- © 2011 American Society of Plant Biologists
Abstract
Leaf senescence, as the last stage of leaf development, is regulated by diverse developmental and environmental factors. Jasmonates (JAs) have been shown to induce leaf senescence in several plant species; however, the molecular mechanism for JA-induced leaf senescence remains unknown. In this study, proteomic, genetic, and physiological approaches were used to reveal the molecular basis of JA-induced leaf senescence in Arabidopsis (Arabidopsis thaliana). We identified 35 coronatine-insensitive 1 (COI1)-dependent JA-regulated proteins using two-dimensional difference gel electrophoresis in Arabidopsis. Among these 35 proteins, Rubisco activase (RCA) was a COI1-dependent JA-repressed protein. We found that RCA was down-regulated at the levels of transcript and protein abundance by JA in a COI1-dependent manner. We further found that loss of RCA led to typical senescence-associated features and that the COI1-dependent JA repression of RCA played an important role in JA-induced leaf senescence.
Leaf senescence, as the last stage of leaf development, proceeds through a highly regulated program in order to remobilize the nutrients from areas where it is no longer required to areas of cell development in the plant (Buchanan-Wollaston, 1997; Quirino et al., 2000). The onset of leaf senescence is age dependent but also can be stimulated by diverse developmental signals, sugar, plant hormones, and environmental stresses, including energy deprivation, darkness, excess light, drought, salinity, nutrient limitation, and wounding (Schippers et al., 2007; Balazadeh et al., 2008). The progression of leaf senescence is accompanied by the rapid loss of chlorophyll, the decreased abundance of photosynthesis-related proteins (Bate et al., 1991), and the increased expression of senescence-associated genes (Nam, 1997). The regulation of leaf senescence also involves numerous transcription factors, such as NAC domain-containing protein, WRKY DNA-binding protein, MYB domain protein, C2H2-type zinc finger, basic leucine-zipper, and APETALA2/ethylene-responsive element binding protein family genes, which control the expression of different senescence-related genes (Balazadeh et al., 2008).
Jasmonates (JAs), as plant growth regulators and defense signals, control many plant developmental and growth processes and mediate plant responses to abiotic and biotic stresses (McConn et al., 1997; Rao et al., 2000; Sasaki et al., 2001; Cheong and Choi, 2003; Farmer et al., 2003; Howe, 2004; Schilmiller and Howe, 2005; Wasternack, 2007; Cheng et al., 2009; Kim et al., 2009; Koo and Howe, 2009; Ren et al., 2009; Shan et al., 2009). JA also functions in the induction of leaf senescence in many plant species (Ueda and Kato, 1980; Weidhase et al., 1987; Reinbothe et al., 2009). In Arabidopsis (Arabidopsis thaliana), exogenous application of JA promotes leaf senescence (He et al., 2002) and regulates the expression of various genes that are involved in leaf senescence (Buchanan-Wollaston et al., 2005; Jung et al., 2007). However, the molecular mechanism for JA-induced leaf senescence is not clear.
The F-box protein coronatine-insensitive 1 (COI1; Xie et al., 1998) is a key regulator in the JA signal pathway. It directly binds to JA-Ile and functions as a JA receptor (Yan et al., 2009). COI1 assembles the SCFCOI1 complex (Xu et al., 2002; Liu et al., 2004; Ren et al., 2005; Wang et al., 2005) to recruit the jasmonate ZIM-domain proteins (JAZs) for degradation by the 26S proteasome (Chini et al., 2007; Thines et al., 2007; Katsir et al., 2008) and subsequently regulates various plant developmental and growth processes. The null mutant coi1-1 (Feys et al., 1994), with the premature stop codon at Trp-467, is male sterile, insensitive to JA-inhibitory root growth, defective in JA-regulated gene expression, and supersensitive to insect attack and necrotrophic pathogen infection (Feys et al., 1994; Xie et al., 1998; Reymond et al., 2000). The coi1-2 mutant, a leaky allele with the missense mutation L245F, is resistant to JA but is partially fertile and able to produce a small quantity of seeds (Xu et al., 2002). The coi1 mutant plants also exhibit relatively delayed senescence phenotypes, including elongated flowering time and relatively higher chlorophyll content (Xiao et al., 2004). However, it remains to be elucidated how COI1 regulates leaf senescence.
In this study, 35 proteins were identified as COI1-dependent JA-regulated proteins by two-dimensional difference gel electrophoresis (2-D DIGE) coupled with matrix-assisted laser desorption inoization-time of flight (MALDI-TOF) mass spectrometry. Further study on Rubisco activase (RCA), one of these 35 identified proteins, revealed that RCA was down-regulated at the levels of transcript and protein abundance by JA in a COI1-dependent manner. Molecular, genetic, and physiological analyses showed that mutation in RCA led to typical senescence-related symptoms and that the COI1-dependent JA repression of RCA played an important role in JA-induced leaf senescence.
RESULTS
Identification of COI1-Dependent JA-Regulated Proteins by 2-D DIGE
To examine the requirement of COI1 for JA-regulated gene expression at the posttranscriptional level, 2-week-old wild-type and coi1-1 mutant plants were drenched in a solution containing 100 μm methyl jasmonate (MeJA) for 2 d and subsequently harvested to perform 2-D DIGE analysis (Supplemental Fig. S1A). The comparative image analysis of JA-treated wild-type with JA-treated coi1-1 mutant plants identified 61 protein spots that changed significantly in abundance with P < 0.05. We performed peptide mass fingerprinting via MALDI-TOF mass spectrometry on these 61 protein spots and successfully generated protein assignments for 43 spots, which represented 35 unique proteins (Fig. 1; Table I). In JA-treated wild-type plants, 21 out of the 35 proteins were up-regulated whereas 14 were down-regulated compared with JA-treated coi1-1 (Fig. 1; Table I), suggesting that both JA treatment and COI1 existence are required for the regulation of these 35 proteins.
