Differentially Delayed Root Proteome Responses to Salt Stress in

Article pubs.acs.org/jpr

Differentially Delayed Root Proteome Responses to Salt Stress in Sugar Cane Varieties Cinthya Mirella Pacheco,† Maria Clara Pestana-Calsa,† Fabio Cesar Gozzo,‡ Rejane Jurema Mansur Custodio Nogueira,§ Marcelo Menossi,∥ and Tercilio Calsa Junior*,† †

Laboratory of Plant Genomics and Proteomics, Department of Genetics, Center for Biological Sciences, Universidade Federal de Pernambuco, Recife, PE, Brazil ‡ Institute of Chemistry and ∥Institute of Biology, Universidade Estadual de Campinas, Campinas, SP, Brazil § Laboratory of Plant Physiology, Department of Biology, Universidade Federal Rural de Pernambuco, Recife, PE, Brazil S Supporting Information *

ABSTRACT: Soil salinity is a limiting factor to sugar cane crop development, although in general plants present variable mechanisms of tolerance to salinity stress. The molecular basis underlying these mechanisms can be inferred by using proteomic analysis. Thus, the objective of this work was to identify differentially expressed proteins in sugar cane plants submitted to salinity stress. For that, a greenhouse experiment was established with four sugar cane varieties and two salt conditions, 0 mM (control) and 200 mM NaCl. Physiological and proteomics analyses were performed after 2 and 72 h of stress induction by salt. Distinct physiological responses to salinity stress were observed in the varieties and linked to tolerance mechanisms. In proteomic analysis, the roots soluble protein fraction was extracted, quantified, and analyzed through bidimensional electrophoresis. Gel images analyses were done computationally, where in each contrast only one variable was considered (salinity condition or variety). Differential spots were excised, digested by trypsin, and identified via mass spectrometry. The tolerant variety RB867515 showed the highest accumulation of proteins involved in growth, development, carbohydrate and energy metabolism, reactive oxygen species metabolization, protein protection, and membrane stabilization after 2 h of stress. On the other hand, the presence of these proteins in the sensitive variety was verified only in stress treatment after 72 h. These data indicate that these stress responses pathways play a role in the tolerance to salinity in sugar cane, and their effectiveness for phenotypical tolerance depends on early stress detection and activation of the coding genes expression. KEYWORDS: salinity, proteome, root, tolerance, Saccharum

■

INTRODUCTION

The need to increase sugar cane yields in Brazil represents a great challenge to genetic breeding programs, since to meet such demand means to obtain varieties (commercial hybrids) simultaneously productive and adapted to several problems associated with environmental factors in the country.1 Among the main problems that negatively affect the crop yield, especially its growth and development in cultivated areas, are drought and soil salinity and also high or low temperature, high ultraviolet radiation, flood, and pollutants.2

Sugar cane cultivation in Brazil is noteworthy in the worldwide scenario, not only in terms of its products exportation, sugar and bioethanol, but mainly due to favorable climate for its development. However, to achieve ideal cultivation conditions nowadays, it is essential to synchronize between edaphoclimatic conditions and the applied known technologies that lead to higher yield in adverse conditions. Facing the observed, coming, or predicted climate changes, such as global warming, and the concern about cost and availability of fossil fuels, world demand for biofuels has increased and favors the expansion of the sugar cane industry to arid and semiarid regions. © 2013 American Chemical Society

Received: June 30, 2013 Published: November 19, 2013 5681

dx.doi.org/10.1021/pr400654a | J. Proteome Res. 2013, 12, 5681−5695

Journal of Proteome Research

Article

salinity tolerance. Also, it may complement the understanding of gene expression regulation networks, increasing the potential of existing varieties or helping the development of new ones based on genes and mechanisms associated with physiological responses to salinity stress. In this work, the purpose was to identify proteins/peptides differentially expressed in roots of sugar cane submitted to salinity stress in order to propose a model of the tolerance response mechanisms in sugar cane.

