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Acid and Elevated Expression of Jasmonate-Responsive Genes in Arabidopsis. Qingzhe Zhai 1, 2, .... For permissions, please email: [email protected]. 1061 ..... covering the HY1 coding region were amplified through.
Plant Cell Physiol. 48(7): 1061–1071 (2007) doi:10.1093/pcp/pcm076, available online at www.pcp.oxfordjournals.org ß The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]

Phytochrome Chromophore Deficiency Leads to Overproduction of Jasmonic Acid and Elevated Expression of Jasmonate-Responsive Genes in Arabidopsis Qingzhe Zhai 1, 2, 5, Chang-Bao Li 1, 3, 5, Wenguang Zheng 1, 2, Xiaoyan Wu 1, 3, Jiuhai Zhao Guoxin Zhou 4, Hongling Jiang 1, Jiaqiang Sun 1, Yonggen Lou 4 and Chuanyou Li 1, *

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State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, PR China 2 Graduate School of Chinese Academy of Sciences, Beijing 100039, PR China 3 The State Key Laboratory of Crop Biology, Agronomy College, Shandong Agricultural University, Taian 271018, PR China 4 Institute of Insect Sciences, Zhejiang University, Hangzhou 310029, PR China

An Arabidopsis mutant line named hy1-101 was isolated because it shows stunted root growth on medium containing low concentrations of jasmonic acid (JA). Subsequent investigation indicated that even in the absence of JA, hy1-101 plants exhibit shorter roots and express higher levels of a group of JA-inducible defense genes. Here, we show that the hy1-101 mutant has increased production of JA and its jasmonate-related phenotype is suppressed by the coi1-1 mutation that interrupts JA signaling. Gene cloning and genetic complementation analyses revealed that the hy1-101 mutant contains a mutation in the HY1 gene, which encodes a heme oxygenase essential for phytochrome chromophore biosynthesis. These results support a hypothesis that phytochrome chromophore deficiency leads to overproduction of JA and activates COI1-dependent JA responses. Indeed, we show that, like hy1-101, independent alleles of the phytochrome chromophore-deficient mutants, including hy1-100 and hy2 (CS68), also show elevated JA levels and constant expression of JA-inducible defense genes. We further provide evidence showing that, on the other hand, JA inhibits the expression of a group of light-inducible and photosynthesis-related genes. Together, these data imply that the JA-signaled defense pathway and phytochrome chromophore-mediated light signaling might have antagonistic effects on each other. Keywords: Arabidopsis — hy1 — hy2 — Jasmonic acid — Light signaling—Phytochrome chromophore. Abbreviations: ALA, aminolevulinic acid; BAC, bacterial artificial chromosome; bHLH, basic helix–loop–helix; BV, biliverdin; CAPS, cleaved amplified polymorphic sequence; coi1, coronatine insensitive1; EMS, ethyl methanesulfonate; GC-MS, gas chromatography–mass spectrometry; GUS, b-glucuronidase; hy1 and hy2, long hypocotyl mutant1/2; JA, jasmonic acid; MeJA, methyl jasmonic acid; phyA and phyB, phytochrome photoreceptor A/B; RT–PCR, reverse transcription–PCR; VSP1, vegetative storage protein1.

Introduction The jasmonate family of oxylipins, which include jasmonic acid (JA), methyl jasmonic acid (MeJA) and other bioactive derivatives (collectively referred here as JAs) are important signaling molecules in the plant kingdom. JAs are well characterized for their role in regulating defense responses against biotic stresses such as herbivore attack and pathogen infection (Farmer and Ryan 1990, Farmer and Ryan 1992, Li et al. 2002, Ryan and Moura 2002, Turner et al. 2002, Stratmann 2003, Howe 2004, Schilmiller and Howe 2005). Accumulating evidence indicated that JAs are also involved in control of plant responses to abiotic stimuli such as mechanical wounding, ozone exposure, salt stress and water deficit (Mason and Mullet 1990, Hildmann et al. 1992, Penninckx et al. 1998, Overmyer et al. 2000, Rao et al. 2000, Kong et al. 2002, Howe 2004, Schilmiller and Howe 2005). In addition to defense, JAs are also implicated as regulators for plant growth and development. Significantly, the fundamental role of JA in reproduction has been revealed by Arabidopsis and tomato mutants impaired in JA production or signaling, and, as a consequence, are male or female sterile (for reviews, see Creelman and Mullet 1997, Turner et al. 2002, Wasternack and Hause 2002, Devoto and Turner 2003, Browse 2005, Liechti and Farmer 2006, Liechti et al. 2006). Mutant-based genetic analysis provides a powerful approach to dissect phytohormone signaling in the model system of Arabidopsis. After treatment with exogenous JA, Arabidopsis seedlings undergo several morphological changes including inhibition of primary root elongation. This phenotype has been used extensively for genetic screens to identify Arabidopsis mutants affected in JA responses. The majority of the JA-insensitive mutants, including jar1 (Staswick et al. 1992, Staswick et al. 1998),