A representative 2-D DIGE image of Cy2-labeled JA-treated wild-type and JA-treated coi1-1 pooled internal standard proteome map. Proteins were resolved first on a 24-cm pH 4 to 7 IPG strip and further separated on a 12.5% SDS-PAGE gel. Proteins with altered expression between JA-treated wild-type and JA-treated coi1-1 plants were identified by MALDI-TOF mass spectrometry and marked with spot numbers (their identities are shown in Table I). The spots marked with white dashed and black solid arrows indicated increase or decrease in JA-treated wild-type plants over JA-treated coi1-1 plants, respectively.
A total of 35 COI1-dependent JA-regulated proteins were classified based on the biological process in which the gene product participated, and their corresponding spots are shown on Figure 1. Col-0, Ecotype Columbia.
To verify whether JA treatment is essential for the regulation of these 35 proteins, we compared the protein profiles of wild-type and coi1-1 mutant plants (Supplemental Fig. S1B). We found that, without JA treatment, the expression levels of these 35 proteins had no significant differences (P > 0.05) between the wild type and the coi1-1 mutant (Table I), indicating that JA treatment is indispensable for the regulation of these proteins. We further compared the protein profiles between coi1-1 and JA-treated coi1-1 to verify whether COI1 is required for the JA-regulated expression of these 35 proteins (Supplemental Fig. S1C). We found that, without COI1, the expression levels of these 35 proteins in the coi1-1 mutant had no obvious differences (P > 0.05) irrespective of whether JA was applied or not (Table I), suggesting that COI1 is essential for the JA-regulated expression of these proteins. Collectively, these data demonstrate that these 35 proteins are regulated by JA in a COI1-dependent manner.
These 35 proteins, including 21 COI1-dependent JA-induced proteins and 14 COI1-dependent JA-repressed proteins, were classified based on the biological process in which the gene product participated (Table I). Most of these proteins encode enzymes that potentially mediate specific cellular and physiological processes, such as JA biosynthesis, amino acid metabolism, photosynthesis/chlorophyll metabolism, cellular respiration, and defense/stress responses (Table I).
By comparison of our proteomics data with previous transcriptomics data (Sasaki et al., 2001; Sasaki-Sekimoto et al., 2005; Jung et al., 2007), we found that 15 of these 35 JA-regulated proteins were identified as JA-regulated RNAs in previous microarray studies (Table I). Qualitative changes in RNA levels of these genes were consistent with those in protein levels upon JA treatment. In the remaining 20 proteins, we analyzed the expression patterns of four genes, TGG2 (for thioglucoside glucohydrolase 2; At5g25980), SBPASE (for sedoheptulose-1,7-bisphosphatase; At3g55800), NQR (for NADPH:quinine oxidoreductase; At3g27890), and CSY4 (for citrate synthase; At2g44350), under JA treatment to further compare the proteomics and transcriptomics data using semiquantitative reverse transcription (RT)-PCR. We found that the expression patterns of TGG2 and SBPASE at the transcript level correlated well with that at the protein level (Fig. 2, A and B; Table I). However, the transcript patterns of NQR and CSY4 did not correlate with their protein expression patterns. The transcript levels of NQR and CSY4 were similar in wild-type and coi1-1 mutant plants irrespective of whether JA was applied or not (Fig. 2C), whereas proteomics data showed that NQR protein was up-regulated and CSY4 protein was down-regulated by JA in the wild type (Table I), which might result from posttranscriptional processes such as mRNA splicing, mRNA degradation, mRNA translation, and protein modification. These data indicate that the posttranscriptional regulation might also function in JA signaling, yet it remains relatively understood when compared with the transcriptional regulation mechanism. As proteins, not RNAs, are the functional elements in diverse biological processes, the proteomics data generated in our study may be more relevant to understanding JA signaling.
Semiquantitative RT-PCR for the indicated genes in 2-week-old wild-type and coi1-1 mutant plants upon treatment with MeJA (+) or water (−) for 2 d. The genes are indicated on the right side of each panel. The amplified actin1 is shown as an internal control. Col-0, Ecotype Columbia.
The Expression of RCA Protein Was Down-Regulated by JA in a COI1-Dependent Manner
Among these 35 proteins, RCA was identified as a COI1-dependent JA-repressed protein (Table I). RCA was reported to catalyze the light activation of Rubisco (Portis, 2003) and to function in plant photosynthesis and growth (Mate et al., 1993; Jiang et al., 1994; Eckardt et al., 1997). To verify the 2-D DIGE data, we examined the RCA expression pattern using our previously generated RCA antibody (Jiang et al., 2001), which could detect 43- and 47-kD protein bands, two isoforms of RCA arising from mRNA alternative splicing (Salvucci et al., 1987; Shen et al., 1991).
Similar to our 2-D DIGE data, western-blot analysis with RCA antibody showed that the expression level of RCA protein was down-regulated by JA in a COI1-dependent manner. The amount of RCA protein declined gradually in response to JA treatment and was reduced significantly upon JA treatment for 5 d in the wild type but not obviously in the coi1-2 mutant (Fig. 3, A and B). The RCA protein level in the wild type was decreased 9.4%, 18.8%, 34.1%, 84.8%, and 93.5% under JA treatment for 1, 2, 3, 4, and 5 d, respectively (Fig. 3A).
The expression of RCA protein was down-regulated by JA in a COI1-dependent manner. A, Western blot for RCA (top) and quantitative analysis of RCA protein level (bottom) in 6-week-old wild-type leaves upon treatment with MeJA (+) or water (–; CK) for the indicated time periods. The immunoblot was detected with GST antibody as a protein-loading control (top). The RCA protein level in the wild type upon water treatment for 1 d was set to 1, and the relative RCA protein levels in other samples were calculated accordingly (bottom). B, Western blot for RCA in 6-week-old coi1-2 mutant leaves upon treatment with MeJA (+) or water (−) for the indicated time periods. The immunoblot was detected with GST antibody as a protein-loading control. C, Schematic drawing of the pRCA-RCA::GUS construct (see “Materials and Methods”). Abbreviations not defined in the text: LB, left border; HYG, hygromycin gene; Nos, nopaline synthase terminator; RB, right border. D, RCA::GUS fusion protein was degraded after JA treatment in the stem (1), silique (2), leaf (3), and floral bud (4) of the transgenic plants. The transgenic plants were treated with MeJA (+) or water (−) for 5 d and subsequently harvested for GUS staining.
