Molecular Biology of Action of Gibberellins, Cytokinins, Brassinosteroids, and Jasmonates

C H A P T E R

24 Molecular Biology of Action of Gibberellins, Cytokinins, Brassinosteroids, and Jasmonates 5. SECTION SUMMARY

SECTION I. MOLECULAR BIOLOGY OF GIBBERELLIN (GA) ACTION 592 1. GA RESPONSES 592 2. GA-BINDING PROTEINS 592 3. SITE OF GA PERCEPTION 592 4. GA-INDUCED GENE EXPRESSION 594 4.1. Aleurone Tissue 594 4.2. Gene Regulation in Stem Elongation and Flowering 595 5. GA RESPONSE MUTANTS 597 5.1. GAI Locus 598 5.2. GRS and RGA: Genes Similar to GAI 599 5.3. Structure of GAI/RGA Proteins 601 5.4. Rhtl and Rhtd Genes in Wheat and d8 Maize Encode Functional Orthologs of GAI/RGA 601 5.5 Slender (sin) Mutatns of Barley and Rice 602 5.6. SPY Locus 602 5.7. Other Mutant Loci 603 6. A WORKING HYPOTHESIS FOR THE GA RESPONSE PATHWAY 604 7. GA/ABA INTERACTION 605 8. GA INTERACTION WITH ENVIRONMENTAL FACTORS 606 9. GA RECEPTOR(S) COULD BE A MEMBER OF A SMALL GENE FAMILY 607 10. SECTION SUMMARY 607

611

SECTION III. MOLECULAR BIOLOGY OF BRASSINOSTEROID (BR) ACTION 611 1. BR-INDUCED RESPONSES 611 2. BR-INDUCED GENE EXPRESSION 612 3. BR RESPONSE MUTANTS 612 3.1. Cloning and Sequencing of BRIl 612 3.2. BR Interaction with Other Hormones 614 3.3. BR Interaction with Light 615 4. SECTION SUMMARY 615 SECTION IV. MOLECULAR BIOLOGY OF JASMONATE (JA) ACTION 615 1. ROLES OF JAs 615 2. DEFENSE-RELATED SIGNALING 615 3. JA-INDUCED GENE EXPRESSION 616 4. JA RESPONSE MUTANTS 617 5. SECTION SUMMARY 617 REFERENCES 618

C h a p t e r s 21-23 p r e s e n t e d c u r r e n t information on the molecular aspects of signaling b y ethylene, auxins, a n d abscisic acid (ABA). These three h o r m o n e s w e r e singled o u t for separate t r e a t m e n t b e c a u s e the inform a t i o n o n each is u n i q u e in s o m e w a y s . Signaling b y a p l a n t h o r m o n e is better a n d m o r e completely k n o w n for ethylene t h a n that for a n y other h o r m o n e . Fiorm o n e - b i n d i n g p r o t e i n s are better characterized for auxin t h a n for other h o r m o n e s , a n d m o r e is k n o w n a b o u t ABA-regulated gene expression t h a n for other h o r m o n e s . In c o m p a r i s o n , our information on the r e m a i n i n g h o r m o n e s , gibberellins (GAs), cytokinins (CKs), brassinosteroids (BRs), a n d j a s m o n a t e s (JAs),

SECTION 11. MOLECULAR BIOLOGY OF CYTOKININ (CK) ACTION 607 1. CK-INDUCED RESPONSES 607 2. CK-BINDING PROTEINS 608 3. CK-INDUCED GENE EXPRESSION 608 4. CK RESPONSE MUTANTS 609 4.1. Screen for Altered Morphology 609 4.2. Screen for Cell Division and Growth 610 4.3. Screen for Ethylene Synthesis in Dark-Grown Seedlings 610

591

592

IV. Molecular Basis of H o r m o n e Action

3. SITE OF G A PERCEPTION

is still fragmentary. This chapter summarizes that information.

SECTION I. MOLECULAR BIOLOGY OF GIBBERELLIN (GA) ACTION 1. G A RESPONSES As explained in earlier chapters, gibberellins play major roles in the elongation growth of stems, petioles, and leaves; the flowering response; seed germination; and mobilization of reserve foods in grass seeds. They also act as general stimulators of fruit growth, probably by creating a sink for photoassimilate, and in expression of the male sex in flowers. This chapter first considers GA-binding proteins and the GA site of perception; then the cis sequences and transcription factors involved in GA-induced gene expression; and finally GA response mutants and how GA signaling might be affected.

While there is considerable evidence for GA-binding activity in soluble fractions from a number of plants, there is also increasing evidence that GA perception occurs at the outer surface of the plasma membrane. Richard Hooley and associates at Long Ashton Research Station, University of Bristol, have synthesized gibberellins that are impermeable across membranes. For instance, GA4 was derivatized at C-17 and attached via a hydrophobic spacer arm to 120-|jLm-diameter Sepharose beads such that only the unmodified end, which included the biologically active A and B rings of the GA molecule, could permeate the plasma membrane to a depth of about 1.95 nm (Fig. 24-1). Such derivatized GA4 could still elicit expression of a-amylase genes in isolated aleurone protoplasts, albeit to a much less extent than the parent, underivatized GA4 (Table 24-1). However, the intact aleurone layers, with cell walls, could not be reached by the derivatized GA4 and, as expected, gave little response.

S(CH2)3SCH2CH(OH)CH20-SEPHAROSE6B

2. G A - B I N D I N G PROTEINS - 1.22 nm •

Soluble proteins from elongating tissues of several plants (pea epicotyls, cucumber and Azuki bean hypocotyls, maize and rice leaves) have been shown to bind radiolabeled GAi or GA4 with high affinity and specificity. These assays have utilized both in vivo and in vitro-binding assays. In general, there is good agreement between the biological activities of various GAs and GA derivatives and their abilities to displace radiolabeled GAs from binding sites. The binding sites have been shown to be proteins, and the optimal pH for binding is about 7.0-7.5. The calculated K^ values range from about 700 pM to 140 nM, a range that agrees with the GA concentrations required for elongation growth in these systems. The proteins have not been purified, however, and it is unclear whether binding is to a GA receptor or to one of the enzymes in GA metabolism. Other studies using photoaffinity-labeled GA4 have identified binding proteins in plasma membrane fractions from oat aleurone protoplasts, as well as pea and Arabidopsis stem tissue. The binding was competed for by biologically active GAs, but not inactive GAs. The identity of proteins is still unknown, but one 18-kDa protein was partly sequenced, which may lead to its identification.

HOOC(CH2)2SCH2CH(OH)CH20-SEPHAROSE 6B (control)

B plasma membrane cytoplasm

FIGURE 24-1 Derivatized GA4 and its penetration into the plasma membrane of oat aleurone protoplasts. (A) Structure of the 17-thiol derivative of GA4 (top row) and control Sepharose 6B (bottom row). (B) A model of plasma membrane and derivatized GA4 linked to a Sepharose bead (not drawn to scale). The derivatized GA molecule cannot enter the plasma membrane except to a depth of about 2.0 nm. Adapted from Hooley et al. (1991).

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24. Molecular Biology of G A s , CKs, BRs, and JAs

TABLE 24-1 a-Amylase Production by Underivatized GA4 and Derivatized GA4 in Oat (Avena fatua) Aleurone Protoplasts^ Total a-amylase (units) GA concentration (M) GA4

GA4-17-S-(CH2)3-SH

48.1

1.0 X 1 0 ' ^ 1.0 X 10-6

51.2

44.3

1.0 X 1 0 ' ^

53.6

31.3

1.0 X 10-8

43.1

16.7

1.0 X 1 0 ' ^

30.7

2.8

From Hooley et al (1991). ^Minus GA control (1.07 Units).

Similar experiments with GA4 17-sulfonic acid, which has a pKa of 0.6 and therefore occurs mostly as an impermeable anion, gave identical results. In other studies, Russell Jones and associates at University of California, Berkeley, used barley aleurone protoplasts, which were immobilized by embedding in a thin film of agarose. Microinjection of GA3 into the protoplasts elicited none of the three GA-induced responses, i.e., increased vacuolation, secretion of a-amylase, and expression of a reporter gene under the control of a barley a-amylase promoter. The addition of GA3 to the external medium, however, stimulated all three responses. Additional evidence that GA is perceived at the cell surface comes from studies involving transmembrane receptors, albeit of two different kinds. As explained in Chapter 25, one class of plasma membrane-based receptors works in concert with heterotrimeric GTPbinding proteins. The receptor, on perceiving the signal, undergoes a conformational change, which is sensed by the neighboring heterotrimeric G-protein complex, which in turn hydrolyzes GTP to GDP and starts a signaling cascade. The cascade has multiple effects, which range from opening or closing of ion channels to gene expression. Some agonists, such as the peptide Mastoparan 7 (Mas7), can trigger the Gprotein cascade in the absence of an external signal. Using oat aleurone protoplasts and layers, it was shown that supplying Mas7, in the absence of any exogenous GA, is sufficient to trigger the expression of the reporter gene, GUS, under the control of an aamylase promoter (Fig. 24-2). A dwarfl mutant of rice is GA insensitive in both aamylase production by embryoless half-seeds and stem elongation of seedlings. The Dzuarfl gene encodes the a subunit of a heterotrimeric G protein, which supports the concept that GA is perceived by a plasma

membrane-based receptor and involves signaling via a G protein. The second type of plasma membrane-based receptors are transmembrane receptor-like kinases. Deepwater rice shows enormous elongation of young internodes in response to flooding, and the response has been shown to be due to GA (see Chapter 15). A gene encoding one of the receptor-like kinases {OsTMKl for Oryza sativa trans-membrane kinase) has been isolated. The expression of the gene is upregulated by GA in zones of cell division and cell elongation. In summary, studies on aleurone tissue, dwarfl rice, and deepwater rice present strong arguments for an extracellular site for GA perception. However, GA-

Mas7

MasCP

-GA

+GA

a'Amy2/54:GUS FIGURE 24-2 Expression of a-Amy2/54::GUS construct in oat aleurone protoplasts. Freshly prepared aleurone protoplasts were transformed by the construct and incubated for 5 days with Mastoparan 7 (Mas7), its inactive analog (MasCP), both 3 |ULM or GAi (50 n M), or without GAi. Mas7 could induce the expression of GUS in the absence of exogenous GAi. From Jones et al (1998).

594

IV. Molecular Basis of H o r m o n e Action

binding studies using GA responsive stem or leaf tissues favor an intracellular site for GA perception. While it is possible that gibberellins are perceived differently in different systems, the balance favors a surface perception of GA. The issue cannot be resolved, however, without the identification and isolation of the GA receptor(s).

considerable homologies within each subgroup, but not between the two subgroups. The third a-amylase subgroup, a-Amj/3, is expressed in wheat only during seed development, not during germination. In rice, however, several a-Amy3 genes are expressed in aleurone cells and in the scutellum during germination. 4,1.1. cis Elements in a-Amylase

4. G A - I N D U C E D GENE EXPRESSION Gibberellin-induced stem and leaf elongation, flower development, seed germination, and mobilization of seed food reserves all involve gene expression. Among these, the best studied is gene expression in the aleurone tissue of cereal grains. GA-induced production and secretion of a-amylases and other hydrolases in cereal grains were explained in Chapter 19. This section deals with the regulation of gene expression in the aleurone system, followed by gene expression in two other systems: stem elongation and flower development. 4.1. Aleurone tissue Among the various genes upregulated by gibberellins in the aleurone system, a-amylase genes in barley, wheat, and rice are the best characterized, and this account pertains specifically to them. The a-amylase gene family is divided into three subgroups based on amino acid homology and gene structure (i.e., the number and placement of introns). In wheat and barley, two subgroups are expressed in aleurone tissue during seed germination; the high pi (a-Amyl) and low pi (a-Amyl) groups. Both are induced by GA, but their temporal pattern of expression differs. In rice, there is only a single a-Amy2 representative, and it is expressed at a very low level. Sequence comparisons in the proximal promoters of genes in both high p i and low pi subgroups in wheat and barley show

Genes

Studies using deletion constructs from promoters of a-amylase genes in all three subgroups have shown that the proximal promoter, to -300 bp from the transcription initiation site, is important for GA-regulated gene expression. Within this region, sequence comparisons among high pi and low pi genes of wheat and barley have led to an identification of three conserved sequences, including the TAACAA/GA core element, which is the GA response element (GARE) (the sequences are numbered 1-3, Fig. 24-3). Mutations in this core element cause severe disruption of reporter gene expression in transient assays. In low pi or a-Amy2 genes in barley, wheat, and oat, functionally important cis elements also occur upstream from the three boxes mentioned earlier (see Fig. 24-3). These include the Opaque-2 element, which forms part of box 2. (The Opaque 2 element, in combination with an ACGT core-containing ABRE, can give ABA-specific expression.) The relative placement or order and orientation of these sequences, although not the spacing between them, are conserved. Functional analysis using aleurone layers (or protoplasts) and transient GUS expression reveals that while the GARE is essential for GA3induced a-amylase expression in both high pi and low pi a-amylase promoters, it works in concert with other elements. For the high pi a-amylase genes in barley, the GARE, together with the most proximal element, the TATCCAC/T box, is sufficient for full GAs-induced expression in aleurone protoplasts. In the intact aleurone layers, however, all three boxes seem to be required. Thus, the elements that constitute the GA

1

Hh

•v/—rf^ Box A2

/Box 2/Op2 \\ C CT T Box / ^ TTGACTTGACC

/ GARE

\

/

\

TAACAA/GA' ' TATCGAC/T

F I G U R E 24-3 Functionally important cis elements in high p / and low p i a-amylase genes. High pi a-amylase genes have three elements: a TAACAA/GA element, which is the GA response element, a distal pyrimidine box with a preponderance of CT bases, and a proximal TATCCAC/T box. Low p / a-amylase genes have several other boxes 5' upstream of the pyrimidine box, including box 2 (which includes Opaque 2 element) and box A2. Sequences are numbered from 1 to 5 starting from the 3' end. Adapted from Huttly and Phillips (1995) and Wilmott et al. (1998) with kind permission from Kluwer.

595

24. Molecular Biology of G A s , CKs, BRs, a n d JAs

response complex (GARC) vary depending on the tissue context, i.e., whether isolated protoplasts or intact aleurone layers are used. In low p i a-amylase genes, GARE, together with box 2 (sequence #4 in Fig. 24-3), is sufficient for high expression. 4.1.2. cis Elements in Other GA-Regulated the Aleurone System

,-

Genes in

Considerably less is known about the cis elements in other GA-regulated genes in the aleurone system. The promoter of a barley cysteine endoproteinase gene, EPB-1, has a GARE, a pyrimidine box, and an upstream element, which are required for high-level GA-induced expression. The barley (1-3, l-4)-p-glucanase promoter also contains a recognizable GARE motif. However, the promoters of many other genes, such as the aleurain (a protease) from barley, a wheat carboxypeptidase, and, conspicuously, rice a-AmyS genes, do not possess the TAACAA/GA sequence. 4.1.3. Transcription

100

Factors

Specific binding of nuclear proteins to cis sequences referred to in Section 4.1.1 has been shown by in vitro footprinting, and a few transcription factors have been identified. As mentioned in Appendix 1, MYB-type transcription factors in plants contain two conserved amino acid repeats (R2 and R3) that are important for DNA binding (see Fig. Al-lOE). Since the TAACAA/GA element had some similarity to the DNA-binding sequence in MYB-type proteins, Jake Jacobsen and associates at CSIRO, Canberra, Australia, used an oligonucleotide probe containing parts of the R2 and R3 coding sequences to fish out a clone from a cDNA library from barley aleurone and then used polymerase chain reaction to obtain a full-length cDNA clone, which they called GAmyb (henceforth called GAMYB). GA-MYB has been shown to be a transcriptional regulator of high pi a-amylase, as well as several other GA-induced genes, including the EPB-1 gene, in barley aleurone. It binds to a DNA sequence containing the GA response element in vitro, specifically to the core-AAC- in the TAACAA/GA sequence. Its mRNA is induced by GA3 treatment of aleurone layers prior to induction of a-amylase mRNA (Fig. 24-4) and is not inhibited by cycloheximide, which suggests that GAMYB is a primary response gene. Moreover, the constitutive expression of GA-MYB in transgenic plants can activate GA-induced genes, such as a-amylase or cysteine proteinase (EPB-l) genes, in the absence of exogenous GA (Fig. 24-5), a characteristic exhibited by many transcription factors.