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These authors contributed equally to this work. *Corresponding author: E-mail, [email protected]; Fax, þ8610-6487-3428. 1061

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coi1 (Feys et al. 1994), jin1 and jin4 (Berger et al. 1996), were previously identified by screening for JA-insensitive root elongation on MS medium containing relatively high concentrations of exogenous JA (50–100 mM). A significant advance in our understanding of JA signaling came from the analysis of the Arabidopsis (Arabidopsis thaliana) coi1 (coronatine insensitive 1) mutant that is insensitive to JA. The identification of COI1 encoding an F-box protein (Xie et al. 1998) and the demonstration that COI1 interacts with Skp1 and Cullin1 to assemble a functional SCFCOI1 ubiquitin ligase complex in vivo (Devoto et al. 2002, Xu et al. 2002) suggests that a ubiquitin-mediated protein degradation machinery is involved in JA signaling. Compared with coi1, the jar1 (Staswick et al. 1992) and jin1 (Berger et al. 1996, Lorenzo et al. 2004) mutants exhibit a relatively weak phenotype with respect to JA-induced inhibition of root growth. JAR1 encodes an enzyme that has JA adenylation activity to form JA–amino acid conjugates, suggesting that covalent modification of JA plays a key role in its action (Staswick et al. 2002). JIN1 encodes a nuclear-localized basic helix– loop–helix (bHLH)-type transcription factor known as AtMYC2 (Lorenzo et al. 2004). In order to identify negative regulators of JA signaling, several groups conducted genetic screens for constitutive JA response mutants (Ellis and Turner 2001, Hilpert et al. 2001, Xu et al. 2001). To date, only one of these genes (CEV1) had been identified. The finding that CEV1 encodes a cellulose synthase suggests a link between cell wall biosynthesis and JA signaling (Ellis et al. 2002). In order to identify additional components involved in JA signaling, we set up a genetic screen by using low doses of JA. A mutant line named B311 was identified because it shows stunted root growth on MS medium containing a low concentration of JA. Subsequent investigation indicated that even in the absence of JA, B311 plants exhibit shorter roots and express higher levels of a group of JA-inducible defense genes. Here, we show that the B311 mutant has increased production of JA and its jasmonate-related phenotype is suppressed by the coi1-1 mutation that interrupts JA signaling. Gene cloning and genetic complementation analyses revealed that the B311 mutant contains a mutation in the HY1 gene, which encodes a heme oxygenase essential for phytochrome chromophore biosynthesis. Genetic analysis indicated that the B311 mutant is allelic to the hy1-100 mutant (Chory et al. 1991, Terry 1997, Davis et al. 1999, Muramoto et al. 1999, Muramoto et al. 2002). Given that a wealth of genetic and biochemical evidence had demonstrated that the hy1 mutant was defective in phytochrome chromophore production (Parks and Quail 1991, Kohchi et al. 2005), the finding that B311 is a new allele of hy1 raises an interesting speculation that the JA-related phenotype of the B311

mutant results, in fact, from phytochrome chromophore deficiency. It is therefore reasonable to predict that the classic phytochrome chromophore-deficient mutants, including hy1 and hy2 (Kohchi et al. 2001), might also show overproduction of JA and constant expression of JA-inducible defense genes. Indeed, we show that the phytochrome chromophore-deficient hy1-100 and hy2 mutants also have increased JA levels and constitutive expression of JA-inducible genes. These results led us to hypothesize that phytochrome chromophore deficiency leads to overproduction of JA and activates COI1-dependent JA responses. We further provide evidence showing that, on the other hand, JA inhibits the expression of a group of light-inducible and photosynthesis-related genes. Together, these data imply that the JA-signaled defense pathway and phytochrome chromophore-mediated light signaling might have antagonistic effects on each other.

Results The B311 mutant line of Arabidopsis shows constant expression of JA-inducible defense genes In order to identify novel JA-related mutants, we set up a genetic screen for Arabidopsis plants showing altered responses to relatively low concentrations of JA using the widely applied root growth inhibition assays (see Materials and Methods). An Arabidopsis mutant line named B311 was initially isolated because it develops short roots on MS medium containing 10 mM JA (Fig. 1B, F). Further investigation indicates that, even in the absence of JA, the roots of the B311 seedlings were shorter than those of the wild type (Fig. 1A, F). In the presence of a range of concentrations of JA, the roots of B311 were generally shorter (Fig. 1G). However, the JA-mediated root growth inhibition effect in B311 was not significantly stronger than that in the wild type. In contrast to short roots, the mutant seedlings exhibited significantly longer hypocotyls (Fig. 1A, B), which was very similar to the phenotype of the well-characterized long hypocotyl mutants such as hy1 and hy2 (Koornneef et al. 1980). Soil-grown B311 plants also showed other phenotypes typical of the photomorphogenetic mutants including reduced stature (Fig. 1C, D), yellow-green leaves (Fig. 1D) and early flowering (Fig. 1E). The short root and stunted growth phenotype of B311 suggested that this mutant might have constitutive expression of defense-related genes (Hilpert et al. 2001, Xu et al. 2001). To test this, the expression levels of JA-inducible marker genes including vegetative storage protein1 (VSP1, At5g24780) and Thionin (Thi2.1, At1g72260) were compared between B311 and wild-type plants using Northern blot analysis. As shown in Fig. 2A, the transcripts of VSP1

