Proteomic Analysis of Wheat Seed in Response to Drought Stress

Transcription

1 Journal of Integrative Agriculture Advanced Online Publication: 2013 Doi: /S (13) Proteomic Analysis of Wheat Seed in Response to Drought Stress ZHANG Yu-feng, HUANG Xiu-wen, WANG Li-li, WEI Liu, WU Zhi-hui, YOU Ming-shan and LI Baoyun 1 Beijing Key Laboratory of Crop Genetic Improvement/Key Laboratory of Crop Genomics and Genetic Improvement, Ministry of Agriculture/Key Laboratory of Crop Heterosis & Utilization, Beijing, P.R.China Abstract Drought stress is one of the major factors affecting in wheat yield and grain quality. In order to investigate how drought stress might influence wheat quality during grain filling, three wheat cultivars were subjected to drought stress during the grain filling stage. Neither globulin and glutenin, nor the relative percentage of amylose significantly changed following drought treatments, whereas of albumin and gliadin concentrations did. The SDS-sedimentation, which has a strong linear correlation with wheat baking quality was markedly decreased following drought stress. These results indicated that drought had an adverse effect on wheat quality. In order to investigate the protein complexes in the wheat flour, the date from native PAGE and SDS-PAGE were combined and a total of 15 spots were successfully identified, and of these eight protein types were determined to be potential complex forming proteins. Key words: wheat seed, drought stress, two-dimensional electrophoresis (2-DE) INTRODUCTION It has long been known that drought stress significantly affects common wheat ( aestivum L.) yield and quality, especially the composition of seed storage proteins that form during the grain filling stage. In fact, the baking quality of spring wheat flour can be negatively affected by drought stress during the development period (Day 1971). Under drought stress, the protein content, SDS-sedimentation value, and wet gluten of winter wheat have been demonstrated to increase by 18.1, 16.5 and 21.9% from germination to maturation, whereas they increased only by 8.3, 8.7 and 10.8% respectively during grain filling (Ozturk 2004). However, Gooding et al. (2002) found that the SDS-sedimentation value decreased under drought stress, Yang et al. (2011) found that α-gliadin and γ-gliadin decreased in response to drought stress, and Attila et al. (2011) found that the A-type starch increased, whereas the B-type starch actually decreased. Proteomics is an effective method to investigate changes in wheat seed protein concentration in response to drought stress. Willian et al. (2005) analyzed the differences in wheat seed proteins between DAP (days after pollination). In this survey, 250 differentially expressed proteins were found, and were involved in 13 different biological processes. Analysis of protein content revealed that most endosperm proteins were related to carbohydrate metabolism, transcription, translation, protein synthesis at 10 DAP, and carbohydrate metabolism and protein synthesis at 36 DAP, but stress, defense and storage proteins predominated, (e.g., heat shock, granule-bound starch synthase, β-amylase, glucose-1-phosphate adenyltransferase, etc.). Correspondence LI Bao-yun, libaoyun310@yahoo.com.cn

