the role of arabidopsis rubisco activase inthe role of arabidopsis rubisco activase in...

The Role of Arabidopsis Rubisco Activase inJasmonate-Induced Leaf Senescence1[W]

Xiaoyi Shan2, Junxia Wang2, Lingling Chua, Dean Jiang, Wen Peng, and Daoxin Xie*

MOE Key Laboratory of Protein Science, School of Life Sciences, Tsinghua University, Beijing 100084, China(X.S., W.P., D.X.); Institute of Molecular and Cell Biology, Singapore 138673 (J.W., L.C.); and College of LifeSciences, Zhejiang University, Hangzhou 310029, China (D.J.)

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 forJA-induced leaf senescence remains unknown. In this study, proteomic, genetic, and physiological approaches were used toreveal 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 RCAwas down-regulated at the levels of transcript and protein abundance by JA in a COI1-dependent manner. We further found that loss ofRCA led to typical senescence-associated features and that the COI1-dependent JA repression of RCA played an important rolein JA-induced leaf senescence.

Leaf senescence, as the last stage of leaf develop-ment, proceeds through a highly regulated program inorder to remobilize the nutrients from areas where it isno longer required to areas of cell development in theplant (Buchanan-Wollaston, 1997; Quirino et al., 2000).The onset of leaf senescence is age dependent but alsocan be stimulated by diverse developmental signals,sugar, plant hormones, and environmental stresses, in-cluding energy deprivation, darkness, excess light,drought, salinity, nutrient limitation, and wounding(Schippers et al., 2007; Balazadeh et al., 2008). Theprogression of leaf senescence is accompanied by therapid loss of chlorophyll, the decreased abundance ofphotosynthesis-related proteins (Bate et al., 1991), andthe increased expression of senescence-associatedgenes (Nam, 1997). The regulation of leaf senescencealso involves numerous transcription factors, such asNAC domain-containing protein, WRKY DNA-bind-ing protein, MYB domain protein, C2H2-type zincfinger, basic leucine-zipper, and APETALA2/ethyl-ene-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 anddefense signals, control many plant developmentaland growth processes and mediate plant responses toabiotic and biotic stresses (McConn et al., 1997; Raoet 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; Kimet al., 2009; Koo and Howe, 2009; Ren et al., 2009;Shan et al., 2009). JA also functions in the induction ofleaf senescence in many plant species (Ueda and Kato,1980; Weidhase et al., 1987; Reinbothe et al., 2009). InArabidopsis (Arabidopsis thaliana), exogenous applica-tion of JA promotes leaf senescence (He et al., 2002)and regulates the expression of various genes that areinvolved in leaf senescence (Buchanan-Wollastonet al., 2005; Jung et al., 2007). However, the molecularmechanism 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 signalpathway. It directly binds to JA-Ile and functions as aJA receptor (Yan et al., 2009). COI1 assembles theSCFCOI1 complex (Xu et al., 2002; Liu et al., 2004; Renet al., 2005; Wang et al., 2005) to recruit the jasmonateZIM-domain proteins (JAZs) for degradation by the26S proteasome (Chini et al., 2007; Thines et al., 2007;Katsir et al., 2008) and subsequently regulates variousplant developmental and growth processes. The nullmutant coi1-1 (Feys et al., 1994), with the prematurestop codon at Trp-467, is male sterile, insensitive to JA-inhibitory root growth, defective in JA-regulated geneexpression, and supersensitive to insect attack andnecrotrophic pathogen infection (Feys et al., 1994; Xieet al., 1998; Reymond et al., 2000). The coi1-2 mutant, a

1 This work was supported by the Ministry of Science andTechnology (973 Program grant no. 2011CB915404), the Ministry ofAgriculture (National Key Program for Transgenic Breeding grantno. 2008ZX08009–003), the National Natural Science Foundationof China (grant nos. 91017012 and 30800593), and the Ministry ofEducation (grant nos. 20070003046 and 20070003038).

2 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Daoxin Xie ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.110.166595

Plant Physiology�, February 2011, Vol. 155, pp. 751–764, www.plantphysiol.org � 2010 American Society of Plant Biologists 751 www.plantphysiol.orgon March 24, 2020 - Published by Downloaded from

Copyright © 2011 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

leaky allele with the missense mutation L245F, isresistant to JA but is partially fertile and able to pro-duce a small quantity of seeds (Xu et al., 2002). The coi1mutant plants also exhibit relatively delayed senes-cence phenotypes, including elongated flowering timeand relatively higher chlorophyll content (Xiao et al.,2004). However, it remains to be elucidated how COI1regulates leaf senescence.

In this study, 35 proteins were identified as COI1-dependent JA-regulated proteins by two-dimensionaldifference gel electrophoresis (2-D DIGE) coupled withmatrix-assisted laser desorption inoization-time offlight (MALDI-TOF) mass spectrometry. Further studyon Rubisco activase (RCA), one of these 35 identifiedproteins, revealed that RCAwas down-regulated at thelevels of transcript and protein abundance by JA in aCOI1-dependent manner. Molecular, genetic, and phys-iological analyses showed that mutation in RCA led totypical senescence-related symptoms and that theCOI1-dependent JA repression of RCA played an im-portant role in JA-induced leaf senescence.

RESULTS

Identification of COI1-Dependent JA-Regulated Proteinsby 2-D DIGE

To examine the requirement of COI1 for JA-regu-lated gene expression at the posttranscriptional level,2-week-old wild-type and coi1-1 mutant plants weredrenched in a solution containing 100 mM methyljasmonate (MeJA) for 2 d and subsequently harvestedto perform 2-D DIGE analysis (Supplemental Fig.S1A). The comparative image analysis of JA-treatedwild-type with JA-treated coi1-1 mutant plants identi-fied 61 protein spots that changed significantly inabundance with P, 0.05. We performed peptide massfingerprinting via MALDI-TOF mass spectrometry onthese 61 protein spots and successfully generatedprotein assignments for 43 spots, which represented35 unique proteins (Fig. 1; Table I). In JA-treated wild-type plants, 21 out of the 35 proteins were up-regu-lated whereas 14 were down-regulated comparedwithJA-treated coi1-1 (Fig. 1; Table I), suggesting that bothJA treatment and COI1 existence are required for theregulation of these 35 proteins.

To verify whether JA treatment is essential for theregulation of these 35 proteins, we compared theprotein profiles of wild-type and coi1-1 mutant plants(Supplemental Fig. S1B). We found that, without JAtreatment, the expression levels of these 35 proteinshad no significant differences (P . 0.05) between thewild type and the coi1-1 mutant (Table I), indicatingthat JA treatment is indispensable for the regulation ofthese proteins. We further compared the protein pro-files between coi1-1 and JA-treated coi1-1 to verifywhether COI1 is required for the JA-regulated expres-sion of these 35 proteins (Supplemental Fig. S1C). Wefound that, without COI1, the expression levels of

these 35 proteins in the coi1-1 mutant had no obviousdifferences (P . 0.05) irrespective of whether JA wasapplied or not (Table I), suggesting that COI1 isessential for the JA-regulated expression of these pro-teins. Collectively, these data demonstrate that these35 proteins are regulated by JA in a COI1-dependentmanner.

These 35 proteins, including 21 COI1-dependentJA-induced proteins and 14 COI1-dependent JA-repressed proteins, were classified based on the bio-logical process in which the gene product participated(Table I). Most of these proteins encode enzymes thatpotentially mediate specific cellular and physiologicalprocesses, such as JA biosynthesis, amino acid metab-olism, photosynthesis/chlorophyll metabolism, cellu-lar respiration, and defense/stress responses (Table I).