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Fig. 1 Phenotypic characterization of the B311 mutant (labeled as hy1-101). (A) and (B) Root elongation phenotype of hy1-101 in response to JA. Seven-day-old wild-type plants (left) and hy1-101 mutants (right) grown on MS medium (A) or MS medium containing 10 mM JA (B); arrows point to the bases of the hypocotyls of the seedlings. (C) and (D) Three-week-old wild-type (C) and hy1-101 mutant plants (D) grown in soil. (E) Four-week-old wild-type (left) and hy1-101 (right) plants. (F) Statistical analysis of root elongation of hy1-101 in response to JA. Root lengths of the seedlings as given in A and B were measured. Data represent the mean  SD of 12 plants per genotype. An asterisk indicates significant difference between wild-type and hy1-101 plants (P50.0001, t-test). (G) Comparison of root length between hy1-101 and wildtype seedlings in different concentrations of JA. Each data point represents the mean  SD of 12 plants per genotype. Three independent experiments were conducted and similar results were obtained.

and Thi2.1 were barely detectable in untreated wild-type plants. In untreated B311 plants, however, the VSP1 and Thi2.1 transcripts were abundant, indicating that the mutants express JA-responsive genes constitutively. JA treatment induced the expression of VSP1 and Thi2.1 in wild-type as well as in B311 plants. Significantly, the overall expression levels of these transcripts were substantially higher in B311, as compared with those in the wild type (Fig. 2A).

Fig. 2 Analysis of JA-induced gene expression in wild-type and hy1-101 plants. (A) Two-week-old wild-type (Col-0) and hy1-101 seedlings were treated with 50 mM MeJA or not treated (0), and tissues were harvested for RNA extraction at the indicated times after treatment. RNA blots were hybridized to 32P-labeled cDNA probes for LOX2, AOS, AOC, OPR3, VSP1 and Thi2.1. A duplicate gel stained with ethidium bromide was used as RNA loading control. (B) GUS expression driven by the VSP1 promoter in wildtype and hy1-101 seedlings. Two-week-old seedlings of the VSP1::GUS transgenic line in the wild-type (Col-0) and hy1-101 background were treated with 50 mM MeJA or left untreated, and then stained for GUS activity.

To examine further the expression of VSP1 in the B311 mutant, the GUS (b-glucuronidase) reporter gene driven by a VSP1 promoter (Ellis et al. 2002) was introduced into B311 and wild-type plants (see Materials and Methods). In the absence of JA, constitutive VSP1::GUS expression was detected in the apical meristem and along the petioles of transgenic mutant plants, whereas in wild-type plants the expression of VSP1::GUS was barely detectable (Fig. 2B). Upon treatment with JA, both the mutant and wild-type plants strongly expressed VSP1::GUS (Fig. 2B). The B311 mutant has elevated levels of endogenous JA The B311 mutant showed constitutively high expression of JA-responsive VSP1 and Thi2.1. We speculate that

Interaction between jasmonate and light signaling

The JA-related phenotype of B311 results from a defect in HY1, a heme oxygenase required for phytochrome chromophore biosynthesis We conducted a set of crosses to determine the genetic basis of the mutation defined by the B311 mutant. B311 was crossed to the wild type (Col-0), and the resulting F1 plants exhibited the wild-type phenotype. F1 plants were selfpollinated and an F2 population consisting of 852 individuals was scored. The segregation ratio of wild-type plants to mutant plants is 646 : 206, which was very close to 3 : 1 (2 ¼ 0.3, P40.5). These results indicated that the phenotype of B311was caused by a single recessive mutation in a nuclear gene. A map-based cloning approach was used to identify the target gene. A total of 603 F2 plants showing the B311 phenotype were selected and used for mapping with simple sequence length polymorphism (SSLP) and InDel (insert/deletion) markers. Initially the target locus was mapped between molecular markers ciw3 and nga1126 on chromosome 2 (Fig. 4A). Fine mapping with five genetic markers delimited the target gene to a region containing four bacterial artificial chromosome (BAC) clones (Fig. 4A). Since previous studies had demonstrated that the photomorphogenesis-related long hypocotyl mutant hy1, which is morphologically similar to B311, also mapped to the same interval (Koornneef et al. 1980, Xu et al. 2001), we therefore considered HY1 (At2g26670) as a candidate for the target gene. Genomic DNA fragments covering the HY1 coding region were amplified through PCR from both B311 and the wild type. DNA sequencing