2 The protein expression profiles of heat-sensitive (cv. Wyuna), and heat-tolerant (cv. Fang) wheat cultivars have been compared following heat shock (Skylas et al. 2002). In this study, 48 proteins were differentially expressed, and of these 17 were identified as heat shock proteins. This is consistent with that found by Majoul et al. (2003, 2004) in which 25 proteins were differentially expressed proteins following heat shock, among which 24 were up-regulated, yet only one was down-regulated. Some of these were enzymes involved in different plant metabolic pathways, such as granule-bound starch synthase and glucose-1-phosphate adenyltransferase (both involved in the starch synthesis), β-amylase, (involved in carbohydrate metabolism) and the ATP synthase β-chain that was related to four heat-decreased proteins. Non-prolamin proteins of Durum wheat were affected by heat stress during grain filling, and 132 differentially expressed spots were revealed. Of these 47 were HSPs, and proteins involved in glycolysis, carbohydrate metabolism and stress-related proteins were analyzed by MALDI-TOF and MALDI-TOF-TOF MS. Sancho et al. (2008) examined the influence of genotype and environment on the accumulation of soluble wheat dough liquor protein (Gliadin) under hot/dry, and cool/wet regimes using 2-DE gel technology. In such, the 7S globulin (vicilin-like), R-globulin families, some protective proteins including members of the serpin, chitinase families were differentially expressed. The combination of water deficit and high-temperature resulted in differential expression of primary metabolism, storage, and stress response related proteins (e.g. late embryogenesis abundant proteins, peroxiredoxin, a-amylase inhibitors, and proteins, etc., Yang et al. (2011). In addition, the effect of different nitrogen and sulfur levels on tris-soluble, and glutenin protein fractions have been determined via use of these 2-DE gels (Grove et al. 2009). In such, 20 tris-soluble and 16 types of glutenin were differentially expressed. Although this study demonstrated that a sulfur deficiency during grain filling might result in changes in protein composition, this was not evident in Dupont s et al. (2006) investigation. This study used a degeneration condition in which albumin and globulin were separated into single subunits, with most functional proteins containing multiple subunits. The individual proteins, which were separated by SDS-PAGE, are often associated with each other, forming temporary, or stable larger protein complexes. Thus it is difficult to ascertain which proteins form a complex through IEF-SDS-PAGE alone. In the present study, proteins were separated by native PAGE, which does not break apart protein complexes in the first dimension (Fig. 1). Followed by the second dimension, protein complexes are separated by SDS-PAGE, in which some flour protein complexes could be identified, and thus provided a more in-depth understanding of flour protein interactions. Fig. 1 Protein complexes electrophoresis diagram. A, first, native-page. B, second, SDS-PAGE.

3 RESULTS Changes in protein composition and starch content in response to drought stress Four seed storage proteins (e.g., albumin, globulin, gliadin, and glutenin) were determined in the cultivar Gaocheng 8901 to assess if their content was affected by drought stress. To analyze the accumulation of different storage protein classes, seed protein was extracted from mature grains and separated into albumin, globulin, gliadin, and glutenin. The albumin and gliadin concentrations increased significantly under drought stress, whereas globulin and glutenin concentrations did not (Fig. 2). Fig. 2 Albumin, globulin, gliadin, and glutenin under drought stress in Gaocheng 8901 flour. *, P<0.05, **, P<0.01. The relative percentage of amylose in Gaocheng 8901 flour under drought stress and normal growth conditions were determined. The relative percentage of amylose content was 11.4 and 13.0% within seeds of drought stressed wheat and normally grown wheat, respectively. Though no statistically significant differences were observed between the two treatments, the quality of wheat flour may be influenced by the slight decrease within drought-stressed seeds. Fig. 3 The percentage of amylose content in flour of Gaocheng 8901 grown under drought and normal conditons. No significant difference was detected. SDS-sedimentation of wheat flour

4 To evaluate the effect of drought stress on baking quality, flour of three drought-stressed wheat cultivars (Jagger, Nongda 3406 and Gaocheng 8901) were subjected to SDS-sedimentation. Results of the SDS-sedimentation at 5, 10, 15, and 20 min are summarized in Fig. 4. The SDS-sedimentation values of these three cultivars under drought stess were significantly lower than that observed for those under normal conditions. The results suggested that drought stress may have a negative effect on the final baking quality of wheat. Fig. 4 SDS-sedimentation of wheat cultivars under drought stress and normal condition. Drot and Norm, for drought and normal, respectively. Ptotein profiling of wheat flour in response to drought stress Fist dimensional native PAGE can be used to separate protein complexes, while a second dimensional SDS PAGE is used to further resolve subunits of protein complexes, which may fail to separate in the first dimension. Albumin and globulin were extracted by KCl solution buffer (Fig. 5). Table 1 proteins identified by MS-MS Spot Protein Species Accession Mw (kda) A1 beta amylase aestivum gi A2 beta amylase Score

