J. AMER.SOC.HORT.SCI. 136(3):219–226. 2011. Heat-induced Proteome Changes in Tomato Leaves Suping Zhou1, Roger J. Sauve,´ Zong Liu, Sasikiran Reddy, and Sarabjit Bhatti Department of Agricultural Sciences, School of Agriculture and Consumer Sciences, Tennessee State University, 3500 John A. Merritt Boulevard, Nashville, TN 37209 Simon D. Hucko, Yang Yong, Tara Fish, and Theodore W. Thannhauser Plant, Soil and Nutrition Research Unit, USDA-ARS, Tower Road, Ithaca, NY 14853-2901 ADDITIONAL INDEX WORDS. Solanum lycopersicum, heat stress, proteomics, photosynthesis, methionine and SAM bio- synthesis, glycolate shunt, Rubisco activase, transketolase ABSTRACT. Three tomato (Solanum lycopersicum) cultivars [Walter LA3465 (heat-tolerant), Edkawi LA 2711 (un- known heat tolerance, salt-tolerant), and LA1310 (cherry tomato)] were compared for changes in leaf proteomes after heat treatment. Seedlings with four fully expanded leaves were subjected to heat treatment of 39/25 8C at a 16:8 h light–dark cycle for 7 days. Leaves were collected at 1200 HR, 4 h after the light cycle started. For ‘Walter’ LA3465, heat-suppressed proteins were geranylgeranyl reductase, ferredoxin-NADP (+) reductase, Rubisco activase, trans- ketolase, phosphoglycerate kinase precursor, fructose–bisphosphate aldolase, glyoxisomal malate dehydrogenase, catalase, S-adenosyl-L-homocysteine hydrolase, and methionine synthase. Two enzymes were induced, cytosolic NADP-malic enzyme and superoxide dismutase. For ‘Edkawi’ LA2711, nine enzymes were suppressed: ferredoxin- NADP (+) reductase, Rubisco activase, S-adenosylmethionine synthetase, methioine synthase, glyoxisomal malate dehydrogenase, enolase, flavonol synthase, M1 family peptidase, and dihydrolipoamide dehydrogenase. Heat-induced proteins were cyclophilin, fructose-1,6-bisphosphate aldolase, transketolase, phosphoglycolate phosphatase, ATPase, photosystem II oxygen-evolving complex 23, and NAD-dependent epimerase/dehydratase. For cherry tomato LA1310, heat-suppressed proteins were aminotransferase, S-adenosyl-L-homocysteine hydrolase, L-ascorbate peroxidase, lactoylglutathione lyase, and Rubisco activase. Heat-induced enzymes were glyoxisomal malate de- hydrogenase, phosphoribulokinasee, and ATP synthase. This research resulted in the identification of proteins that were induced/repressed in all tomato cultivars evaluated (e.g., Rubisco activase, methionine synthase, adenosyl-L- homocysteine hydrolase, and others) and those differentially expressed (e.g., transketolase). Temperature is a key factor determining optimal growth and pollination, resulting in unfertilized embryos and aborted fruits productivity of plants. In recent decades, many parts of North (Berry and Rafique-Ud-Din, 1988). America have been experiencing an increase in the number of Previous research has demonstrated that there is a strong unusually hot days and nights. Continued global warming is correlation between heat stress and fruit yield in tomato (Berry likely to result in an increase in frequency and intensity of heat and Rafique-Ud-Din, 1988). Membrane thermostability and waves and drought (U.S. Global Change Research Program, heat-induced increase in chlorophyll a:b ratio and decrease in 2008). Leaves function as the primary manufacturer of many chlorophyll:carotenoids ratio are directly associated with the metabolites used during plant growth. Both the integrity of the level of thermotolerance in tomato cultivars (Camejo et al., machinery and functionality of enzymes associated with 2005; Saeed et al., 2007). Individual genes encoding for heat photosynthetic activity are sensitive to heat stress (Berry and stress transcription factors (Chan-Schaminet et al., 2009; Bjo¨rkman, 1980; Murakami et al., 2000). Significant inhibition Scharf et al., 1998; Schultheiss et al., 1996), heat shock and of photosynthesis occurs at temperatures only a few degrees chaperonin proteins (Port et al., 2004), and other functional pro- above the optimum, resulting in a considerable loss of potential teins [e.g., kinases, reactive oxygen species scavengers, en- productivity. zymes associated with sugar metabolism (Frank et al., 2009; Tomato is one of the most important vegetable species and Link et al., 2002)] play key roles in modulating thermotolerance cash crops in the world. Daytime temperatures consistently in tomato. above 32 °C and evening temperatures that stay above 24 °C are Damage resulting from heat stress is present in multiple considered excessive and are detrimental to tomato plant forms such as oxidative burst (Camejo et al., 2006), metabolic growth and fruit development. Tomato leaves exposed to pro- toxicity, membrane disorganization, inhibition of photosynthe- longed heat stress conditions experience starch depletion as a sis, and