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this might result from the elevated levels of endogenous JA. To test this possibility, the expression levels of genes involved in JA biosynthesis, including LOX2 (At3g45140), AOS (At5g42650), AOC1 (At3g25760) and OPR3 (At2g06050), were compared between B311 and wild-type plants. As expected, our Northern blot analysis demonstrated that the basal and MeJA-induced expression levels of these genes were significantly higher in B311 than in the wild type (Fig. 2A). To determine further if the increased expression level of JA biosynthetic genes leads to higher JA production, we used gas chromatography–mass spectrometry (GC-MS) to measure internal JA levels in wild-type and mutant plants (Fig. 3A). The results showed that the JA levels in untreated wild-type and B311 plants were 24.8  4.6 and 618.0  89.5 ng g1 FW, respectively. One hour after mechanical wounding, the JA levels in the two genotypes increased to 1047.6  82.0 and 1581.6  174.6 ng g1 FW, respectively. This finding indicates that the mutation in B311 leads to overproduction of JA, which could lead to constant and increased expression of JA-responsive genes in the B311 mutant.

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Fig. 4 Molecular identification of the target gene. (A) Map-based cloning. The genetic locus defined by the hy1-101 mutant was initially mapped between markers ciw3 and nga1126 on chromosome 2. Fine mapping with five genetic markers delimited the target gene to a region containing four BAC clones. (B) Schematic structure of the HY1 gene and its mutation in the hy1-101 plant. The start codon (ATG) and stop codon (TAG) are indicated. (C) DNA polymorphism between hy1-101 and wild-type plants. The G to A mutation of HY1/At2g26670 in the hy1-101 genomic DNA destroys an NlaIV recognition site. The DNA fragments flanking the NlaIV site were amplified from the wild-type and hy1-101 plants, digested with NlaIV, and separated on an agarose gel. (D) Examination of HY1/At2g26670 expression in the hy1-101 mutant by RT–PCR. The Arabidopsis UBQ5 gene was amplified as a control.

and comparison revealed a single G to A nucleotide substitution in the B311-derived HY1 genomic DNA (Fig. 4B). Because this base pair change destroys an NlaIV recognition site in the mutant, we developed a cleaved amplified polymorphic sequence (CAPS) marker to verify the mutation in B311 (Fig. 4C). This polymorphism changed the codon TGG, which specifies a tryptophan, to the stop codon TGA, resulting in premature termination of the protein translation. Our reverse transcription–PCR (RT–PCR) analysis indicated that this mutation impaired the steady-state levels of HY1 transcripts in the B311 mutant (Fig. 4D). Consistent with previous studies (Ellis and Turner 2001, Zheng et al. 2006), HY1 transcripts were found to be abundant in wild-type seedlings, but significantly reduced to undetectable levels in B311 seedlings

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(Fig. 4D). This observation is consistent with the welldocumented existence of RNA surveillance mechanisms that target mRNAs containing premature translational termination codons for rapid degradation (Hilleren and Parker 1999). Taken together, these results support a hypothesis that the mutation we identified might abolish the function of HY1 and, as a consequence, could account for the versatile phenotype of the B311 mutant. We performed two experiments to test this hypothesis. First, a 3.9 kb genomic DNA fragment containing the entire HY1 coding region and the 1.1 kb upstream sequence was introduced into the B311 genetic background using Agrobacterium tumefaciens-mediated transformation. Positive transformants (which were confirmed by measuring HY1 gene expression with RT–PCR, Fig. 5D) grew and developed in the same way as wild-type plants (Fig. 5A–C). Furthermore, gene expression analysis indicates that the wild-type HY1 gene also rescued the B311 phenotype in terms of constant expression of JA-responsive genes (data not shown). These results demonstrate that the developmental as well as JA-related phenotypes of B311 result from loss of function of HY1, which encodes a heme oxygenase, a rate-limiting phytochrome chromophore biosynthetic enzyme (Davis et al. 1999, Muramoto et al. 1999). Secondly, an allelic test shows that the B311 and hy1-100 mutants (Davis et al. 1999) cannot complement each other, because all the F1 plants derived from a cross between B311 and hy1-100 exhibit the long hypocotyl phenotype (Fig. 5E). This result indicates that the B311 mutant we described herein is actually a new allele of the well-known hy1 mutant, which is one of the most extensively characterized photomorphogenetic mutants in Arabidopsis. We henceforth refer to B311 as hy1-101. The hy1 and hy2 mutants show overproduction of JA and constant expression of JA-inducible defense genes The finding that B311 is allelic to hy1-100 prompted us to examine the JA levels in the classic hy1 and hy2 mutants. Our GC-MS analysis indicated that the JA level in untreated hy1-100 leaves was 4566.2  80.4 ng g1 FW, while that in untreated wild type was 524.7  4.6 ng g1 FW (Fig. 3B). The wound-induced JA level in hy1-100 was also higher than that of the wild type. Similarly, leaves of the untreated hy2 mutant also had far higher levels of JA than those of wild-type plants (Fig. 3C). These results support the hypothesis that phytochrome chromophore deficiency leads to overaccumulation of JA. The expression levels of JA-responsive marker genes such as LOX2 and VSP1 were also compared between the hy mutants and their corresponding wild-type plants. Our Northern blot analyses indicated that, like hy1-101, the hy1-100 and hy2 mutants expressed LOX2 and VSP1 constantly (Fig. 6). In the case of wound treatment,