• n

GA-MYB a-amylase

12

hours FIGURE 24-4 Time course of GA-MYB and a-amylase gene expression in G A3-treated barley aleurone layers. RNAs were isolated at specified times and hybridized with gene-specific probes. The hybridization signal was normalized to that of rRNA before being plotted. From Gubler et al (1995).

Plants have many MYB genes, including CI, which is involved in regulating anthocyanin biosynthesis in maize kernels (see Chapter 23). It could be shown in the experiment just described that only GA-MYB, and not CI, could activate the AMYr.GUS construct. Some other proteins that bind to the GARE or other cis sequences have been identified, but their precise roles are still unclear. Their relationship to GA-MYB is also unknown. Data currently available on cis sequences in a-amylase genes and nuclear proteins that bind to them are summarized in Fig. 24-6.

4.2. Gene Regulation in Stem Elongation and Flowering As mentioned in Chapter 15, genes for a- and ptubulin, as well as for a water channel protein in tonoplast, 7-TIP, have been shown to be upregulated during GA-induced stem elongation, but how GA regulates their transcription is unknown. Flowers of many plants accumulate anthocyanins in their petals (corolla), which are responsible for their brilliant colorations. The synthesis of anthocyanins is regulated by a variety of factors, including light, temperature, pathogens, and hormones. The development and pigmentation of petals in flowers of Petunia hybrida are apparently under the control of gibberellins. If stamens are surgically removed from Petunia flowers at a young stage, the corolla develops poorly and lacks pigmentation. However, if the young, emasculated flowers are treated with or placed in a solution of GA3, corolla development and pigmentation are

596

IV. Molecular Basis of H o r m o n e Action

effector construct Actini promoter

GA-MYB cDNA

N0S3' I I

ACTIN1::GA-MYB

reporter construct a Amylase promoter

ADH1 intron

1

GUS

T

N0S3' AMY::GUS

B

AMY::GUS ACTIN1::GA-MYB FIGURE 24-5 Transactivation of the high p7 a-amylase promoter by GA-MYB in barley aleurone layers, (A) Two constructs were made: one construct consisted of an Actini promoter plus the GA-MYB-coding sequence {ACT::GA-MYB) and the other, a reporter construct, consisted of the proximal region of the aamylase promoter fused to the GUS-coding sequence (via an intron from an alcohol dehydrogenase gene). Both constructs had a 3' end from the nos gene of Agrobacterium T-DNA. Barley half-grains were cobombarded with the two constructs, and GUS expression was monitored without (control) and plus GA3 treatment. (B) A comparison of the control bars shows clearly that the ACT::GA-MYB construct by itself, in the absence of exogenous GA3, is sufficient to raise the expression of the reporter gene by about 10-fold, an expression that is not increased any further by exogenous GA3. Modified from Gubler et al (1995).

CGATGG

COATGG / TAACAGA\ TTGACTTGACC

FIGURE 24-6 A summary of current data concerning transcription of high pi and low p i a-amylase genes in aleurone cells of cereal grains. Two transcription factors, a MYB type and a WRKY type, are shown bound to GARE and box 2, respectively. In low pi a-amylase genes, the GA response complex includes the GARE and box 2, whereas in high pi a-amylase genes, it includes the GARE and the more proximal cis element. Proteins whose identity is unknown are shown bound to the TATCCAC/T box and to sequences distal to box 2. Adapted from Wilmott et al. (1998) with kind permission from Kluwer.

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24. Molecular Biology of GAs, CKs, BRs, and JAs

In Chapter 7, it was mentioned that GA20 OXIDASE genes in Arabidopsis and some other plants are regulated negatively by biologically active GAs. An analysis of their regulatory cis sequences and transcription factors that bind to them is likely to add substantially to our knowledge of GA-induced gene expression.

5. G A RESPONSE M U T A N T S GA response mutants fall into two phenotypic categories: GA-insensitive dwarfs and elongated slender mutants (see Table 24-2). Dwarf mutants have a phenotype similar to that of weak or 'Teaky" alleles of GA-deficient biosynthesis mutants, except that exogenous treatment with GA does not restore them to the wild type. This class includes the well-known Rht (for reduced height) wheat lines used to develop the high-yield, short-statured wheat varieties that led to the "green revolution'' in countries like Mexico and India (Fig. 24-8A). Other members include dwarfS (dS) maize and GA-insensitive (gai) Arabidopsis. Mutant alleles identified for these GA-insensitive dwarfs are all dominant or semidominant over the wild type and have pleiotropic effects. For instance, the Rht3 mutant in wheat in the homo- or heterozygous state shows reduced stem height and leaf length compared to the wild type, and the aleurone does not produce a-amylase or a-amylase mRNA in response to GA3. The pleiotropic nature of these

FIGURE 24-7 Effect of GA3 on the development of corolla and accumulation of anthocyanins in Petunia. Courtesy of David Weiss, The Hebrew University of Jerusalem, Rehovot, Israel.

normal (Fig. 24-7). These observations suggest that corolla development and pigmentation are regulated by gibberellins, which are synthesized in the stamens. In support, GA synthesis genes have been shown to be expressed in young stamens. Some 15 structural genes encoding various enzymes mediating anthocyanin biosynthesis are coordinately expressed; some are expressed early, whereas others are expressed later, and GA has been shown to be involved in the expression of both sets. Although cis elements in promoters of many of these genes are known, there is little information on their GA-induced regulation. Apparently, Myb-type transcription factors are involved, and some are induced very early in response to GA and their synthesis does not require prior protein synthesis.

TABLE 24-2 Selected GA Response Mutants'" Plant

Mutant^

WT gene (nature of encoded protein)

Ref.

Dwarf phenotype Triticum aestivum (wheat) Zea mays (maize) Arabidopsis thaliana Silene armeria Oryza sativa (rice) Slender phenotype Hordeum vulgare (barley)

reduced height (Rht) (sd)

rht-1 (transcription factor)

Gale and Yousefian (1985), Peng et al. (1999)

dwarf8 (dS) (sd)

D8 (transcription factor)

Peng et al (1999), Harberd and Freeling (1989)

gibberellic acid-insensitive (gai) (sd)

GAI (transcription factor)

Peng et al (1997)

dwarf Silene (r)

—

Suttle and Zeevaart (1979)

dwarfl

a subunit of a heterotrimeric G protein

Mitsunaga et al (1994)

slender (sin) (r)

Foster (1977) SLN (transcription factor)

Oryza sativa (rice)

slender (sin) (r)

Pisum sativum (pea)

la cry^ double mutant (r)

Potts et al (1985)

Lycopersicon esculentum (tomato)

procera (pro) (r)

Jones (1987)

A thaliana

spindly (spy) (r)

SPY (a GlcNAc transferase)

Ikeda et al (2001)

Jacobsen et al (1996)

In wheat, the wild-type gene is written with lowercase letters, whereas for mutant alleles, the first letter is written in uppercase. Dominance: sd, semidominant or dominant alleles, r, recessive.

598

IV. Molecular Basis of Hormone Action

ff

rht L Rht1 m Flht2MRht1+2t |R/7ft+2i Rht3 mRht2+3 FIGURE 24-8 (A) Near-isogenic lines of wheat seedlings containing different reduced height {Rht) dwarfing genes in the background tall (rht) variety. Maris Huntsman (for usage of Rht and rht, see legend for Table 24-2). Modem bread wheat is hexaploid {2n — 42), containing A, B, and D genomes derived from three diploid ancestors, each containing seven pairs of homoeologous chromosomes. The semidwarfing genes, Rhtl and Rhtl, are present on chromosomes 4BS and 4DS, respectively. The more potent dwarfing gene, Rht?>, is an alternative allele at the same locus as Rhtl, and the combination of Rhtl + Rht?> leads to an even more severe dwarf phenotype. The extent of the block to responsiveness to applied GA is proportional to the degree of dwarfism with the semidwarf lines Rhtl and RJntl showing slight GA responsiveness, whereas the more extreme dwarf lines, Rht?> and Rhtl + R}it?>, are nonresponsive to the hormone. Courtesy of John Lenton, ICAR, Long Ashton Research Station, Bristol University, UK. (B) Wild-type and slender barley seedlings, sin Seedlings are homozygous segregants in the progeny of heterozygous plants. Courtesy of Peter Chandler, CSIRO, Canberra, Australia.

mutants suggests that they act at early steps in GA perception/signal transduction. The endogenous content of biologically active C19 GAs in these dwarf mutants is higher than in the wild-type plants, possibly because they are unable to regulate GA synthesis by feedback inhibition of GA 20-oxidases involved in GA biosynthesis (see Chapter 7). In contrast to GA-insensitive dwarfs, slender mutants behave as if the GA response pathway is constitutively activated. All are recessive mutants, but there are differences between them, slnl Homozygous barley plants have elongated stems and leaves (see Fig. 24-8B), a constitutive synthesis and secretion of a-amylases and other enzymes in aleurone tissue, and are male sterile. The supply of exogenous GA or treatment with GA synthesis inhibitors has no effect on the sin phenotype. The la crxf' double mutant in pea, likewise, is unaffected by GA levels, but shows a slender phenotype only in the double mutant; individual alleles at the two loci, even in the homozygous state, show the wild phenotype. The s^y (Arabidopsis) and pro (tomato) mutants partially respond to exogenous GA. In contrast to dominant or semidominant dwarfs, recessive mutants show a lower endogenous content of biologically active C19 GAs than the isogenic wild-type plants. The explanation seems to be that constitutive GA signaling represses GA 20-oxidase and, hence, synthesis of active GAs. Constitutive signaling is also likely to activate GA 2-oxidase, thus lowering the concentration of active GAs still further.

Many of the mutants just described are in species (wheat, barley, pea) that do not lend themselves easily to map-based cloning of genes and genetic manipulation. Hence, molecular genetic work, until very recently, had been done using Arabidopsis. This situation is changing, however, and homology-based cloning using information from the rice genome sequencing programs is being used to isolate genes from more intractable species such as wheat. In the following, we first consider some mutants isolated in screens for GA signaling in Arabidopsis, their wild-type genes and nature of their encoded proteins, and their orthologs in wheat and maize. We next consider some recessive mutations and their genes and encoded proteins before summarizing our present understanding of GA signaling. 5.1. GAI Locus The gai mutant of Arabidopsis was isolated following X-ray irradiation of wild-type seeds. Genetic analysis indicated that it was a single gene semidominant mutation. The phenotype of gai is similar to that of GAdeficient gal mutants, but there are some important differences. Both are dwarf, have darker green foliage, and take longer to flower than the wild type, but the gai alleles produce fertile flowers, whereas the severe mutant alleles at the GAI locus show a drastic curtailment of inflorescence and flower development, especially petals and anthers (Fig. 24-9). The GA

599

24. Molecular Biology of GAs, CKs, BRs, and JAs

5,1.1, Loss-of-Function Tall Phenotype

FIGURE 24-9 Wild-type AmUdopsis (Landsberg "erecta"), a severe GA-deficient synthesis mutant, gal-3, and a GA-insensitive mutant, gai. Note that gal-3 is a dwarf and shows a curtailment of inflorescence and flowers; but gai, although a dwarf, shows inflorescence. From Koornneef et al. (1985).

insensitivity of gai alleles is quantitative, they are not completely unaffected by GA, and still retain a low level of GA responses. Figure 24-9 illustrates an important point about GA-insensitive mutants. The GA insensitivity, depending on mutant alleles, may be well pronounced in vegetative parts, but not extend to reproductive parts. In some of the Rht dwarfs in wheat, such as Rhil and Rhtl, which represent different genetic loci, the GA insensitivity is seen in vegetative parts, but not in reproductive axes or in aleurone tissue. In the more severe Rht3, which is allelic to Rhtl, it extends to aleurone tissue as well. Likewise, in mutants of Silene armeria, GA insensitivity does not extend to flowers. The GAI gene was cloned using a Ds transposon tag and encodes a protein that belongs to a novel class of plant transcription factors (see Sections 5.2 and 5.3). Comparison of GAI and gai sequences indicates that the gai gene has a 51-bp in frame deletion in the GAI open reading frame. As a result, the encoded gene product lacks a segment of 17 amino acids near the N-terminal, known as the DELLA region, named after the first 5 of the 17 amino acids (see Fig. 24-10). The gai protein is still produced, but its function is altered.

GAI Alleles Confer a

Since the gai mutation is dominant, it could not be concluded definitively how the wild type (GAI) or the mutated protein interacts with the GA response pathway. Hence, loss-of-function mutations at the GAI locus were created by 7-irradiation and screening for suppression of the gai phenotype {gai-d mutant); other loss-of-function mutants were available from the Ds transposon tagging used to clone the GAI gene (e.g., gai-t6). These loss-of-function mutations yield tall plants, which, in their phenotype, resemble the wildtype plants (Fig. 24-10). Sequence analysis indicates that the mutations are intragenic and interrupt the open reading frame of the gai gene; hence, they are unlikely to encode functional products. Since a loss of function at the GAI locus confers a phenotype that is similar to the wild type, it may be concluded that GAI is dispensable for the control of growth in normal conditions. However, this is not strictly true. These loss-of-function mutants also show an insensitivity to paclobutrazol, a GA biosynthesis inhibitor (see Chapter 7). At the same concentration of paclobutrazol, mutant plants show greater stem growth and more open flowers and extended petals than the wild type (Fig. 24-11). This increased resistance to paclobutrazol indicates that GAI is involved in the wild-type GA response, perhaps as a negative regulator of GA-mediated growth responses.

5.2. GRS and RGA: Genes Similar to GAI Arabidopsis contains another gene, GRS (for GAIrelated sequence), which encodes a product (GRS) very similar to GAI. Moreover, the GRS gene has been found to be the same as RGA (see later). As explained in Chapter 7, the GAI locus encodes the first enzyme in GA biosynthesis, copalyl synthase, which catalyzes the conversion of geranylgeranyl diphosphate to copalyl diphosphate. To know more about GA signaling, extragenic suppressor mutants for the GA-deficient, gal-3 phenotype (see Fig. 24-9) were generated. Several mutant alleles were obtained, including some at a previously identified SPY locus (see Section 5.6). Other mutant alleles, designated rga (for repressor oi gal-3), defined a new locus, RGA. The rga/gal-3 mutations are recessive; they enhance the stem growth of gal-3 to 30-50% of wild-type levels, but still retain partial male sterility (Fig. 24-12). GAdeficient mutants contain elevated levels of GA4 transcripts (encoding 3p-hydroxylase, which converts C19 GAs to active forms). The rga mutants are deficient in endogenous GA, but, unlike GA-deficient mutants.

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IV. Molecular Basis of H o r m o n e Action

DELIA region GAI

5

FIGURE 24-10 Schematic representation of GAI, dominant gai and loss-of-function alleles, gai-t6 and gai-d, and their phenotypes. From Harberd et al. (1998) and Peng et al. (1997).

suppress the levels of GA4 transcripts. Hence, it is thought that the RGA protein acts in the GA response pathway. Moreover, their deficiency in endogenous GA content, coupled to a partial increase in stem growth, suggests that plants lacking RGA require less GA than normal plants for equivalent growth. Therefore, RGA, like GAI, opposes the effect of GA and functions as a negative regulator of GA-mediated growth responses.