Soil salinity in high concentrations harms the crop yields and causes diversified effects on plants, derived from two main elements: osmotic, which drastically alters water balance of the plant, and ionic, which causes toxic effects by excess ion unbalance.3−5 Additionally, it may lead to secondary oxidative damages through the accumulation of reactive oxygen species (ROS) in harmful levels for plant growth and development. Plants usually detect and respond to salinity by changes in gene expression, resulting in biochemical and physiological alterations to survive in the environment.6 Molecular studies on plants’ response to salinity stress have become more complete, aiming to understand the mechanisms of regulation that lead to tolerance against the adverse condition. Multiple signal transduction pathways are activated on a cellular level to adjust gene expression in plants exposed to stress-causing abiotic factors, but the turning on of these tolerance response pathways is one of the critical steps toward the plant’s survival to such factors.7,8 According to Ciarmiello,9 the effectors that directly modulate or reduce the stress effects are classified as determinants of tolerance, and among them are those that act as regulatory molecules of stress perception, signal transduction, and effector function modulation. However, responses against damage caused by salinity are often more associated with tolerant genotypes than to sensitive ones. In sugar cane, functional genomics approaches have been intensified, based on the SUCEST-FUN transcriptome database (http://sucest-fun.org),10 which has allowed gene expression studies helpful to investigate the tolerance to abiotic stresses in many genotypes. After complete sequencing of the genome of model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) and the development of techniques for protein identification and characterization, proteomics became an important field of functional genomics.11 Many studies on sugar cane functional genomics have focused on elucidating tolerance mechanisms to main biotic and abiotic stresses.12 The vast majority of such research concentrates on transcriptome analysis, especially on sucrose content and through EST,10 microarray and SAGE methods,13−16 and identified genes significantly involved in cell signaling, pest resistance, metabolism, development, and responses to biotic and abiotic stresses. The first studies on sugar cane proteomics were performed to establish protein extraction methods;17−19 however, to our knowledge, no differential approach linked to salinity stress has been reported yet. Some authors relate there is diminished protein synthesis in plants under salinity conditions, since proteolysis is increased,20 but other proteins may accumulate in higher levels, such as those involved in cell membrane stabilization and in signaling of responses to salt stress.21 There is a database of high salt concentration-responsive plant proteins, identified from proteomic approaches, comprising a total of 2,170 proteins from leaves, roots, sprouts, cuttings, seeds, hypocotile, radicule, and panicles of 34 species.22 Since proteins are direct effectors of stress response and are closer to phenotype, the importance of proteomic approaches is evident with stress caused by higher saline concentrations. Most proteins responsive to ion excess are involved in photosynthesis, energy metabolism, ROS scavenging, and ion homeostasis.22 Despite all of the research available on plant response to salinity stress, even focusing on the organ with more direct contact to salt (root) or further metabolic adjustment, such approaches are still relatively few for sugar cane. In this context, proteomic analysis allows finding proteins that may serve as functional molecular markers in sugar cane breeding to improve

■

MATERIALS AND METHODS

Plant Materials and Treatments

The greenhouse experiment was performed in the Laboratory of Plant Physiology of Federal Rural University of Pernambuco (UFRPE), located at latitude 08°00′57″S and longitude 34°57′02″W, in Recife, PE. The sugar cane varieties were RB92579, RB867515 (drought-tolerant) and RB72454, RB855536 (drought-sensitive), kindly provided by the Interuniversity Network for Sugar cane Industry Development (RIDESA). Plants were propagated from stem transversal section fragments containing one lateral bud, planted on washed sand as substrate. Fifteen days after sprouting, the plants most uniform in height were transferred to polyethylene vases, 3 plants per vase, with the same substrate, being irrigated on alternate days with water and half-strength Hoagland and Arnon23 nutrient solution until drainage. Plants were cultivated for 45 days and were then exposed to treatments: control (half-strength nutrient solution) and saline (half-strength nutrient solution with 200 mM NaCl added). Control and saline treatments solutions corresponded to 1.00 and 16.88 dS m−1 of electrical conductivity (EC), respectively. Levels of salinity of substrate solution were kept near constant by measuring EC with a conductivimeter (Waterproof): after drainage of the vases, the substrate solution EC varied from 15.41 to 15.70 dS m−1 after 2 h of saline treatment, and from 16.69 to 17.07 dS m−1 after 72 h of saline treatment; in control, the EC range was between 0.87 and 1.62 dS m−1. Experimental design was completely randomized in a 4 × 2 (varieties × treatments) factorial scheme with three repetitions. Samples were harvest 2 and 72 h after NaClcontaining solution application, for physiological as well as proteomic analyses. At harvest times, the environmental parameters of average temperature and air relative humidity inside the greenhouse were measured with a termohygrometer (Microvelle, LR03): 29.6 °C and 81% after 2 h; and 33.5 °C and 52% after 72 h. On the basis of results from physiological analyses, the more tolerant and the more sensitive sugar cane varieties were selected for proteomics analysis. Physiological Analyses