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Fig. 5 Genetic complementation of hy1-101. (A–C) Three-weekold wild-type (Col-0) (A), hy1-101 mutant (B) and one of the transgenic hy1-101 plants complemented by the wild-type genomic DNA of the HY1 gene (C) grown in soil. (D) RT–PCR analysis of the HY1/At2g26670 gene expression in plants corresponding to (A), (B) and (C), respectively. The Arabidopsis UBQ5 gene was amplified as control. (E) hy1-101 is allelic to hy1-100. Col-0, hy1-101, hy1-100 and an F1 plant derived from a cross between hy1-101 and hy1-100 grown on MS medium for 7 d.

the LOX2 and VSP1 expression levels in these three hy mutants were higher than those in their wild-type counterparts (Fig. 6). Taken together, these results support the hypothesis that phyochrome chromophore deficiency leads to constitutive activation of JA-mediated responses. Constant expression of JA-responsive genes in hy1-101 requires COI1 Since the F-box protein COI1 plays a central role in JA-signaled processes in Arabidopsis, we further investigated whether COI1 is required for the manifestation of the hy1-101 phenotype. For this purpose, a double mutant was constructed between hy1-101 and coi1-1 (Xie et al. 1998). The homozygous hy1-101/coi1-1 double mutant lost the

hy1-101 mutant trait in terms of constant and woundinduced VSP1 expression (Fig. 7C). These results indicated that the constant activation of jasmonate responses in hy1-101 requires the COI1-dependent signal transduction pathway. Interestingly, at the seedling stage, the root length of the hy1-101/coi1-1 double mutant was shorter than that of coi1-1 but longer than that of hy1-101 in JA-containing medium (Fig. 7A). Like coi1-1, the double mutant was also male sterile (Fig. 7B). The phyA, phyB and phyA/phyB double mutant do not show overproduction of JA Because all members (phyA–phyE) of the phytochrome photoreceptor family in Arabidopsis use the same chromophore, a direct effect of chromophore production deficiency in the hy1 and hy2 mutants is reduced activity of all phytochrome species (Terry 1997, Terry and Kendrick 1999). We therefore further investigated whether the phyA, phyB and phyA/phyB double mutants show jasmonate-related phenotypes. Our JA quantification results indicated that phyA, phyB and phyA/phyB double mutants show wild-type levels of JA, in both the presence and absence of mechanical wounding (data not shown and Fig. 3D). JA suppresses the expression of HY1 and other light-related genes The finding that phytochrome chromophore deficiency leads to overproduction of JA and constitutive activation of JA responses prompted us to investigate the role of JA, if any, in the expression of phytochrome chromophore biosynthesis-related genes. Indeed, when plants encountered exogenous JA, the transcript level of HY1 was significantly down-regulated (Fig. 8). We then took a closer look at of our comprehensive microarray data acquired in order to look for JA-regulated genes (Zheng et al. 2006).

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Fig. 7 Effects of the coi1-1 mutation on the hy1-101 phenotype. (A) Seven-day-old wild-type (Col-0), hy1-101, coi1-1 and hy1-101/ coi1-1 plants grown on MS medium or MS medium containing 10 mM JA. (B) Seven-week-old wild-type (Col-0), hy1-101, coi1-1 and hy1-101/coi1-1 plants grown in soil. (C) The coi1-1 mutation suppresses the constant and wound-induced VSP1 expression in the hy1-101 mutant. Two-week-old wild-type (Col-0), hy-101, coi1-1 and hy1-101/coi1-1 plants were wounded with a hemostat or not wounded (0). Samples were harvested 6 h after treatment and RNA was isolated as described above. RNA gel blot was performed using a 32P-labeled VSP1 cDNA fragment. A duplicate gel stained with ethidium bromide was used as RNA loading control.

Significantly, we found that the expression of a group of light-inducible and photosynthesis-related genes was suppressed by JA (Zheng et al. 2006). The JA inhibition effect on the expression of some of these genes, including those encoding the small subunit of ribulose-1,5-bisphosphate carboxylase (RBCS, At1g67090), Rubisco activase (RCA, At1g73110) and Chl a/b-binding protein (CAB, At2g40100), was confirmed by Northern blot analysis (Fig. 8). These findings, which agree well with several previous microarray analyses (Feng et al. 2003), imply that the JA-signaled defense pathways and phytochrome chromophore-mediated

light signaling pathways might have antagonistic effects on each other.