FIGURE 24-11 Growth inhibition in GAI (two plants in front) and gai-t6 (two plants in rear) grown on media supplemented with paclobutrazol (10^^ M). Inflorescence stem growth is suppressed in the wild type, but not to the same extent in the mutant. From Peng et al. (1997).

Although GAI and RGA both seem to be redundant to normal growth, they are expressed throughout the plant.

FIGURE 24-12 Phenotype of Arabidopsis wild type (Landsberg erecta, Ler), rga, and spindly {spy) mutants in^fl/-3 background. Note that rga and spy both enhance shoot growth over gal-3 and that the rga/spy combination restores gal-3 to almost the wild phenotype. From Silverstone et al. (1997).

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24. Molecular Biology of G A s , CKs, BRs, a n d JAs

The RGA gene was cloned and found to be the same gene as GRS. GAI and RGA/GRS (henceforth called only RGA) belong to a novel gene family, which was first defined by SCARECROW {SCR) in Arabidopsis. SCR encodes a putative transcription factor, which regulates the pattern of cell divisions regulating endodermis development in Arabidopsis roots and stems. Members of this gene family, designated GRAS family (acronym for GAI, RGA, SCR), occur extensively in plants. Nineteen members are known from Arabidopsis alone (in addition to the 3 just described, 16 others are known from expressed sequence tags). Orthologs of SCR are known from maize and pea. Another member, LATERAL SUPPRESSOR (LS), is known from tomato where it encodes a protein thought to regulate the origin of axillary branches at the vegetative shoot meristem (see Chapter 14). So far, members of this family have been found in plants only. 5.3. Structure of GAI/RGA Proteins The GRAS family of proteins shows a variable Nterminal region, but a common C-terminal region with several conserved domains. These domains include two leucine heptad repeat regions (LHRl and LHR2), a highly conserved VHIID domain, which typifies the family, and two domains designated PYFRE and SAW toward the carboxy terminus (Fig. 24-13). The sequence of these domains, although not the spacing between them, is highly conserved. The functions of most of these domains are still unknown, although the LHR regions are thought to be involved in proteinprotein interactions and formation of multimeric complexes. GAI/RGA differ from most of the GRAS family members in their N termini. Both have two regions of closely related sequence (regions I and II, the first five amino acids of region I are DELTA; hence, this region is referred to as the DELTA sequence). These regions are lacking in other members of the GRAS family.

including SCR and Ls, which indicates that these regions are probably important for GA signaling, a conclusion supported by the fact that mutations in this region render plants insensitive to applied or endogenous GA (e.g., gai mutant). In common with SCR, they contain nuclear localization signals (NLSs). Moreover, a fusion protein between RGA and green fluorescent protein (reporter) is localized to the nucleus in onion epidermal cells, indicating that the NLSs are functional. 5.4. Rhtl and Rhtd G e n e s in Wheat and d8 Maize Encode Functional Orthologs of GAI/RGA The dominant or semidominant mutants, Rht (for reduced height) wheat and dwarfS (d8) maize, have been known for decades, but the cloning of their wild-type genes was difficult because, as indicated in the legend for Fig. 24-8, cultivated wheat is hexaploid with three homoeologous chromosome sets (the A, B, and D genomes), and isolating a maize gene via gene tagging is a laborious process. Nicholas Harberd at John Innes Centre, Norwich, United Kingdom, and associates used a data base search of rice expressed sequence tags to identify a DNA sequence that could encode a polypeptide with the DELLA sequence of amino acids. Since cereal genomes are well characterized and show substantial conservation of gene order (colinearity), they could use the rice sequence to isolate a wheat cDNA and ultimately the wheat rht genes located on 4B and 4D chromosomes, as well as the maize d8 gene. The encoded products of wheat Rhtl and Rht2 and maize d8 genes show 88% amino acid identity and map to the same region of cereal genome; hence, they are thought to be the same gene. The deduced amino acid sequences of the wild type wheat Rhtl and maize D8 proteins contain all the conserved domains in GAI/RGA proteins, including the N-terminal regions I (DELLA region) and II, the

DELLA

LHR1

VHIID

LHR2

PFYRE

SAW

FIGURE 24-13 Schematic structure of GAI/RGA/SCR-type proteins. Conserved domains in the carboxy terminus are designed by single letter code for amino acids. LHR regions are probably involved in proteinprotein interactions, but the functions of other domains, VHIID, PYFRE, and SAW, are unknown. The N termini of GAI and RGA proteins differ from SCR in that they contain two closely related sequences— regions I (DELLA region) and II—which are thought to be specific for GA signaling. Adapted with permission from Pysh et ah (1999), © Blackwell Science Ltd.

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IV. Molecular Basis of H o r m o n e Action

9^1

i!;!::

Dd-1

Rht-D1b

FIGURE 24-14 Schematic representation of the N- terminal regions of proteins encoded by dominant GAinsensitive mutant alleles: gai Arabidopsis, D8-1 maize, and Rht-Dlb wheat. The conserved regions, DELLA I and II, are underlined, and deletions (including substitutions) are highlighted in white. All mutations alter the N-terminal region of their encoded proteins. Modified with permission from Peng et al. (1999).

NLSs, and the other domains in the carboxy terminus. The semidominant mutations in wheat and maize, allelic to Rhtl and d8, confer differing severities of dwarfism. Like gai, they are not completely insensitive to GA. For instance, maize d8-l mutants treated with paclobutrazol show enhanced dwarfism, which is corrected to original dwarf stature by simultaneous GA3 treatment. Sequence analysis of these alleles indicates that they all contain mutations in the N terminus, particularly the DELLA region (Fig. 24-14), but seem to produce truncated proteins that are still active. Moreover a loss-of-function allele of wheat Rhtl, which lacks the DNA sequence encoding the DELLA region, confers a tall phenotype, which is GA responsive. Thus, it is concluded that Rhtl and Rht2 wheat and D8 maize are functional orthologs of Arabidopsis GAI/RGA. 5.5. Slender (sin) Mutants of Barley and Rice Although the sin mutant of barley (see Table 24-2 and Fig. 24-8B) has been known for a long time, its gene has not yet been cloned. Recently, a slender (slnl) mutation in rice was obtained, it is a recessive mutation which, like its barley counterpart, shows a constitutive GA response; the plants are inordinately tall as if saturated with GA, insensitive to applied paclobutrazol, and their aleurone tissue produces a-amylase without an exogenous GA treatment. Since slnl is a loss-of-function mutation, the SLNl locus negatively regulates the GA responses. The SLNl gene was cloned and found to encode an ortholog of GAI/ RGA/Rht/D8, with all the conserved domains including the DELLA sequence, an NLS, the leucine heptad repeats, and the VHIID domain. Several slnl alleles occur in rice. The slnl-1 allele has a premature stop codon and is a null allele that does not produce a protein.

These data from Arabidopsis, wheat, maize, and rice indicate that GAI/RGA/Rht/D8/SLN represent a protein belonging to the GRAS family of transcriptional regulators, which is specific to GA responses. If the protein is mutated in the DELLA region it produces a defective protein which makes the system mostly, though not completely, GA insensitive. By contrast, if the protein is not expressed at all because of a null mutation, it gives a constitutive GA response. The data from slender rice also confirm the earlier results that loss-of-function intragenic suppressor mutations in the GAI/Rht genes result in a tall phenotype. 5.6. SPY Locus Since seed germination in Arabidopsis depends on endogenous GA (more correctly, a balance between endogenous GA and ABA), GA-insensitive mutants can be isolated by screening for seeds that germinate under conditions of reduced endogenous GA. The spy (for spindly) mutations were obtained by mutagenizing seeds and plating them on a medium with 1 JJLM paclobutrazol. Several independent recessive mutants were isolated; all were alleles at the SPY locus. As expected, these mutants have a reduced requirement for GA in seed germination. They also have longer internodes and display early flowering, partial male sterility, and increased parthenocarpy in fruit (siliques), traits shown by wild-type plants when supplied with an excess of biologically active GAs. However, spy mutants, unlike slender mutant in barley, still respond to exogenous GA, which suggests that they are not constitutive to GA responses. Since spy alleles are recessive, the SPY locus is postulated to encode a negative regulator of the GA response. Double mutational analysis indicates that spy alleles partially suppress the phenotype associated with GA

603

24. Molecular Biology of G A s , CKs, BRs, a n d JAs WT

gai

_

spy-4

^

reporter

effectors

AMY|I GUS NOS

UBI NOS

AMY::GUS

UBI UBI HvSPY NOS UBI::HvSPY

B

120 100 h

ga 1 spy-4

ga 1 gai spy••4

gai spy-4

FIGURE 24-15 Phenotypes of adult wild type, gal, gai, and spyA mutants of Arabidopsis. The phenotypes of spy-4 in gal, gai, or gal gai backgrounds are also shown, spy-4 is a strong allele and shows a near-normal phenoype; it also restores gal and gai partially to wild type. Courtesy of Neil Olszewski, University of Minnesota, St. Paul.

80 \o

(/)

60 h

O 0)

40 h

.> iS

deficiency (e.g., gal alleles) or GA insensitivity {gai alleles) (Fig. 24-15), They also enhance the suppression of the gal-3 phenotype conferred by rga alleles (see Fig. 24-12). Mutant alleles at the SPY locus are epistatic to rga and gai, which suggests that SPY acts downstream of RGA and GAI or that it modifies those proteins. The aleurone system is a well-characterized system for GA-induced gene expression. Accordingly, a barley homologue of SPY, HvSPY, was cloned. The coding sequence of HvSPY under a constitutive promoter was coexpressed in aleurone tissue along with a high pi aamylase::GUS construct. The constitutive expression of HvSPY reduced the GUS expression in GAs-treated aleurone layers, which confirms that SPY acts as a negative regulator of the GA response pathway (Fig. 24-16). SPY and HvSPY genes are orthologs. They encode a protein that is structurally similar to a class of enzymes in animals that transfer N-acetylglucosamine to target proteins. These enzymes, known as O-GlcNAc transferases (OGTs), transfer a single GlcNAc from UDPGlcNAc to specific serine a n d / o r threonine residues via an O linkage. Such O-GlcNAc modification of regulatory proteins, similar to protein phosphorylation, is believed to play a role in signal transduction pathways. The SPY protein contains 10 copies of a tetratricopeptide repeat (TPR) motif at the N-terminal and a C-terminal catalytic domain, both features seen in cytosolic OGTs from animal systems (Fig. 24-17). The TPR motif is involved in protein-protein interactions. Whether the SPY protein acts as an OGT in plant systems is unknown, but recent data suggest that it is involved in several roles in plant development including a negative regulation of GA responses.

20 h

0)

reporter: AMY::GUS effector: UBI UBI::HvSPY

+ + -

FIGURE 24-16 Functional analysis of HvSPY protein. (A) Structure of the constructs. Effector constructs were either a ubiquitin promoter fused to the HvSPY-coding sequence {UBI::HvSPY) or a blank casette consisting of the UBI promoter only (control). The reporter construct consisted of a barley high pi a-amylase promoter fused, via an intron (I), to the GUS-coding sequence {AMY::GUS). Each construct was terminated with the 3' region of the NOS gene from Agrobacterium. (B) Barley half-grains were cobombarded with the reporter gene plus either of the two effector constructs and incubated in a medium plus or minus 10"^ M GA3. Relative GUS activity is expressed as a percentage of total GUS activity in aleurone layers bombarded with the blank UBI casette and the reporter gene construct. The means and standard error are shown. From Robertson et al. (1998).

5.7. Other Mutant Loci Other mutant loci identified in Arabidopsis include SLEEPY (SLY) and PICKLE (PKL). Recessive slyl mutations were identified in a suppressor screen for an ABA-insensitive, abil-1, mutation. The mutants have a reduced GA requirement for seed germination. They also have a phenotype similar to that of GA-deficient mutants, but it is not corrected by exogenous GA. The recessive pkl mutations, identified in a screen for affected root development, show some features reminiscent of GA-deficient or -insensitive mutants combined with primary roots, which have swollen tips and retain the characteristics of an embryonic root. These loci await further characterization before their role in GA signaling can be assessed.

604

IV. Molecular Basis of Hormone Action SPY Catalytic domain

TPR domain 1

2

3

4

5

6

7

8

9

10

1

2

3

4

5

6

7

8

9

10

ocn 11

FIGURE 24-17 Schematic representation of Arabidopsis SPINDLY (SPY) protein. From Thornton et al (1999) with permission from Elsevier Science.

6. A WORKING HYPOTHESIS FOR THE G A RESPONSE PATHWAY Data reviewed so far suggest a working hypothesis for GA signaling with several unconnected pieces in the puzzle. The balance of evidence favors a cell surface perception of the GA signal by a transmembrane receptor, which could be a receptor-like kinase (RLK) or a G-protein-coupIed receptor. The downstream elements for signaling are unknown. For RLK-type signaling, a type 2C protein phosphatase may be involved; for the G-protein-coupled receptor, secondary messengers (e.g., cyclic GMP, Ca^+) and Ca^+calmodulin-activated protein kinases have been postulated. Molecular genetic data from response mutants in Arabidopsis, wheat, maize, and rice indicate that GA responses are negatively regulated by a protein or a protein complex. A model for GA signaling is shown in Fig. 24-18. According to the model, elongation growth via the GA response pathway is repressed by an inhibitor protein represented in Fig. 24-18 by GAI (it could also be RGA/Rht/D8/SLN). This repression is relieved when endogenous or supplied GA is present at a concentration above a threshold. A mutation in the N-terminal DELLA sequences in the repressor protein confers a dominant phenotype; an altered function protein is produced which cannot be derepressed by GA, thus providing GA-insensitivity. By contrast, a loss-of-function mutation in the gene, which results in a lack of the protein, allows constitutive GA responses. Additional studies indicate that not only elongation growth, but most GA-regulated processes are controlled through a negatively acting GA-signaling pathway. The concept that plants contain an endogenous inhibitor of growth and that GA acts by inhibiting the activity of this inhibitor is not new. In fact, it was proposed by Percy Brian in 1957.