To further verify the JA-repressed RCA protein expression, we made transgenic plants harboring the pRCA-RCA::GUS construct, in which the GUS reporter was translationally fused to the RCA protein under the control of the RCA endogenous promoter fragment extending from −1,982 to −1 (relative to the RCA translational start ATG; Fig. 3C). Upon JA treatment for 5 d, RCA::GUS fusion protein was almost absent in the transgenic plants (Fig. 3D), whereas a strong expression of the RCA::GUS fusion protein, without JA treatment, was detected in the leaf, stem, floral bud, and silique of the transgenic plants (Fig. 3D). These results further demonstrate that RCA protein is down-regulated under JA treatment.
The Decrease of RCA Transcripts Preceded the Reduction of RCA Protein upon JA Treatment
As changes of protein level may not necessarily be reflected at the RNA level (Griffin et al., 2002; Huber et al., 2004; Tian et al., 2004; Fig. 2), we analyzed the RCA RNA expression pattern in comparison with its protein expression pattern in the same tissues analyzed by 2-D DIGE. RT-PCR analysis showed that the level of RCA transcripts was down-regulated by JA in the wild type but not in the coi1-1 mutant (Fig. 4A), suggesting that the down-regulation of RCA protein by JA in a COI1-dependent manner is also reflected at its RNA level.
The decrease of RCA transcripts preceded the reduction of RCA protein upon JA treatment. A, Semiquantitative RT-PCR for RCA in 2-week-old wild-type and coi1-1 mutant plants upon treatment with MeJA (+) or water (−) for 2 d. The amplified actin1 is shown as an internal control. B, Northern blot for RCA (top) and quantitative analysis of RCA RNA level (bottom) in 6-week-old wild-type leaves upon treatment with MeJA (+) or water (–; CK) for the indicated time periods. Ethidium bromide staining of rRNA was used as a loading control (top). The RCA RNA level in the wild type upon water treatment for 1 d was set to 1, and the relative RCA RNA levels in other samples were calculated accordingly (bottom).
Through comparison of RCA expression patterns at the protein level (Fig. 3A) and the RNA level (Fig. 4B) under JA treatment for various time periods, we found that the RCA transcripts were decreased more quickly than its protein level in response to JA treatment. When the wild type was treated by JA for 1 and 3 d, the RCA transcripts exhibited 60.7% and 89.5% reduction, respectively (Fig. 4B), while the RCA protein displayed only 9.4% and 34.1% reduction, respectively (Fig. 3A). Thus, the decrease of RCA transcripts precedes the reduction of RCA protein upon JA treatment, which is different from other chloroplast proteins that are decreased more rapidly than their transcripts under JA treatment (Reinbothe et al., 1993, 1994, 1997).
The COI1-Dependent JA Repression of RCA Correlated with JA-Induced Leaf Senescence
As shown in Figures 3A and 5B, the RCA protein content was significantly reduced when wild-type leaves were treated with JA for 5 d. Correlated with the loss of RCA, the wild-type leaves presented severe senescence, especially yellowing under JA treatment for 5 d (Fig. 5A).
The COI1-dependent JA repression of RCA correlated with JA-induced leaf senescence. A, Phenotypes of 6-week-old wild-type and coi1-2 mutant leaves upon treatment with MeJA (+) or water (−) for 5 d. B, Western blot for RCA in 6-week-old wild-type and coi1-2 mutant leaves upon treatment with MeJA (+) or water (−) for 5 d. The immunoblot was detected with GST antibody as a protein-loading control.
In contrast, the leaves of the coi1-2 mutant were still green or only turned a little yellow even after JA treatment for 5 d (Fig. 5A). Simultaneously, we found that the RCA protein level was not greatly affected in the coi1-2 mutant (Fig. 5B). These data indicate that COI1 is essential for JA-induced leaf senescence and that the COI1-dependent JA repression of RCA correlates with JA-induced leaf senescence.
Identification of the rca Mutants
To investigate whether the loss of RCA causes leaf senescence, we identified two rca mutant alleles, rca-1 (SALK_118831), with a T-DNA insertion in the first intron, 339 bp downstream of the start codon (Fig. 6A), and rca-2 (SALK_003204), with a T-DNA insertion in the 5′ untranslated region, 163 bp upstream of the start codon (Fig. 6A).
Identification of the rca-1 and rca-2 mutants. A, Schematic diagram of the T-DNA insertions into the Arabidopsis RCA gene (At2g39730.1). Numbers indicate the positions of T-DNA insertions relative to the translational start codon ATG. The open triangles show the T-DNA insertions. B, Semiquantitative RT-PCR for RCA in the wild type, rca-1, rca-2, the rca-1 mutant complemented with the wild-type RCA gene (referred to as RF1), and the rca-2 mutant complemented with the wild-type RCA gene (referred to as RF2). The amplified actin1 is shown as an internal control. C, Western blot for RCA in wild-type, rca-1, rca-2, RF1, and RF2 plants. The immunoblot was detected with GST antibody as a protein-loading control. D, Phenotypes of wild-type, rca-1, rca-2, RF1, and RF2 plants at different developmental stages (top, 3 weeks old; bottom, 9 weeks old).
Semiquantitative RT-PCR showed that the RCA transcripts were present in the wild type but not in the rca-1 mutant (Fig. 6B). Western-blot analysis indicated that the RCA protein bands could be detected in the wild type but not in the rca-1 mutant (Fig. 6C). Thus, the rca-1 mutant represents a true null or severe loss-of-function mutation for the RCA gene, while the rca-2 mutant is a leaky allele with an obvious decrease in the RCA transcripts and RCA protein level (Fig. 6, B and C).
The rca-1 mutant seedlings were yellow, seriously stunted, and unable to grow further to set seeds (Fig. 6D), while the rca-2 mutant seedlings were yellow-green, displayed slight reduction in vegetative growth, and could grow further to set a small quantity of seeds (Fig. 6D).