By comparison of our proteomics data with previ-ous transcriptomics data (Sasaki et al., 2001; Sasaki-Sekimoto et al., 2005; Jung et al., 2007), we found that15 of these 35 JA-regulated proteins were identifiedas JA-regulated RNAs in previous microarray studies(Table I). Qualitative changes in RNA levels of thesegenes were consistent with those in protein levelsupon 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 oxidoreduc-tase; At3g27890), and CSY4 (for citrate synthase;At2g44350), under JA treatment to further comparethe proteomics and transcriptomics data using semi-quantitative reverse transcription (RT)-PCR. We found

Figure 1. A representative 2-D DIGE image of Cy2-labeled JA-treatedwild-type and JA-treated coi1-1 pooled internal standard proteomemap. Proteins were resolved first on a 24-cm pH 4 to 7 IPG strip andfurther separated on a 12.5% SDS-PAGE gel. Proteins with alteredexpression between JA-treated wild-type and JA-treated coi1-1 plantswere identified by MALDI-TOF mass spectrometry and marked withspot numbers (their identities are shown in Table I). The spots markedwith white dashed and black solid arrows indicated increase ordecrease in JA-treated wild-type plants over JA-treated coi1-1 plants,respectively.

Shan et al.

752 Plant Physiol. Vol. 155, 2011 www.plantphysiol.orgon March 24, 2020 - Published by Downloaded from



Table I. The COI1-dependent JA-regulated proteins

A total of 35 COI1-dependent JA-regulated proteins were classified based on the biological process in which the gene product participated, andtheir corresponding spots are shown on Figure 1. Col-0, Ecotype Columbia.

FunctionSpot

No.Gene Locus Protein Name

JA-Treated

coi1-1/ JA-

Treated Col-0

JA-Treated

coi1-1/Water-

Treated coi1-1

Water-Treated

coi1-1/Water-

Treated Col-0Mowse

Score

No. of

Matching

Peptidesb

Protein

Coveragec

Ratioa P Ratioa P Ratioa P

%Storage protein 1 At5g24780d,e Vegetative storage protein 1 230.71 1.80E-08 21.76 5.60E-01 22.05 2.10E-01 3.43E+06 13 38

2 At5g24770d Vegetative storage protein 2 244.80 1.80E-07 21.75 6.20E-01 22.71 2.10E-01 1.31E+06 10 40

JA biosynthesis

protein

3 At3g45140d,f,g Lipoxygenase 2 28.44 2.20E-05 21.07 7.90E-01 2.30 5.70E-01 5.38E+09 19 25



6 At3g25770d,e Allene oxide cyclase 2 24.55 2.00E-05 21.14 6.50E-01 21.09 3.70E-01 6.83E+03 6 32

Jacalin lectin

family protein

7 At3g16420d Jacalin-related

lectin 30

22.26 3.50E-05 21.51 2.30E-01 21.25 2.30E-01 3.38E+06 11 52

8 At3g16450d Jacalin lectin family protein 21.92 1.90E-03 21.19 5.20E-01 21.18 1.60E-01 1.66E+05 7 36

9 At3g16460d Jacalin lectin

family protein

21.86 2.10E-02 1.16 4.80E-01 21.09 1.58E+00 2.28E+04 6 13

Amino acid

synthesis and

modification

protein

10 At4g23600d Cystine lyase 26.98 1.50E-05 1.27 3.40E-01 1.23 5.70E-02 8.63E+05 10 25

11 At4g14880 Cytosolic

O-acetylserine(thiol)lyase

22.38 8.40E-05 21.04 8.90E-01 1.11 3.50E-01 2.78E+05 8 30

12 At3g54640d Trp synthase a-subunit 21.50 5.30E-03 1.06 5.10E-01 1.05 6.40E-01 5.10E+05 7 36

13 At5g17920 Met synthase 20.64 7.60E-03 21.04 7.40E-01 3.04 6.30E-02 1.08E+13 19 33

14 At4g33010 Gly decarboxylase P-protein 1 1.99 4.90E-04 1.95 5.40E-01 2.44 1.40E-01 7.79E+09 17 25

15 At4g33010 Gly decarboxylase P-protein 1 1.70 6.10E-04 1.12 6.70E-01 1.94 1.70E-01 4.55E+08 14 21

Cellular

respiration

protein

16 At2g44350f Citrate synthase 1.16 2.00E-02 1.21 5.70E-01 1.21 2.80E-01 5.52E+11 17 48

17 At1g42970d,f Glyceraldehyde-3-phosphate

dehydrogenase B subunit

1.53 1.50E-04 1.33 2.80E-01 1.30 2.50E-01 7.70E+06 14 31

18 At5g11670 Putative malate oxidoreductase 1.38 1.00E-02 1.06 7.00E-01 1.47 1.20E-01 4.64E+10 17 35

Defense-related

protein

19 At1g19570d,f,g Dehydroascorbate reductase 22.89 1.20E-06 21.17 3.60E-01 21.18 1.70E-01 1.01E+07 8 59

20 At3g12490 Protein with Cys proteinase

inhibitor activity

21.66 7.50E-04 21.40 3.50E-01 21.58 1.50E-01 6.33E+04 6 39

21 At1g52400d b-Glucosidase homolog 1 24.94 8.50E-06 21.24 9.70E-01 21.27 1.04E+00 4.88E+07 12 37

22 At1g52400d b-Glucosidase homolog 1 22.44 1.40E-04 21.12 7.80E-01 21.27 1.20E-01 1.88E+05 8 24

23 At5g16970 2-Alkenal reductase 1.72 3.20E-05 1.64 8.40E-02 1.05 9.40E-01 4.01E+05 8 33

Translation

elongation

factor protein

24 At1g56070 Translation elongation factor

2-like protein

1.68 1.60E-03 1.44 6.00E-01 1.04 1.80E-01 1.21E+09 16 22

25 At1g56070 Translation elongation factor

2-like protein

2.01 1.10E-03 1.14 7.10E-01 2.99 1.10E-01 5.71E+10 22 26

Photosynthesis-

and

chlorophyll-

related

protein

26 At3g27890 NAD(P)H:quinone reductase 21.73 3.30E-05 21.30 2.10E-01 21.02 9.50E-01 1.76E+04 6 33

27 At3g04790 Ribose 5-phosphate

isomerase-related

21.34 2.00E-03 21.30 5.20E-01 21.03 9.00E-01 1.57E+06 9 50

28 AtCG00480 b-Subunit of ATP synthase 22.62 7.40E-05 1.40 1.60E-01 1.32 5.10E-01 1.24E+09 13 36

29 At3g60750f Putative transketolase 1.46 3.30E-03 1.26 4.90E-01 2.08 1.00E-01 2.04E+10 16 27

30 At1g32060 Phosphoribulokinase 1.81 2.40E-03 21.11 9.40E-01 1.25 1.20E-01 2.23E+09 12 46

31 AtCG00490f Rubisco large chain 1.58 9.10E-04 1.30 4.90E-01 1.14 6.20E-01 4.20E+08 16 32

32 AtCG00490f Rubisco large chain 1.68 3.50E-02 1.59 2.00E-01 1.14 5.50E-01 1.59E+05 10 24

33 At2g39730f Rubisco activase 1.50 3.40E-04 1.24 2.60E-01 21.03 9.20E-01 1.80E+08 11 31

34 At3g55800g Sedoheptulose-1,7-

bisphosphatase

1.57 3.60E-04 21.14 3.80E-01 1.20 1.80E-01 2.53E+07 13 36

Other protein 35 At5g24420d Glucosamine/galactosamine-

6-phosphate

isomerase-related

22.35 2.60E-02 1.15 6.00E-01 21.27 8.70E-01 1.43E+04 5 27

36 At5g25980 Myrosinase (thioglucoside

glucohydrolase)