5 SDS-PAGE SDS-PAGE B1 B2 B3 B4 C1 C2 serpin cytosolic kinase serpin serpin oligopeptidase A-like UDP-glucose pyrophosphorylase 3-phosphoglycerate aestivum gi aestivum gi aestivum gi aestivum gi aestivum gi Brachypodium distachyon gi Oryza sativa gi D like protein GF14-6 Zea mays gi D protein aestivum gi E1 alpha amylase inhibitor aestivum gi E2 alpha amylase inhibitor aestivum gi F1 beta amylase aestivum gi F2 UDP-glucose Oryza sativa pyrophosphorylase gi Protein complexes, which failed to separate following the second dimension SDS-PAGE were excised, and identified by MALDI-TOF-TOF. Fifteen proteins were successfully identified (Table 1). Those identified included metabolic enzymes (beta amylase, UDP-glucose pyrophosphorylase), signal transduction protein ( protein), and stress related proteins (alpha amylase inhibitor and serpin). Native-PAGE Native-PAGE Normal Drought Fig. 5 Native PAGE-SDS PAGE images in drought sample and normal sample.

6 The beta amylase was composed of subunits (A1 and A2). There may be interaction between beta amylase and UDP-glucose pyrophosphorylase (H1 and H2). The interaction between UDP-glucose pyrophosphorylase and oligopeptidase A-like (C1 and C2) may also exist. D1 and D2 both were protein (Fig. 6 and Table 1). Fig. 6 Enlargement of the Native PAGE-SDS PAGE images. Some subunits (B1, B2) of serpin, a supposedly stress-related protein might have interaction with cytosolic 3-phosphoglycerate kinase (B2, Fig. 7 and Table 1). Fig. 7 Enlargement of the Native PAGE-SDS PAGE images As illustrated in Fig. 8, E2 was identified as a 27K protein, and both F1 and F2 were identified as alpha amylase inhibitors. These results suggest that alpha amylase inhibitor may consist of subunits, and these subunits combine to form the final functional complex. Fig. 8 Enlargement of the Native PAGE-SDS PAGE images.

7 DISCUSSION Wheat quality is susceptible to prevailing environmental conditions; of which drought stress is one such factor. During grain filling, drought stress affects grain carbohydrates, storage protein synthesis and accumulation, subsequently negatively affecting yield and quality (Jiang et al. 2007). Previous studies indicated that the protein content, SDS-sedimentation value, and wet gluten of winter wheat were increase by 18.1, 16.5 and 21.9%, respectively under drought stress during from germination to maturation, and protein content, sedimentation value and wet gluten content were increased by 8.3, 8.7 and 10.8%, respectively during grain filling (Ozturk and Aydin 2004). These results are similar to those obtained this study using Gaocheng Albumin and gliadin contents increased under drought stress, whereas globulin and glutenin did not change appreciably. We did find that all protein contentrations increased in seed of plants exposed to drought stress. For example, drought stress results in a significant increase in total seed protein, and grain glutenin/gliadin protein ratio (Tao Lan and Dong Jiang et al. (2004), Dai and Hui Zhao et al. (2006)) Although the total protein content increases under drought stress, protein production decrease due grain production declined (Fan et al. 2004). Protein complexes research is available for studying adversity stress in wheat. MATERIALS AND METHODS Plant material and drought treatment Wheat cultivars Gaocheng 8901, Jagger and Nongda 3406 were grown in 50 cm (L) 30 cm (H) 40 cm plastic boxes at 25 and a soil water content of 20-25%, and moved into phytotron chambers until anthesis. The growing condition was at 20 (13 h d)/12 (11 h night). Soil water content was controlled as in Aprile (2009) in which normal was considered to 20-25%, and drought was 7-12%, the normal condition was at 20-25%. Soil water was controlled as described by Aprile et al. (2009), the soil water content in drought condition was at 7-12%, the normal condition was at 20-25%. Protein and Amylose content determination Albumin, globulin, gliadin, and gluten were extracted as previously described (Tian, 2006). Briefly, mature grain was milled with a Brabender D-4100, and the resulting flour (100 mg) suspended in 1 ml distilled H 2O. These suspensions were incubated at 50 for 30 min with intermittent mixing, and then centrifuged at 4000 r min -1 for 20 min at 4, repeated three times. This process resulted in the albumin being retained in the soluble fraction. Remaining pellets were suspended in 1 ml 10% NaCl, and incubated, mixed and centrifuged as in above, resulting in globulin retained in the soluble fraction. The remaining pellets were again then suspended in 1 ml 75% EtOH, treated as in above, and any gliadin was retained in the soluble fraction. Lastly, the remaining pellets were suspended in 1 ml 0.2% NaOH, again treated as in above, and any remaining gluten retained in the soluble fraction. Protein content was determined by the Bradford method. Amylose was extracted and determined by suspending 100 mg of flour in 1 ml EtOH, to which was added 9 ml NaOH (1 mol L -1 ). Suspensions were subsequently incubated in boiling (100 ) H 2O for 10 min,