We further introduced a RCA genomic fragment, containing its endogenous promoter region and the RCA entire coding region, into the rca-1 and rca-2, mutant plants respectively. The rca-1 and rca-2 mutant plants transgenic for this RCA genomic fragment (referred as to RF1 and RF2), in which the RCA transcripts and RCA protein level were similar to that in wild-type plants, displayed the wild-type-like phenotypes with green leaves and normal growth (Fig. 6, B–D). These results demonstrate that the T-DNA insertions within the RCA gene are indeed responsible for the phenotypes of the rca mutants.
Mutation in RCA Led to Typical Senescence-Associated Features
The 3-week-old rca-1 and rca-2 mutant leaves both showed chlorotic phenotypes to different degrees: the leaves from the rca-1 mutant exhibited yellowing (Fig. 7A), while the leaves from the rca-2 mutant displayed yellow-green coloring (Fig. 7A). Measurement of the chlorophyll content, a typical senescence-associated physiological marker (Yoshida et al., 2002), showed that the relative chlorophyll contents in 3-week-old rca-1 and rca-2 mutant leaves were only 42.5% and 78.0% of that in wild-type leaves, respectively (Fig. 7B).
Mutation in RCA led to typical senescence-associated features. A, Phenotypes of 3-week-old wild-type, rca-1, and rca-2 leaves. B, Relative chlorophyll contents of 3-week-old wild-type, rca-1, and rca-2 leaves. The chlorophyll content in the wild type was set to 100%, and the relative chlorophyll contents in the rca-1 and rca-2 mutants were calculated accordingly. C, Quantitative analysis of SEN4, SAG13, and SAG21 relative expression levels in 3-week-old wild-type, rca-1, and rca-2 plants. The expression level in the wild type was set to 1, and the relative expression levels in other samples were calculated accordingly. D, Quantitative analysis of CAB1, CAB2, and RBCS relative expression levels in 3-week-old wild-type, rca-1, and rca-2 plants. The expression level in the wild type was set to 1, and the relative expression levels in other samples were calculated accordingly.
As leaf senescence could also be assessed by examining the changes of senescence marker gene expression, we further analyzed the expression pattern of senescence marker genes, including three senescence-induced genes, SAG21 (for senescence-associated gene 21), SAG13, and SEN4 (for senescence 4; Park et al., 1998; Weaver et al., 1998), and three senescence-reduced genes, CAB1 (for chlorophyll a/b-binding protein 1), CAB2, and RBCS (for Rubisco small subunit; Park et al., 1998; Weaver et al., 1998), in 3-week-old rca mutants. The expression of SAG21, SAG13, and SEN4 was significantly up-regulated, whereas the expression of CAB1, CAB2, and RBCS was obviously down-regulated, in the null mutant rca-1 compared with the wild type (Fig. 7, C and D). Similar to rca-1, the leaky allele rca-2 also exhibited up-regulation of the senescence-induced genes and down-regulation of the senescence-reduced genes, although at a moderate level (Fig. 7, C and D).
These results showed that mutation in RCA led to various features typically associated with leaf senescence, such as leaf yellowing, loss of chlorophyll, up-regulation of senescence-induced genes, and down-regulation of senescence-reduced genes. Together with the observations shown in Figure 5, our data suggest that the COI1-dependent JA repression of RCA plays an important role in JA-induced leaf senescence.
Mutation in RCA Led to a Decrease in JA-Induced Expression of COR1, PDF1.2, and Thi2.1
Having shown a clear role of RCA in JA-induced leaf senescence, we attempted to investigate whether RCA functioned in other JA-regulated responses. As the rca mutants showed defects in growth and development, they were unavailable for analyzing JA responses by a physiological index such as root growth measurement. Thus, we detected the expression patterns of JA-regulated genes, including COR1 (for coronatine-induced protein 1), PDF1.2 (for plant defensin 1.2), Thi2.1 (for thionin 2.1), and VSP (for vegetative storage protein), in the 3-week-old rca mutants.
As shown in Figure 8, the expression of COR1, PDF1.2, Thi2.1, and VSP genes was induced by JA in the wild type but not in the coi1-1 mutant, which was consistent with previous studies (Benedetti et al., 1995, 1998; Xu et al., 2002).
Mutation in RCA led to a decrease in JA-induced expression of COR1, PDF1.2, and Thi2.1. A, Semiquantitative RT-PCR for COR1, PDF2.1, and Thi1.2 in 3-week-old plants upon treatment with MeJA (+) or water (−) for 8 h. The amplified actin1 is shown as an internal control. B, Northern blot for PDF1.2 and Thi2.1 in 3-week-old plants upon treatment with MeJA (+) or water (−) for 8 h. Ethidium bromide staining of rRNA was used as a loading control. C, Northern blot for VSP in 3-week-old plants upon treatment with MeJA (+) or water (−) for 8 h. Ethidium bromide staining of rRNA was used as a loading control. D, Semiquantitative RT-PCR for IAA5 in 3-week-old plants upon treatment with IAA (+) or water (−) for 1 h. The amplified actin1 is shown as an internal control.
We found that the JA-induced expression of COR1, PDF1.2, and Thi2.1 genes was significantly down-regulated in the null mutant rca-1 compared with that in the wild type (Fig. 8, A and B). In the RF1 transgenic plants, the JA-induced expression recovered to a similar level to the wild type (Fig. 8, A and B). We further found that the leaky allele rca-2 also displayed an obvious reduction in JA-induced expression of these three genes and that the decrement was almost similar to that in the rca-1 mutant (Fig. 8, A and B). However, mutation in RCA did not block the JA-induced expression of VSP (Fig. 8C), indicating that the regulation mechanism of VSP might be different from that of COR1, PDF1.2, and Thi2.1 under JA treatment. Previous studies also showed that VSP was positively regulated, whereas PDF1.2 was negatively regulated, by AtMYC2 and AtERF4 upon JA treatment (Lorenzo et al., 2004; McGrath et al., 2005; Pré, 2006; Memelink, 2009).
Our results showed that the disruption of RCA led to a decrease in JA-induced expression of PDF1.2 and Thi2.1, which were previously identified as JA-regulated defense genes (Penninckx et al., 1996; Vignutelli et al., 1998), implying a possible role for RCA in JA-mediated defense responses. Consistent with our data, it has been reported that the tobacco (Nicotiana tabacum) RCA gene was involved in herbivore resistance (Giri et al., 2006; Mitra and Baldwin, 2008). The tobacco RCA was decreased at the levels of transcript and protein abundance by herbivore damage (Giri et al., 2006). Furthermore, down-regulation of this RCA gene caused reduced defense against herbivore attack (Mitra and Baldwin, 2008).