21.87 3.00E-03 1.15 6.60E-01 21.00 9.70E-01 1.20E+10 16 33

37 At3g16470d,g JA-inducible protein isolog 23.43 2.90E-05 21.03 9.30E-01 21.11 8.10E-01 7.32E+05 8 34



40 At3g14540 Terpene synthase/cyclase

family protein

22.34 3.60E-08 21.39 3.30E-01 21.18 9.00E-01 9.64E+03 5 14

41 At5g65020d Annexin Arabidopsis 2 21.64 2.40E-04 21.02 7.50E-01 1.55 7.50E-01 5.13E+04 8 26

42 At4g01870 tolB protein-related 2.22 7.20E-04 3.40 1.10E-01 1.95 9.50E-02 1.03E+09 12 32

43 At3g13470 GroEL-like chaperone 1.31 5.00E-03 1.01 9.30E-01 1.48 8.20E-02 1.37E+08 13 30

aAverage volume ratio quantified by Decyder 6.5 biological variation analysis module. bNumber of peptide masses matching the top hit fromMS-Fit peptide mass fingerprinting. cAmino acid sequence coverage for the proteins. dJA-regulated genes identified previously in Arabidopsisby microarray analysis. eJA-regulated proteins identified previously in Arabidopsis. fSimilar JA-regulated proteins identified in rice. gOPDA-regulated proteins identified previously in Arabidopsis.

Role of RCA in JA-Induced Leaf Senescence

Plant Physiol. Vol. 155, 2011 753 www.plantphysiol.orgon March 24, 2020 - Published by Downloaded from



that the expression patterns of TGG2 and SBPASE atthe transcript level correlated well with that at theprotein level (Fig. 2, A and B; Table I). However, thetranscript patterns of NQR and CSY4 did not correlatewith their protein expression patterns. The transcriptlevels ofNQR and CSY4were similar in wild-type andcoi1-1 mutant plants irrespective of whether JA wasapplied or not (Fig. 2C), whereas proteomics datashowed that NQR protein was up-regulated and CSY4protein was down-regulated by JA in the wild type(Table I), which might result from posttranscriptionalprocesses such as mRNA splicing, mRNA degrada-tion, mRNA translation, and protein modification.These data indicate that the posttranscriptional regu-lation might also function in JA signaling, yet itremains relatively understood when compared withthe transcriptional regulation mechanism. As proteins,not RNAs, are the functional elements in diversebiological processes, the proteomics data generatedin our study may be more relevant to understandingJA signaling.

The Expression of RCA Protein Was Down-Regulated by

JA in a COI1-Dependent Manner

Among these 35 proteins, RCA was identified as aCOI1-dependent JA-repressed protein (Table I). RCAwas reported to catalyze the light activation of Rubisco(Portis, 2003) and to function in plant photosynthesisand growth (Mate et al., 1993; Jiang et al., 1994; Eckardtet al., 1997). To verify the 2-D DIGE data, we examinedthe RCA expression pattern using our previouslygenerated RCA antibody (Jiang et al., 2001), whichcould detect 43- and 47-kD protein bands, two iso-forms of RCA arising from mRNA alternative splicing(Salvucci et al., 1987; Shen et al., 1991).

Similar to our 2-D DIGE data, western-blot analysiswith RCA antibody showed that the expression levelof RCA protein was down-regulated by JA in a COI1-dependent manner. The amount of RCA protein de-clined gradually in response to JA treatment and wasreduced significantly upon JA treatment for 5 d in thewild type but not obviously in the coi1-2 mutant (Fig.3, A and B). The RCA protein level in the wild typewas 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).

To further verify the JA-repressed RCA proteinexpression, we made transgenic plants harboring thepRCA-RCA::GUS construct, in which the GUS reporterwas translationally fused to the RCA protein under thecontrol of the RCA endogenous promoter fragmentextending from 21,982 to 21 (relative to the RCAtranslational start ATG; Fig. 3C). Upon JA treatmentfor 5 d, RCA::GUS fusion protein was almost absent inthe transgenic plants (Fig. 3D), whereas a strongexpression of the RCA::GUS fusion protein, withoutJA 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 RCATranscripts Preceded the Reduction

of RCA Protein upon JA Treatment

As changes of protein level may not necessarily bereflected at the RNA level (Griffin et al., 2002; Huberet al., 2004; Tian et al., 2004; Fig. 2), we analyzed theRCA RNA expression pattern in comparison with itsprotein expression pattern in the same tissues ana-lyzed by 2-D DIGE. RT-PCR analysis showed that thelevel of RCA transcripts was down-regulated by JA inthe wild type but not in the coi1-1 mutant (Fig. 4A),suggesting that the down-regulation of RCA proteinby JA in a COI1-dependent manner is also reflected atits RNA level.

Through comparison of RCA expression patterns atthe protein level (Fig. 3A) and the RNA level (Fig. 4B)under JA treatment for various time periods, we foundthat the RCA transcripts were decreased more quicklythan its protein level in response to JA treatment.When the wild type was treated by JA for 1 and 3 d, the

Figure 2. Semiquantitative RT-PCR for the indicated genes in 2-week-old wild-type and coi1-1 mutant plants upon treatment with MeJA (+)or water (2) for 2 d. The genes are indicated on the right side of eachpanel. The amplified actin1 is shown as an internal control. Col-0,Ecotype Columbia.

Shan et al.




RCA transcripts exhibited 60.7% and 89.5% reduction,respectively (Fig. 4B), while the RCA protein dis-played only 9.4% and 34.1% reduction, respectively(Fig. 3A). Thus, the decrease of RCA transcripts pre-cedes the reduction of RCA protein upon JA treatment,

which is different from other chloroplast proteins thatare decreased more rapidly than their transcriptsunder JA treatment (Reinbothe et al., 1993, 1994, 1997).

The COI1-Dependent JA Repression of RCA Correlatedwith JA-Induced Leaf Senescence

As shown in Figures 3A and 5B, the RCA proteincontent was significantly reduced when wild-typeleaves were treated with JA for 5 d. Correlated withthe loss of RCA, the wild-type leaves presented severesenescence, especially yellowing under JA treatmentfor 5 d (Fig. 5A).

In contrast, the leaves of the coi1-2 mutant were stillgreen or only turned a little yellow even after JA treat-ment for 5 d (Fig. 5A). Simultaneously, we found thatthe RCA protein level was not greatly affected in thecoi1-2 mutant (Fig. 5B). These data indicate that COI1is essential for JA-induced leaf senescence and that theCOI1-dependent JA repression of RCA correlates withJA-induced leaf senescence.

Figure 3. The expression of RCA protein was down-regulated by JA in aCOI1-dependent manner. A, Western blot for RCA (top) and quantita-tive analysis of RCA protein level (bottom) in 6-week-old wild-typeleaves upon treatment with MeJA (+) or water (–; CK) for the indicatedtime periods. The immunoblot was detected with GST antibody as aprotein-loading control (top). The RCA protein level in the wild typeupon water treatment for 1 d was set to 1, and the relative RCA proteinlevels in other samples were calculated accordingly (bottom). B,Western blot for RCA in 6-week-old coi1-2 mutant leaves upontreatment with MeJA (+) or water (2) for the indicated time periods.The immunoblot was detected with GST antibody as a protein-loadingcontrol. C, Schematic drawing of the pRCA-RCA::GUS construct (see“Materials andMethods”). Abbreviations not defined in the text: LB, leftborder; HYG, hygromycin gene; Nos, nopaline synthase terminator;RB, right border. D, RCA::GUS fusion protein was degraded after JAtreatment in the stem (1), silique (2), leaf (3), and floral bud (4) of thetransgenic plants. The transgenic plants were treated with MeJA (+) orwater (2) for 5 d and subsequently harvested for GUS staining.

