore it is a complex process, which involves a variety of mechanisms to keep it going particularly in the long term,

at least some long term requirements are distinct from the early processes of elongation following auxin application

The prolonged elongation of coleptiles involves additional factors., as the cells need to adjust to their new size with protein synthesis, and also are differentiating at this time. It also requires the present of solutes, so that the enlarging cells can maintain their osmotic potential.

(Cleland calls this PGR(prolonged growth response) vs IGR (immediate growth response))

what kind of mechanism could be proposed to explain this?

in pea stem sections, turgor pressure (P) doesnt change after addition of auxin (based on the measurement of P by micropressure probe.

in contrast, the indirect measurements of m by different physical stress tests on the stem (via instron, stress-relaxation, creep tests, bending assays, turgor relaxation), all show an increase in m associated with auxin-induce elongation growth

problems in study of specific changes resulting in increased elasticity.

cell wall bonds that must be broken aren't well characterized

-cellulases don't promote cell elongation

-some evidence exists that hemicelluloses such as xyloglucans break during elongation

-new polysaccharides may be formed to intercalate into cell wall

The only factors that have been shown to loosen cell wall are protons.

-decreasing the pH of the cell wall induces elongation

How does auxin stimulate growth?

Probably via a combination of rapid stimulation of cell wall expansion by relaxation of cell wall structure, and by expression of batteries of new genes required for long-term cell growth.

Rapid stimulation of cell wall expansion: the acid growth theory (Cleland)

-auxin directly or indirectly induces the generation of acidic conditions in the cell wall

-this enables cell walls to elongate by activation of enzymes required for wall extensibility, such as those that break down hemicellulose.

-The change in pH might occur via an ATPase proton pump located in the plasma membrane, such an ATPase is known to be present in PM and is inhibited by vanamycin, also an inhibitor of auxin-induced growth.

This response may be mediated by ABP1.

Auxin-induced changes in gene expression (Theologis, pea epicotyl; Hagen and Guilfoyle, soybean hypocotyl)

Soybean hypocotyl, in planta, responds to auxin by increase in length and girth; excised basal sections only elongate in response to auxin

pea epicotyl also elongated in response to auxin

Short range responses to auxins include expression of ^{early auxin response genes}_:

(occur less than 20 min after application of auxin) several mRNAs accumulate specifically, and rapidly in hypocotyls and epicotyls in response to auxin. mRNAs also accumulate following exposure of callus tissue to auxin. Some of these mRNAs are the same and some different than hypocotyl RNAs induced by auxin. (level of other RNAs decrease)

· SAURS accumulate 2.5-5 min after auxin applied to soybean, precede cell elongation response

9-10 kd proteins with regions of high homology to one another

Promoter-deletion-GUS fusions indicate a motif (AXRe) common to many SAUR promoters is needed for auxin-induced expression.

Transcription factors (AGE) may bind to AXRe, repress transcription.

· IAA genes (1-20 in Arabidopsis)

Accumulate in 4-10 min, have half-lives of 6-8 min

Mostly different in sequence but overlapping expression patterns

Nuclear proteins with 4 conserved domains.

Many have been isolated independently in mutant screen for auxin-independent growth.

Domains III and IV involved in inter-protein binding. These domains are homologous to ARF domains II and IV.

Longer range response (2-48h):

de novo synthesis of new mRNAs and proteins required for cell enlargement (Cell wall synthesis proteins, proton pump proteins, etc).

Any protein that is required at a particular concentration will need to increase associated with an increase in cell volume. Thus many proteins that show an increase do not play a causal role in cell enlargement (such as proteins needed for energy)

^{Auxin response mutants:}

^{Many are in ubiquitination pathway which rapidly degrades proteins that repress early auxin response genes.}

^{Includes genes selected as both auxin response mutants and auxin transport mutants!}

^{axr1, rub, eir1, ecr1, tir1, ask, ATcul1.}

^{Includes genes that are auxin early response genes (IAA genes) or that regulate them.}

^{AGE2 represses expression of a subset of early auxin response genes}

^{Other genes identified in screens for various mutants that are altered in auxin response.}

^{Monopteros: expressed in embryos, plant. Needed for normal polarization of embryo, vascular development, floral development, needed for induction of early genes that mediate auxin transport.}

^{N03 increases activity of a MADs box protein that increases activity of AXR4. Auxin also increases activity of AXR4. AXR4 is important for elongation of lateral root apices.}