Comparative phosphoproteomics analysis of saltresponsive phosphoproteins regulated the MKK9MPK6 cascade in Arabidopsis.pdf

plant science 241 (2015) 138150 contents lists available at sciencedirect plant science journal homepage: comparative phospho-proteomics analysis of salt-responsive phosphoproteins regulated by the mkk9-mpk6 cascade in arabidopsis zhenbin liu, yuan li, hanwei cao, dongtao ren state key laboratory of plant physiology and biochemistry, college of biological sciences, china agricultural university, beijing 100193, china a r t i c l e i n f o article history: received 10 july 2015 received in revised form 9 october 2015 accepted 10 october 2015 keywords: salt stress comparative phospho-proteomics pro-q diamond staining mapk cascade rubisco activase a b s t r a c t mitogen-activated protein kinase (mapk) cascades are involved in the salt stress response in plants. however, the identities of specifi c proteins operating downstream of mapks in the salt stress response remain unclear. our studies showed that mkk9 and mpk6 null mutant seedlings are hyposensitive to salt stress. moreover, we showed that mpk6 was activated by salt stress, indicating that the mkk9-mpk6 cascade mediated the salt stress response in arabidopsis. to identify phosphoproteins downstream of the mkk9-mpk6 cascade in the salt stress response pathway, we performed two-dimensional electrophoresis (2-de) with pro-q phosphoprotein staining and matrix-assisted laser desorption ionization time of fl ight mass spectrometry (maldi-tof ms) to identify phosphoproteins induced by salt treatment in mkk9, mpk6, and wild-type seedlings. phosphorylation of 4 proteins, including rubisco activase (rca), plastid ribosomal protein s 1 (prps1), plastid division protein (ftsz2-2), and tortifolia2 (tor2), was found to be regulated by activation of mkk9-mpk6 cascade. further phospho-proteomics analysis of mkk9ddmutant seedlings revealed that rca phosphorylation was up-regulated as a result of mkk9 activation. the fi nding that the mkk9-mpk6 cascade functions in the salt stress response by regulating phosphorylation of rca, prps1, ftsz2-2, and tor2, provides a novel insight into the mapk-related mechanisms underlying the salt stress response in plants. 2015 elsevier ireland ltd. all rights reserved. 1. introduction high soil salinity is a major obstacle to agricultural develop- ment 1,2. high soil salinity induces osmotic stress, ionic stress, and secondary oxidative stress in plant cells, eventually affecting plant growth and development 3. photosynthesis is a major fac- tor in the determination of plant growth. salt stress results in the decreased stomatal opening, and thereby restricts the diffusion of co2into chloroplast and reduces the leaf photosynthetic rate 4,5. salt stress can also inhibit photosynthesis by affecting the activity of photosynthesis-related proteins. for example, salt treat- abbreviations: 2-de, two-dimensional electrophoresis; aaa+, atpase associ- ated with various cellular activities; bam1, beta-amylase 1; dex, dexamethasone; ftsz2-2, plastid division protein; ipgam, 2, 3-biphosphoglycerate-independent phosphoglycerate mutase; lc, liquid culture; maldi-tof ms, matrix-assisted laser desorption ionization time of fl ight mass spectrometry; mapk, mitogen activated protein kinase; mbp, myelin basic protein; prps1, plastid ribosomal protein s1; rca, rubisco activase; roc4, cyclophilin 20-3; tca, trichloroacetic acid; tor2, tortifolia2. corresponding author at: 2 west yuanming yuan rd., haidian district, beijing 100193, china. fax: +86 010 62733794. e-mail address: ren (d. ren). ments inhibit the activity of and reduce the content of rubisco, a crucial enzyme in co2 fi xation process 6,7. plants are generally immobile and unable to select their environments. therefore, in order to adapt to the environment, plants have evolved molecular mechanisms to perceive and respond to various biotic and abi- otic stresses. protein phosphorylation by kinases is an important post-translational modifi cation that regulates cellular signal trans- duction in response to changes in the environment. in eukaryotes, activation of mitogen-activated protein kinase (mapk) signaling cascades is a general mechanism through which external stimuli, including biotic and abiotic stresses, are translated into cellular responses. mapk cascades are highly conserved signaling processes in eukaryotes that function downstream of sensors and receptors by converting