Figure 4. The decrease of RCA transcripts preceded the reduction ofRCA protein upon JA treatment. A, Semiquantitative RT-PCR for RCA in2-week-old wild-type and coi1-1 mutant plants upon treatment withMeJA (+) or water (2) for 2 d. The amplified actin1 is shown as aninternal control. B, Northern blot for RCA (top) and quantitativeanalysis of RCA RNA level (bottom) in 6-week-old wild-type leavesupon treatment with MeJA (+) or water (–; CK) for the indicated timeperiods. Ethidium bromide staining of rRNA was used as a loadingcontrol (top). The RCA RNA level in the wild type upon water treatmentfor 1 d was set to 1, and the relative RCA RNA levels in other sampleswere calculated accordingly (bottom).





Identification of the rca Mutants

To investigate whether the loss of RCA causes leafsenescence, we identified two rca mutant alleles, rca-1(SALK_118831), with a T-DNA insertion in the firstintron, 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 startcodon (Fig. 6A).

Semiquantitative RT-PCR showed that the RCA tran-scripts were present in the wild type but not in the rca-1mutant (Fig. 6B). Western-blot analysis indicated thatthe RCA protein bands could be detected in the wildtype but not in the rca-1mutant (Fig. 6C). Thus, the rca-1mutant represents a true null or severe loss-of-functionmutation for the RCA gene, while the rca-2 mutant is aleaky allele with an obvious decrease in the RCAtranscripts and RCA protein level (Fig. 6, B and C).

The rca-1 mutant seedlings were yellow, seriously stun-ted, 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 couldgrow further to set a small quantity of seeds (Fig. 6D).

We further introduced a RCA genomic fragment,containing its endogenous promoter region and theRCA entire coding region, into the rca-1 and rca-2,mutant plants respectively. The rca-1 and rca-2 mutantplants transgenic for this RCA genomic fragment(referred as to RF1 and RF2), in which the RCAtranscripts and RCA protein level were similar tothat in wild-type plants, displayed the wild-type-likephenotypes with green leaves and normal growth (Fig.6, B–D). These results demonstrate that the T-DNAinsertions within the RCA gene are indeed responsiblefor the phenotypes of the rca mutants.

Mutation in RCA Led to TypicalSenescence-Associated Features

The 3-week-old rca-1 and rca-2 mutant leaves bothshowed chlorotic phenotypes to different degrees: the

Figure 5. The COI1-dependent JA repression of RCA correlated withJA-induced leaf senescence. A, Phenotypes of 6-week-old wild-typeand coi1-2mutant leaves upon treatment with MeJA (+) or water (2) for5 d. B,Western blot for RCA in 6-week-old wild-type and coi1-2mutantleaves upon treatment with MeJA (+) or water (2) for 5 d. The immu-noblot was detected with GST antibody as a protein-loading control.

Figure 6. Identification of the rca-1 and rca-2mutants. A, Schematic diagram of the T-DNAinsertions into the Arabidopsis RCA gene(At2g39730.1). Numbers indicate the positionsof T-DNA insertions relative to the translationalstart codon ATG. The open triangles show theT-DNA insertions. B, Semiquantitative RT-PCR forRCA in the wild type, rca-1, rca-2, the rca-1 mu-tant complemented with the wild-type RCA gene(referred to as RF1), and the rca-2 mutant com-plemented with the wild-type RCA gene (referredto as RF2). The amplified actin1 is shown as aninternal control. C, Western blot for RCA in wild-type, rca-1, rca-2, RF1, and RF2 plants. Theimmunoblot was detected with GST antibody asa protein-loading control. D, Phenotypes of wild-type, rca-1, rca-2, RF1, and RF2 plants at differentdevelopmental stages (top, 3 weeks old; bottom,9 weeks old).

Shan et al.




leaves from the rca-1 mutant exhibited yellowing(Fig. 7A), while the leaves from the rca-2 mutantdisplayed yellow-green coloring (Fig. 7A). Measure-ment of the chlorophyll content, a typical senescence-associated physiological marker (Yoshida et al., 2002),showed that the relative chlorophyll contents in3-week-old rca-1 and rca-2 mutant leaves were only42.5% and 78.0% of that in wild-type leaves, respec-tively (Fig. 7B).As leaf senescence could also be assessed by exam-

ining the changes of senescence marker gene expres-sion, we further analyzed the expression pattern ofsenescence marker genes, including three senescence-induced genes, SAG21 (for senescence-associated gene21), 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 pro-tein 1), CAB2, and RBCS (for Rubisco small subunit;Park et al., 1998; Weaver et al., 1998), in 3-week-oldrca mutants. The expression of SAG21, SAG13, andSEN4 was significantly up-regulated, whereas the ex-pression of CAB1, CAB2, and RBCS was obviouslydown-regulated, in the null mutant rca-1 comparedwith the wild type (Fig. 7, C and D). Similar to rca-1,the leaky allele rca-2 also exhibited up-regulation ofthe senescence-induced genes and down-regulationof the senescence-reduced genes, although at a mod-erate level (Fig. 7, C and D).

These results showed that mutation in RCA led tovarious features typically associated with leaf senes-cence, such as leaf yellowing, loss of chlorophyll,up-regulation of senescence-induced genes, and down-regulation of senescence-reduced genes. Together withthe observations shown in Figure 5, our data suggestthat the COI1-dependent JA repression of RCA playsan 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 leafsenescence, we attempted to investigate whether RCAfunctioned in other JA-regulated responses. As the rcamutants showed defects in growth and development,they were unavailable for analyzing JA responses by aphysiological index such as root growth measurement.Thus, we detected the expression patterns of JA-regu-lated genes, including COR1 (for coronatine-inducedprotein 1), PDF1.2 (for plant defensin 1.2), Thi2.1 (forthionin 2.1), and VSP (for vegetative storage protein), inthe 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 inthe wild type but not in the coi1-1 mutant, which wasconsistent with previous studies (Benedetti et al., 1995,1998; Xu et al., 2002).

Figure 7. Mutation in RCA led to typical senes-cence-associated features. A, Phenotypes of3-week-old wild-type, rca-1, and rca-2 leaves.B, Relative chlorophyll contents of 3-week-oldwild-type, rca-1, and rca-2 leaves. The chloro-phyll content in the wild type was set to 100%,and the relative chlorophyll contents in the rca-1and rca-2 mutants were calculated accordingly.C, Quantitative analysis of SEN4, SAG13, andSAG21 relative expression levels in 3-week-oldwild-type, rca-1, and rca-2 plants. The expressionlevel in the wild type was set to 1, and the relativeexpression levels in other samples were calcu-lated accordingly. D, Quantitative analysis ofCAB1, CAB2, and RBCS relative expression levelsin 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 sampleswere calculated accordingly.





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 withthat in the wild type (Fig. 8, A and B). In the RF1transgenic plants, the JA-induced expression recov-ered to a similar level to the wild type (Fig. 8, A and B).We further found that the leaky allele rca-2 alsodisplayed an obvious reduction in JA-induced expres-sion of these three genes and that the decrement wasalmost similar to that in the rca-1mutant (Fig. 8, A andB). However, mutation in RCA did not block the JA-induced expression of VSP (Fig. 8C), indicating thatthe regulation mechanism of VSP might be differentfrom that of COR1, PDF1.2, and Thi2.1 under JAtreatment. Previous studies also showed that VSPwas positively regulated, whereas PDF1.2 was nega-tively regulated, by AtMYC2 and AtERF4 upon JAtreatment (Lorenzo et al., 2004; McGrath et al., 2005;Pre, 2006; Memelink, 2009).