8 and cooled in ice water. Aliquots of the each suspension (20 ml) were transferred to 50 ml centrifuge tubes and incubated with 10 ml petroleum ether for 10 min, heated at 25 for 15 min, repeated three times. The upper fraction contained amylose solution, and the amylose content was determined using the iodine-potassium iodide method. The upper fraction was amylase solution, and amylose content was determined by Iodine-potassium iodide method. The experimental results were analyzed by Excel2010 software. SDS-sedimentation analysis Wheat flour samples (2g) were suspended in 16.7 ml H 2O. (containing 100 ppm bromophenol blue), and incubated at room temperature ( 25 ) for 5 min; 16.7 ml of SDS solution (2% SDS, mol L -1 lactic acid) was then added to each sample, and each was again incubated at room temperature for an additional 5 min. Sedimentation volumes were recorded every 5 min for a total of 20 min. Protein electrophoresis The first-dimensional electrophoresis was conducted on a 10 % native PAGE, at 300V per gel, and 16 for 3-4 h until the dye front exited the gel. Gel strips containing protein were cut from the gel and equilibrated in mol L -1 Tris-HCl, ph 6.8, containing 4% w/v SDS, 0.02% w/v bromophenol blue, and 0.9% w/v beta-mercaptoethanol for 20 min. For the second-dimensional electrophoresis, gel strips were transferred to 12.5% SDS-PAGE, at 300V per gel and 16 for 3-4 h until the dye front exited the gel. Proteins were then visualized by silver staining as in Song et al. (2009). Protein identification by MALDI-TOF-TOF Proteins of interest were manually excised from the silver-stained gels, and subjected to in-gel trypsin digestion. Following digestion, peptides were extracted twice with 0.1% trifluoracetic acid (TFA) in 50% Acetonitrile. Extracts were pooled, dried completely in a Speed Vac, and mixtures re-dissolved in 0.1% Trifluoracetic acid. Peptide solutions (0.8 μl) were mixed with 0.4 μl of matrix (α-cyano-4-hydroxycinnamic acid (CHCA) in 30% ACN, 0.1% TFA) prior to spotting on the target plate. Proteins were identified on an AB SCIEX MALDI TOF-TOF 5800 Analyzer (AB SCIEX, Foster City, CA) equipped with a neodymium: yttrium-aluminum-garnet laser (349 nm). The TOF/TOF calibration mixtures (AB SCIEX) were used to calibrate the spectrum to a mass tolerance of within 150 ppm. The raw MS and MS/MS spectra data were processed using GPS Explorer TM software. Proteins with score confidence intervals (C.I.) above 95% (protein score > 66) were considered confident identifications. Identified proteins were then matched to specific biological processes, or molecular functions using the Gene Ontology database ( Acknowledgements This work was supported by the National Nature Foundation of China ( ),the High-Tech R&D