The position of SPY in this model is unclear. It could modulate GAI/RGA activity by the addition of Olinked GlcNAC residues or modulate the activity of some other protein downstream of GAI and RGA. It may also be part of an alternate signaling pathway that converges with the GA-signaling pathway. In Fig. 24-18, a single protein is shown as the inhibitor, but it could also be a complex of several proteins including GAI/RGA or their orthlogs in other plants. An altered function protein produced by gai/rga "poisons" the complex rendering it insensitive to GA, whereas a lack of the protein allows constitutive GA signaling. A negative regulation of the GA response pathway is somewhat similar to ethylene signaling (see Chapter 21). In ethylene signaling, ethylene binding to its receptor(s) derepresses the ethylene signaling pathway. However, GAI/RGA and, by analogy, their orthologs in wheat, maize and rice are not plasma membrane-based receptor proteins, like ETRl. Structurally, they contain no motifs, which suggest an association with membranes; instead, they are localized to the nucleus and have the hallmarks of transcription activators. Their biochemical functions and target genes are unknown. They could regulate transcription factors such as GA-MYB, which, in turn, could, lead to GA-specific gene expression, but such regulation still has to be shown. Their upstream signaling elements are also unknown. The cereal aleurone systen\ has long been considered a paradigm for hormone-mediated gene expression. GA signaling in this system has been associated with surface perception of the GA signal, participation by heterotrimeric G proteins, and secondary messenger-activated protein kinases. The availability of Rht in wheat and SLN in rice, makes it possible to test the roles of these proteins in GA signaling in the aleurone system. In this connection, it has been shown already that overexpression of GAI or gai in rice suppresses the GA-induced a-amylase production, while lack of SLN expression in rice allows

605

24. Molecular Biology of GAs, CKs, BRs, and JAs GAI is a repressor of plant growth and occurs in 2 forms

B

wild type

gai

GA-deficient

\ GAor IGA signal \ GAor 1 JGAsignall

^^^^A repressing form

non-repressing form

no repression

repression

1 1 1 GROWTH 1

1 GROWTH!

^

repression

i 1 1 GROWTHI

(1

NORMAL GROWTH

REPRESSED GROWTH

REPRESSED GROWTH

GA de-repressible

notGA de-repressible

FIGURE 24-18 Derepression model for regulation of plant stem elongation by gibberellin (GA). GA (possibly via a signaling intermediate) opposes the activity of GAI, a protein that represses plant growth. (A) GAI exists in two forms. Interaction between GAI and GA (or the GA signal) results in conversion of the growth-repressing form of GAI into a nonrepressing form. (B) Normal (wild-type) plants grow tall because endogenous GA converts GAI to the nonrepressing form. GA-deficient plants are dwarfed because they lack sufficient GA to allow conversion of GAI, which therefore remains in the repressing form, gai Mutant plants are dwarfed because the mutant gai protein cannot interact with GA (or GA signal) and thus is not converted to the nonrepressing form. Reprinted with permission from Harberd et al. (1998).

constitutive a-amylase production. The wheat system is particularly instructive because rht alleles offer different degrees of GA insensitivity. Rhtl and Rht2, which represent different genetic loci, are GA insensitive in vegetative parts, but not in aleurone tissue, whereas Rht3 (allelic to Rhtl) shows GA insensitivity in the aleurone tissue as well.

7. GA/ABA INTERACTION Many, although not all, GA-induced responses are antagonized by ABA, and vice versa. For instance, GA promotes cell division and cell elongation in young growing tissues. Associated with cell division, it stimulates the accumulation of cdc2 mRNA and some cyclins, both of which are inhibited by ABA. Cell elongation is accompanied by a predominantly

transverse orientation of microtubules in the parietal cytoplasm and enhanced activities of xyloglucan endotransglycosylases (XETs) that play a role in wall loosening. GA promotes these processes, while ABA inhibits them. Leaf development in aquatic plants is affected in opposite ways by GA and ABA. Leaves initiated while the shoot apex is underwater are dissected and filiform, whereas those initiated when the shoot apex is in air tend to be entire or much less lobed (see Fig. 1-2 in Chapter 1). GA supplied to leaves, which are developing while submerged, accentuates the dissected and filiform morphology, whereas ABA given under similar circumstances promotes the development of an aerial type leaf. ABA induces seed dormancy and can inhibit germination, whereas GA promotes seed germination and may be involved in breaking seed dormancy. A graphic demonstration of ABA/GA antagonism is seen in the induction of genes in the cereal aleurone system. GA induces the tran-

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IV. Molecular Basis of H o r m o n e Action

scription of a-amylase and several other protease and nuclease genes and suppresses the expression of the aamylase inhibitor, alcohol dehydrogenase, and some other genes. ABA has the opposite effect. The question arises — where does this antagonism occur? Do the two hormones interfere with each other at the receptor level, in the signaling pathway, or at the gene transcription level? It does not seem likely that the two hormones interfere with each other at the receptor level. In i;/fro-binding assays using [^H]GAi or [ H]GA4 show no competition by ABA. Also, examples there are that the two signaling pathways can proceed independently of each other. For example, ABA induction of anion current in the guard cells is not interfered with by GAs. In the slender mutant of barley, which is a constitutive GA responder, a-amylase gene expression is inhibited by ABA, but ABAand dehydration-induced gene expression in roots remains unaffected. Gene transcription in the aleurone system is affected by both GA and ABA. Hence, the promoters of genes induced by GA or by ABA in this system have been studied for clues. As mentioned earlier, the ABA and GA response elements are different and presumably bind different transcription factors. The response elements work in concert with other cis elements with which they form response complexes. At least one cis element, Opaque2, in a low pi a-amylase promoter in barley, is able to form a response complex with either an ABRE or a GARE and be responsive to either ABA or GA, respectively. Thus a reasonable postulate would be that ABA acts on the regulated gene by interfering with the binding of transcription factors to GARC. It is unlikely that ABA per se causes this interference, rather it would be some component of the ABA signal transduction that would so interfere. There are indications that such interference by a component in ABA signaling occurs further upstream on the GA signaling pathway. For instance, GAMyb constitutively expressed is sufficient to drive the expression of a-amylase and several other genes, including the gene for a cysteine proteinase in barley (EPB-l). While ABA suppresses the GA-induced gene expression, it does not suppress the GAMyb-induced expression; hence ABA must operate at a level upstream of GAMyb, as shown schematically in Fig. 24-19A. The role of protein kinases and protein phosphatases in signal transduction is discussed in Chapter 25. However, it is appropriate to mention here that a protein kinase that is specifically induced by ABA (PKABAl for protein kinase responsive to ABA), if expressed constitutively in barley aleurone tissue, suppresses the GA-induced expression of low p7 and high pi a-amylase genes, without exogenous ABA,

ABA GA-

GAMyb

- ^ [a-amylase, I cysteine proteinase] I

B ABAGA

PKABA1

±

[a-amylase]

FIGURE 24-19 Schematic illustration of cross talk between GA and ABA signaling in cereal aleurone. (A) GA induces the synthesis of GAMyb, which, in turn, regulates the expression of GA-induced genes, such as a-amylase. ABA has no effect on GA responsive gene expression, however, if GAMyb is expressed constitutively. Hence, ABA inhibits GA signal transduction at a step before the synthesis of GAMyb. (B) ABA induces synthesis of a protein kinase, PKABAl, in wheat embryo. Constitutive expression of PKABAl in barley aleurone inhibits GA-induced a-amylase gene expression, indicating that ABA mediates its effect on GA-induced gene expression via a protein kinase. Based on data in Cercos et al. (1999) and GomezCadenas et al (1999).

which suggests that ABA exercises inhibition over GA-induced responses via a protein ]<:inase (Fig. 2419B). Such constitutive expression of PKABAl has little effect on expression of an ABA-specific LEA gene, HVAl, however, implying that HVAl is regulated by ABA in some other manner. Another ABA and salt stress-induced protein icinase in salt-tolerant wild wheatgrass, Lophopyrum elongatum, has been found to inhibit a GA-induced low pi a-amylase gene expression in barley aleurone tissue.

8. G A INTERACTION WITH ENVIRONMENTAL FACTORS In addition to ABA, GAs are also known to interact with environmental factors, such as light and temperature. Seeds of many plants, while in a wet state, require an exposure to light in order to germinate. These seeds are referred to as photoblastic seeds. Some of the common examples include Arabidopsis and certain cultivars of lettuce (e.g., Lactuca saliva cv Grand Rapids). The phenomenon has been well studied for lettuce seeds, which have an absolute requirement for exposure to red light at 680 nm while imbibed. It is a phytochrome-regulated phenomenon and will be dealt with in Chapter 26. Here, it is important to point out that this red light requirement can be completely bypassed by exposure of seeds to a solution of GA. Similarly, many winter annuals, biennials, and perennials growing in temperate climates have a requirement for exposure to cold temperature, a phenomenon known as vernalization, before they will flower. Weeks and

24. Molecular Biology of GAs, CKs, BRs, and JAs months may elapse between the perception of temperature signal and the flowering response. Examples include winter wheat, winter rye, and biennials such as spinach or rape. In these plants, an exposure to exogenous GA has been shown to bypass the cold temperature requirement. While the molecular mechanisms by which GA bypasses these requirements are not understood, it is clear that the GA signaling pathway must intersect and cross talk with other signaling pathways related to phytochrome or cold temperature.

9. G A RECEPTOR(S) COULD BE A MEMBER OF A SMALL GENE FAMILY It is possible that there are different isoforms of a GA receptor in different groups of plants, and also possibly for different responses, such as cell elongation, flowering, production of hydrolytic enzymes in seed tissue, and production of antheridia in fern gametophytes. Biological activities of C-3, 13-dihydroxy GAs, such as GAi or GA3, and those of the C-3 single hydroxylated GAs, such as GA4 or GA7, differ in different families of plants. For example, GAi or GA3 has higher activities in members of the legume and grass families than in cucurbits or Arabidopsis, whereas the reverse is true for GA4 or GA7. In ferns and many gymnosperms, GA4 or GA7 often has higher activities than GA1/GA3. In some long-day plants, such as Lolium, some polyhydroxylated GAs are more active in promoting flowering than in stem elongation. In contrast, GAs that are active in stem elongation, such as the monohydroxy GA4, GA7, or dihydroxy GAi, GA3 have very little promotive activity in flowering. The promotion of antheridia formation in gametophytes of certain ferns also involves a GA-type structure different from that of GAs that promote stem elongation. These observations are based on bioassays and are not conclusive, but they suggest that different isoforms of the GA receptor may be present among different taxa and possibly at different developmental stages. Such isoforms would represent different members of a small gene family.

607

is also substantial evidence for a cell surface perception of the GA signal by a G-protein-coupled receptor or a transmembrane receptor-like kinase. Studies on gene expression in the aleurone system have revealed many cis sequences in GA-regulated promoters, which seem to bind nuclear proteins. A MYB-type transcription factor specific to GA signaling mediates the expression of many GA responsive genes in the aleurone system and possibly other GA responses. Some other putative transcription factors are known but have not been characterized. The upstream signaling components that these transcription factors interact with are unknown. Molecular genetic studies using response mutants from Arahidopsis, wheat, and rice maize indicate that GA responses are negatively regulated by a protein or protein complex (e.g., GAI/RGA in Arabidopsis or their orthologs in wheat, and rice maize). The inhibitory protein(s) represses the normal response(s), and GA acts not by directly stimulating growth or another response, but by derepressing the inhibitor protein. GAI/RGA-type proteins are unique to GA signaling, but share structural motifs in their carboxy terminus, with other members of the GRAS family, a family of plant transcription factors of diverse functions. Other repressors of GA action, such as SPY in Arabidopsis and its ortholog in barley, are O-GlcNAc transferases, which may act by the modification of GAI/RGA-type proteins or some other proteins in the signal transduction pathway. GA interaction with ABA and environmental factors, such as light and temperature, underscores the complexity of signal transduction pathways, their interconnections, and possibilities of cross talk between them. Some data indicate that cross talk among GA and ABA-signaling pathways occurs in later steps of signal transduction, but prior to the synthesis of GA-specific transcription factors. There is increasing evidence for a role for Gprotein signaling and of calcium and phosphorylations/dephosphorylations in early events in GA signaling. These aspects are covered in Chapter 25.

SECTION 11. MOLECULAR BIOLOGY OF CYTOKININ (CK) ACTION 1. CK-INDUCED RESPONSES

10. SECTION SUMMARY Hormone-binding studies have indicated the presence of soluble and membrane-bound proteins in GA responsive systeras, but the proteins have not been purified and their identity remains unknown. There

The major effects of cytokinins on plant growth include the promotion of cell division in cell and tissue culture, promotion of lateral branching, inhibition of root growth, and delaying of senescence and abscission of leaves, petioles, and flowers. In dark-grown

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IV. Molecular Basis of Hormone Action

seedlings, cytokinins promote expression of the photomorphogenetic program, i.e., inhibition of hypocotyl growth, differentiation of chloroplasts from proplastids, greening, and expression of CAB and RBCS genes. Cytokinins are also active in mosses (e.g., Physcomitrella patens), where they promote bud (gametophore) formation on protonema. Many of the effects of cytokinins, such as regulation of lateral bud growth and apical dominance, and root-shoot ratio in tissue culture are mediated in association with auxin. Higher cytokinin to auxin ratios promote shoot over root growth and promote outgrowth of lateral buds and a bushier habit.

2. CK-BINDING PROTEINS Cytokinin-binding studies have been carried out using ^^C or -^H-labeled N^-adenine cytokinins (e.g., benzyladenine, kinetin, zeatin, or isopentenyladenine) and extracts from various sources, cells in tissue culture, embryos, mature leaves, etc. Specific and exchangeable binding has been reported for proteins in the soluble fraction, particulate fraction including ribosomes, and thylakoid membranes from chlorolplasts. Photoaffinity-labeled cytokinins have also been used. Several proteins have been purified, but either the affinities for natural cytokinins have been too low (KD values usually more than 10"^ M) or the protein has been present in such large abundance that a case that one is dealing with a receptor protein cannot be made. For instance, a cytokinin-binding protein from wheat embryos is well characterized; its affinity for natural cytokinins is in the range of ~ 10~^ M, but it occurs in large abundance (~10% of soluble protein) and has amino acid sequence similarity to 7S globulin-type storage proteins. A protein complex in tobacco with at least two subunits of 57 and 36 kDa is reported to bind cytokinins. The 57-kDa-binding protein is interesting because it is a homologue of S-adenosyl-L-homocysteine hydrolase (SAH hydrolase). As explained in Chapter 4, DNA methylation plays an important role in cell differentiation and in maintenance of the determined state; in contrast, loss of methylation is correlated with dedifferentiation and somatic embryogenesis. The intracellular source of methyl groups is Sadenosylmethionine (SAM). SAM provides methyl groups for the synthesis of ethylene as well as polyamines (see Chapter 11); it is also the source of methyl groups for methylation of DNA and proteins by methyl transferases. SAH hydrolase is a product of methyl transfer from SAM and, thus, is a competitive

inhibitor of SAM-dependent methyl transferase reactions on DNA and proteins and probably ethylene biosynthesis. Interestingly, SAH hydrolase activity is typically high in cells in culture and in root tips. Antisense inhibition of SAH hydrolase yields transgenics, half of which are stunted, probably because cell division is inhibited, and a three times higher CK content as judged from a bioassay of root exudate. Since cytokinins promote cell division, it is interesting that they should show binding affinity toward an enzyme that inhibits DNA methylation. Many synthetic diphenyl urea derivatives, despite their vastly different structures from adenine-type cytokinins, show greater activity, on a molar basis, than adenine-type cytokinins. Hence, tritiated diphenyl urea derivatives have also been used to isolate cytokininbinding proteins. A partially purified protein from mung bean seedlings had a XD of ~ 10~^ - 10"^ M for zeatin or benzyladenine. Free diphenyl-type molecules and the N^ adenine cytokinins competed freely, but cytokinin ribosides did not (a difference between cytokinins and their ribosides, which is not seen in several other cytokinin-binding proteins). Anticytokinins act as competitive inhibitors of cytokinin-induced responses, i.e., their effect can be overcome by higher concentrations of cytokinins (see Chapter 8). This suggests that they compete for the receptor site (or some other site early in signal transduction) and can be used to isolate the receptor protein or a nearby element in signal transduction. A fluorescent analog of a pyrolo [2,3-rf]pyrimidine was used as a probe for cytokinin-binding proteins. Several candidate proteins from cytokinin-dependent tobacco tissue culture were reported. One protein had a reasonably high affinity for cytokinins (10"^ M), but was not characterized in detail. In summary, several approaches have been used to isolate cytokinin-binding proteins. Several proteins have been purified or partially purified and show high-affinity, exchangeable binding to natural and synthetic cytokinins, but in no case has it been shown conclusively that the binding is to a cytokinin receptor.