Our results showed that the disruption of RCA ledto a decrease in JA-induced expression of PDF1.2 andThi2.1, which were previously identified as JA-regu-lated defense genes (Penninckx et al., 1996; Vignutelliet 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 taba-cum) RCA gene was involved in herbivore resistance(Giri et al., 2006; Mitra and Baldwin, 2008). The to-bacco RCA was decreased at the levels of transcriptand protein abundance by herbivore damage (Giriet al., 2006). Furthermore, down-regulation of thisRCA gene caused reduced defense against herbivoreattack (Mitra and Baldwin, 2008).

To investigate whether the reduction of JA-inducedgene expression in the 3-week-old rca mutants is ageneral stress response to hormone overexposure fortheir growth defects, we examined the indole-3-aceticacid (IAA)-induced IAA5 expression in these plants.The induction of IAA5 expression in the rca-1 and rca-2mutants was comparable to that in the wild type uponIAA treatment (Fig. 8D), suggesting that the defectiveof JA-induced gene expression in the rca mutants is aspecific response to JA signal rather than a generalresponse to hormone overexposure.

Down-Regulation of RCA Protein Was Involved inDark-Induced Senescence

Previous studies showed that diverse developmen-tal signals and various environmental stresses includ-

Figure 8. Mutation in RCA led to a decrease inJA-induced expression of COR1, PDF1.2, andThi2.1. A, Semiquantitative RT-PCR for COR1,PDF2.1, and Thi1.2 in 3-week-old plants upontreatment with MeJA (+) or water (2) for 8 h. Theamplified actin1 is shown as an internal control.B, Northern blot for PDF1.2 and Thi2.1 in3-week-old plants upon treatment with MeJA (+)or water (2) for 8 h. Ethidium bromide staining ofrRNA was used as a loading control. C, Northernblot for VSP in 3-week-old plants upon treatmentwith MeJA (+) or water (2) for 8 h. Ethidiumbromide staining of rRNA was used as a loadingcontrol. D, Semiquantitative RT-PCR for IAA5 in3-week-old plants upon treatment with IAA (+) orwater (2) for 1 h. The amplified actin1 is shown asan internal control.

Shan et al.




ing dark treatment were able to specifically inducesenescence in Arabidopsis (Schippers et al., 2007). Toinvestigate whether dark-induced senescence also cor-relates with the reduction of RCA protein, we exam-ined the RCA protein in whole seedlings or detachedleaves of Arabidopsis wild-type plants under darktreatment.As shown in Figure 9, dark incubation induced

senescence in detached leaves after 10 d of treatmentand in whole seedlings after 15 d of treatment. Visibleyellowing was observed in detached leaves (Fig. 9A),and the chlorotic phenotypes were also found in leavesand stems of whole seedlings (Fig. 9C). Correlatedwith dark-induced senescence, the protein abundanceof RCA was severely reduced in detached leaves orwhole seedlings (Fig. 9, B and D). These data suggestthat down-regulation of RCA protein is also involvedin dark-induced senescence.

DISCUSSION

Leaf senescence is a developmental program that isregulated by intrinsic factors such as hormones anddevelopmental age and environmental factors such astemperature, light, nutrients, and pathogens (Schipperset al., 2007; Balazadeh et al., 2008). The JA-induced leafsenescence has been known for a long time (Ueda andKato, 1980). In this study, we specially investigated themolecular mechanism underlying JA induction of leafsenescence. We identified RCA as a COI1-dependentJA-repressed protein using 2-D DIGE coupled withMALDI-TOF mass spectrometry. We found that

RCA was down-regulated in response to JA treatmentat levels of transcript and protein abundance in aCOI1-dependent manner. Further genetic and physio-logical analyses showed that the COI1-dependentJA repression of RCA correlated with JA-inducedsenescence and that loss of RCA led to typicalsenescence-associated features. Our results suggestthat the COI1-dependent JA repression of RCA playsan important role in JA-induced leaf senescence. Inaddition, we showed that dark-induced senescencecorrelated with the reduction of RCA protein, indicat-ing that down-regulation of RCA protein is also in-volved in dark-induced senescence.

Identification of 35 COI1-Dependent JA-RegulatedProteins Provided New Insights into JA Signaling

Previously, transcriptomics analyses revealed thatJA was essential for the regulation of various genesthat were involved in a variety of physiological eventsand that COI1 was required for the transcription ofmany JA-regulated genes (Sasaki et al., 2001; Devotoet al., 2005; Sasaki-Sekimoto et al., 2005; Jung et al.,2007). In this study, we identified 35 COI1-dependentJA-regulated proteins (Fig. 1; Table I). Of these, 21proteins were induced while 14 were repressed by JAin a COI1-dependent manner (Fig. 1; Table I). To ourknowledge, our report is the first proteome study on aCOI1 requirement in Arabidopsis JA responses. Thisstudy is also, to our knowledge, the first application of2-D DIGE-based proteomics to identify the JA-regu-lated proteins in Arabidopsis.

There were not many studies of protein expressionmapping upon JA treatment in Arabidopsis, except forthe identification of several proteins at redox statusunder JA treatment using two-dimensional gel elec-trophoresis (2-DE) coupled with monobromobimanelabeling (Alvarez et al., 2009). Only two proteins (VSP1and allene oxide cyclase 2), which displayed signifi-cant increases in abundance under JA treatment, wereidentified by both studies (Alvarez et al., 2009; Table I).

Using a proteomics approach, Rakwal and Komatsu(2000) and Mahmood et al. (2007) found that 13proteins 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 inrice. Seven Arabidopsis homologs of these proteinswere found in our study (Table I). Among these sevenrice proteins, the expression patterns of three proteins(glyceraldehyde-3-phosphate dehydrogenase B sub-unit, putative transketolase, and RCA) were inconsis-tent in different studies (Cho et al., 2007; Mahmoodet al., 2007), which needs to be verified. The remainingfour proteins (lipoxygenase, dehydroascorbate reduc-tase, citrate synthase, and Rubisco large chain) showedsimilar trends in expression patterns upon JA treat-ment in rice and Arabidopsis (Rakwal and Komatsu,2000; Cho et al., 2007; Mahmood et al., 2007; Table I).These results suggest that characterization of theArabidopsis JA-responsive proteins would have broad

Figure 9. Down-regulation of RCA protein was involved in dark-induced senescence. A, Phenotypes of 3-week-old wild-type detachedleaves upon dark treatment (+) or not (2) for 10 d. B, Western blot forRCA in 3-week-old wild-type detached leaves upon dark treatment (+)or not (2) for 10 d. The immunoblot was detected with GSTantibody asa protein-loading control. C, Phenotypes of 3-week-old wild-typewhole seedlings upon dark treatment (+) or not (2) for 15 d. D,Western blot for RCA in 3-week-old wild-type whole seedlings upondark treatment (+) or not (2) for 15 d. The immunoblot was detectedwith GST antibody as a protein-loading control.





implications for understanding JA actions in otherhigher plants.

In addition, the differential 2-DE analysis of the JAprecursor 12-oxophytodienoic acid (OPDA)-treatedArabidopsis leaves revealed 37 differentially ex-pressed proteins (Dueckershoff et al., 2008). Four ofthese 37 OPDA-regulated proteins were also identifiedas JA-regulated proteins in our study: three proteins(lipoxygenase 2, dehydroascorbate reductase, and JA-responsive 1) were up-regulated both in OPDA andMeJA treatments (Dueckershoff et al., 2008; Table I),while the remaining one (sedoheptulose-1,7-bisphos-phatase) was up-regulated by OPDA (Dueckershoffet al., 2008) but down-regulated by MeJA (Table I).These data indicate that JA and its precursor OPDAmight play overlapping yet distinct roles in the regu-lation of gene expression.