9 Program of China (2012AA ) and the Key Basic Research Program of China (2009CB118300). References Altenbach Susan B, Tanaka Charlene K, Hurkman William J, et al Differential effects of a post-anthesis fertilizer regimen on the wheat flour proteome determined by quantitative 2-DE. Proteome Science, 9, 46. Aprile Alessio, Mastrangelo Anna M, de Leonardis Anna M, et al Transcriptional profiling in response to terminal drought stress reveals differential responses along the wheat genome. BMC Genomics, 10, 279. AttilaFa bia ń, Ja ger Katalin, Rakszegi Mariann, et al Embryo and endosperm development in wheat ( aestivum L.) kernels subjected to drought stress. Plant Cell Reports, 30, Day A D, Barmore M A Effects of soil moisture stress on the yield and quality of flour from wheat ( aestivum L.). Agronomy Journal, 63, Dai Yanbo, ZhaoHui, JingQi et al Effects of high temperature and water stress during grain filling on grain protein and starch formation in winter wheat. Acta Ecologica Sinica, 26, Dupont F M, Hurkmana W J, Vensel W H, et al Differential accumulation of sulfur-rich and sulfur-poor wheat flour proteins is affected by temperature and mineral nutrition during grain development. Joural of Cereal Science, 44, Fan Xuemei, Jiangdong, Dai Yan et al Effects of post-anthesis and waterlogging on the quality of grain formation in different wheat varieties. Acta Ecologica Sinica, 26, Grove Harald, Hollung Kristin, Moldestad Anette et al Proteome Changes in Wheat Subjected to Different Nitrogen and Sulfur Fertilizations. Journal of Agricultural and Food Chemistry, 57, Gooding M J, Ellis R H, Shewry P R, et al Effects of restricted water availability and increased temperature on the grain filling, drying and quality of winter wheat. Journal of Cereal Science, 37, Hajheidari Mohsen, Eivaz Alirezai, Buchanan Bob B et al Proteomics Uncovers a Role for Redox in Drought Tolerance in Wheat. Journal of Proteome Research, 6, HurkmanWilliam J, Vensel William H, Tanaka Charlene K et al Effect of high temperature on albumin and globulin accumulation in the endosperm proteome of the developing wheat grain. Journal of Cereal Science, 49, Jiang D Y, Yu Z W Effects of soil water on yield and grain quality of wheat. Journal of nuclear agriculturae sciences, 21, Laino Paolo, Shelton Dale, Finnie Christine et al. 2010,.Comparative proteome analysis of metabolic proteins from seeds of durum wheat (cv. Svevo) subjected to heat stress. Proteomics, 10, Majoul Thouraya, Bancel Emmanuelle, TriboïEugène et al Proteomic analysis of the effect of heat stress on hexaploid wheat grain: Characterization of heat-responsive proteins from total endosperm. Proteomics, 3, Majoul Thouraya, Bancel Emmanuelle, TriboïEugène et al Proteomic analysis of the effect of heat stress on hexaploid wheat grain: Characterization of heat-responsive proteins from non-prolamins fraction. Proteomis, 4, Nadaud Isabelle, Girousse Christine, Debiton Cle ḿent et al Proteomic and morphological analysis of early stages of wheat grain development. Proteomics, 10, Ozturk A, Aydin F Effect of water stress at various growth stages on some quality characteristics of winter wheat. Journal of Agronomy and Crop Science, 190, Sancho Ana I, Gillabert Muriel, Tapp Henri et al Effect of environmental stress during grain filling on the soluble proteome of wheat ( aestivum) dough liquor. Journal of Agriculture and Food Chemistry, 56,

10 Skylas D J, Cordwell S J, Hains P G et al Heat shock of wheat during grain filling: proteins associated with heat-tolerance. Journal of Cereal Science, 35, Song J M, Liu A F, Wu X Y et al Composition and content of high-molecuiar-weight giutenin dubunitsand their reiations with wheat quaiity. Scientia Agricultura Sinica, 36, Tian J C, et al Grain Quality Testing Theory and Method. Science Press, Beijing. Vensel William H, Tanaka Charlene K, Cai Nick et al Developmental changes in the metabolic protein profiles of wheat endosperm. Proteomics, 5, Yang Fen, Jørgensen Anders D, Li Huawei et al Implications of high-temperature events and water deficits on protein profiles in wheat ( aestivum L. cv. Vinjett) grain. Proteomics, 11,