Cell membrane integrity level was estimated by electrolyte leakage analysis24 after 2 and 72 h of stress induction by salt. The percent of electrolyte leakage (DM, %), mainly proportional to plasma membrane damages, was estimated in the leaves +2 (according to van Dillewijn and Waltham25), by measurements of the electrical conductivity of leaf discs immersed in deionized water at room temperature and after being boiled at 100 °C for 1 h, by using the relation DM = 100(C1/C2) . Leaf water potential (Ψw) was measured on leaves “0” by using a Scholander pressure chamber.26 Relative water content (RWC) was determined for the same leaf samples used for DM estimation; defined areas of the leaf blade were weighed to obtain the fresh weight, then were submerged in deionized water, cooled to 8 °C for 24 h, and then weighed again to obtain the turgid weight. Soon after, these leaf samples were stored in paper bags and submitted to drying in an 5682



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accumulation of spots, as well as exclusive spots, between treatments and varieties. Differential spots that presented a percent volume (% vol) ratio ≥1.5 and ANOVA test value ≤0.05 were considered DEPs and selected for identification.

oven for 72 h to obtain the dry matter weight. Finally, RWC was calculated by the formula RWC = [(fresh mass − dry mass)/ (total mass − dry mass)] × 100.27 The physiological parameters of net photosynthesis (A), transpiration (E), and stomatal conductance (gs) were measured in the leaves +1 after 2 and 72 h of application of salt, by using infrared CO2 analyzer ADC model LCi Pro (Hoddesdon, U.K.) according to the manufacturer’s recommended procedures. Physiological data were submitted to variance analysis, and the averages were compared by Tukey test (5% probability) by using the Assistat 7.5 Beta program.28

Digestion, Sequencing, and Identification of the DEPs

Selected single-peak spots were manually excised from gels, transferred in sterile conditions to microtubes containing sterile deionized water at −20 °C until digestion, performed with trypsin (Promega), and postprocessed for mass spectrometry (MS) according to Shevchenko et al.31 Peptides were submitted to analysis by ultrapure liquid chromatography linked to electrospray MS (UPLC-ESI-Q-ToF), and spectra selection was performed by using Distler program (Matrix Science). Putative identification of peptides was achieved through peptide mass fingerprinting via Mascot (Matrix Science; www. matrixscience.com/search_form_select.html) considering SwissProt database (www.uniprot.org/), organism group Viridiplantae, and post-translational modifications of methionine oxidation and carboxymethylation. Significant matched peptides were also searched for putative identification by alignment against the UniProt/Viridiplantae protein database (http://www.uniprot. org/?tab=blast) with significant E-value ≤0.05 and Max ident =100%; the NCBI publicly available protein nonredundant data set from tribe Andropogoneae; and the Saccharum transcript database available in TIGR Gene Index (http://compbio.dfci. harvard.edu/cgi-bin/tgi/gimain.pl?gudb=s_officinarum).

Protein Extraction and Labeling

For proteome analysis, root samples were harvested from plants and washed in sterile tap water to remove substrate particles, avoiding mechanical injury. Excess water was quickly dried out from the roots, which were immersed in liquid nitrogen and stored at −80 °C. Three distinct biological replicates were harvested from each saline condition after 2 and 72 h. Soluble total protein extraction was performed according to Hurkman and Tanaka,29 adjusted to 2 mL tubes. Extracted proteins were quantified through the method described by Bradford,30 and their integrity was checked after 12.5% SDS-PAGE. Two-Dimensional Electrophoresis

Isolelectrical focusing (IEF) was performed on IPG dry-strips 11 cm/pH 3−10 (GE Life Sciences) for each of three biological replicates from each treatment. Approximately 60 μg of protein was dissolved in IPG buffer pH 3−10 nonlinear (GE Life Sciences; 7 M urea, 2 M thiourea, 2% CHAPS, 19.4 mM DTT, 0.5% ampholines) and 0.005% (m/v) bromophenol blue and applied on IPG strips. Rehydration step was made in an IEF dispositive (IPGbox, GE Life Sciences) at room temperature for 16 h. IEF was performed in three stages, in the following conditions: 300−30 V/h, 3500−2900 V/h, 3500−6170 V/h; constant current at 2 mA, power at 5 W and temperature of 10 °C. After IEF, strips could be stored at −80 °C until second dimension. IPG strips were equilibrated with solution containing 50 mM Tris-HCI (pH 8,8), 6 M urea, 30% (v/v) glycerol, 2% (m/v) SDS, 1% DTT, and 2.5% IAA; the last two were added separately in two steps of 20 min under orbital rotation. The second dimension (electrophoresis) was performed in a Multiphor II Electrophoresis Unit (GE Life Sciences) set, in precast 12.5% ExcelGel homogeneous polyacrylamide gel (GE Life Sciences) at 15 °C. Gels were stained with 0.02% Coomassie PhastGel blue R (GE Life Sciences) in methanol, acetic acid, and deionized water at 3:1:6 volume proportion, according to manufacturer instructions. After destaining, gels were scanned in an Image Scanner III (GE Life Sciences), calibrated for optical density, by using the LabScan 6.0 program (GE Life Sciences).