3. CK-INDUCED GENE EXPRESSION The list of genes induced or upregulated by cytokinins is long. It includes genes involved in photosynthesis, such as those encoding chl a / b protein (CAB), a small subunit of RUBISCO (RBCS); genes involved in anthocyanin biosynthesis, such as chalcone synthase (CHS) and dihydroflavonol reductase (DFR); genes

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24. Molecular Biology of G A s , CKs, BRs, a n d JAs

encoding cyclins, especially cyclin D3, cyclin-dependent kinases (CDKs), and ribosomal RNA, all involved in cell division; and genes encoding various metabolic enzymes, such as nitrate reductase (NR), phosphoenolpyruvate carboxylase (PEPC), hydroxypyruvate reductase, and the gene encoding the SAH hydrolase mentioned in the previous section. Other upregulated genes encode products that probably participate in metabolic partitioning across cell boundaries. These include an extracellular form of acid invertase, which hydrolyzes sucrose, a carrier protein for glucose transport across membranes, and several early nodulin (ENOD) genes, which are activated in roots of leguminous plants in response to nodulation factors secreted by nodulating bacteria, such as Rhizobium. Some of these genes have been shown to be regulated by cytokinins at the transcriptional level (e.g., cyclin D3, NR genes), whereas for others, cytokinins have been reported to enhance mRNA stability (e.g., CAB, RBCS genes). Many of these genes are also induced by other factors, such as light, auxin, nitrate supply, wounding, and other stresses; and most are induced several hours, even days, after treatment with cytokinins. Thus, it is unclear whether they are induced primarily by cytokinins. Using the differential display technique, two genes, called INDUCED BY CYTOKININ {IBC6 and IBC7) have been identified in Arabidopsis. These genes are induced rapidly by benzyladenine (Fig. 24-20) and some urea cytokinins, which have strong activity in bioassays, e.g., thidiazuron (N-phenyl-N '-1,2,3-thidiazol-5-urea) and forchlorofenuron [N-(2-chloro-4-pyridyl)-N-phenylurea] (for structures, see Fig. 8-11 in Chapter 8). Curiously, zeatin and diHZ showed only about half as much induction as BA, and other adenine cytokinins, such as 2iP, or kinetin, showed much less induction. These genes were not induced by other factors, such as light, or other hormones, such as GA, ABA, or 2,4-D. Moreover, their induction did not require prior synthesis of another protein; hence, they are primary response genes. Both IBC6 and IBC7 are single copy genes. The IBC6 gene encodes a protein {Mj. 20.1 kDa, p i 4.8) with sequence similarity to response regulators in the bacterial two-component systems, specifically to the CheY protein in Escherichea coli, which serves as the response regulator in the system for chemotaxis (for two component signaling systems, see Chapter 21). The three highly conserved residues of response regulators are all conserved in IBC6, including the putative phosphorylation site at Asp^^. The predicted sequence of IBC7 protein is similar, but it has an acidic C-terminal extension found in many transcriptional activators.

12.5

10.0 \-

c O 3 •D

o

10

100

1000

time (min) FIGURE 24-20 Kinetics of induction of the IBC6 gene in Arabidopsis. Dark-grown seedlings were treated with benzyladenine (5 |JLM). At indicated times, RNA was extracted and hybridized with IBC6 cDNA probe. Maximum levels of IBC6 mRNA were reached within 40 min; the levels then progressively declined to 6 h, although even at that time the levels were about four fold higher than basal levels. Modified from Brandstatter and Kieber (1998).

The precise functions of these proteins in cytokinininduced responses are still unclear. As yet, there is little information on either the response elements in proraoters of cytokinin-induced genes or the transcription factors that bind to them.

4. CK RESPONSE M U T A N T S Cytokinin response mutants have been selected using several different screens. 4.1. Screen for Altered Morphology Since cytokinins cause increased branching, loss of apical dominance, and inhibit root growth, these morphological criteria have been used to screen for mutants that are resistant or tolerant to high levels of cytokinins. The cyrl (for cytokinin resistant) mutant of Arabidopsis was selected for resistance of root growth to cytokinins. The mutant also displays incomplete cotyledon and leaf expansion, limited shoot development, reduced chlorophyll production, absence of anthocyanin, and an inability to develop mature, fertile flowers. The pleiotropic nature of this mutant suggests that CYRl may play a role in cytokinin signaling. The stpl (for stunted plant 1) mutant oi Arabidopsis was isolated in a screen for decreased root growth under normal growth conditions and was later found to be cytokinin resistant. Cytokinin insensitivity is, how-

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IV. Molecular Basis of H o r m o n e Action

ever, restricted to root growth; thus, STPl may have only a limited role in cytokinin signaling. The zeaS (for zeatin) mutant of Nicotiana plumbaginifolia was selected for its ability to germinate on zeatin levels, which completely inhibit germination of wild-type plants. The effect of zea3 mutation also seems to be restricted to germination. All of these are monogenic recessive mutations, but the wild-type genes have not been cloned and it is not clear whether they are linked directly to cytokinin perception or signaling. Another mutant from Arabidopsis, ampl (for altered meristem program), is an overproducer of cytokinins and is considered elsewhere (see Chapter 8). 4.2. Screen for Cell D i v i s i o n and Growth Since cytokinins promote cell divisions in cell/tissue culture, cytokinin autonomous mutants have been isolated by screening for cells/cell lines that are able to proliferate in cytokinin-depleted medium or in the absence of exogenous cytokinin. Several cki mutants (for cytokinin independent) from Arabidopsis were isolated after T-DNA activation tagging (for activation tagging, see Appendix 1). These mutants were able to grow in culture without exogenous cytokinins and form shoots. One mutant, ckil-1, went on to form whole plants, flower, and set seed. It carried a single T-DNA insertion and allowed the cloning of the gene. The CKll gene encodes a protein with an extracellular domain and an intracellular region, which combines the histidine kinase domain and the receiver domain with a conserved Asp, typical of the two-component signaling systems. It is similar to ETRl, one of the receptors for ethylene (see Chapter 21), although CKIl has two membrane-spanning domains, whereas ETRl has three such domains (Fig. 24-21). CKIl has been postulated as a cytokinin receptor because overexpression of the CKIl gene in transgenic plants results in typical cytokinin-type responses. Cytokinin-binding studies using CKIl protein, such as those for ETRl expressed in yeast cells, have not been performed, and the precise role of CKIl in cytokinin signaling is still unknown.

Asp

His CKI1

|-

,J

His ETR1

Asp

-Hf

FIGURE 24-21 Structure of CKIl in Arabidopsis compared with that of ETRl. Reprinted with permission from Kakimoto (1996), © American Association for the Advancement of Science.

Another cytokinin response mutant, crel (for cytokinin response 1), has been isolated in a screen using lack of cell proliferation and shoot histogenesis in tissue culture in response to added cytokinins. Mutant plants are also much less sensitive to inhibition of root elongation by cytokinins. The wild-type gene CREl encodes a protein that is predicted to have an N-terminal, extracellular region flanked by two transmembrane segments and an intracellular region with a histidine kinase domain and a response regulator domain at the C terminus. The gene is identical to ]NOLl and to AHK4 in Arabidopsis, which have been shown to regulate cell proliferation and proper formation of vascular tissues in roots (see Chapter 3) and to serve as the cytokinin receptor in roots, respectively. Transgenic expression of CREl in the mutants crel1 and crel-2 restored their responsiveness to added cytokinins in tissue culture. Yeast strains lacking a functional SLNl protein involved in osmosensing (see Box 21-1, Chapter 21) are lethal. Expression of CREl in the slnlA mutant by itself did not suppress lethality, but the addition of cytokinins, such as transzeatin, to the medium restored the mutant cells to normal growth, thus providing a direct link between cytokinin perception by CREl and a biological response. Together these data provide convincing proof that CREl is a cytokinin receptor and that plant cells probably perceive cytokinins at the cell surface and cytokinin signaling involves a phosphorelay. These results are striking for another reason—they show that yeast cells respond to trans-zeatin, one of the few examples of a plant hormone eliciting a response in a different kingdom. Significantly, czs-zeatin was ineffective in this assay. 4.3. Screen for Ethylene Synthesis in Dark-Grown Seedlings Cytokinins induce the synthesis of ethylene in darkgrown seedlings by activating ACC synthase, the key regulatory enzyme in ethylene biosynthesis. In Arabidopsis, five ACC synthase genes {ACS1-ACS5) have been identified, three of which encode functional enzymes and are differentially induced by different signals, including auxin and wounding (see Chapter 11). Low levels of cytokinins (e.g., 0.5 [xM benzyladenine or less) enhance the stability of ACS5 mRNA in dark-grown seedlings, and ethylene produced is sufficient in amount to cause the triple response (see Chapter 21). The induction of triple response occurs within 3 days and, thus, provides a rapid screen for sensitivity or insensitivity to applied cytokinin. Arabidopsis seeds were mutagenized, germinated in the

24. Molecular Biology of GAs, CKs, BRs, and JAs presence of 0.5 |JLM benzyladenine, and screened for lack of triple response. The screen yielded several mutants that were either defective in ethylene production or ethylene insensitive. It also yielded some mutants that were insensitive to applied cytokinins but not to ethylene. These latter mutants, cinl-cin4 (for cytokinin insensitive), show the normal triple response to ethylene (Fig. 24-22). Moreover, they produce low levels of ethylene at low concentrations, but normal levels of ethylene at high concentrations of applied cytokinins, indicating clearly that different isoforms of ACC synthase are involved in ethylene production in the dark and that low levels of cytokinins affect only the expression of the ACS5 gene. cin mutations may prove to be important in the identification of elements necessary for cytokinin signaling to ACS5.

WS

611

5. SECTION S U M M A R Y Cytokinin perception and signaling are still very much a mystery, although some important advances have been made recently. Despite much effort, little is known about cytokinin-binding proteins and cytokinin-induced gene expression. There is still no information on cytokinin response elements or on transcription factors that bind to them. This picture may change with the identification of two genes in Arabidopsis, IBC6 and IBC7, which are induced within minutes of benzyladenine treatment and which are primary response genes and encode proteins similar to bacterial response regulators. Several response mutants, cyrl, cinl, and cinl in Arabidopsis, have been characterized, but the wild-type genes have not been cloned and it is not known what part they play in cytokinin signaling. Despite these handicaps, progress is being made in the identification of cytokinin receptors. Two genes in Arabidopsis, CKIl and CREl, encode proteins that suggest that CK perception and signaling proceed via a two-component signaling system. Both proteins are similar to ETRl, an ethylene receptor, and combine features of a sensor protein and a response regulator. While the role of CKIl is still unclear, evidence for CREl being a cytokinin receptor is strong. Mutations in the CREl gene render plants or plant tissues nonresponsive to cytokinins, and expression of CREl in crel mutants reestores their responsiveness. Moreover, expression of CREl in a lethal mutant of yeast defective in osmosensing restores the mutant cells to normal growth if cytokinin is added to the medium. These data indicate that cytokinin perception in plant cells occurs extracellularly and that signaling proceeds via a phosphorelay. With the identification of CREl as a putative cytokinin receptor in Arabidopsis, an important breakthrough has been made. Future studies will undoubtedly focus on identifying elements in the signaling pathway, including relationships, if any, between CREl and proteins, such as CKIl, IBC6, and IBC, and on identification of proteins similar to CREl in other plants.

SECTION III. MOLECULAR BIOLOGY OF BRASSINOSTEROID (BR) ACTION FIGURE 24-22 Cytokinin-insensitive mutants of Arabidopsis. The mutants cinl and cin2 (in the wild ecotype WS background) and cin3 and cin4 (in Col background) in air, after cytokinin, and after ethylene treatment. Note that after cytokinin treatment, WT plants show a triple response, whereas mutants do not. In ethylene, all plants show the triple response. From Vogel et al. (1998).

1. BR-INDUCED RESPONSES Brassinosteroids play important and unique roles in extension growth of stems or petioles in dicot seedlings and in grass leaves. In dark-grown seedlings.

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IV. Molecular Basis of Hormone Action

exogenous application of BRs has little apparent effect, but BR synthesis mutants clearly show that BRs are needed for normal etiolated growth in the dark. These mutants display a partial light-grown habit, which indicates that BRs repress a precocious expression of photomorphogenesis in dark-grown seedlings or in seeds germinating under soil. BRs inhibit root growth. They also seem to have a role in flowering, pollen tube growth, tracheary cell differentiation, senescence, and expression of some stress-related genes.

2. BR-INDUCED GENE EXPRESSION Brassinosteroids have come to be recognized as hormones in their own right only since the early 1990s. Published studies on binding of radiolabeled BRs to cell fractions a n d / o r on brassinosteroidinduced gene expression are very few. A gene called BRUl (for brassinosteroid upregulated) was isolated from soybean {Glycine max) seedlings. The encoded protein shows sequence similarity to xyloglucan endotransglycosylases from other plant species (e.g., Arabidopsis), and the expressed protein shows XET activity in vitro. Gene encoding a putative p-tubulin subnit and an effector involved in actin polymerization are also reported to be induced However, the cis sequences and transcription factors regulating its expression are unknown.

3. BR RESPONSE M U T A N T S Several screens have been used to isolate brassinosteroid-insensitive mutants. All mutants identified to date are from Arabidopsis. Since brassinosteroids inhibit root growth, stimulate stem elongation in light, and suppress the expression of the photomorphogenic program in dark-grown seedlings, these features can be used to screen for BR-insensitive mutants. One mutant, bril (for brassinosteroid-insensitive), was identified by germinating EMS-mutagenized seeds in media supplemented with exogenous 24-epibrassinolide (10~^ or 10~^M) and screening for seedlings for lack of inhibition of root growth. Three ebb (for cabbage morphology) mutants were isolated by screening for the dwarf phenotype from a mutant collection generated by insertional mutagenesis. While two of the ebb mutants proved to be brassinolide synthesis mutants, one mutant, ebb2, could not be rescued by exogenous brassinolide and turned out to be a brassinosteroid-insensitive mutant. Still other mutants.

called bin (for brassinosteroid-insensitive), were identified by screening mutagenized seedlings grown in dark for expression of photomorphogenic program, i.e., a phenotype similar to that of det, eop mutants (see Chapter 26). Subsequently, a secondary screen separated the mutants isolated into those that could be rescued by applied brassinosteroids from those that could not be so rescued. The bin mutants, 18 in number, all were found to be alleles of the same gene, called binl. Moreover, binl turned out to be an allele of bril, which is also allelic to cbb2. In short, using three different mutant screens, only a single brassinosteroid signaling gene has been identified. Following the rules of nomenclature, the gene is BRU (or CBB2), with 20 mutant alleles. bril Is a recessive mutation with pleiotropic effects, which phenotypically resembles BR-deficient mutants, except that it cannot be rescued by an exogenous supply of brassinolide. Grown in darkness, mutant plants show short, thick hypocotyls, open, expanded cotyledons, development of primary leaf buds, and accumulation of anthocyanins. Grown in light the mutant is an extreme dwarf, less than one-tenth the height of the wild type (Fig. 24-23A). It has smaller, darker green leaves than the wild type, shows reduced apical dominance and enhanced lateral branching, and reduced male fertility. It also exhibits a delay in flowering and senescence. The bril mutation is specific to brassinosteroid signaling because the mutant plants retain their sensitivities to other hormones, lAA, cytokinins (benzyladenine and kinetin), GA3, and ABA (Fig. 24-23B). The Ika mutant in pea is another BR-insensitive mutant. It is a dwarf and, in contrast to Ikb, which is a BR synthesis mutant, is not rescued by application of brassinolide or castasterone (Fig. 24-24). Like some GA-insensitive mutants, both bril and Ika accumulate higher levels of endogenous BRs than the wild type, probably due to lack of feedback control over their synthesis. 3.1. Cloning and Sequencing of BRIl The BRIl gene encodes a large (Mr -^ 130 kDa) receptor-like kinase (RLK) (see Chapter 25). These kinases have an extracellular domain, a transmembrane domain, and an intracellular domain, which serves as a serine/threonine protien kinase. RLKs vary in their extracellular domains; some have several tandem copies of an amino acid sequence rich in leucine, known as leucine-rich repeats (LRRs). BRIl is somewhat unique in that the extracellular domain not only shows an extensive LRR repeat region, but also a 70 amino acid island between two LRRs; it also

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24. Molecular Biology of GAs, CKs, BRs, and JAs

heterodimers; the LRR repeat motif is believed to be involved in protein-protein interactions; and the intracellular kinase domain is thought to play a role in signal transduction. BRIl mRNA is expressed ubiquitously and constitutively in light- or dark-grown seedlings and throughout plant development. Several facts suggest that BRIl plays a role in BR signaling and that it is a receptor kinase. All 20 mutant alleles, using three different screening strategies, are mutations in a single gene, which suggests that BRIl is a component in the BRsignaling pathway. Second, the 70 amino acid island between two leucine-rich repeats in the extracellular region is unique for LRRs, and mutations in this island disrupt the functioning of BRIl. Third, in vitro binding studies have shown that the extracellular domain of

B

0.5 !iM lAA

0.5 [iM 0.5 |iM 0.5 |iM BA KINETIN ABA

0.5 |iM 100|aM GA3 GA3

FIGURE 24-23 The bril mutant of Arahidopsis and the effect of various hormones on root growth. (A) Phenotypes of a 2-month-old bril mutant (left) and a wild-type plant (middle). Also shown is a 4month-old mutant plant (right). The mutant is an extreme dwarf with small dark green leaves and enhanced lateral branching. Sacle, the fruits (siliques) in the wild type plant are ~ 1cm long. (B) Comparison of effects of various hormones on root growth in the wild type and the bril mutant. Plants were treated with IA A, benzyladenine (BA), kinetin, ABA, and GA3, all at 0.5 |JLM; and GA3 also at 100 jxM. Root length is plotted as percent of control, that is, root length in wild-type plants without any hormonal treatment. Note that there is little difference between the wild type and the mutant in their responses to lAA and cytokinins; there is a little more inhibition in the mutant by ABA, and a little less by GA. From Clouse et al. (1996).

shows a leucine zipper motif (Fig. 24-25). The leucine zipper motif suggests that the protein forms homo- or

.^^^->-

•

.