signals generated at the sensors/receptors into cellu- lar responses 810. mapk cascades are minimally composed of 3 types of kinase, map kinase kinase kinases (mapkkk), map kinase kinases (mapkk), and mapk (denoted mkkk, mkk, and mpk in ara- bidopsis, according to accepted systematic nomenclature), which are linked in various ways to upstream receptors and downstream targets 8,10. numerous mapk pathways that respond to a variety /10.1016/j.plantsci.2015.10.005 0168-9452/ 2015 elsevier ireland ltd. all rights reserved. z. liu et al. / plant science 241 (2015) 138150 139 of external stimuli have been characterized in yeast, animals, and plants 11. plant mapk cascades have previously showed to be important for the regulation of salt stress responses. simk (salt stress-induced mapk) and simkk (simk kinase) in alfalfa cells are activated by salt stress 12,13. salt and osmotic stress can enhance the activity of sipk (salicylic acid-induced protein kinase) in tobacco protoplasts 14. three salt stress-induced mapks have been identifi ed in zea mays: zmmpk3, zmmpk5, and zmsimk1 12,15,16. in arabidop- sis, the most completely characterized mapk cascade functioning in response to abiotic stress is the mekk1-mkk2-mpk4/mpk6 cas- cade, which is activated by salt or cold stress 17. previous studies demonstrated that activation of mkk9 enhanced the sensitivity of transgenic seedlings to salt stress 18,19. however, it is unclear how mkk9, mpk6, and other kinases regulate the response to salt stress. to understand how kinases mediate the salt stress response, it is vital to identify their downstream components. phospho-proteomics is a powerful tool that can be used to iden- tify mapk substrates and downstream proteins, because it allows unbiased localization and site-specifi c quantifi cation of many phos- phorylated proteins in a single in vivo experiment. we used pro-q diamond phosphoprotein gel stain 2023 to study protein phos- phorylation in arabidopsis under salt stress 24. the intensity of the pro-q diamond stain was proportional to the degree of phosphorylation of each phosphoprotein, but not to the protein concentration 25. in this study, we found that mkk9 and mpk6 mutants were hyposensitive to salt stress in comparison with wild-type plants. the fresh shoot weight of mkk9 and mpk6 seedlings was signifi - cantly heavier than that of the wild-type seedlings. comparative analysis of the two-dimensional electrophoresis (2-de) patterns in wild-type seedlings, mkk9 and mpk6 mutants under salt treatment was used to identify proteins with different phosphorylation sta- tuses among the groups. twenty salt- responsive phosphoproteins were identifi ed with a high level of confi dence in arabidopsis wild- type seedlings. four of these proteins was found to be regulated by activation of mkk9-mpk6 cascade. 2. material and methods 2.1. plant materials and treatments wild-type (col-0) and mutant arabidopsis thaliana (ecotype columbia) seeds were surface-sterilized. after cold treatment in the dark for 2 d at 4c, the seeds were germinated and grown on murashige and skoog medium (containing 2.5% sucrose) with 0.5% phytagel agar (sigmaaldrich, st. louis, mo, usa) plates. for the investigation of the mutant phenotype, wild-type and mutant seedlings were transferred to new plates containing 100 mm nacl and grown at 22c in a growth room with a 12-h photoperiod (pho- ton fl ux density, 100 ?e/m2/s1). three biological replicates were done for each treatment. for kinase assays and phospho-proteomics studies, seedlings were transferred to liquid culture medium (0.5 murashige and skoog medium containing 0.025% 2-(n-morpholino) ethanesul- fonic acid (mes) and 0.25% sucrose, ph 5.7) 18 and grown at 22c under continuous light (photon fl ux density, 70 ?e/m2/s1). two-week-old seedlings were treated with liquid culture medium containing nacl to obtain fi nal concentration of 100 mm. the con- trol group was treated with liquid culture medium (lcm) only. the mkk9 active mutant (mkk9dd), mkk9 inactive mutant (mkk9kr), and mkk9dd/mpk6 crossed seedlings were treated with 2 ?m dex- amethasone (dex) for 4 and 8 h 26,27. plant samples were collected at different time points, fl ash-cooled in liquid nitrogen, and stored at 80c. t-dna insert mutants (mkk9, salk 060 h06; mpk3, salk 100651; mpk6, salk 062471) were obtained from the arabidopsis biologi- cal resource center. mkk9kr, mkk9dd, and mkk9dd/mpk6 mutant plants were generated as previously described 18. 