To investigate whether the reduction of JA-induced gene expression in the 3-week-old rca mutants is a general stress response to hormone overexposure for their growth defects, we examined the indole-3-acetic acid (IAA)-induced IAA5 expression in these plants. The induction of IAA5 expression in the rca-1 and rca-2 mutants was comparable to that in the wild type upon IAA treatment (Fig. 8D), suggesting that the defective of JA-induced gene expression in the rca mutants is a specific response to JA signal rather than a general response to hormone overexposure.
Down-Regulation of RCA Protein Was Involved in Dark-Induced Senescence
Previous studies showed that diverse developmental signals and various environmental stresses including dark treatment were able to specifically induce senescence in Arabidopsis (Schippers et al., 2007). To investigate whether dark-induced senescence also correlates with the reduction of RCA protein, we examined the RCA protein in whole seedlings or detached leaves of Arabidopsis wild-type plants under dark treatment.
As shown in Figure 9, dark incubation induced senescence in detached leaves after 10 d of treatment and in whole seedlings after 15 d of treatment. Visible yellowing was observed in detached leaves (Fig. 9A), and the chlorotic phenotypes were also found in leaves and stems of whole seedlings (Fig. 9C). Correlated with dark-induced senescence, the protein abundance of RCA was severely reduced in detached leaves or whole seedlings (Fig. 9, B and D). These data suggest that down-regulation of RCA protein is also involved in dark-induced senescence.
Down-regulation of RCA protein was involved in dark-induced senescence. A, Phenotypes of 3-week-old wild-type detached leaves upon dark treatment (+) or not (−) for 10 d. B, Western blot for RCA in 3-week-old wild-type detached leaves upon dark treatment (+) or not (−) for 10 d. The immunoblot was detected with GST antibody as a protein-loading control. C, Phenotypes of 3-week-old wild-type whole seedlings upon dark treatment (+) or not (−) for 15 d. D, Western blot for RCA in 3-week-old wild-type whole seedlings upon dark treatment (+) or not (−) for 15 d. The immunoblot was detected with GST antibody as a protein-loading control.
DISCUSSION
Leaf senescence is a developmental program that is regulated by intrinsic factors such as hormones and developmental age and environmental factors such as temperature, light, nutrients, and pathogens (Schippers et al., 2007; Balazadeh et al., 2008). The JA-induced leaf senescence has been known for a long time (Ueda and Kato, 1980). In this study, we specially investigated the molecular mechanism underlying JA induction of leaf senescence. We identified RCA as a COI1-dependent JA-repressed protein using 2-D DIGE coupled with MALDI-TOF mass spectrometry. We found that RCA was down-regulated in response to JA treatment at levels of transcript and protein abundance in a COI1-dependent manner. Further genetic and physiological analyses showed that the COI1-dependent JA repression of RCA correlated with JA-induced senescence and that loss of RCA led to typical senescence-associated features. Our results suggest that the COI1-dependent JA repression of RCA plays an important role in JA-induced leaf senescence. In addition, we showed that dark-induced senescence correlated with the reduction of RCA protein, indicating that down-regulation of RCA protein is also involved in dark-induced senescence.
Identification of 35 COI1-Dependent JA-Regulated Proteins Provided New Insights into JA Signaling
Previously, transcriptomics analyses revealed that JA was essential for the regulation of various genes that were involved in a variety of physiological events and that COI1 was required for the transcription of many JA-regulated genes (Sasaki et al., 2001; Devoto et al., 2005; Sasaki-Sekimoto et al., 2005; Jung et al., 2007). In this study, we identified 35 COI1-dependent JA-regulated proteins (Fig. 1; Table I). Of these, 21 proteins were induced while 14 were repressed by JA in a COI1-dependent manner (Fig. 1; Table I). To our knowledge, our report is the first proteome study on a COI1 requirement in Arabidopsis JA responses. This study is also, to our knowledge, the first application of 2-D DIGE-based proteomics to identify the JA-regulated proteins in Arabidopsis.
There were not many studies of protein expression mapping upon JA treatment in Arabidopsis, except for the identification of several proteins at redox status under JA treatment using two-dimensional gel electrophoresis (2-DE) coupled with monobromobimane labeling (Alvarez et al., 2009). Only two proteins (VSP1 and allene oxide cyclase 2), which displayed significant increases in abundance under JA treatment, were identified by both studies (Alvarez et al., 2009; Table I).
Using a proteomics approach, Rakwal and Komatsu (2000) and Mahmood et al. (2007) found that 13 proteins were regulated by JA in rice (Oryza sativa). Cho et al. (2007) also identified 52 (from shoot) and 56 (from root) nonredundant JA-regulated proteins in rice. Seven Arabidopsis homologs of these proteins were found in our study (Table I). Among these seven rice proteins, the expression patterns of three proteins (glyceraldehyde-3-phosphate dehydrogenase B subunit, putative transketolase, and RCA) were inconsistent in different studies (Cho et al., 2007; Mahmood et al., 2007), which needs to be verified. The remaining four proteins (lipoxygenase, dehydroascorbate reductase, citrate synthase, and Rubisco large chain) showed similar trends in expression patterns upon JA treatment in rice and Arabidopsis (Rakwal and Komatsu, 2000; Cho et al., 2007; Mahmood et al., 2007; Table I). These results suggest that characterization of the Arabidopsis JA-responsive proteins would have broad implications for understanding JA actions in other higher plants.
In addition, the differential 2-DE analysis of the JA precursor 12-oxophytodienoic acid (OPDA)-treated Arabidopsis leaves revealed 37 differentially expressed proteins (Dueckershoff et al., 2008). Four of these 37 OPDA-regulated proteins were also identified as JA-regulated proteins in our study: three proteins (lipoxygenase 2, dehydroascorbate reductase, and JA-responsive 1) were up-regulated both in OPDA and MeJA treatments (Dueckershoff et al., 2008; Table I), while the remaining one (sedoheptulose-1,7-bisphosphatase) was up-regulated by OPDA (Dueckershoff et al., 2008) but down-regulated by MeJA (Table I). These data indicate that JA and its precursor OPDA might play overlapping yet distinct roles in the regulation of gene expression.