:

^

-

FIGURE 24-24 The Ika mutant of pea {Pisum sativum). The Ika plant (left, control) and 3 days after the application of 100 ng of brassinolide to the third leaf (right). From Nomura et al. (1997).

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IV. Molecular Basis of H o r m o n e Action

3.2. BR Interaction w i t h Other Hormones Leucine zipper

Leucine-rich repeats

extracellular region island

"r intracellular region

membrane

f

kinase domain

FIGURE 24-25 Schematic structure of BRIl in Arabidopsis. BRIl has an extracellular domain, which consists of a leucine zipper motif, followed by 25 tandem leucine-rich repeats (LRRs), with a 70 amino acid island between LRR 21 and 22. The extracellular domain is followed by a transmembrane span and finally by an intracellular serine/threonine kinase domain. Based on data in Li and Chory (1997).

BRIl binds brassinolide specifically, and the number of binding sites is proportional to the amount of BRIl protein. Moreover, treatment of Arabidopsis seedlings with brassinolide induces autophosphorylation of BRIl. Fourth and finally, the extracellular and membrane-spanning domain has been shown to initiate a signaling cascade which brings about a biological response. This domain was fused to the serine-threonine kinase domain of a receptor kinase known to be involved in plant defence reactions, and the construct was used to transform rice cell lines. The chimeric receptor initiated defence responses in rice cells upon treatment with brassinolide. The above data provide convincing proof that BRIl is a receptor kinase that transduces steroid signals across the plasma membrane. However, there are still some gaps in our knowledge. It is not known whether BRIl occurs as part of a receptor complex and, if so, the identity of the interacting partners. The downstream signaling components are also unknown.

A new mutant, saxl (for hypersensitive to abscisic acid and to auxin), of Arabidopsis was isolated in a screen for inhibition of root growth at auxin concentrations that are not normally inhibitory (~10nM NAA). The mutant later proved to be hypersensitive to ABA as well in root growth and stomatal closure (although not in other ABA-specific responses). The mutant also does not show GA-induced hypocotyl elongation growth in light, a significant difference from the other BR synthesis mutants (Fig. 24-26). It also seems insensitive to ethylene, as shown by a lack of response to added ACC. The addition of 24-epibrassinolide (10~^M in root; 10~^M in hypocotyl) to the growth medium restores the wild-type sensitivity in root growth to lAA and ABA and for hypocotyl growth to GA3. However, the sensitivity to ethylene is not restored by epi-BL. The SAXl locus has been identified by feeding intermediates and rescue experiments and appears to be a new locus in an alternate pathway of BR biosynthesis between campesterol and 6-deoxocathasterone (for the synthetic pathway, see Fig. 9-5 in Chapter 9). These results are interesting because they provide a link between BRs and auxin, ABA, GA, and ethylene signaling pathways. Since BRs restore a wildtype sensitivity to auxin, ABA, and GA3 in the growth of saxl seedlings, it appears that three different signaling pathways controlling cell elongation may be regulated by a fourth pathway involving BRs.

240 220 (0

200

WT sax1 WT + ep/-BR sax1 + epi-BR

180

o o o Q, JO

I

160 140 120 100 -80

10-6

-10-5

10-4

[GA3] (M) FIGURE 24-26 Hypocotyl growth in GAa-treated wild type Arabidopsis and saxl seedlings in light. Inhibition of growth in saxl seedlings in restored by the addition of epibrassinolide. From Ephritikhine et al (1999).

24. Molecular Biology of GAs, CKs, BRs, and JAs 3.3. BR Interaction w i t h Light Brassinosteroids are involved in the maintenance of the etiolated habit in dark-grown seedlings and also interact with red and blue light receptors. These topics are covered elsewhere (see Chapter 26, Section IV, 1.3).

4. SECTION S U M M A R Y In summary, relatively few genes have been shown so far to be transcriptionally induced by BRs, and cissequences and transcription factors involved in BRinduced gene expression are unknown. But our information on BR signaling has taken a quantum leap forward with the cloning and characterization of the BRIl gene. The BRIl gene encodes a transmembrane receptor kinase which binds brassinolide in its extracellular domain. Such binding results in autophosphorylation of the intracellular domain and likely sets up a signaling cascade which involves phosphogroup transfer via the histidine kinase domain. The receptor function of BRIl has been demonstrated, but it is not known whether it is part of a multimeric complex and what its downstream signaling components might be. The discovery of a novel BR synthesis mutant, saxl, indicates that BRs are involved in cross talk with signaling by auxins, ABA, and GA, but the nature and sites of such cross talk are obscure. BR signaling plays a role in maintenance of the etiolated phenotype in dark-grown seedlings; it also interacts with signaling by photoreceptors, such as phytochrome.

SECTION IV. MOLECULAR BIOLOGY OF JASMONATE (JA) ACTION 1. ROLES OF JAS Jasmonates play important roles in plant defenses against wounding caused by physical injury or chewing by insects, attack by microbial pathogens, and some abiotic stresses such as exposure to ozone. They are involved in the synthesis of vegetative storage proteins (VSPs), coiling of tendrils, fruit ripening, and acceleration of senescence and abscission of plant parts such as leaves. They are also reported to play a role in the production of viable pollen and in the formation of tubers, bulbs, and storage roots. Among the responses, just given, the role of jasmonates in

615

defense against wounding and microbial attack and the synthesis of VSPs have been the most studied, and most data on jasmonate-induced gene expression and signaling come from these sources.

2. DEFENSE-RELATED SIGNALING Since one of the major causes for jasmonate production is wounding or exposure to microbial elicitors, plant defense responses to pests and pathogens involving jasmonates are in fact a two-stage process. In the first stage, plants perceive the signal of wounding or microbial challenge and start a signaling cascade, which results in the synthesis of jasmonates via the octadecanoid pathway. In the second stage, jasmonates trigger the activation of defense-related genes (see Fig. 12-3 in Chapter 12). The perception of the wound signal or microbial elicitors in the first stage is still a mystery, but the subsequent events, i.e., the activation of prosystemin gene, cleavage and transport of systemin, its perception at the surface of target cells, and release of linolenic acid from cell membranes, are much better understood, mostly due to the efforts of Clarence Ryan at Washington State University, Pullman, WA, and associates. The second stage involves the perception of jasmonates by their receptor and start of the second signaling cascade, which ends in the transcription of defense-related genes. This part is mostly unknown, but a few pieces in the puzzle seem to be falling in place. This chapter is mostly concerned with the second stage, which deals with jasmonate signaling. For the first stage, which includes systemin signaling, the interested reader is referred to the reference section. It should be pointed out that transmission of the wound or elicitor signal to distant sites in the first stage proceeds extremely rapidly. Chapter 25 describes signaling events from cell surface receptors to gene expression and the roles of secondary messengers and protein kinases and phosphatases in signal transduction. It is sufficient to say here that a mitogenactivated protein kinase in Arabidopsis, called WIPK (wound-induced protein kinase), is induced within minutes of wounding and has been proposed to activate the enzymes involved in jasmonic acid biosynthesis. The perception of systemin by its receptor on the surface of target cells is also reported to start a signaling cascade, which includes ion transport, and an increase in intracellular Ca^^ and Ca^'^/calmodulin-activated protein kinases.

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IV. Molecular Basis of Hormone Action 3. JA-INDUCED GENE EXPRESSION

There are no hormone-binding studies using radiolabeled jasmonates. Expression of many genes, however, is known to be affected by jasmonates. Most of these genes encode defense-related proteins (see below); others encode enzymes involved in the synthesis of jasmonate or related compounds in the octadecanoid pathway (e.g., lipoxygenase, allene oxide synthase, and hydroperoxy lyase). Still others encode vegetative storage proteins (see Chapters 12 and 20). The senescence-related breakdown of chlorophyll involves removal of the phytol chain from the porphhyrin nucleus by an enzyme chlorophyllase (see Chapter 20). In Arahidopsis, two genes encoding chlorophyllase, AtCHLl and AtCHLZ, have been cloned and are differentially activated. Treatment with MeJA activates the transcription of AtCHLl, but not that of AtCHLl, which is constitutively expressed at low levels. ABA and jasmonates share some responses. Both can inhibit growth, inhibit seed germination, and promote senescence. Both are also known to induce some genes encoding seed storage proteins, as well as an oil body protein (oleosin) in Brassica napus. However, many ABA-regulated genes are not affected by JA (e.g., LEA genes). The common ice plant {Mesembryanthemum crystallinum), a succulent, shows Crassulacean acid metabolism (CAM) and expresses one specific isoform of phosphoenolpuryvate carboxylase. Induction of the gene encoding this isoform (Ppcl) is regulated by ABA, not by JA. The plant response to biotic stresses involves complex interactions between a variety of signals. For example, defense responses to attack by pests and pathogens are mediated via ABA, ethylene, and jasmonate. Responses to pathogens are also mediated via salicylic acid. To separate which genes are regulated by which hormone, use is made of synthesis inhibitors a n d / o r synthesis mutants for specific hormones. A comprehensive study on regulation of the leucine aminopeptidase (LapA) gene in tomato {Lycopersicon esculentum) by jasmonates, ABA, and salicylic acid utilized some of these techniques. It was shown that LapA is induced by jasmonates and ABA (although not by water stress or salinity) and that the two hormones act synergistically. The induction of proteinase inhibitor 2 {pin!) gene, in contrast, required jasmonate but not exogenous ABA. The Le4 gene in tomato encodes a dehydrin; its expression is upregulated by ABA, water deficit, and salinity, but not JA. Finally, the genes for two pathogenesis-related proteins in tomato, PR1 (pathogenesis-related 1) and GluB (basic (3-1, 3-glu-

canase), were induced by jasmonate independently of systemin or wounding; also, their induction did not require exogenous ABA. Among the PR genes in Arahidopsis, some are regulated by salicylic acid independently of JA, whereas others are regulated by JA independently of salicylic acid. Moreover, the two pathways act antagonistically to each other. Arabidopsis plants infected with the fungus Alternaria bmssicicola or healthy plants treated with exogenous MeJA, but not salicylic acid, show the induction of the PDF1.2 gene, which encodes an antifungal peptide belonging to the family of plant defensins. However, PDF1.2 expression is not under the exclusive control of jasmonates; it requires the simultaneous availability of ethylene. In mutants that are defective in the synthesis of jasmonates or the response pathway of either hormone the gene is not expressed. Many chitinases are induced by ethylene or jasmonates independently. For example, the BjCHI (for Brassica juncea chitinase) gene is induced by wounding and by JA, but not by ABA, ethylene, or salicylic acid. There is also evidence for antagonism between ABA and JA signaling. For instance, JA-induced accumulation of PR proteins in rice (Oryza sativa) roots is negatively regulated by ABA, whereas JA negatively affects ABA-induced transcript accumulation of the LEA3 gene in rice {OsLEA3). In summary, defense-related genes are regulated by a variety of signals, some genes are regulated exclusively by one signal (e.g., JA induction of some wound-specific PIN genes) and others by more than one signal (e.g., PDF1.2 gene). These results highlight the complexities of plant responses to biotic stresses and indicate that there must be extensive cross talk among signaling pathways of jasmonates, ABA, ethylene, and salicylic acid. As explained in Chapter 23, the responses to abiotic stresses (e.g., water deficit, salinity) are mediated separately and jointly by the stress and by ABA. Jasmonate-induced gene expression also occurs in responses to abiotic stresses, such as exposure to salinity or ozone, but the details of such gene regulation are unknown. Although several genes are now known to be induced by jasmonates, the cis elements in promoters of these genes and transcription factors that bind to them are mostly known. A G-box sequence (CACGTG) has been identified in the promoters of the PinZ gene in tomato and the VSPp> gene in soybean, but it is unclear whether it is able to confer induction by jasmonates. For the VSP gene in soybean, other cis elements responsive to nutritional factors, such as carbon or phosphorus availability, seem to act in concert with the G box. Transcription factors binding to cis elements in

24. Molecular Biology of GAs, CKs, BRs, and JAs the promoters have not been identified. Considering that some promoters have G boxes, it is possible that bZIP proteins bind to these sequences.