Discussion Previous studies indicated the existence of cross-talk between the JA pathway and light signaling. For example, light induces the expression of JA biosynthetic genes such as allene oxide synthase (AOS) in a phytochrome-dependent manner in rice (Haga and Iino 2004), and JA biosynthesis deficiency leads to impaired light response in a rice mutant (Riemann et al. 2003). The bHLH protein MtMYC2/JIN1, a transcription factor in jasmonate signaling (Boter et al. 2004, Lorenzo et al. 2004), was recently shown to be a negative regulator of blue light-mediated photomorphogenetic growth (Yadav et al. 2005). There were also reports showing that jasmonate affects the expression of lightregulated genes (Wierstra and Klopstech 2000). However, relatively little is known about the mechanism of how these two pathways are connected. Through characterization of an additional allele of the Arabidopsis hy1 mutant (B311, renamed here hy1-101), we show here that the hy1-101 mutant, which is predicted to be deficient in phytochrome chromophore production, has far higher levels of endogenous JA and expresses JA-inducible defense genes constitutively. These results provided new information on the connection between JA biosynthesis and light signaling.

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Disruption of chromophore biosynthesis leads to overproduction of JA and constant activation of COI1-dependent JA signaling hy1 and hy2 are well-characterized photomorphogenetic Arabidopsis mutants that lost their ability to synthesize the phytochrome chromophore (Parks and Quail 1991, Terry 1997). The genes responsible for hy1 and hy2 were cloned based on their map positions, and their biochemical functions in the phytochrome chromophore were elucidated. HY1 encodes a heme oxygenase that catalyzes the oxygenation of heme to produce biliverdin (BV) IXa, an essential intermediate in phytochrome chromophore biosynthesis (Davis et al. 1999, Muramoto et al. 1999, Muramoto et al. 2002). HY2 encodes PB synthase, a ferredoxin-dependent BV reductase that is responsible for the final step in phytochrome chromophore 3E-phytochromobilin (PPhiB) biosynthesis (Kohchi et al. 2001). Previous biochemical analysis showed that both hy1 and hy2 mutants were defective in phytochrome chromophore production (Parks and Quail 1991, Kohchi et al. 2005). Gene cloning and genetic analyses demonstrated that the hy1-101 mutation is allelic to hy1-100. These results led us to the hypothesis that phytochrome chromophore deficiency results in overproduction of JA and leads to the hy1-101 mutant showing constant expression of JAinducible defense genes. Indeed, JA measurement and gene expression analyses indicated that, like hy1-101, independent alleles of the phytochrome chromophoredeficient mutants, including hy1-100 and hy2 (CS68), also show elevated JA levels (Fig. 3) and constant expression of JA-inducible defense genes (Fig. 6). Furthermore, double mutant analysis indicates that hy1-101/coi1-1 loses its ability to express constant and JA-induced defense genes (Fig. 7C), which demonstrates that the activation of jasmonate responses in phytochrome chromophoredeficient mutants strictly requires the COI1-dependent signal transduction pathway. The above-mentioned findings raise the interesting question of how phytochrome chromophore deficiency leads to activation of the JA signaling pathway. Based on current knowledge, phytochrome chromophore and chlorophyll are both synthesized in the plastid from aminolevulinic acid (ALA) and share a common pathway between the intermediates ALA and protoporphyrin IX (Terry 1997, Terry and Kendrick 1999). A wealth of evidence indicated that disruption of chromophore biosynthesis in the hy1 and hy2 mutants leads to two consequences. The first and direct consequence of chromophore deficiency is lack of phytochrome photoreceptors. Because all members (phyA– phyE) of the phytochrome photoreceptor family in Arabidopsis use the same chromophore, so the deficiency of chromophore results in plants showing reduced activity

of all phytochrome species (Terry 1997, Terry and Kendrick 1999). The second and indirect consequence of chromophore deficiency is reduced Chl synthesis, because the hy1 and hy2 mutations block the degradation of the physiologically active pool of heme, which in turn results in feedback inhibition of the biosynthesis of ALA, a common precursor for heme and chlorophyll production (Terry and Kendrick 1999). To help understand whether phytochrome receptorrelated deficiency or cholorophyll-related deficiency in the hy mutants contributes to the activation of JA responses, we tested the jasmonate-related phenotypes of the phyA, phyB and phyA/phyB double mutant. Our JA measurement results indicated that the JA levels in control and wounded phyA/phyB double mutants were not significantly higher that those in wild-type plants (Fig. 3D). However, these data were not able to rule out the possibility that the jasmonate-related phenotype of hy1 and hy2 mutants resulted from reduced photoactive phytochrome, because of the existence of other photoreceptor members (i.e. phyC– phyE) in the phyA/phyB double mutant. The other speculation was that the elevated jasmonate responses in hy1 and hy2 resulted from the role of indirect effects, i.e. deficiency in chlorophyll synthesis. We are currently investigating the JA-related responses of a collection of chlorophyll-deficient mutants. The other possibility was that both direct and indirect effects of chromophore deficiency contributed to the elevated JA responses in hy1 and hy2. In this scenario, a combination of misregulated tetrapyrrole-binding proteins and chlorophyll-binding proteins might lead to increased photooxidative stress in the light, which potentially mimics wounding and, therefore, activates JA signaling in the mutants. JA inhibits the expression of a group of light-inducible and photosynthesis-related genes Based on characterization of the hy1 and hy2 mutants, we found that phytochrome chromophore deficiency leads to overproduction of JA and constitutive activation of COI1-dependent defense responses. A reasonable explanation for this phenomenon is that, in wild-type plants, the phytochrome chromophore-mediated signaling pathways act negatively to repress the JA-mediated signaling pathways. Chromophore biosynthesis deficiency in the hy1 and hy2 mutants may result in the relief of the repression of JA signaling and, consequently, leads to constant JA production and responses. These findings prompted us to investigate the possible effects of JA on the phytochrome chromophore-mediated signaling pathways. Microarray (Zheng et al. 2006) and Northern blot analyses indicate that JA treatment indeed inhibits the expression of a group of light-inducible and photosynthesis-related genes (Fig. 8). Consistent with our findings, it was previously shown that