Bioassays SOD

Superoxide dismutase (SOD) was selected among the proteins presenting the highest expression differences between saline treatments, potentially associated with tolerance to such stress, in order to determine its activity. SOD activity was measured through spectrophotometry at 560 nm in accordance with the protocol of Giannopolitis and Ries,32 with minor modifications.

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RESULTS AND DISCUSSION

Physiological Analysis and Variety Selection for Proteomics

After 2 h of NaCl stress exposure, sugar cane varieties did not present significant increase in electrolyte leakage percentage, DM, related to control (Table 1). With longer exposure to NaCl, after 72 h the RB92579 variety (drought-tolerant) presented an increase of 24%, while the sensitive one, RB855536, had a 55% increase in DM. The highest values of RWC, 90% and 88%, were observed for RB867515 (tolerant) and RB72454 (sensitive), respectively (Table 1), but with no significant differences. On the other hand, after 72 h of saline exposure a significant decrease of 17% was observed only in RB72454. Similarly to RWC, no significant differences in leaf water potential, Ψw, were verified after 2 h of stress in all analyzed varieties, compared to control (Table 1). However, after 72 h of stress, all varieties presented a significant decrease in their Ψw, where the most negative value of −1.53 MPa was observed for RB867515 and RB72454 varieties. Despite presenting reduction in net photosynthesis (A) after 2 h of saline stress, such decrease was not significant (Table 1). After 72 h of stress, only RB867515 did not show a significant reduction in A and among the varieties with significant decrease in A, the highest reduction was observed in RB92579 (89%). For leaf transpiration (E), after 2 h of stress there were decreases of 38% and 29% in the sugar cane drought-tolerant varieties RB92579 and RB867515, respectively. However, after 72 h, E was reduced by 76% only in RB92579 (Table 1). This variety, drought-tolerant, was the only one to present significant

Two-Dimensional Gels Image Analysis

Differentially expressed proteins/peptides (DEPs) were selected through comparative analysis of 2D-PAGE digital images, by using the Image Master 2D Platinum v7.05 (IMP, GE Life Sciences), according to manufacturer instructions. Three biological replicates were considered for each saline condition, so that the gel images were grouped in three series of three comparisons (matches), resulting in a total of nine comparisons; in each series, only one parameter (treatment or variety) was considered as variable. Replicates presenting a correlation coefficient (R2) equal to or higher than 0.7 were considered for analysis. Based on normalization and statistical analyses performed in IMP, it was possible to verify the differential 5683



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RB867515 (salinity-tolerant) and RB855536 (salinity-sensitive) under 200 mM NaCl salt stress.

Table 1. Average Values of Electrolyte Leakage (DM), Relative Water Content (RWC), Leaf Water Potential (Ψw), Net Photosynthesis (A), Leaf Transpiration (E), and Stomatal Conductance (gs) in the Four Sugarcane Varieties RB92579, RB867515 (Drought-Tolerant) and RB72454, RB855536 (Drought-Sensitive) Cultivated in a Greenhouse, after 2 and 72 h of stress with 200 mM NaCla

Two-Dimensional Gel Image Analysis and Spots Selection

The R2 of biological replicates was determined, with supposedly minor technical variations due to simultaneous parallel IEF in uniform dry-strips and electrophoresis in the same precast uniform gels (GE Life Sciences). The correlation coefficient between 2D gels of three biological replicates in the same treatment was significant, ranging from 0.7057 to 0.8701. IMPidentified spots presented a molecular mass distribution between 14.4 and 97.0 kDa, and a more frequent concentration in pI range between 5 and 7 (Figure 1; Supplementary Figure 1). In the series of comparisons where saline condition was adopted as variable, the differential expression of spots was verified in both varieties. Thus, a total of 83 differentially expressed proteins (DEPs) was selected (Figure 2) for both varieties, of which 47 were from the tolerant variety (RB867515) and 36 were from the sensitive one (RB855536), including commonly and exclusively expressed spots. Only in 0 mM × 200 mM (72 h) comparison for RB855536, no common DEPs were identified. In the comparison between varieties in the same saline condition (200 mM NaCl after 2 or 72 h of stress) several DEPs were detected (Figure 2), with a higher amount of exclusive spots found in the salinitytolerant at both stress exposure times.