4. JA RESPONSE M U T A N T S Four jasmonate-insensitive mutants are known from Arabidopsis. Three of them, jarl (for jasmonate resistant), jinl, and jin4 (for jasmonate-insensitive), were isolated from mutagenized seeds grown in the presence of concentrations of jasmonic acid (~ 10 mM) that inhibit root growth. A fourth mutant, coil (for coronatine insensitive), was identified by its resistance to coronatine, a chlorosis-inducing bacterial toxin from Pseudomonas syringae, which is similar in structure and activity to jasmonic acid. Both coronatine and JA inhibit root growth, cause anthocyanin production, and stimulate the expression of a gene encoding a VSP. All four mutants are single gene recessives and respond to exogenous ethylene and ABA like the wild-type plants; hence, they are not mutants in either ethylene or ABA response pathways, but they differ in the range of their insensitivities to exogenous jasmonates. jinl and ;m4 show insensitivity to jasmonates in root growth, but produce fertile flowers and accumulate JA-induced VSPs in floral structures. The jarl also fails to accumulate two VSPs similar to VSPa/p in soybean when sprayed with 50 |JLM MeJA. The coil mutant shows the most pleiotropic effects. It is phenotypically similar to the jasmonate-deficient triple fad (fatty acid desaturase) mutant (fad 3-2 fad 7-2 fad 8) of Arabidopsis. The triple fad mutant is unable to synthesize linolenic acid, the precursor fatty acid for jasmonate biosynthesis (see Chapter 12). The mutant has little defense against herbivorous insects; it also produces nonviable pollen. However, both protection against insect pest and pollen sterility are rescued by an exogenous application of jasmonates. The coil mutant also shows a decreased resistance to insect attack, a reduced response to wound damage, and produces nonviable pollen, but the defects are not cured by exogenous jasmonates. Hence, the COIl gene is believed to function in the JA signaling pathway. Moreover, the pleiotropic nature of the coil mutation suggests that it may be defective in an early step in jasmonate perception or signaling. The coil gene was cloned using a map-based strategy. The encoded COIl is a cytosolic protein because it lacks any domains, suggesting a signal peptide or membrane-spanning domains. It shows two significant motifs, a F box and 16 leucine-rich repeats (LRRs). Both LRR and F box occur in some proteins

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in yeast and mammals and are involved in proteinprotein interactions and recruitment of targeted proteins for polyubiquitination and eventual proteolysis. A similar protein, TIRl, is known from Arabidopsis and has been proposed to recruit repressors of auxin signaling for proteolytic degradation (see Fig. 22-15, Chapter 22). Occurrence of the F box and LRR motifs in two proteins, which on genetic evidence are involved in jasmonate or auxin signaling, suggests that selective ubiquitination of repressor proteins may be a more common theme in plant signaling than hitherto realized. The target proteins for recruitment by COIl or other components with which COIl interacts are unknown. Recently, a loss-of-function recessive mutation, cevl (for constitutive expression of VSPl) was obtained by targeted genetics. The promoter of the JA-induced VSPl gene of Arabidopsis was fused to the coding sequence of LUCIFERASE reporter gene, and the construct used to transform Arabidopsis seedlings. The progeny seeds were mutagenized, and the mutant plants screened for luciferase activity without treatment with exogenous methyl jasmonate. The cevl seedlings show a constitutive expression of both JAinduced and ethylene-induced phenotypes. The plants were stunted, had short roots, an excess of root hairs (phenotypes typical of ethylene-treated seedlings); they also accumulated anthocyanins and expressed JA-induced VSPl and VSP2 genes constitutively. They also showed a constitutive expression of two genes, PDFl-2 and CHITINASE-B, which are regulated by both JA and ethylene. CEVl, therefore, seems to function at an early step in both signaling pathways.

5. SECTION SUMMARY Although jasmonates are clearly involved in several morphogenetic as well as defense responses in plants, there is still very little known about the molecular basis of their action. Jasmonates induce the expression of several genes, some independently and some in combination with either ABA or ethylene. Some genes induced by jasmonates are inhibited by ABA or salicylic acid. The existence of separate (or parallel) and cross signaling pathways among four hormones, jasmonates, ABA, ethylene, and salicylic acid, and possibly some abiotic factors, such as salinity or water stress, makes the elucidation of jasmonate signaling difficult and challenging. Some cis elements responsive to JA have been reported, but the details of regulation, including transcription factors that bind to them, are unknown. A pleiotropic mutant, coil, that

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shows insensitivity to jasmonates in several responses has been described. The COIl gene encodes a protein that seems to be involved in the recruitment of repressor proteins targeted for proteolysis. A similar protein is also known for auxin signaling. The identity of proteins that repress jasmonate signaling is unknown; also unknown are the proteins that interact with COIl. Another mutant, cevl, defines a locus that is common to both JA and ethylene signaling. References SECTION I. MOLECULAR BIOLOGY OF GIBBERELLIN (GA) ACTION Book and Reviews Harberd, N. P., King, K. E., Carol, P., Cowling, R. J., Peng, J., and Richards, D. E. (1998). Gibberellin: Inhibitor of an inhibitor of . . . . ? BioEssays 20, 1001-1008. Hooley, R. (1994). Gibberellins: Perception, transduction and responses. Plant Mol. Biol. 26, 1529-1555. Jacobsen, J. V., Gubler, P., Chandler, P. M. (1995). Gibberellin action in germinated cereal grains. In "Plant Hormones, Physiology, Biochemistry and Molecular Biology" (P. J. Davies, ed.) 2nd Ed., pp. 246-271. Kluwer, Boston. Lovegrove, A., and Hooley, R. (2000). Gibberellin and abscisic acid signalling in aleurone. Trends Plant Sci. 5, 102-110. Richards, D. E., King, K. E., Ait-ali, T., and Harberd, N. P. (2001). How gibberellin regulates plant growth and development: A molecular genetic analysis of gibberellin signaling. Annu. Rev. Plant Physiol Plant Mol. Biol. 52, 67-88. Sun, T.-p. (2000). Gibberellin signal transduction. Curr. Opin. Plant Biol. 3, 374-380. Swain, S. M., and Olszewski, N. E. (1996). Genetic analysis of gibberellin signal transduction. Plant Physiol. Ill, 11-17. 2. GA-Binding Proteins Hooley, R., Smith, S. J., Beale, M. H., and Walker, R. P. (1993). In vivo photoaffinity labelling of gibberellin-binding proteins in Avena fatua aleurone. Aust. J. Plant Physiol. 20, 573-584. Keith, B., Brown, S., and Srivastava, L. M. (1982). In vitro binding of gibberellin A4 to extracts of cucumber measured by using DEAEcellulose filters. Proc. Natl. Acad. Sci. USA 79, 1515-1519. Keith, B., and Rappaport, L. (1987). In vitro gibberellin Ai binding in Zea mays. Plant Physiol. 85, 934-941. Lovegrove, A., Barratt, D. H. P., Beale, M. H., and Hooley, R. (1998). Gibberellin photoaffinity labelling of two polypeptides in plant plasma membrane. Plant J. 15, 311-320. Nakajima, M., Takita, K., Wada, H., Mihara, K., Hasegawa, M., Yamaguchi, I., and Murofushi, N. (1997). Partial purification and characterization of a gibberellin-binding protein from seedlings of Azukia angularis. Biochem. Biophys. Res. Commun. 241, 782-786. Stoddart, J. L., Breidenbach, W. R., and Rappaport, L. (1974). Selective binding of [^Hjgibberellin Ai by protein fractions from dwarf pea epicotyls. Proc. Natl. Acad. Sci. USA 71, 3255-3259. 3. Site of GA Perception Ashikari, M., Wu, J., Yano, M., Sasaki, T., and Yoshimura, A. (1999). Rice gibberellin-insensitive dwarf mutant gene Dzuarfl encodes the a-subunit of GTP-binding protein. Proc. Natl. Acad. Sci. USA 96, 10284-10289.

Gilroy, S., and Jones, R. L. (1994). Perception of gibberellin and abscisic acid at the external face of the plasma membrane of barley (Hordeum vulgare L.) aleurone protoplasts. Plant Physiol. 104, 1185-1192. Hooley, R., Beale, M. H., and Smith, S. J. (1991). Gibberellin perception at the plasma membrane oi Avena fatua aleurone protoplasts. Planta 183, 274-280. Jones, H. D., Smith, S. J., Desikan, R., Plakidou-Dymock, S., Lovegrove, A., and Hooley, R. (1998). Heterotrimeric G proteins are implicated in gibberellin induction of a-amylase gene expression in wild oat aleurone. Plant Cell 10, 245-253. Mitsunaga, S., Tashiro, T. and Yamaguchi, J. (1994). Identification and characterization of gibberellin-insensitive mutants selected from among dwarf mutants of rice. Theor. Appl. Genet. 87,705-712. van der Knaap, E., Song, W.-Y., Ruan, D.-L., Sauter, M., Ronald, P. C , and Kende, H. (1999). Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase. Plant Physiol. 120, 559-569. 4. GA-Induced Gene Expression Cercos, M., Gomez-Cadenas, A., and Ho, T.-D. (1999). Hormonal regulation of a cysteine proteinase gene, EPB-1, in barley aleurone layers: cis- and trans-acting elements involved in the coordinated gene expression regulated by gibberellins and abscisic acid. P/flnf 7.19,107-118. Gubler, F., Kalla, R., Roberts, J. K., and Jacobsen, J. V. (1995). Gibberellin-regulated expression of a tnyb gene in barley aleurone cells: Evidence for myb transactivation of a high-pl a-amylase gene promoter. Plant Cell 7, 1879-1891. Gubler, F., Raventos, D., Keys, M., Watts, R., Mundy, J., and Jacobsen, J. V. (1999). Target genes and regulatory domains of the GAMYB transcriptional activator in cereal aleurone. Plant ]. 17,1-9. Huttly, A. K., and Phillips, A. J. (1995). Gibberellin-regulated plant genes. Physiol. Plant 95, 310-317. Huttiy, A. K., Phillips, A. L., and Tregear, J. W. (1992). Localization of cis elements in the promoter of a wheat a-amyl gene. Plant Mol. Biol. 19, 903-911. Jacobsen, S. E., and Olszewski, N. E. (1996). Gibberellins regulate the abundance of RNAs with sequence similarity to proteinase inhibitors, dioxygenases and dehydrogenases. Planta 198, 78-86. Lanahan, M. B., Ho, T.-H. D., Rogers, S. W., and Rogers, J. C. (1992). A gibberellin response complex in cereal a-amylase gene promoters. Plant Cell 4, 203-211. Raventos, D., Skriver, K, Schlein, M, Karnahl, K, Rogers, S. W. Rogers, J. and C, Mundy, J. (1998). HRT, a novel zinc finger, transcriptional repressor from barley. /. Biol Chem. 273, 23313-23320. Rushton, P. J., Hooley, R., and Lazarus, C. M. (1992). Aleurone nuclear proteins bind to similar elements in the promoter regions of two gibberellin-regulated a-amylase genes. Plant Mol Biol 19, 891-901. Rushton, P. J., MacDonald, H., Huttly, A. K., Lazarus, C. M., and Hooley, R. (1995). Members of a new family of DNA-binding proteins bind to a conserved c/s-element in the promoters of a-Amy2 genes. Plant Mol Biol 29, 691-702. Tanida, I., Kim, J.-K., and Wu, R. (1994). Functional dissection of a rice high pl a-amylase gene promoter. Mol. Gen. Genet. 244, 127-134. Weiss, D., van Tunen, A. J., Halevy, A. H., Mol, J. N. M., and Gerats, A. G. M. (1990). Stamens and gibberellic acid in the regulation of flavonoid gene expression in the corolla of Petunia hybrida. Plant Physiol 94, 511-515. Wilmott, R. L., Rushton, P. J., Hooley, R., and Lazarus, C. M. (1998). DNasel footprints suggest the involvement of at least three types

24. Molecular Biology of GAs, CKs, BRs, and JAs of transcription factors in the regulation of a-Amy!/A by gibberellin. Plant Mol Biol. 38, 817-825. 5. GA Response Mutants Chandler, P. M. (1988). Hormonal regulation of gene expression in the ''slender" mutant of barley {Hordeum vulgare L.). Planta 175, 115-120. Croker, S. J., Hedden, P., Lenton, J. R., and Stoddart, J. L. (1990). Comparison of gibberellins in normal and slender barley seedlings. Plant Physiol. 94, 194-200. Foster, C. A. (1977). Slender: An accelerated extension growth mutant of barley. Barley Genet Newslett 7, 24-27. Fu, X., Sudhakar, D., Peng, J., Richards, D. E., Christou, P., and Harberd, N. P. (2001). Expression of Arabidopsis GAI in transgenic rice represses multiple gibberellin responses. Plant Cell 13, 1791-1802. Gale, M. D., and Yousefian, S. (1985). Dwarfing genes in wheat. In "Progress in Plant Breedin - 1" (G. E. Russell, ed.), pp. 1-35. Butterworth & Co., London. Harberd, N. P., and Freeling, M. (1989). Genetics of dominant gibberellin-insensitive dwarfism in maize. Genetics 111, 827-838. Ikeda, A., Ueguchi-Tanaka, M., Sonoda, Y., Kitano, H., Koshioka, M., Futsuhara, Y., Matsuoka, M., and Yamaguchi, J. (2001). slender Rice, a constitutive gibberellin response mutant, is caused by a null mutation of the SLNl gene, an ortholog of the heightregulating gene GAI/RGA/RHT/D8. Plant Cell 13, 999-1010. Jacobsen, S. E., Binkowski, K. A., and Olszewski, N. E. (1996). SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis. Proc. Natl. Acad. Sci. USA 93, 9292-9296. Jones, M. G. (1987). Gibberellins and the procera mutant of tomato. Planta 172, 280-284. Koornneef, M., Elgersma, A., Hanhart, C. J., van Loenen-Martinet, E. P., van Rijn, L., and Zeevaart, J. A. D. (1985). A gibberellin insensitive mutant of Arabidopsis thaliana. Physiol. Plant 65, 33-39, Lanahan, M. B., and Ho, D. T.-H. (1988). Slender barley: A constitutive gibberellin-response mutant. Planta 175, 107-114. Ogas, J., Cheng, J.-C, Sung, Z. R., and Somerville, C. (1997). Cellular differentiation regulation by gibberellin in the Arabidopsis thaliana pickle mutant. Science 277, 91-93. Peng, J., Carol, P., Richards, D. E., King, K. E., Cowling, R. J., Murphy, G. P., and Harberd, N. P. (1997). The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses. Genes Dev. 11, 3194-3205. Peng, J., Richards, D. E., Hartley, N. M., Murphy, G. P., Devos, K. M., Flintham, J. E., Beales, J., Fish, L. J., Worland, A. J., Pelica, F., Sudhakar, D., Christou, P., Snape, J. W., Gale, M. D., and Harberd, N. P. (1999). "Green revolution" genes encode mutant gibberellin response modulators. Nature 400, 256-261. Peng, J., Richards, D. E., Moritz, T., Cano-Delgado, A., and Harberd, N. P. (1999). Extragenic suppressors of the Arabidopsis gai mutation alter the dose-response relationship of diverse gibberellin responses. Plant Physiol. 119, 1199-1207. Potts, W. C , Reid, J. B., and Murfet, I. C. (1985). Internode length in Pisum: Gibberellins and the slender phenotype. Physiol Plant 63, 357-364. Pysh, L. D., Wysocka-Diller, J. W., Camilleri, C , Bouchez, D., and Benfey, P. N. (1999). The GRAS family in Arabidopsis: Sequence characterization and basic expression analysis of the SCARECROW-like genes. Plant]. 18,111-119. Robertson, M., Swain, S. M., Chandler, P. M., and Olszewski, N. E. (1998). Identification of a negative regulator of gibberellin actin, HvSPY, in barley. Plant Cell 10, 995-1007.