The COI1-Dependent JA Repression of RCA Played an Important Role in JA-Induced Leaf Senescence
Previous studies showed that JA induced leaf senescence in Arabidopsis (He et al., 2002). In this study, we found that the JA-treated Arabidopsis wild-type plants exhibited severe reduction in RCA protein level and simultaneously displayed leaf senescence (Fig. 5), whereas the coi1 mutant was still green and its RCA protein level was not greatly affected after JA treatment for 5 d (Fig. 5). These results suggest that the COI1-dependent JA repression of RCA is interrelated with JA-induced leaf senescence. We further found that the T-DNA insertional mutation in RCA (Fig. 6) led to diverse senescence-related symptoms, manifesting as yellowing leaf, lower chlorophyll content, increased expression of senescence-induced genes (SAG13, SAG21, SEN4), and decreased expression of senescence-reduced genes (CAB1, CAB2, RBCS; Fig. 7). Our results indicate that the COI1-dependent JA repression of RCA plays a clear role in JA-induced leaf senescence.
Furthermore, we found that the RCA protein was significantly down-regulated in the dark-induced senescent Arabidopsis leaves and seedlings (Fig. 9), implying that the reduction in RCA protein is also interrelated with dark-induced senescence. Thus, down-regulation of RCA protein might be involved in many types of senescence, including JA- and dark-induced senescence.
In this study, we found that the expression level of RCA protein was down-regulated under JA treatment in a COI1-dependent manner. Although COI1 is required for the JA-repressed RCA protein expression, RCA is not the direct target of the SCFCOI1 complex. We found that RCA was unable to specifically interact with COI1 protein by coimmunoprecipitation assay (data not shown). We also found that the JA-repressed RCA protein expression was not affected by treatment of MG132, a specific inhibitor of the 26S proteasome (data not shown). These results imply that the SCFCOI1-26S proteasome-mediated proteolysis is not involved in the COI1-dependent JA-repressed RCA protein expression.
We further found that the RCA transcripts were down-regulated in response to JA in a COI1-dependent manner (Fig. 3) and that the decrease of RCA RNA preceded the reduction of RCA protein under JA treatment (Fig. 4). These data suggest that the JA-repressed RCA protein expression is mainly through the JA-repressed RCA RNA expression.
Previous studies showed that the expression of various JA pathway downstream genes was regulated by some JA-responsive transcription factors (Memelink, 2009). Several cis-acting elements bound by these transcription factors, including the GCC motif, the G-box, and TGACG (as-1-type) sequences, have been identified in the regulatory regions of these JA pathway downstream genes (Memelink, 2009). We found that the G-box (at positions −251 to −246) and TGACG (at positions −276 to –272 and −28 to –24) sequences also existed in the regulatory region of the RCA gene (Fig. 10), indicating that some JA-responsive transcription factors might bind with these motifs and function as transcription repressors (or activators) to repress (or activate) the expression of RCA RNA.
A model for JA function in Arabidopsis leaf senescence. Upon JA treatment, COI1 recruits the JAZs to the SCFCOI1 complex for ubiquitination and degradation through the 26S proteasome. The JAZ-interacting proteins (JAPs) are then released to directly or indirectly activate the JA-responsive transcription repressors (TRs), which might bind with the G-box and TGACG sequences in the regulatory region of the RCA gene to repress the expression of RCA RNA. Alternatively, the JAZ-interacting proteins are released to repress the JA-responsive transcription activators (TAs) essential for the expression of RCA RNA. The down-regulation of RCA RNA leads to the reduction of RCA protein, resulting in JA-induced leaf senescence. Down-regulation of RCA is also involved in dark-induced senescence.
We speculate that these JA-responsive transcription repressors (or activators) might be directly or indirectly activated (or repressed) by some JAZ-interacting proteins (Fig. 10). Upon JA treatment, COI1 recruits JAZs to SCFCOI1 for ubiquitination and degradation through the 26S proteasome. The JAZ-interacting proteins are released to activate (or repress) these JA-responsive transcription repressors (or activators), which leads to the down-regulation of RCA RNA. The RCA protein level is subsequently reduced, resulting in various typical senescence-associated features, including leaf yellowing, loss of chlorophyll, up-regulation of senescence-induced genes, and down-regulation of senescence-reduced genes (Fig. 10). Down-regulation of RCA might also be involved in dark-induced senescence (Fig. 10). Whether RCA expression is the primary signal for all the senescence-related changes remains to be elucidated. Identification of the JA-responsive transcription factors would provide new insights into further understanding of leaf senescence.
MATERIALS AND METHODS
Plant Materials
The Arabidopsis (Arabidopsis thaliana) mutants rca-1 and rca-2 were isolated from the T-DNA-tagged pools produced at the Salk Institute T-DNA Express (http://signal.salk.edu/cgi-bin/tdnaexpress). The Arabidopsis mutants coi1-1 (Feys et al., 1994) and coi1-2 (Xu et al., 2002) were described previously.
Seeds were surface sterilized, chilled at 4°C for 3 d, plated, and grown on Murashige and Skoog (MS) medium (Sigma) supplemented with 1% Suc under a 16-h-light (23°C–25°C)/8-h-dark (17°C–20°C) photoperiod. Soil-grown plants were under the same photoperiod.
For senescence assay in detached leaves upon JA treatment (Fig. 5A), leaves were cut from 6-week-old plants grown in soil and placed onto 100 μm MeJA (Aldrich) for 5 d.
For senescence assay in detached leaves upon dark treatment (Fig. 9A), leaves were cut from 3-week-old plants grown on MS medium and placed onto 3 mm MES buffer (pH 5.8) in the dark for 10 d.
For senescence assay in whole seedlings upon dark treatment (Fig. 9C), 3-week-old plants grown on MS medium were placed in the dark for 15 d.