The COI1-Dependent JA Repression of RCA Played anImportant Role in JA-Induced Leaf Senescence

Previous studies showed that JA induced leaf se-nescence in Arabidopsis (He et al., 2002). In this study,we found that the JA-treated Arabidopsis wild-typeplants exhibited severe reduction in RCA protein leveland simultaneously displayed leaf senescence (Fig. 5),whereas the coi1 mutant was still green and its RCAprotein level was not greatly affected after JA treat-ment for 5 d (Fig. 5). These results suggest that theCOI1-dependent JA repression of RCA is interrelatedwith JA-induced leaf senescence. We further foundthat the T-DNA insertional mutation in RCA (Fig. 6)led to diverse senescence-related symptoms, manif-esting as yellowing leaf, lower chlorophyll content,increased expression of senescence-induced genes(SAG13, SAG21, SEN4), and decreased expression ofsenescence-reduced genes (CAB1, CAB2, RBCS; Fig. 7).Our results indicate that the COI1-dependent JA re-pression of RCA plays a clear role in JA-induced leafsenescence.

Furthermore, we found that the RCA protein wassignificantly down-regulated in the dark-induced se-nescent Arabidopsis leaves and seedlings (Fig. 9),implying that the reduction in RCA protein is alsointerrelated with dark-induced senescence. Thus,down-regulation of RCA protein might be involvedin many types of senescence, including JA- and dark-induced senescence.

In this study, we found that the expression level ofRCA protein was down-regulated under JA treatmentin a COI1-dependent manner. Although COI1 is re-quired for the JA-repressed RCA protein expression,RCA is not the direct target of the SCFCOI1 complex. Wefound that RCA was unable to specifically interactwith COI1 protein by coimmunoprecipitation assay(data not shown). We also found that the JA-repressedRCA protein expression was not affected by treatmentof 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 proteinexpression.

We further found that the RCA transcripts weredown-regulated in response to JA in a COI1-depen-dent manner (Fig. 3) and that the decrease of RCARNA preceded the reduction of RCA protein under JAtreatment (Fig. 4). These data suggest that the JA-repressed RCA protein expression is mainly throughthe JA-repressed RCA RNA expression.

Previous studies showed that the expression ofvarious JA pathway downstream genes was regulatedby some JA-responsive transcription factors (Memelink,2009). Several cis-acting elements bound by these tran-scription factors, including the GCC motif, the G-box,and TGACG (as-1-type) sequences, have been identi-fied in the regulatory regions of these JA pathwaydownstream genes (Memelink, 2009). We found thatthe G-box (at positions 2251 to 2246) and TGACG

Figure 10. A model for JA function in Arabidopsis leaf senescence.Upon JA treatment, COI1 recruits the JAZs to the SCFCOI1 complex forubiquitination and degradation through the 26S proteasome. The JAZ-interacting proteins (JAPs) are then released to directly or indirectlyactivate the JA-responsive transcription repressors (TRs), which mightbind with the G-box and TGACG sequences in the regulatory region ofthe RCA gene to repress the expression of RCA RNA. Alternatively, theJAZ-interacting proteins are released to repress the JA-responsivetranscription activators (TAs) essential for the expression of RCA RNA.The down-regulation of RCA RNA leads to the reduction of RCAprotein, resulting in JA-induced leaf senescence. Down-regulation ofRCA is also involved in dark-induced senescence.

Shan et al.




(at positions 2276 to –272 and 228 to –24) sequencesalso existed in the regulatory region of the RCA gene(Fig. 10), indicating that some JA-responsive transcrip-tion factors might bind with these motifs and functionas transcription repressors (or activators) to repress (oractivate) the expression of RCA RNA.We speculate that these JA-responsive transcription

repressors (or activators) might be directly or indi-rectly activated (or repressed) by some JAZ-interactingproteins (Fig. 10). Upon JA treatment, COI1 recruitsJAZs to SCFCOI1 for ubiquitination and degradationthrough the 26S proteasome. The JAZ-interacting pro-teins 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, re-sulting in various typical senescence-associated fea-tures, including leaf yellowing, loss of chlorophyll,up-regulation of senescence-induced genes, anddown-regulation of senescence-reduced genes (Fig.10). Down-regulation of RCA might also be involvedin dark-induced senescence (Fig. 10). Whether RCAexpression is the primary signal for all the senescence-related changes remains to be elucidated. Identifica-tion of the JA-responsive transcription factors wouldprovide new insights into further understanding ofleaf senescence.

MATERIALS AND METHODS

Plant Materials

The Arabidopsis (Arabidopsis thaliana) mutants rca-1 and rca-2 were iso-

lated from the T-DNA-tagged pools produced at the Salk Institute T-DNAExpress

(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 mM

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 mM 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% b-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 at220�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 ammo-

nium acetate in methanol, two times with 80% (v/v) acetone, and once with

70% (v/v) ethanol. The mixture was incubated at 220�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 mg

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 mL 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

mL21 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 mL21 dithiothreitol

and thenwith 0.025 gmL21 iodoacetamide, each for 15min. 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 mm 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 detec-

tion, 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 Stu-

dent’s t test (n = 3). We used a spot abundance ratio of greater than 1.15 or less

than 21.15 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 Solu-

tion 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

Themass spectrum data were used to search for protein candidates usingMS-

Fit in theNational 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 ho-

mologous 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 Supple-

mental 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

MSmediumwere drenched in solution containing 100 mM 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

mM 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 onMSmediumwere

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 mM 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 mM 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 semiquan-

titative 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 mM 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 onMS

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 onMSmediumwere 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 antibodywas 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 trans-

genic plants harboring the pRCA-RCA::GUS construct grown in soil, placed

onto solution containing 100 mM 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, semi-

quantitative RT-PCR, and vector construction.

ACKNOWLEDGMENTS

We thank the Arabidopsis Biological Resource Center for providing the

rca-1 and rca-2 mutant seeds.

Received September 29, 2010; accepted December 7, 2010; published Decem-

ber 20, 2010.

LITERATURE CITED

Alvarez S, ZhuM, Chen S (2009) Proteomics of Arabidopsis redox proteins

in response to methyl jasmonate. J Proteomics 73: 30–40

Balazadeh S, Riano-Pachon DM, Mueller-Roeber B (2008) Transcription

factors regulating leaf senescence in Arabidopsis thaliana. Plant Biol

(Stuttg) (Suppl) 10: 63–75

Bate NJ, Rothstein SJ, Thompson JE (1991) Expression of nuclear and

chloroplast photosynthesis-specific genes during leaf senescence. J Exp

Bot 42: 801–811

Benedetti CE, Costa CL, Turcinelli SR, Arruda P (1998) Differential

expression of a novel gene in response to coronatine, methyl jasmonate,

and wounding in the coi1 mutant of Arabidopsis. Plant Physiol 116:

1037–1042

Benedetti CE, Xie D, Turner JG (1995) Coi1-dependent expression of an

Arabidopsis vegetative storage protein in flowers and siliques and in

response to coronatine or methyl jasmonate. Plant Physiol 109: 567–572

Buchanan-Wollaston V (1997) The molecular biology of leaf senescence. J

Exp Bot 48: 181–199

Buchanan-Wollaston V, Page T, Harrison E, Breeze E, Lim PO, Nam HG,

Lin JF, Wu SH, Swidzinski J, Ishizaki K, et al (2005) Comparative

transcriptome analysis reveals significant differences in gene expression

and signalling pathways between developmental and dark/starvation-

induced senescence in Arabidopsis. Plant J 42: 567–585

Cheng H, Song S, Xiao L, Soo HM, Cheng Z, Xie D, Peng J (2009)

Gibberellin acts through jasmonate to control the expression of MYB21,

MYB24, and MYB57 to promote stamen filament growth in Arabidopsis.