altered nutrient acquisition (Ismail and Hall, 1999; result of enhanced hydrolysis and reduced biosynthesis Karim et al., 1999; Wahid et al., 2007). Tolerant plants have a activities (Dinar et al., 1983). Short-term heat stress affects higher capacity to maintain homeostasis under stress by the activation of stress perception and signaling pathways, antiox- idant capacity, gene expression regulation pathways, and al- Received for publication 7 Jan. 2011. Accepted for publication 7 Mar. 2011. teration of metabolic cycles (Bohnert et al., 2006, and This project is supported by the Agriculture and Food Research Initiative references therein). As a result of the complexity of plant competitive grant no. 2010-65114-20405 from the USDA National Institute of response to heat stress, very few tolerant cultivars have been Food, and Agriculture, 1890 Capacity Building Program, and Evans-Allen produced using traditional breeding protocols. Genetic trans- Research Funds. We thank Drs. Nick Gawel and Daifeng Hui at Tennessee State University for formation techniques have been of little use as a result of critical review of the manuscript. limited knowledge and availability of genes with known effects 1Corresponding author. E-mail: [email protected]. on plant heat-stress tolerance (Foolad, 2005; Wahid et al., J. AMER.SOC.HORT.SCI. 136(3):219–226. 2011. 219 2007). In this project, heat-induced changes in the whole pro- the protein extractions used in the experiment was used to teomes of tomato leaves were identified using a proteomics normalize across the multiple gels (Alban et al., 2003), which approach. The objective of this research was to determine greatly reduces variation in the samples as a result of electro- candidate genes and pathways that should be investigated when phoresis and loading. The dye:protein ratio for the experiments breeding tolerant tomato cultivars. was 200 pmol dye:50 mg total protein. The analytical gels were run using 50 mg of protein from each labeled sample. A Materials and Methods preliminary analysis on a limited number of samples was done to conduct a power analysis to facilitate the design of the large- PLANT GROWTH AND HEAT TREATMENT. Three tomato culti- scale analysis. The pilot study demonstrated that three vars {Walter LA3465 [heat-tolerant (Swift, 2008)], Edkawi biological replicates were sufficient to identify differentially LA2711 [unknown heat tolerance, salt-tolerant], and LA1310 expressed proteins with greater than a 1.5-fold change at a sta- [cherry tomato]} were compared. Seed stocks were obtained tistical power of 0.85 or greater. Therefore, subsequent exper- from the C.M. Rick Tomato Genetics Resource Center at the iments had three biological replicates per treatment. University of California, Davis. Seeds were propagated in a For running the gels, a protein sample was first subjected to greenhouse at Tennessee State University (Nashville). For this isoelectric focusing (IEF) on the 24-cm Immobiline DryStrip project, tomato seeds were germinated in seed cubes (Smithers- pH 3-10 NL (GE Healthcare). At the completion of the IEF run, Oasis, Kent, OH), and seedlings were grown to the four fully proteins were reduced and alkylated (Zhang et al., 2003). Strips expanded mature leaf stage in a greenhouse. Before heat were transferred onto 12.52% acrylamide–sodium dodecyl treatment, tomato seedlings were transferred into two illumi- sulphate gels, which were prepared using 41.75% (v/v) of pro- nated incubators (Thermo Fisher Scientific, Pittsburgh, PA), togel (National Diagnostics, Atlanta, GA), and the gels (255 · which were programmed at 25 °C and light cycle of 16/8 h (day/ 196 · 1 mm) were run on a Hoefer SE900 vertical slab gel night). After 1 week, the incubator for heat treatment was electrophoresis unit (Hoefer, Holliston, MA) using the follow- reprogrammed to 39/25 °C (16/8 h day/night) and the other ing protocol: 20 °C at 20 mA for 30 min and then 50 mA for 12 h incubator for control was kept at 25 °C. Cool-white fluorescent until the bromophenol blue front dye reached the bottom of tubes provided a photosynthesis photon flux intensity of 500 the gel. mmolÁm–2Ás–1. Five plants from each cultivar were placed on Gels were scanned on the Typhoon 9300 Variable Mode each shelf, and three layers of shelves (removing the top and the Imager (GE Healthcare) at 100 dpi according to the manufac- third shelves) were used in an incubator. The upper three fully turer’s specifications for Cy Dyes (GE Healthcare) and Colloi- expanded leaves were collected at 1200 HR, 4 h after the light dal Coomassie Blue (Invitrogen, Carlsbad, CA) -stained gels cycle was initiated. Leaf tissues collected from the five plants were visualized with the 632.8 nm helium–neon laser with no on each shelf were pooled as one