saline conditions (NaCl) variety

0 mM

RB92579 RB867515 RB72454 RB855536

19.02 aC 14.92 aA 20.75 aB 24.71 aAB

RB92579 RB867515 RB72454 RB855536

89.0 aA 94.0 aA 94.0 aA 84.0 aA

RB92579 RB867515 RB72454 RB855536

−0.83 bcA −0.68 baA −0.52 aA −0.9 cA

RB92579 RB867515 RB72454 RB855536

14.73 aAB 17.06 aA 13.61 aAB 16.27 aA

RB92579 RB867515 RB72454 RB855536

2.04 aB 1.88 aBC 1.77 aA 1.50 aA

RB92579 RB867515 RB72454 RB855536

0.19 aA 0.12 bA 0.12 bA 0.14 abA

200 mM (2 h)

0 mM

DM (%) 23.34 aBC 33.55 aAB 18.88 aA 23.59 aA 25.94 aB 32.87 aAB 20.02 aB 24.39 aB RWC (%) 78.0 aA 76.0 aA 90.0 aA 82.0 aA 88.0 aAB 80.0 aBC 85.0 aA 77.0 aA Ψw (MPa) −0.87 cA −1.02 aA −0.65 abA −1.05 aB −0.45 aA −1.04 aB −0.85 bcA −0.98 aA A (μmol CO2 m‑2 s‑1) 10.55 aB 17.20 aA 11.60 aA 17.21 aA 10.80 aAB 14.82 aA 13.91 aA 18.30 aA E (mmol H2O m‑2 s‑1) 1.26 aBC 2.94 aA 1.47 aC 2.76 aA 1.66 aA 1.97 bA 1.53 aA 1.67 bA gs (mol H2O m‑2 s‑1) 0.12 aB 0.17 aAB 0.1 aAB 0.14 aA 0.12 aA 0.13 aA 0.14 aA 0.17 aA

200 mM (72 h) 41.71 aA 27.47 bA 42.11 aA 37.76 abA 83.0 aA 82.0 aA 73.0 aC 79.0 aA −1.44 aB −1.53 aC −1.53 aC −1.42 aB

MS Identification of DEPs 1.82 bC 13.64 aA 7.32 abB 7.45 abB

Among the 47 spots from the tolerant variety (RB867515) selected for digestion and identification through mass spectrometry, 17 could be putatively annotated (Table 2; Figures 3 and 4), including two spots corresponding to phosphoglycerate-kinase, two to methionine-synthase, and two matched to the unknown protein Sb01g036580 from Sorghum bicolor. From a total of 36 spots from the sensitive variety (RB855536), 22 had significant similarity to proteins from grass species, including some from the same tribe of sugar cane, Andropogoneae (Table 2; Figures 3 and 4). The DEPs were distributed in functional groups according to the plant metabolic processes: growth, root formation and cell division and expansion, glycolysis and carbon sequestration, energy supply, protein biosynthesis, general defense, transport and signaling, biosynthesis of compatible osmolytes, secondary metabolism, antioxidant system, and a group with unknown functions. Considering the putatively annotated peptides, they all had significant matches to proteins correlated to abiotic stresses responses. Both varieties presented DEPs related to growth, root formation and cell division and expansion groups. However, it was observed that the varieties contrasting for salinity tolerance expressed distinct DEPs from such groups. Variety RB867515 (tolerant) showed higher expression of ADF3 (actin depolymerizing factor 3) and CDC48 (similar to a cell division cycle protein), after 2 h of exposure to the stress. Both have important roles in root formation33 and cell division and expansion and in organs’ growth process as in roots of fast-growing plants, and ADF3 has already been associated with acclimation to stress by cold.34 On the other hand, RB855536 (sensitive) presented differential expression of the enzymes fructokinase-2 (FrK2) and S-adenosylmethionine synthase (SAMS), which increased their expression after 2 h of exposure to the salt. These proteins are related to root growth inhibition, as observed in tomato mutants,35 and to the higher deposition of lignin in vascular tissues of plants under saline stress, as reported for tomato plants roots submitted to salt stress.36 Besides, the high expression of SAMS in the initial phase of exposure to salt stress could activate an ethylene-signaling pathway that promotes senescence or cell

0.71 bC 2.28 aAB 2.36 aA 1.75 aA 0.02 bC 0.06 abB 0.09 aA 0.07 abB

a

Identical lowercase letters between varieties and identical uppercase between treatments indicate no significant difference using the Tukey test at 5% probability.

reduction of 37% in stomatal conductance (gs) compared to control after 2 h of stress, and this decrease was more drastic after 72 h (88%), being the highest gs reduction among the varieties studied here (Table 1). Among the drought-sensitive varieties, only RB855536 had significant reduction of 59% in gs compared to control after 72 h of stress. RB867515 was selected as the most tolerant to salinity, even more than RB92579. This one, although also tolerant to drought, seemed to have coped with salinity stress activating mechanisms with high energy and growth costs, with almost total stomatal closure, lowering E and A. Such mechanisms may help to maintain the water status in cells, as seen in Ψw and RWC results, but very likely at a high photosynthetic expense. Besides, RB92579 and RB855536 presented the highest DM, suggesting a probable deficiency in excess ion compartmentalization in vacuoles, for example. From these results, RB855536 was selected as the most sensitive to salinity. Based on these physiological data, the proteomic analyses were restricted to 5684