Interaction between jasmonate and light signaling

application of JA to plant leaves not only reduces the expression of the small subunit of Rubisco, but also reduces translation and induces degradation of Rubisco (Parthier 1990). In addition to Rubisco, a series of photosynthesis- and growth-related genes were also reported to be repressed by JA at the transcription and protein levels (Reinbothe et al. 1994). Recent microarray analysis also found that JA generally stimulates the expression of genes involved in stress responses while it suppresses the expression of genes involved in photosynthesis and energy generation (Feng et al. 2003). Taken together, these findings imply that the JA-signaled defense pathways and phytochrome chromophore-mediated light signaling pathways might have antagonistic effects on each other. The antagonistic interactions between JA-mediated defense signaling and chromophore-mediated light signaling might be consistent with the current understanding of the biological functions of the JA response pathway. JA is best recognized for its role in regulating plant defense responses against mechanical trauma, herbivore attack and pathogen infections (Penninckx et al. 1996, Creelman and Mullet 1997, McConn et al. 1997, Berger 2002, Turner et al. 2002). Because these JA-mediated defenses are costly in terms of energy and resources, plants have to limit their resource allocation with respect to chromophore-mediated processes including photosynthesis and light signaling. This phenomenon, which is called ‘fitness cost’, has been well recognized at the physiological level in the field of plant–insect interactions (Baldwin 1998, Heil and Baldwin 2002). However, the molecular mechanism of ‘fitness cost’ is poorly understood because of methodological problems (Heil and Baldwin 2002). In this regard, the interaction between phytochrome chromophore-mediated signaling pathways and JA-mediated signaling pathways might provide a proper starting point for in-depth studies, and the hy1, hy2 and other related mutants might provide valuable genetic materials for these studies.

Materials and Methods Plant material and growth conditions The JA response mutant coi1-1 was kindly provided by Dr. Daoxin Xie (Tsinghua University, Beijing, PR China). The hy1-100 mutant was described previously (Yang et al. 2000). The hy2 (CS68), phyA, phyB and phyA/phyB double mutant were ordered from the Arabidopsis Biological Resource Center (http://www.arabidopsis.org/abrc/). For seed harvest and crosses, Arabidopsis plants were grown in potting soil mixture (rich soil : vermiculite 2 : 1, v/v) and kept in a growth chamber at 228C with a 16 h light/8 h dark cycle. For JA response analysis, 2-week-old seedlings grown on 0.5 Murashige and Skoog (MS) medium were sprayed with solutions containing MeJA or wounded with a hemostat and then incubated in a growth