2.2. protein extraction samples were ground to a fi ne powder in liquid nitrogen and transferred to a tube with precooled 10% tca/acetone and 1% ?-mercaptoethanol, vortexed briefl y, and incubated overnight at 20c. after centrifugation at 10,000 g for 20 min at 4c, the supernatants were discarded and the pellets were washed twice with precooled acetone containing 0.1% ?-mercaptoethanol. after incubation for 30 min at 20c, the samples were cen- trifuged at 10,000 g for 20 min at 4c. the fi nal pellets were freezedried under vacuum and solubilized in lysis buffer (7 m urea, 2 m thiourea, 4% chaps detergent, 60 mm dithiothreitol (dtt), and 0.8% ipg buffer) to extract proteins. the solution was centrifuged (13,000 g for 10 min at 25c) and the supernatants were collected for 2-de. total protein concentrations were estimated using 2d quant kits (ge healthcare life sciences). 2.3. 2-de and pro-q staining total protein samples (1 mg) in rehydration buffer (8 m urea, 2% chaps detergent, 60 mm dtt, 0.8% ipg buffer, and a trace amount of bromophenol blue) were loaded onto 18-cm ief linear strips (ph 47, ge healthcare life sciences). rehydration was performed at room temperature for 18 h. the ief voltage was 100 v for 3 h, 300 v for 3 h, 1000 v for 1.5 h, 3000 v for 1.5 h, and fi nally 8000 v for a total of 75,000 vh. when ief was completed, the strips were equilibrated as previously described 28. proteins in the strips were separated on 11% polyacrylamide gels, fi xed overnight in 500 ml of a solution of 50% methanol and 10% acetic acid, washed in 500 ml deionized water for 30 min, and stained with 3-fold-diluted pro-q stain for 2 h. after staining, the gels were destained with 250 ml of a solution of 50 mm sodium acetate (ph 4.0) in 20% acetonitrile (acn) 4 times for 30 min. all steps from staining to washing were performed in a dark room. gels were scanned with a typhoon 9410 fl uorescence scanner, stained with a blue-silver staining method as previously described 29, scanned with a umax 2000 scanner, and analyzed with pd-quest 8.0 software. three biological replicates were done for each condition. protein spots were considered credible when they were detected in at least three biological replicates. 2.4. protein identifi cation by maldi-tof ms in-gel protein digestion was performed using trypsin (roche) as previously described with minor modifi cations 28. first, excess trypsin solution was removed, after which the gel pieces were sub- merged in working solution (1 mm cacl2and 25 mm nh4hco3). supernatants were collected after incubation in the working solu- tion for 12 h at 37c. the gel pieces were extracted twice with 70% acetonitrile and 0.1% trifl uoroacetic acid for 15 min with sonica- tion. supernatants were pooled and concentrated by freezedrying. samples were loaded onto an anchor chip target plate (bruker dal- tonics). matrix solution (1 mg/ml of a-cyano-4-hydroxycinnamic acid dissolved in 70% acetonitrile and 0.1% trifl uoroacetic acid) was added to the dried peptide samples. after fast evaporation, the pep- tides were washed with 0.1% trifl uoroacetic acid and analyzed using an autoflexii tof/tof mass spectrometer. spectrum masses of 700 to 4000 d were acquired. all mass spectra were externally cal- ibrated with a peptide calibration standard. monoisotopic peaks were collected and used for peptide fi ngerprinting identifi cation with flex analysis 3.0 software (bruker daltonics). proteins were identifi ed by searching the national center for biotechnology infor- 140 z. liu et al. / plant science 241 (2015) 138150 mation non-redundant database with the mascot search engine using the following search parameters: cleaving enzyme, trypsin; peptide mass tolerance, 30 ppm; and one missed cleavage allowed. carbamido methylation of cys and oxidation of met were set as fi xed modifi cations. 2.5. in-gel kinase assays the in-gel kinase assay was performed as described previously 27. total protein samples (10 ?g per lane) were separated on 10% sds-poly acrylamide gels with 0.1 mg/ml myelin basic pro- tein (sigma) as a kinase substrate. after electrophoresis, the gels were washed 3 times at room temperature with washing buffer (25 mm tris, 0.5 mm dtt, 0.1 mm na3vo4, 5 mm naf, 0.5 mg/ml bovine serum albumin, and 0.1% tritonx-100 (ph 7.5) to remove sds. the gels were incubated in a solution of 25 mm tris, 1 mm dtt, 0.1 mm na3vo4, and 5 mm naf (ph 7.5) at 4c overnight, with 3 changes of the buffer to re-nature the kinases. after incubation in reaction buffer (25 mm tris, 2 mm egta, 12 mm mgcl2, 1 mm dtt, 0.1 mm na3vo4(ph 7.5) with 200 nm atp and 50 ?ci ?