Extraction of Total Proteins for 2-D DIGE
For 2-D DIGE analysis (Fig. 1; Supplemental Fig. S1), 2-week-old plants grown on MS medium were drenched in solution containing 100 μm MeJA or water for 2 d and then harvested for protein extraction.
Protein extracts were prepared by homogenizing Arabidopsis tissues in ice-cold extraction buffer (0.7 m Suc, 0.1 m KCl, 0.5 m Tris-HCl, pH 7.4, 50 mm EDTA, 1% [w/v] polyvinylpolypyrrolidone, 1 mm phenylmethylsulfonyl fluoride, and 0.2% β-mercaptoethanol) supplemented with the protease inhibitor cocktail (Roche). The protein extracts were placed on ice for 30 min, mixed with an equal volume of ice-cold phenol (Tris-HCl, pH 8.0, buffered), and incubated for 30 min at 4°C with shaking. After centrifugation at 5,000g for 20 min, the upper phenol phase was recovered. The proteins were allowed to precipitate with 5 volumes of cold 0.1 m ammonium acetate in methanol at −20°C overnight and then pelleted by centrifugation at 5,000g for 20 min. The pellet was then washed two times with ice-cold 0.1 m ammonium acetate in methanol, two times with 80% (v/v) acetone, and once with 70% (v/v) ethanol. The mixture was incubated at −20°C for 20 min between each wash. The pellet was then dissolved in the lysis buffer (7 m urea, 2 m thiourea, 4% [w/v] CHAPS, and 30 mm Tris-HCl, pH 8.0, adjusted to pH 9). The proteins were then cleaned up using the 2D Clean Up Kit (GE Healthcare Bio-Sciences) and redissolved in the lysis buffer after the pH was adjusted to about 8.5. Proteins were labeled with DIGE-specific Cy3 or Cy5 according to the manufacturer’s instructions (GE Healthcare Bio-Sciences). A total of 50 μg of proteins was mixed with 400 pmol of CyDye and incubated on ice in the dark for at least 30 min. The reaction was quenched by adding 1 μL of 10 mm Lys for 10 min under the same conditions. The pooled sample internal standard was always Cy2 labeled.
2-DE
Labeled samples to be separated on the same gel were mixed together with an equal volume of rehydration solution (8 m urea, 2% [w/v] CHAPS, 0.002 g mL−1 dithiothreitol, and 1% immobilized pH gradient [IPG] buffer) before performing 2-DE. The first dimension isoelectric focusing (IEF) was carried out on an Ettan IPGphor IEF system (GE Healthcare Bio-Sciences) according to the 2-DE manual of Amersham Pharmacia Biotech with a 24-cm strip, pH gradient from 4 to 7, for a total of 84,250 Vh. After IEF, IPG strips were equilibrated in the equilibration buffer (6 m urea, 30% [w/v] glycerol, 2% [w/v] SDS, 50 mm Tris-HCl, pH 8.0, and 1% bromphenol blue), first with 0.01 g mL−1 dithiothreitol and then with 0.025 g mL−1 iodoacetamide, each for 15 min. The strips were then run on 12.5% SDS-PAGE gels using the Ettan Dalt six apparatus (GE Healthcare Bio-Sciences). Gels were run at 1 W per gel for 1 h followed by 17 W per gel at 15°C until the bromphenol blue dye front had run off the bottom of the gels. For each condition analysis, three replicate gels were prepared from three pairs of independent samples.
Image Scanning and Spot Analysis
Labeled gels were scanned at a resolution of 100 μm using a Typhoon laser scanner (GE Healthcare Bio-Sciences). Cy2-, Cy3-, and Cy5-labeled images of each gel were acquired at excitation/emission values of 488/520 nm, 532/580 nm, and 633/670 nm, respectively. Gel analysis was carried out using the Decyder version 6.5 software (GE Healthcare Bio-Sciences). Gels were first processed by the Decyder differential in-gel analysis module for spot detection, spot volume quantification, and volume ratio normalization of different samples on the same gel. Gel-to-gel matching and statistical analysis were carried out using the Decyder biological variation analysis software module. The statistical significance of quantitative data was determined using Student’s t test (n = 3). We used a spot abundance ratio of greater than 1.15 or less than −1.15 and P < 0.05 as a threshold to identify differentially expressed proteins in subsequent studies. Differentially expressed protein spots between pair samples were then visually confirmed, marked, excised for trypsin digestion, and peptide mass fingerprinting analyzed on a Voyager DE STR MALDI-TOF mass spectrometer (Applied Biosystems) with Proteomics Solution 1 software (Applied Biosystems). The obtained spectrum was analyzed with the Data Explorer software. The standard peptide mixtures (angiotensin II, angiotensin I, substance P, bombesin, adrenocorticotropic hormone clip 1–17, adrenocorticotropic hormone clip 18–39, and somatostatin 28) were used for external mass calibration, while self-degraded fragments of trypsin were used for internal calibration.
Protein Identification and Database Search
The mass spectrum data were used to search for protein candidates using MS-Fit in the National Center for Biotechnology Information nonredundant database. MS-Fit searching parameters were as follows: species searched, Arabidopsis; molecular mass searched, from 1,000 to 100,000 D; pI searched, from 0 to 14; enzyme, trypsin; maximum missed cleavages, 1; N terminus, hydrogen (H); C terminus, free acid (OH); fixed modifications, carbamidomethylation of Cys (C); considered modifications, phosphorylation of Ser (S), Thr (T), and Tyr (Y); minimum number of peptides to match, 4; mass accuracy, 50 ppm.
Plasmid Construction and Arabidopsis Transformation
A 4,280-bp genomic fragment containing the RCA promoter region and the RCA coding sequence without the stop codon was amplified using RCA1 forward (RCA1-F) and reverse (RCA1-R) primers and then cloned into the pCAMBIA1305.2 vector at SmaI-NcoI sites, resulting in the pRCA-RCA::GUS construct (Fig. 3C). The pRCA-RCA::GUS construct was transferred into wild-type plants by the floral dip method of in planta Agrobacterium tumefaciens-mediated transformation (Clough and Bent, 1998).