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Figure 1. Partial 2D-PAGE images from RB867515 (tolerant; A, B, C) and RB855536 (sensitive; D, E, F) sugar cane varieties roots stained with Coomassie blue. Proteins were separated in IPG strips of 11 cm, pH 3 to 10. (A, D) 0 mMNaCl; (B, E) after 2 h exposure to 200 mM NaCl; (C, F) after 72 h exposure to 200 mM NaCl.

Figure 2. Venn diagrams of the total number of spots with significant differential expression (DEPs) in roots of sugar cane varieties RB867515 (tolerant) and RB855536 (sensitive) cultivated in a greenhouse under 200 mM NaCl stress. DEPs were selected for MS identification when % vol ratio ≥1.5 and ANOVA ≤ 0.05.

5685


0.001

0.029

inhibited

inhibited

d

d

d

1.522

160

123

74

5686

0.006

0.006

0.00005

induced

inhibited

d

d

d

induced

inhibited

150

157

124

0.001

0.0005

d

d

induced

155

0.002

d

141

induced

113

0.001

0.005

d

induced

151

0.024

d

1.554

2

0.032

d

1.799

86

ANOVAc

fold change 200 mM (2 h)b

match ID


GMLQPHQIIAEYNSAIPEAEREK

LVVDEATNDDNSVVALHPDTMER

AAQDIALAELAPTHPIR

ALAGQKDEAYFAANAAAQASR

TLTSLSGVTAYGFDLVR

VATPAQAQEVHASLR

NPEEIPWGEAGADYVVESTGVFTDKDK

KPFAAIVGGSK

LASVADLYVNDAFGTAHR

ALHGGNFQGTPIGVSMDNAR

EIVVDQVGDR

NILSTINPELLK

peptide

Sb01g033060

CDC48

14-3-3

MetS

MetS

TPI

GAPDH

PGK

PGK

PAL

ADF 3

RB867515 ATPA

abbrev ATPase subunit 1 ATP synthase subunit α ATP synthase subunit α Actin-depolymerizing factor 3 Actin-depolymerizing factor3 Actin-depolymerizing factor3 Phenylalanine ammonia-lyase Phenylalanine ammonia-lyase Phenylalanine ammonia-lyase Phosphoglycerate kinase Phosphoglycerate kinase Phosphoglycerate kinase Phosphoglycerate kinase Phosphoglycerate kinase Phosphoglycerate kinase Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Triosephosphate isomerase Triosephosphate isomerase Triosephosphate isomerase Methionine synthase 2 Methionine synthase 2 Methionine synthase Methionine synthase Uncharacterized protein Methionine synthase 14-3-3-like protein 14-3-3-like protein 14-3-3-like protein Cell division cycle protein 48-like protein Putative uncharacterized protein Cell division cycle protein 48-like protein Sucrose synthase 2-like protein Putative uncharacterized protein Sb01g033060 Sucrose synthase2

annotation (Mascot/Uniprot/TIGR Sugar cane)

540 178 118

TC128630/SCACHR1041G04.g

174 117

B6UD10 TC122450/SCCCSD2093G09.g XP_002465161.1 C5WXJ1

202 107 72 85 121 83 235 147 44 215 132 84 346

148

TC137517/SCACST3158A04.g ACG24648.1 B4FY90 TC127396/SCJFHR1C06H05 CAJ01714.1 Q4LB12 TC127370/SCRLFL4110H03.g AAL33589.1 M0WNE9 TC133431/SCCCNR1002G04.g AAP48904.1 B6TDI1 TC115550/SCJLFL4183H01.b XP_002467173.1

205

118 89 58 57 75 49 55 148 107 145 131 91 139 76 55 478

score

Q0H425

YP_588408.1 H0I4G4 TC145810/SCCCLB1004B02.g NP_001105474.1 Q41764 TC124458/SCJFRZ2033E05.g ABM63378.1 D5M8H8 TC134702/SCRLSB1044C03.g NP_001142404.1 Q850M6 TC135669/SCRLFL1013B07.g AAO32643.1 K7AFS6 TC135669/SCRLFL1013B07.g P26517

access

Table 2. Putative Annotation of Sugarcane RB867515 (Tolerant) and RB855536 (Sensitive) Varieties Root DEPs Expressed under Salinity Conditiona