-32p- atp (3000ci/mmol) at room temperature for 60 min, the gels were washed several times in wash buffer (5% trichloroacetic acid and 1% sodium pyrophosphate) for at least 5 h to remove unincorporated ?-32p-atp. finally, the gels were dried and exposed to kodak x-ray fi lm. 3. results 3.1. mkk9 and mpk6 null mutants were hyposensitive to salt stress in comparison with wild-type plants the mkk9-mpk3/mpk6 cascade participates in regulation of the biosynthesis of ethylene and camalexin 18. in order to further understand whether mpk3 and mpk6 are downstream of mkk9 in the salt stress response, we designed an experiment to test the tolerance phenotypes of mkk9, mpk3, and mpk6 null mutant plants under treatment with 100 mm nacl. as shown in fig. 1a, mkk9 and mpk6 seedlings were hyposensitive to nacl treatment in compar- ison with wild-type seedlings (col-0). shoots of mkk9 and mpk6 seedlings were bigger than those of wild-type seedlings after salt stress. the mpk3 seedlings were slightly more sensitive to salt than the mkk9 and mpk6 seedlings. the weight of shoot of mkk9 and mpk6 seedlings was signifi cantly heavier than that of the wild-type seedlings (fig. 1b). the weight of the roots of the seedlings was not signifi cantly different after salt treatment. these results sug- gest that mkk9 and mpk6 negatively regulate shoot growth under conditions of salt stress. to further understand the functions of mkk9 and mpk6 in the cellular response to salt stress, we used an in-gel kinase assay to detect changes of mapk activity in seedling after salt treatment. the results showed that mpk6 was strongly activated in wild- type, mpk3, and mkk9 seedlings by salt treatment, while mpk3 was slightly activated in mpk6 seedlings (fig. 1c). these results sug- gest that mpk3 and mpk6 participate together in the salt stress response in plants with at least some degree of functional redun- dancy. salt stress also activated another mapk (fig. 1c), which was tentatively identifi ed as mpk4 based on its size. a previous study demonstrated that mpk4 in arabidopsis was activated by salt stress. 17. activation of mpk6 was more signifi cant than that of mpk3 and mpk4, suggesting a major role for mpk6 in the salt stress response. mpk6 activity in mkk9 seedlings was reduced but not abolished in comparison with that of wild-type control seedlings. these results suggest that mkk9 is an up-stream mapkk of mpk6 in the salt stress response pathway and other mkks also may be involved in this pathway. based on these results, we conclude that the mkk9-mpk6 cascade participates in the salt response pathway and negatively regulates shoot growth under salt stress conditions. 3.2. identifi cation of differentially phosphorylated proteins in wild-type and mutant seedlings without nacl treatment before analyzing the differentially phosphorylated proteins induced by salt treatment, we compared pro-q stained 2-de images of untreated wild-type, mkk9, and mpk6 seedlings to allow us to fi l- ter out non-salt-responsive proteins from the phospho-proteomic results collected after salt exposure. after tca/acetone extraction and pro-q phosphoprotein staining for proteomic analysis, approx- imately 200 distinct spots were detected on the 2-de image. some protein spots in the image showed different intensities in the mkk9 and mpk6 mutant seedlings and the wild-type seedlings, indicat- ing different degrees of phosphorylation. in untreated seedlings (0 h), spots 83, 84, 85, 89, and 109 were composed of proteins with increased phosphorylation in the mkk9 and mpk6 seedlings in comparison with those of the wild-type seedlings, indicating that mkk9 and mpk6 might involve some processes which regulate de- phosphorylation of these proteins (fig. 2). the protein spot 42 was down-regulated in the mpk6 seedlings. the expression levels of the proteins comprising spots 42, 83, 84, 85, 89, and 109 did not dif- fer signifi cantly in the null mutant and wild-type seedlings (fig. s1). the identities of the protein spots are shown in table 1. the enzyme 2, 3-biphosphoglycerate-independent phosphoglycerate mutase (ipgam) (spots 83, 85, and 86) is involved in glycolysis and catalyzes the reversible interconversion of 3-phosphoglycerate to 2-phosphoglycerate. in addition, ipgam plays a critical role in sto- matal movement 30. ipgam phosphorylation w