PLoS Genet 5: e1000440

Shan et al.




Cheong JJ, Choi YD (2003) Methyl jasmonate as a vital substance in plants.

Trends Genet 19: 409–413

Chini A, Fonseca S, Fernandez G, Adie B, Chico JM, Lorenzo O, Garcıa-

Casado G, Lopez-Vidriero I, Lozano FM, Ponce MR, et al (2007) The

JAZ family of repressors is the missing link in jasmonate signalling.

Nature 448: 666–671

Cho K, Agrawal GK, Shibato J, Jung YH, Kim YK, Nahm BH, Jwa NS,

Tamogami S, Han O, Kohda K, et al (2007) Survey of differentially

expressed proteins and genes in jasmonic acid treated rice seedling

shoot and root at the proteomics and transcriptomics levels. J Proteome

Res 6: 3581–3603

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agro-

bacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:

735–743

Devoto A, Ellis C, Magusin A, Chang HS, Chilcott C, Zhu T, Turner JG

(2005) Expression profiling reveals COI1 to be a key regulator of genes

involved in wound- and methyl jasmonate-induced secondary metab-

olism, defence, and hormone interactions. Plant Mol Biol 58: 497–513

Dueckershoff K, Mueller S, Mueller MJ, Reinders J (2008) Impact of

cyclopentenone-oxylipins on the proteome of Arabidopsis thaliana.

Biochim Biophys Acta 1784: 1975–1985

Eckardt NA, Snyder GW, Portis ARJ Jr, Orgen WL (1997) Growth and

photosynthesis under high and low irradiance of Arabidopsis thaliana

antisense mutants with reduced ribulose-1,5-bisphosphate carboxyl-

ase/oxygenase activase content. Plant Physiol 113: 575–586

Farmer EE, Almeras E, Krishnamurthy V (2003) Jasmonates and related

oxylipins in plant responses to pathogenesis and herbivory. Curr Opin

Plant Biol 6: 372–378

Feys B, Benedetti CE, Penfold CN, Turner JG (1994) Arabidopsis mutants

selected for resistance to the phytotoxin coronatine are male sterile,

insensitive to methyl jasmonate, and resistant to a bacterial pathogen.

Plant Cell 6: 751–759

Giri AP, Wunsche H, Mitra S, Zavala JA, Muck A, Svatos A, Baldwin IT

(2006) Molecular interactions between the specialist herbivore Manduca

sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata.

VII. Changes in the plant’s proteome. Plant Physiol 142: 1621–1641

Griffin TJ, Gygi SP, Ideker T, Rist B, Eng J, Hood L, Aebersold R (2002)

Complementary profiling of gene expression at the transcriptome and

proteome levels in Saccharomyces cerevisiae. Mol Cell Proteomics 1:

323–333

He Y, Fukushige H, Hildebrand DF, Gan S (2002) Evidence supporting a

role of jasmonic acid in Arabidopsis leaf senescence. Plant Physiol 128:

876–884

Howe GA (2004) Jasmonates as signals in the wound response. J Plant

Growth Regul 23: 223–237

Huber M, Bahr I, Kratzschmar JR, Becker A, Muller EC, Donner P,

Pohlenz HD, Schneider MR, Sommer A (2004) Comparison of proteo-

mic and genomic analyses of the human breast cancer cell line T47D

and the antiestrogen-resistant derivative T47D-r. Mol Cell Proteomics 3:

43–55

Jiang CZ, Quick WR, Alred R, Kliebenstein D, Rodermel SR (1994)

Antisense RNA inhibition of Rubisco activase expression. Plant J 5:

787–798

Jiang DA, Weng XY, Lu Q (2001) Quantitation of Rubisco activase by single

radial immunodiffusion. J Zhejiang Univ (Agric Life Sci) 27: 255–258

Jung C, Lyou SH, Yeu S, Kim MA, Rhee S, Kim M, Lee JS, Choi YD,

Cheong JJ (2007) Microarray-based screening of jasmonate-responsive

genes in Arabidopsis thaliana. Plant Cell Rep 26: 1053–1063

Katsir L, Schilmiller AL, Staswick PE, He SY, Howe GA (2008) COI1 is a

critical component of a receptor for jasmonate and the bacterial viru-

lence factor coronatine. Proc Natl Acad Sci USA 105: 7100–7105

Kim EH, Kim YS, Park SH, Koo YJ, Choi YD, Chung YY, Lee IJ, Kim JK

(2009) Methyl jasmonate reduces grain yield by mediating stress signals

to alter spikelet development in rice. Plant Physiol 149: 1751–1760

Koo AJ, Howe GA (2009) The wound hormone jasmonate. Phytochemistry

70: 1571–1580

Liu F, Ni W, Griffith ME, Huang Z, Chang C, Peng W, Ma H, Xie D (2004)

The ASK1 and ASK2 genes are essential for Arabidopsis early develop-

ment. Plant Cell 16: 5–20

Lorenzo O, Chico JM, Sanchez-Serrano JJ, Solano R (2004) JASMONATE-

INSENSITIVE1 encodes a MYC transcription factor essential to dis-

criminate between different jasmonate-regulated defense responses in

Arabidopsis. Plant Cell 16: 1938–1950

Mahmood T, Kakishima M, Komatsu S (2007) Proteomic analysis of

jasmonic acid-regulated proteins in rice leaf blades. Protein Pept Lett 14:

311–319

Mate CJ, Hudson GS, von Caemmerer S, Evans JR, Andrews TJ (1993)

Reduction of ribulose biphosphate carboxylase activase levels in to-

bacco (Nicotiana tabacum) by antisense RNA reduces ribulose biphos-

phate carboxylase carbamylation and impairs photosynthesis. Plant

Physiol 102: 1119–1128

McConn M, Creelman RA, Bell E, Mullet JE, Browse J (1997) Jasmonate is

essential for insect defense in Arabidopsis. Proc Natl Acad Sci USA 94:

5473–5477

McGrath KC, Dombrecht B, Manners JM, Schenk PM, Edgar CI, Maclean

DJ, Scheible WR, Udvardi MK, Kazan K (2005) Repressor- and activator-

type ethylene response factors functioning in jasmonate signaling and

disease resistance identified via a genome-wide screen of Arabidopsis

transcription factor gene expression. Plant Physiol 139: 949–959

Memelink J (2009) Regulation of gene expression by jasmonate hormones.

Phytochemistry 70: 1560–1570

Mitra S, Baldwin IT (2008) Independently silencing two photosynthetic

proteins in Nicotiana attenuata has different effects on herbivore resis-

tance. Plant Physiol 148: 1128–1138

Nam HG (1997) The molecular genetic analysis of leaf senescence. Curr

Opin Biotechnol 8: 200–207

Park JH, Oh SA, Kim YH, Woo HR, NamHG (1998) Differential expression

of senescence-associated mRNAs during leaf senescence induced by

different senescence-inducing factors in Arabidopsis. Plant Mol Biol 37:

445–454

Penninckx IA, Eggermont K, Terras FR, Thomma BP, De Samblanx GW,

Buchala A, Metraux JP, Manners JM, Broekaert WF (1996) Pathogen-

induced systemic activation of a plant defensin gene in Arabidopsis

follows a salicylic acid-independent pathway. Plant Cell 8: 2309–2323

Portis AR Jr (2003) Rubisco activase: Rubisco’s catalytic chaperone. Photo-

synth Res 75: 11–27

Pre M (2006) ORA EST: functional analysis of jasmonate-responsive AP2/

ERF domain transcription factors in Arabidopsis thaliana. PhD thesis.