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Silverstone, A. L., Ciampaglio, C N., and Sun, T.-P. (1998). The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway. Plant Cell 10,155-169. Silverstone, A. L., Mak, P. Y. A., Martinez, E. C , and Sun, T.-P. (1997). The new RGA locus encodes a negative regulator of gibberellin response in Arabidopsis thaliana. Genetics 146,1087-1099. Steber, C. M., Cooney, S. E., and McCourt, P. (1998). Isolation of the GA-response mutant slyl as a suppressor of ABIl-1 in Arabidopsis thaliana. Genetics 149, 509-521. Suttle, J. C , and Zeevaart, J. A. D. (1979). Stem growth, flower formation, and endogenous gibberellins in a normal and dwarf strain of Silene armeria. Planta 145,175-180. Swain, S. M., Tseng, T.-s., and Olszewski, N. E. (2001). Altered expression of SPINDLY affects gibberellin response and plant development. Plant Physiol. 126, 1174-1185. Thornton, T. M., Swain, S. M., and Olszewski, N. E. (1999). Gibberellin signal transduction presents the SPY who O-GlcNAc'd me. Trends Plant Sci. 4, 424-428. Wilson, R. N., and Somerville, C. R. (1995). Phenotypic suppression of the gibberellin-insensitive mutant {gai) of Arabidopsis. Plant Physiol. 108, 495-502. 7. GA/ABA Interaction Gomez-Cadenas, A., Verhey, S. D., Holappa, L. D., Shen, Q., Ho, T.-H. D., and Walker-Simmons, M. K. (1999). An abscisic acidinduced protein kinase, PKABAl, mediates abscisic acid-suppressed gene expression in barley aleurone layers. Proc. Natl. Acad. Sci. USA 96, 1767-1772. Shen, W., Gomez-Cadenas, A., Routly, E. L., Ho, T.-H. D., Simmonds, J. A., and Gulick, P. J. (2001). The salt stress-inducible protein kinase gene, Esi47, from the salt-tolerant wheatgrass Lophopyrum elongatum is involved in plant hormone signaling. Plant Physiol. 125, 1429-1441. 8. GA Interaction with Environmental Factors

See Chapter 26. SECTION II. MOLECULAR BIOLOGY OF CYTOKININ (CK) ACTION 2. CK-Binding Proteins Brault, M., Maldiney, R., and Miginiac, E. (1997). Cytokinin-binding proteins. Physiol. Plant 100, 520-527. Brinegar, A. C , Cooper, G., Stevens, A., Hauer, C. R., Shabanowitz,}., et al. (1988). Characterization of a benzyladenine binding-site peptide isolated from a wheat cytokinin-binding protein: Sequence analysis and identification of a single affinity-labeled residue by mass spectrometry. Proc. Natl Acad. Sci. USA 85,5927-5931. Hamaguchi, N., Iwamura, J., and Fujita, T. (1985). Fluorescent anticytokinins as a probe for binding. Eur. J. Biochem. 153, 565-572. Mitsui, S., Wakasugi, T., and Sugiura, M. (1993). A cDNA encoding the 57 kDa subunit of a cytokinin-binding protein complex from tobacco: The subunit has high homology to S-adenosyl-L-homocysteine hydrolase. Plant Cell Physiol 34,1089-1096. Mitsui, S., Wakasugi, T., Hanano, S., and Sugiura, M. (1997). Localization of a cytokinin-binding protein CBP57 / S-adenosyl-L-homocysteine hydrolase in a tobacco root. /. Plant Physiol 150,752-754. Momotani, E., and Tsuji, H. (1992). Isolation and characterization of a cytokinin-binding protein from the water-soluble fraction of tobacco leaves. Plant Cell Physiol. 33, 407-412. Nagata, R., Kawachi, E., Hashimoto, Y., and Shudo, K. (1993). Cytokinin-specific binding protein in etiolated mung bean seedlings. Biochem. Biophys. Res. Commun. 191, 543-549.

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IV. Molecular Basis of H o r m o n e Action

Nogue, F., Mornet, R., and Laloue, M. (1995). Specific photoaffinity of a thylakoid membrane protein with an azido cytokinin agonist. Plant Growth Regiil 18, 51-58. Polya, G. M., and Davis, A. W. (1978). Properties of a high-affinity cytokinin-binding protein from wheat germ. Planta 139,139-147. Romanov, G. A., Taran, V. Y., and Venis, M. A. (1990). Cytokininbinding protein from maize shoots. /, Plant Physiol 136, 208-212. 3. CK-Induced Gene Expression Brandstatter, L, and Kieber, J. J. (1998). Two genes with similarity to bacterial response regulators are rapidly and specifically induced by cytokinin in Arabidopsis. Plant Cell 10,1009-1019. Chen, C.-M., Jin, G., Anderson, B. R., and Ertl, J. (1993). Modulation of plant gene expression by cytokinins. Aiist. J. Plant Physiol. 20, 609-619. Crowell, D. N., and Amasino, R. M. (1994). Cytokinins and plant gene regulation. In "Cytokinins: Chemistry, Activity, and Function" (D. W. S. Mok and M. C , Mok, eds.), pp. 233-242. CRC Press, Boca Raton, FL. Downes, B. P., and Crowell, D. N. (1998). Cytokinin regulates the expression of a soybean p-expansin gene by a posttranscriptional mechanism. Plant Mol. Biol. 37, 437-444. Lu, J.-L., Ertl, J. R., and Chen, C.-M. (1990). Cytokinin enhancement of the light induction of nitrate reductase transcript levels in etiolated barley leaves. Plant Mol. Biol. 14, 585-594. Schmiilling, T., Schafer, S., and Romanov, G. (1997). Cytokinins as regulators of gene expression. Physiol. Plant 100, 505-519. 4. CK Response mutants Baskin, T. I., Cork, A., Williamson, R. E., and Gorst, J. R. (1995). STUNTED PLANT 1, a gene required for expansion in rapidly elongating but not in dividing cells and mediating root growth responses to applied cytokinin. Plant Physiol, 107, 233-234. Deikmen, J., and Ulrich, M. (1995). A novel cytokinin-resistant mutant of Arabidopsis with abbreviated shoot development. Planta 195, 440-449. Faure, J. D., Jullien, M., and Caboche, M. (1994). zea3: A pleiotropic mutation affecting cotyledon development, cytokinin resistance and carbon-nitrogen metabolism. Plant J. 5, 481-491. Inoue, T., Fliguchi, M., Hashimoto, Y., Seki, M., Kobayashi, M., Kato, T., Tabata, S., Shinozaki, K., and Kakimoto, T. (2001). Identification of CREl as a cytokinin receptor from Arabidopsis. Nature 409, 1060-1063. Kakimoto, T. (1996). CKIl, a histidine kinase homolog implicated in cytokinin signal transduction. Science 274, 982-985. Martin, T., Sotta, B., Jullien, M., Caboche, M., and Faure, J. D. (1997). ZEA3: A negative modulator of cytokinin responses in plant seedlings. Plant Physiol. 114, 1177-1185. Miklashevichs, E., and Walden, R. (1997). Plant mutants with altered responses to cytokinins. Physiol. Plant 100, 528-533. Ueguchi, C , Sato, S., Kato, T., and Tabata, S. (2001). The AHK4 gene involved in the cytokinin-signaling pathway as a direct receptor molecule in Arabidopsis thaliana. Plant Cell Physiol. 42, 7b\-755. Vogel, J. P., Schuerman, P., Woeste, K., Brandstatter, I., and Kieber, J. J. (1998). Isolation and characterization of Arabidopsis mutants defective in the induction of ethylene biosynthesis by cytokinin. Genetics 1^9 A17-All. SECTION III. MOLECULAR BIOLOGY OF BRASSINOSTEROID (BR) ACTION Reviews Bishop, G. J., and Yokota, T. (2001). Plant steroid hormones, brassinosteroids: Current highlights of molecular aspects on their syn-

thesis/metabolism, transport, perception, and response. Plant Cell Physiol. 42, 114-120. 2. BR-Induced Gene Expression Oh, M.-H., Romanow, W. G., Smith, R. C , Zamski, E., Sasse, J., and Clouse, S. D. (1998). Soybean BRUl encodes a functional xyloglucan endotransglycosylase that is highly expressed in inner epicotyl tissues during brassinosteroid-promoted elongation. Plant Cell Physiol. 39, 124-130. Zurek, D. M., and Clouse, S. D. (1994). Molecular cloning and characterization of a brassinosteroid-regulated gene from elongating soybean {Glycine max L.) epicotyls. Plant Physiol. 104, 161-170. 3. BR Response Mutants Altmann, T. (1998). A tale of dwarfs and drugs: Brassinosteroids to the rescue. Trends Genet. 14, 490-495. Clouse, S. D., Langford, M., and McMorris, T. C. (1996). A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development. Plant Physiol. I l l , 671-678. Ephritikhine, G., Fellner, M., Vannini, C , Lapous, D., and BarbierBrygoo, H. (1999). The saxl dwarf mutant of Arabidopsis thaliana shows altered sensitivity of growth responses to abscisic acid, auxin, gibberellins and ethylene and is partially rescued by exogenous brassinosteroids. Plant f. 18, 303-314. Ephritikhine, G., Pagant, S., Fujioka, S., Takatsuto, S., Lapous, D., Caboche, M., Kendrick, R. E., and Barbier-Brygoo, H. (1999). The saxl mutation defines a new locus involved in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana. Plant J. 18, 315-320. He, Z., Wang, Z.-Y., Li, J., Zhu, Q., Lamb, C , Ronald, P., and Chory, J. (2000). Perception of brassinosteroids by the extracellular domain of the receptor kinase BRIl. Science 288, 2360-2363. Kauschmann, A., Jessop, A., Koncz, C , Szekeres, M., Willmitzer, L., and Altmann, T. (1996). Genetic evidence for an essential role of brassinosteroids in plant development. Plant J. 9, 701-713. Li, J., and Chory, J. (1997). A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90, 929-938. Nomura, T., Kitasaka, Y., Takatsuto, S., Reid, J. B., Fukami, M., and Yokota, T. (1999). Brassinosteroid/sterol synthesis and plant growth as affected by Ika and Ikb mutations of pea. Plant Physiol. 119, 1517-1526. Nomura, T., Nakayama, M., Reid, J. B., Takeuchi, Y., and Yokota, T. (1997). Blockage of brassionsteroid biosynthesis and sensitivity causes dwarfism in garden pea. Plant Physiol. 113, 31-37. Schumacher, K., and Chory, J. (2000). Brassinosteroid signal transduction: Still casting the actors. Curr. Opin. Plant Biol. 3, 79-84. Wang, Z.-Y., Seto, H., Fujioka, S., Yoshida, S., and Chory, J. (2001). BRIl is a critical component of a plasma-membrane receptor for plant steroids. Nature 410, 380-383. SECTION IV. MOLECULAR BIOLOGY OF JASMONATE (JA) ACTION 2. Defense-Related Signaling Bergey, D. R., and Ryan, C. A. (1999). Wound- and systemin-inducible calmodulin gene expression in tomato leaves. Plant Mol. Biol. 40, 815-823. Blechert, S., Brodschelm, W., Holder, S., Kammerer, L., Kutchan, T. M., Mueller, M. J., Xia, Z.-Q., and Zenk, M. H. (1995). The octadecanoic pathway: Signal molecules for the regulation of secondary pathways. Proc. Natl. Acad. Sci. USA 92, 4099-4105.

24. Molecular Biology of G A s , CKs, BRs, a n d JAs Bogre, L., Ligterink, W., Meskiene, 1., Barker, P. J., Heberle-Bors, E., Huskisson, N. S., and Hirt, H. (1997). Wounding induces the rapid and transient activation of a specific MAP kinase pathway. Plant Cell 9, 75-83. Farmer, E. E. (1994), Fatty acid signalling in plants and their associated microorganisms. Plant Mol Biol. 26,1423-1437. Farmer, E. E., and Ryan, C. A. (1992). Octadecanoid precursors of jasmonic acid activate the synthesis of wound-inducible proteinase inhibitors. Plant Cell 4, 129-134. Heitz, T., Bergey, D. R., and Ryan, C. A. (1997). A gene encoding a chloroplast-targeted lipoxygenase in tomato leaves is transiently induced by wounding, systemin, and methyl jasmonate. Plant Physiol. 114, 1085-1093. Ryan, C. A. (1990). Protease inhibitors in plants: Genes for improving defenses against insects and pathogens. Annu. Rev. Phytopathol. 28, 425-449. Schaller, A., and Ryan, C. A. (1996). Systemin: A polypeptide defense signal in plants. Bioessays 18, 27-33. Scheer, J. M., and Ryan, C. A. (1999). A 160-kD systemin receptor on the surface of Lycopersicon peruvianum suspension-cultured cells. Plant Cell 11,1525-1535. Seo, S., Sano, H., and Ohashi, Y. (1999). Jasmonate-based wound signal transduction requires activation of WIPK, a tobacco mitogen-activated protein kinase. Plant Cell 11, 289-298. Wildon, D. C , Thain, J. F., Minchin, P. E. H., Gubb, I. R., Reilly, A. J., Skipper, Y. D., Doherty, H. M., O^Donnell, P. J., and Bowles, D. J. (1992). Electrical signaling and systemic proteinase inhibitor induction in the wounded plant. Nature 360, 62-65. 3. JA-Induced Gene Expression Berger, S., Bell, E., Sadka, A., and Mullet, J. E. (1995). Arabidopsis thaliana AtVsp is homologous to soybean VspA and VspB, genes encoding vegetative storage protein acid phosphatases, and is regulated similarly by methyl jasmonate, wounding, sugars, light and phosphate. Plant Mol. Biol. 27, 933-942. Chao, W. S., Gu, Y.-Q., Pautot, V., Bray, E. A., and Walling, L. L. (1999). Leucine aminopeptidase RNAs, proteins, and activities increase in response to water deficit, salinity, and the wound signals systemin, methyl jasmonate, and abscisic acid. Plant Physiol. 120, 979-992. Mason, H. S., DeWald, D. B., and Mullet, J. E. (1993). Identification of a methyl jasmonate-responsive domain in the soybean VspB promoter. Plant Cell 5, 241-251. Moons, A., Prinsen, E., Bauw, G., and Van Montagu, M. (1997). Antagonistic effects of abscisic acid and jasmonates on salt stress-inducible transcripts in rice roots. Plant Cell 9, 2243-2259. Pena-Cortes, H., and Wilmitzer, L. (1995). The role of hormones in gene activation in response to wounding. In ''Plant Hormones: Physiology, Biochemistry and Molecular Biology'' (P. J. Davies, ed.), 2nd Ed., pp. 395-^14. Kluwer, Dordrecht.

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Penninckx, I. A. M. A., Thomma, B. P. H. J., Buchala, A., Metraux, J.P., and Broekaert, W. F. (1998). Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell 10, 2103-2113. Reinbothe, S., Reinbothe, C , Lehman, J., Becker, W., Apel, K., and Parthier, B. (1994). JIP60, a methyl jasmonate-induced ribosomeinactivating protein involved in plant stress reactions. Proc. Natl. Acad. Sci. USA 91, 7012-7016. Thomma, B. P. H. J., Eggermont, K., Penninckx, L A. M. A., MauchMani, B., Vogelsang, R., Cammue, B. P. A., and Broekaert, W. F. (1998). Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc. Natl. Acad. Sci. USA 95, 15107-15111. Tsuchiya, T., Ohta, H., Okawa, K., Iwamatsu, A., Shimada, H., Masuda, T., and Takamiya, K.-L (1999). Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: Finding of a lipase motif and the induction by methyl jasmonate. Proc. Natl. Acad. Sci. USA 96,15362-15367. Wasternack, C , and Parthier, B. (1997). Jasmonate-signalled plant gene expression. Trends Plant Sci. 2, 302-307. Zhao, K. J., and Chye, M. L. (1999). Methyl jasmonate induces expression of a novel Brassica juncea chitinase with two chitinbinding domains. Plant Mol. Biol. 40, 1009-1018.

4. JA Response Mutants Benedetti, C. E., Xie, D., and Turner, J. G (1995). COIl-dependent expression of an Arabidopsis vegetative storage protein in flowers and siliques and in response to coronatine or methyl jasmonate. Plant Physiol. 109, 567-572. Berger, S., Bell, E., and Mullet, J. E. (1996). Two methyl jasmonateinsensitive mutants show altered expression of Atvsp in response to methyl jasmonate and wounding. Plant Physiol. I l l , 525-531. Ellis, C , and Turner, J. G (2001). The Arabidopsis mutant cevl has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens. Plant Cell 13,1025-1033. Feys, B. J. F., Benedetti, C E., Tenfold, C N., and Turner, J. G. (1994). Arabidopsis mutants selected for resistance to the phytotoxin coronatine are male sterile, insensitive to methyl jasmonate, and resistant to a bacterial pathogen. Plant Cell 6, 751-759. Staswick, P. E., Su, W., and Howell, S. H. (1992). Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant. Proc. Natl. Acad. Sci. USA 89, 6837-6840. Xie, D.-X., Feys, B. F., James, S., Nieto-Rostro, M., and Turner, J. G (1998). coil: An Arabidopsis gene required for jasmonateregulated defense and fertility. Science 280,1091-1094.