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chamber under continuous light. Tissues were then harvested at the time intervals indicated for RNA extraction. Mutant screening Ethyl methanesulfonate (EMS)-mutagenized M2 seeds (Zheng et al. 2006) of Arabidopsis thaliana ecotype Columbia (Col-0) were grown on 0.5 MS medium containing 10 mM JA and screened for mutants in which root growth was severely inhibited. The mutant line B311 (renamed here hy1-101) was recovered because it shows short roots and stunted growth. Seeds were collected from the original hy1-101 plants and the root growth was retested. The hy1-101 mutant was backcrossed successively to the wild type (Col-0) for three generations before physiological and genetic analyses. Map-based cloning A map-based cloning approach (Lukowitz et al. 2000) was used to identify the genetic basis for the hy1-101 mutation. The hy1-101 (Col-0 background) mutants were crossed to Landsberg erecta (Ler) plants to create mapping populations. A total of 603 individual F2 plants showing the hy1-101 phenotype were selected for genetic mapping. For complementation analysis, a 3.9 kb DNA fragment contains the entire HY1/At2g26670 coding region and a 1.1 kb upstream sequence was amplified from the wild-type (Col 0) by PCR using the primers: 50 -CCGGGTACCCGCTTACAGCTGT AAAAGATG-30 and 50 -CCGGGTACTTGCACCAAGGAGTG AAAG-30 . The PCR product was digested with KpnI and PstI, and inserted into the same sites of pCAMBIA1300. The construct obtained was then introduced into the hy1-101 plants using A. tumefaciens-mediated transformation. Transformants were selected based on their resistance to hygromycin. For the allelic test, the hy1-101 mutant was crossed to the previously characterized hy1-100 mutant (Terry 1997, Muramoto et al. 1999) and the resulting F1 plants were examined. Construction of hy1-101/coi1-1 double mutant To isolate double mutants, homozygous hy1-101 plants were crossed to homozygous coi1-1 plants, and the F2 progeny were scored with CAPS markers to identify plants containing the homozygous coi1-1 allele as well as the homozygous hy1-101 allele. The CAPS marker information to identify coi1-1/coi1-1 was previously reported (Xie et al. 1998). A 0.5 kb DNA fragment containing part of the HY1 or the hy1-101 mutant allele could be amplified by PCR primers P1 (50 -CTAAAACTACATTGCGT GACTTCT-30 ) and P2 (50 -AAGTTGGGAAATTGGAGTCTT GAA-30 ) using genomic DNA as template. NlaIV cleaved the 0.5 kb fragment from the wild-type DNA but not from the hy1-101 mutant, in which the NlaIV recognition site was altered by the G to A mutation (Fig. 4C). This CAPS marker was used to identify the hy1-101/hy1-101 allele. JA measurement Leaves of 2-week-old plants were harvested for JA extraction and measurement, as previously described (Li et al. 2006). For wound treatment, all but the first and second rosette leaves of 2-week-old plants were crushed several times across the middle vein with a hemostat. Plants were incubated for 1 h, and then the leaves were harvested and immediately frozen in liquid nitrogen. JA was extracted and quantified by GC-MS with 190 ng of [1,2-13C]JA (kindly provided by Ian T. Baldwin, Max-Planck Institute of Chemical Ecology, Jena, Germany)

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as internal standard, as described by Heidel and Baldwin (2004). The concentrations of JA were expressed as ng g1 FW. RNA blot analysis Two-week-old plants were treated with MeJA or wounded as described above. Total RNA was prepared by a guanidine thiocyanate extraction method, and RNA gel blot analysis was performed as previously described (Zheng et al. 2006). A 10 mg aliquot of total RNA was separated in an agarose gel containing 10% formaldehyde, blotted onto a Hybond Nþ membrane (Amersham), and probed with the PCR-amplified DNA fragments using the following primers: VSP1F (50 -ATGAAAATCCTCTCA CTTTCA-30 ) and VSP1R (50 -TATCCATATTTAGCGTAG TAGG-30 ); LOX2F (50 -GGATTACGGTAGAAGACTACGC-30 ) and LOX2R (50 -TGTTAATGAGAATGGGCATC-30 ); OPR3F and OPR3R (50 -CGTCAACGAACAAACCAATCTCG-30 ) (50 -TCCCTTGCCTTCCAGACTCTG-30 ); AOC1F (50 -CTGAGC GTGTACGAAATCAATG-30 ) and AOC1R (50 -TTGCTTACAA CTCCACTGGGC-30 ); AOSF (50 -GGCGGGCGGGTCATCAA GT-30 ) and AOSR (50 -TCGCCGGAAAATCTCATCACAA-30 ); Thi2.1F (50 -GTGATCAAACAAGTAAACCAT-30 ) and Thi2.1R (50 -AACAAACCTTCTACGACACAT-30 ); HY1F (50 -CTAAAAC TACATTGCGTGACTTCT-30 ) and HY1R (50 -AAGTTGGGAA ATTGGAGTCTGA-30 ); TCAF (50 -GGTCGGGGCCCAGTAG AAC-30 ) and TCAR (50 -CCGCAAAGCTTATCCGTCAGA-30 ); RBCSF (50 -ATGGCTTCCTCTATGCTCTCT-30 ) and RBCSR (50 -TTAACCGGTGAAGCTTGGTGG-30 ); and CABF (50 -CAA GAACGTGGCGGGTGACAT-30 ) and CABR (50 -GGCCGCCT GAACAGCAAAGAT-30 ). Analysis of GUS activity The JA reporter line containing the promoter of VSP1 fused with GUS in a wild-type (Col-0) background was constructed previously in our laboratory (Zheng et al. 2006). The VSP::GUS reporter was transferred into the hy1-101 background through crossing and selection. Histochemical staining for GUS activity was performed as previously described (Jefferson et al. 1987).

Acknowledgments We gratefully acknowledge Dr. Jianru Zuo (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing) for providing EMS-mutagenized Arabidopsis (Col-0) M2 seeds, and Dr. Daoxin Xie (Tsinghua University, Beijing) for providing coi1-1 seeds. We also thank Dr. Xinnian Dong (Duke University, Durham, NC, USA) and Dr. Edward Farmer (University of Lausanne Lausanne, CH-1015 Switzerland) for critical reading of the manuscript. This work was supported by grants from the Ministry of Science and Technology of China (2006CB102004), the National Natural Science Foundation of China (30425033, 30530440) and the Chinese Academy of Sciences (CXTD-S2005-2).

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(Received April 4, 2007; Accepted June 10, 2007)