Leiden University, Leiden, The Netherlands

Quirino BF, Noh YS, Himelblau E, Amasino RM (2000) Molecular aspects

of leaf senescence. Trends Plant Sci 5: 278–282

Rakwal R, Komatsu S (2000) Role of jasmonate in the rice (Oryza sativa L.)

self-defense mechanism using proteome analysis. Electrophoresis 21:

2492–2500

Rao MV, Lee H, Creelman RA, Mullet JE, Davis KR (2000) Jasmonic acid

signaling modulates ozone-induced hypersensitive cell death. Plant

Cell 12: 1633–1646

Reinbothe C, Parthier B, Reinbothe S (1997) Temporal pattern of jasmo-

nate-induced alterations in gene expression of barley leaves. Planta 201:

281–287

Reinbothe C, Springer A, Samol I, Reinbothe S (2009) Plant oxylipins: role

of jasmonic acid during programmed cell death, defence and leaf

senescence. FEBS J 276: 4666–4681

Reinbothe S, Mollenhauer B, Reinbothe C (1994) JIPs and RIPs: the

regulation of plant gene expression by jasmonates in response to

environmental cues and pathogens. Plant Cell 6: 1197–1209

Reinbothe S, Reinbothe C, Parthier B (1993) Methyl jasmonate-regulated

translation of nuclear-encoded chloroplast proteins in barley (Hordeum

vulgare L. cv. Salome). J Biol Chem 268: 10606–10611

Ren C, Han C, Peng W, Huang Y, Peng Z, Xiong X, Zhu Q, Gao B, Xie D

(2009) A leaky mutation in DWARF4 reveals an antagonistic role of

brassinosteroid in the inhibition of root growth by jasmonate in

Arabidopsis. Plant Physiol 151: 1412–1420

Ren C, Pan J, Peng W, Genschik P, Hobbie L, Hellmann H, Estelle M, Gao

B, Peng J, Sun C, et al (2005) Point mutations in Arabidopsis Cullin1

reveal its essential role in jasmonate response. Plant J 42: 514–524

Reymond P, Weber H, Damond M, Farmer EE (2000) Differential gene

expression in response to mechanical wounding and insect feeding in

Arabidopsis. Plant Cell 12: 707–720

Salvucci ME, Werneke JM, Ogren WL, Portis AR (1987) Purification and

species distribution of Rubisco activase. Plant Physiol 84: 930–936

Sasaki Y, Asamizu E, Shibata D, Nakamura Y, Kaneko T, Awai K, Amagai

M, Kuwata C, Tsugane T, Masuda T, et al (2001) Monitoring of methyl

jasmonate-responsive genes in Arabidopsis by cDNA macroarray: self-

activation of jasmonic acid biosynthesis and crosstalk with other phy-

tohormone signaling pathways. DNA Res 8: 153–161





Sasaki-Sekimoto Y, Taki N, Obayashi T, Aono M, Matsumoto F, Sakurai

N, Suzuki H, Hirai MY, Noji M, Saito K, et al (2005) Coordinated

activation of metabolic pathways for antioxidants and defence com-

pounds by jasmonates and their roles in stress tolerance in Arabidopsis.

Plant J 44: 653–668

Schilmiller AL, Howe GA (2005) Systemic signaling in the wound re-

sponse. Curr Opin Plant Biol 8: 369–377

Schippers J, Jing H, Hille J, Dijkwel P (2007) Developmental and hormo-

nal control of leaf senescence. In S Gan, ed, Senescence Processes in

Plants. Blackwell Publishing, Oxford, pp 145–170

Shan X, Zhang Y, Peng W, Wang Z, Xie D (2009) Molecular mechanism for

jasmonate-induction of anthocyanin accumulation in Arabidopsis. J Exp

Bot 60: 3849–3860

Shen JB, Orozco EMJ Jr, Ogren WL (1991) Expression of the two isoforms

of spinach ribulose 1,5-bisphosphate carboxylase activase and essenti-

ality of the conserved lysine in the consensus nucleotide-binding

domain. J Biol Chem 266: 8963–8968

Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu G, Nomura K, He

SY, Howe GA, Browse J (2007) JAZ repressor proteins are targets of the

SCF(COI1) complex during jasmonate signalling. Nature 448: 661–665

Tian Q, Stepaniants SB, Mao M, Weng L, Feetham MC, Doyle MJ, Yi EC,

Dai H, Thorsson V, Eng J, et al (2004) Integrated genomic and proteomic

analyses of gene expression in mammalian cells. Mol Cell Proteomics 3:

960–969

Ueda J, Kato J (1980) Isolation and identification of a senescence-promoting

substance from wormwood (Artemisia absinthum L.). Plant Physiol 66:

246–249

Vignutelli A, Wasternack C, Apel K, Bohlmann H (1998) Systemic and

local induction of an Arabidopsis thionin gene by wounding and path-

ogens. Plant J 14: 285–295

Wang Z, Dai L, Jiang Z, Peng W, Zhang L, Wang G, Xie D (2005) GmCOI1,

a soybean F-box protein gene, shows ability to mediate jasmonate-

regulated plant defense and fertility in Arabidopsis. Mol Plant Microbe

Interact 18: 1285–1295

Wasternack C (2007) Jasmonates: an update on biosynthesis, signal trans-

duction and action in plant stress response, growth and development.

Ann Bot (Lond) 100: 681–697

Weaver LM, Gan S, Quirino B, Amasino RM (1998) A comparison of the

expression patterns of several senescence-associated genes in response

to stress and hormone treatment. Plant Mol Biol 37: 455–469

Weidhase RA, Kramell HM, Lehmann J, Liebisch HW, Lerbs W, Parthier

B (1987) Methyl jasmonate-induced changes in the polypeptide pattern

of senescing barley leaf segments. Plant Sci 51: 177–186

Xiao S, Dai L, Liu F, Wang Z, Peng W, Xie D (2004) COS1: an Arabi-

dopsis coronatine insensitive1 suppressor essential for regulation of

jasmonate-mediated plant defense and senescence. Plant Cell 16:

1132–1142

Xie DX, Feys BF, James S, Nieto-Rostro M, Turner JG (1998) COI1: an

Arabidopsis gene required for jasmonate-regulated defense and fertil-

ity. Science 280: 1091–1094

Xu L, Liu F, Lechner E, Genschik P, Crosby WL, Ma H, Peng W, Huang D,

Xie D (2002) The SCF(COI1) ubiquitin-ligase complexes are required for

jasmonate response in Arabidopsis. Plant Cell 14: 1919–1935

Yan J, Zhang C, Gu M, Bai Z, Zhang W, Qi T, Cheng Z, Peng W, Luo H,

Nan F, et al (2009) The Arabidopsis CORONATINE INSENSITIVE1

protein is a jasmonate receptor. Plant Cell 21: 2220–2236

Yoshida S, Ito M, Nishida I, Watanabe A (2002) Identification of a novel

gene HYS1/CPR5 that has a repressive role in the induction of leaf

senescence and pathogen-defence responses in Arabidopsis thaliana.

Plant J 29: 427–437

Shan et al.




the role of arabidopsis rubisco activase inthe role of arabidopsis rubisco activase in...

Documents