6.118

5.437

5.570

5.884

5.809

5.64

7.225

5.681

5.717

6.825

5.435

5.844

pI

21933

90717

20313

80341

86489

24996

41495

50204

40713

74206

14012

63636

MW

Journal of Proteome Research Article


0.001

0.043

1.5981

induced

d

d

d

2.6102

10

128

14

5687

0.005

0.004

0.00001

d

inhibited

d

inhibited

d

induced

75

102

76

0.004

d

inhibited

63

0.0003

0.019

0.008

induced

induced

induced

d

97

92

induced

d

93

0.015

0.011

d

inhibited

169

0.002

d

inhibited

130

ANOVAc


match ID


Table 2. continued

LVSWYDNEWGYSNR

ALNEHHVLLEGTLLKPNMVTPGSDSKK

ANSEATLGTYKGDAAADTESLHVK

GDANDEKNVLSLWFDGLK

IFDMESVHGGSPYGAGTFAGDGSR

ASSEWILDCIAHGGDIYGVTTGFGGTSHR

ALVVDDLIATGGTLCAAVK

KLSVETTANQDPLVTK

HAAQLCVLAEDCDQPDYVK

GAEAYLVANPDAYN

FSNQIKDEEGNPAFALVNK

FSNQIKDEEGNPAFALVNK

peptide

GAPDH

FBA

FBA

Frk2

wrbA

PAL

RB855536 APRT

Mn-SOD

RPS12

PR-10

Sb01g036580

RB867515 Sb01g036580

abbrev

Unknown Adenine phosphoribosyl transferase Adenine phosphoribosyl transferase Phenylalanine ammonia-lyase Phenylalanine ammonia-lyase Phenylalanine ammonia-lyase Flavoprotein wrbA-like Flavoprotein wrbA Flavoprotein wrbA Fructokinase-2 Fructokinase-2 Fructokinase-2 Fructose-bisphosphate aldolase Fructose-bisphosphate aldolase Fructose-bisphosphate aldolase Fructose-1,6-bisphosphate aldolase-like Fructose-bisphosphate aldolase Fructose-bisphosphatealdolase Glyceraldehyde-3-phosphate dehydrogenase 2

Sorghum hypothetical protein 01g036580 Uncharacterized protein Sb01g036580 Osr40g2 protein Sorghum hypothetical protein 01g036580 Uncharacterized protein Sb01g036580 Osr40g2 protein Pathogenesis-related protein 10a-like Pathogenesis-related protein 10a Pathogenesis-related protein 10 40S ribosomalprotein S12 40S ribosomalprotein S12 40S ribosomal protein S12 Superoxide dismutase [Mn] precursor Superoxide dismutase [Mn] Superoxide dismutase [Mn]


98 279 142 98 147 104 74 86 148 106 421

TC137001/SCRFSB1024E02.g XP_002465329.1 C5 × 1Q1 TC137001/SCRFSB1024E02.g XP_002468008.1 Q4VQB5 TC133893/SCBFSD2034E05.g ACG24471.1 D3Y1X9 TC130196/SCQSFL3031F01.g XP_002439542.1

42 133 92 382 217 162 163 176 130 270 136 94 171 169 120 444 199 140 556

ACF84896.1 B6U1A1 TC123199/SCEPAM2014F03.g ABM63378.1 K7UX63 TC151535/SCACCL6006A03.g NP_001151964.1 B6U724 TC113999/SCQGST3123G04.g ACG34312.1 K7UH47 TC132154/SCBFRT3092E03.g NP_001105336.1 B6SI53 TC149024/SCQSRT2035H08.b XP_002458944.1 B6SI53 TC125112/SCCCRT1C05F10.g NP_001105700.1