A 4,264-bp genomic fragment containing the RCA promoter region and the RCA coding sequence without the stop codon was amplified using RCA2 forward (RCA2-F) and reverse (RCA2-R) primers and then fused to the flag epitope at a SmaI site in the pFlag vector (Ren et al., 2005), resulting in the pRCA-Flag construct. The pRCA-Flag construct was transferred into rca-1/RCA-1 and rca-2 plants by the floral dip method of in planta A. tumefaciens-mediated transformation (Clough and Bent, 1998). Three independent homologous rca-1 or rca-2 mutants transgenic for this genomic fragment were identified, which all exhibited the same phenotypes. Two representative transgenic lines, RF1 and RF2, are presented in Figures 6 and 8.
The RCA1-F, RCA1-R, RCA2-F, and RCA2-R primers are shown in Supplemental Table S1.
Northern-Blot Analysis and Semiquantitative RT-PCR
For semiquantitative RT-PCR analysis on TGG2, SBPASE, NQR, CSY4, and RCA genes upon JA treatment (Figs. 2 and 4A), 2-week-old plants grown on MS medium were drenched in solution containing 100 μm MeJA or water for 2 d and then harvested for RNA extraction.
For northern blot on the RCA gene upon JA treatment (Fig. 4B), leaves were cut from 6-week-old plants grown in soil, placed onto solution containing 100 μm MeJA or water for various time periods from 1 to 5 d, and then harvested for RNA extraction. We noted that treatment with JA for more than 3 d caused obvious reduction of total rRNA contents. Plant materials treated with JA for 4 and 5 d were not used for RNA extraction.
For semiquantitative RT-PCR on the RCA gene and senescence-related genes (Figs. 6B and 7, C and D), 3-week-old plants grown on MS medium were harvested for RNA extraction.
For RNA analysis on COR1, PDF2.1, Thi1.2, and VSP genes upon JA treatment (Fig. 8, A–C), 3-week-old plants grown on MS medium were drenched in solution containing 100 μm MeJA or water for 8 h in the daytime and then harvested for RNA extraction.
For semiquantitative RT-PCR on the IAA5 gene upon IAA treatment (Fig. 8D), 3-week-old plants grown on MS medium were drenched in solution containing 20 μm IAA or water for 1 h in the daytime and then harvested for RNA extraction.
Total RNAs were isolated with the TRIzol reagent. For northern blot, the probe labeling and RNA gel-blot hybridization were performed as described previously (Xu et al., 2002; Shan et al., 2009). For semiquantitative RT-PCR, the RT and RT-PCR were performed as described previously (Shan et al., 2009). For relative gene expression level assay, northern-blot analysis or semiquantitative RT-PCR was repeated three times. Bio-Rad Quantity One software was used for analysis and quantification. The primers used for probe amplification and gene RT-PCR analysis are shown in Supplemental Table S1.
Western Blot
For western blot on RCA protein upon JA treatment (Figs. 3, A and B, and 5B), leaves were cut from 6-week-old plants grown in soil, placed onto solution containing 100 μm MeJA or water for various time periods from 1 to 5 d, and then harvested for protein extraction.
For western blot on RCA protein (Fig. 6C), 3-week-old plants grown on MS medium were harvested for protein extraction.
For western blot on RCA protein in detached leaves upon dark treatment (Fig. 9B), leaves were cut from 3-week-old plants grown on MS medium, placed onto 3 mm MES buffer (pH 5.8) in the dark for 10 d, and then harvested for protein extraction.
For western blot on RCA protein in whole seedlings upon dark treatment (Fig. 9D), 3-week-old plants grown on MS medium were placed in the dark for 15 d and then harvested for protein extraction.
The western-blot analysis was performed as described previously (Xu et al., 2002). Anti-RCA antibody was generated by Jiang et al. (2001). The crude antisera of anti-glutathione S-transferase (GST) and anti-RCA were used at dilutions of 1:4,000 and 1:20,000, respectively. For relative protein expression level assay, western-blot analysis was repeated three times. The Bio-Rad Quantity One software was used for analysis and quantification. Anti-GST antibody was made by Alpha Diagnostic.
GUS Staining Assay
Leaves, stems, floral buds, and siliques were cut from 6-week-old transgenic plants harboring the pRCA-RCA::GUS construct grown in soil, placed onto solution containing 100 μm MeJA or water for 5 d, and then harvested for GUS staining (Fig. 3D). Histochemical staining for GUS activity assay was performed as described previously (Liu et al., 2004).
Chlorophyll Measurement
Leaves from 3-week-old plants grown on MS medium were harvested for chlorophyll content measurement (Fig. 7B). Chlorophyll measurement was performed as described previously (Xiao et al., 2004). Experiments were repeated three times.
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers At2g39940 (COI1), At2g39730 (RCA), At5g25980 (TGG2), At3g55800 (SPBASE), At3g27890 (NQR), At2g44350 (CSY4), At4g30270 (SEN4), At2g29350 (SAG13), At4g02380 (SAG21), At1g29930 (CAB1), At1g29920 (CAB2), At1g67090 (RBCS), At1g19670 (COR1), At5g44420 (PDF1.2), At1g72260 (Thi2.1), At5g24780 (VSP1), At5g24770 (VSP2), At1g15580 (IAA5), and At2g37620 (actin1).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. 2-D DIGE analysis using the mixed-sample internal standard.
Supplemental Table S1. The primers used for probe amplification, semiquantitative RT-PCR, and vector construction.
Acknowledgments
We thank the Arabidopsis Biological Resource Center for providing the rca-1 and rca-2 mutant seeds.
Footnotes
↵1 This work was supported by the Ministry of Science and Technology (973 Program grant no. 2011CB915404), the Ministry of Agriculture (National Key Program for Transgenic Breeding grant no. 2008ZX08009–003), the National Natural Science Foundation of China (grant nos. 91017012 and 30800593), and the Ministry of Education (grant nos. 20070003046 and 20070003038).
↵2 These authors contributed equally to the article.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Daoxin Xie (daoxinlab{at}tsinghua.edu.cn).
↵[W] The online version of this article contains Web-only data.
- Received September 29, 2010.
- Accepted December 7, 2010.
- Published December 20, 2010.