115 78

142

C5 × 1Q1

M1TAD0 TC114055/SCRFFL5036E06.g

288

score

XP_002465329.1

access

5.746

8.024

7.880

5.566

5.865

6.045

6.453

6.145

5.391

5.558

6.947

6.867

pI

39501

43815

43350

32155

29836

86003

29673

21639

18428

15803

38056

36104

MW



inhibited

inhibited

d

d

100

126

5688

0.005

0.002

0.005

inhibited

d

d

d

induced

d

inhibited

inhibited

inhibited

d

112

64

62

61

88

0.035

0.001

d

inhibited

80

0.0008

0.004

0.005

0.051

d

inhibited

74

0.003

d

inhibited

73

ANOVAc


match ID


Table 2. continued

AAVIDWHTLAPK

GNNLAIADPLTHTSDPYVVLQYGAQK

NPAELAHGANAGLDIAVR

FSNQIKDEEGNPAFALVNK

DGATNPTFLYFSHGLK

ALAGQKDEAYFAANAAAQASR

YGAGIGPGVYDIHSPR

IFSNPEVAAEEPWYGIEQEYTLLQK

HETADINTFSWGVANR

VIHDNFGIIEGLMTTVHAITATQK

peptide

PR-10

Sb02g033760

Sb01g038760

Sb01g036580

PP

MetS

MetS

GS

GS

GAPDH

RB855536

abbrev Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase 1 Glyceraldehyde-3-phosphate dehydrogenase 2 Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase1 Glutamines ynthetase Cytosolic glutamine synthetase alpha Glutamine synthetase Glutamine synthetase Glutamine synthetase Glutamine synthetase Methionine synthase Uncharacterized protein Methionine synthase Methionine synthase Uncharacterized protein Methionine synthase Putative uncharacterized protein Putative uncharacterized protein Translationally controlled tumor protein-like Sorghum hypothetical protein 01g036580 Sorghum hypothetical protein 01g036580 Osr40g2 protein Sorghum hypothetical protein 01g038760 Sorghum hypothetical protein 01g038760 Ascorbate peroxidase Sorghum hypothetical protein 02g033760 Sorghum hypothetical protein 02g033760 Chitinase III-like protein Pathogenesis-related protein 10a-like Pathogenesis-related protein 10a Pathogenesis-related protein 10


122

TC139434/SCRLFL1004E08.g

181 142 98 392 128 91 307 193 132 64 92 64

XP_002465329.1 C5 × 1Q1 TC137001/SCRFSB1024E02.g XP_002468053.1 C5WNL8 TC141093/SCRLFL1004F07.g XP_002462882.1 C5 × 978 TC119228/SCVPRT2080B01.g XP_002468008.1 Q4VQB5 TC119168/SCCCRT2C03D05.g

283 124 89 438 197 134 46 120 91 198 147 44 160 120 89

182

A2IBP5

AAW21273.1 Q94KI3 TC128884/SCVPAM2066E05.g AAW21273.1 Q5MD11 TC141076/SCQGSB1065D04.g AAF74983.1 K7AJK1 TC125720/SCCCCL5001E01.g AAL33589.1 M0WNE9 TC133431/SCCCNR1002G04.g ACF82810.1 B6TLQ6 TC143228/SCEZFL5085E11.g

747

86

TC137690/CCCNR1002A03.g NP_001105700.1

118

score

B6SBH9

access

5.341

6.038

6.204

5.699

4.651

5.679

6.072

5.632

6.096

7.013

pI

16455

25389

22827

31813

19292

83432

71896

40662

38963

44011

MW



0.006

0.002

0.004

d

induced

d

induced

d

inhibited

90

66

IKVLQAQDDLVNK

AVVVHADPDDLGKGGHELSK

KLSVETTANQDPLVTK

LGATFSSHPNELIALFSR

VLVNIEQQSPDIAQGVHGHFTK

peptide

YLP

Cu−Zn−SOD

SOD

SS2

RB855536 SAMS3

abbrev

YLP/vacuolar ATP synthase subunit E Y2-like protein Vacuolar ATP synthase subunit E

S-Adenosylmethionine synthase S-Adenosylmethionine synthase 1 S-Adenosylmethionine synthase 2 Sucroses ynthase Sucrose synthase-2 Sucrose synthase-2 Mn superoxide dismutase Mn superoxide dismutase Superoxide dismutase Superoxide dismutase [Cu−Zn] Superoxide dismutase [Cu−Zn] Superoxide dismutase [Cu−Zn] 2


160 96 62

Q9ZR97 TC115432/SCCCFL5059C04.g

293 165 115 275 131 90 113 115 78 59 143 106

score

Q944U4.1 Q5UML0 TC120734/SCJLRT1015H10.g ACM69042.1 Q6BA98 TC121542/SCEQRZ3089F12.g AAA57130.1 M1TAD0 TC114055/SCRFFL5036E06.g Q9SQL5.1 C0JPM7 TC104633/SUR01−126-H08-A06 ACG31413.1

access

7.077

5.520

5.757

5.949

5.569

pI

37256

22308

23173

90717

47910

MW

Obtained through PMF/Mascot/Viridiplantae, BlastP alignment search to Uniprot/Viridiplante database (http://www.uniprot.org/?tab=blast) and more specifically to Saccharum transcript accesses available in TIGR Gene Index (http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=s_officinarum). bFold change (ratio of % vol) compared to the control treatment (0 mM NaCl). cANOVA compared to the control treatment (0 mMNaCl). dNo significant variation (ratio of % vol

Differentially Delayed Root Proteome Responses to Salt Stress in

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