Proteomic analysis of heat stress resistance of cucumber leaves when grafted onto Momordica rootstock

Abstract

Various biotic and abiotic stresses threaten the cultivation of future agricultural crops. Among these stresses, heat stress is a major abiotic stress that substantially reduces agricultural productivity. Many strategies to enhance heat stress tolerance of crops have been developed, among which is grafting. Here, we show that Momordica-grafted cucumber scions have intrinsically enhanced chlorophyll content, leaf area, and net photosynthetic rate under heat stress compared to plants grafted onto cucumber rootstock. To investigate the mechanisms by which Momordica rootstock enhanced cucumber scions heat stress tolerance, comparative proteomic analysis of cucumber leaves in response to rootstock-grafting and/or heat stress was conducted. Seventy-seven differentially accumulated proteins involved in diverse biological processes were identified by two-dimensional electrophoresis (2-DE) in conjunction with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). The following four main categories of proteins were involved: photosynthesis (42.8%), energy and metabolism (18.2%), defense response (14.3%), and protein and nucleic acid biosynthesis (11.7%). Proteomic analysis revealed that scions grafted onto Momordica rootstocks upregulated more proteins involved in photosynthesis compared to scions grafted onto cucumber rootstocks under heat stress and indicated enhanced photosynthetic capacity when seedlings were exposed to heat stress. Furthermore, the expression of photosynthesis-related genes in plants grafted onto Momordica rootstocks significantly increased in response to heat stress. In addition, increased high-temperature tolerance of plants grafted onto Momordica rootstock was associated with the accumulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and oxygen-evolving enhancer protein 1 (OEE1). Taken together, the data indicated that Momordica rootstock might alleviate growth inhibition caused by heat stress by improving photosynthesis, providing valuable insight into enhancing heat stress tolerance in the global warming epoch.

References

[1] Kotak, S. et al. Complexity of the heat stress response in plants. Curr. Opin. Plant. Biol. 10, 310 (2007).

[2] Allakhverdiev, S. I. et al. Heat stress: an overview of molecular responses in photosynthesis. Photosynth. Res. 98, 541 (2008).

[3] Qaderi, M. M., Kurepin, L. V. & Reid, D. M. Effects of temperature and watering regime on growth, gas exchange and abscisic acid content of canola (Brassica napus) seedlings. Environ. Exp. Bot. 75, 107-113 (2012).

[4] Serbin, S. P., Dillaway, D. N., Kruger, E. L. & Townsend, P. A. Leaf optical properties reflect variation in photosynthetic metabolism and its sensitivity to temperature. J. Exp. Bot. 63, 489 (2012).

[5] Mathur, S., Agrawal, D. & Jajoo, A. Photosynthesis: response to high temperature stress. J. Photochem. Photobiol. B Biol. 137, 116-126 (2014).

[6] Mishra, D., Shekhar, S., Agrawal, L., Chakraborty, S. & Chakraborty, N. Cultivar-specific high temperature stress responses in bread wheat (Triticum aestivum L.) associated with physicochemical traits and defense pathways. Food Chem. 221, 1077 (2017).

[7] Killi, D., Bussotti, F., Raschi, A. & Haworth, M . Adaptation to high temperature mitigates the impact of water deficit during combined heat and drought stress in C3 sunflower and C4 maize varieties with contrasting drought tolerance. Physiol. Plant. 159, 130 (2017).

[8] Shi, J., Yan, B., Lou, X., Ma, H. & Ruan, S. Comparative transcriptome analysis reveals the transcriptional alterations in heat-resistant and heat-sensitive sweet maize (Zea mays L.) varieties under heat stress. BMC Plant Biol. 17, 26 (2017).

[9] Lee, J. M. et al. Current status of vegetable grafting: diffusion, grafting techniques, automation. Sci. Hortic. 127, 93-105 (2010).

[10] Martínez-Ballesta, M. C., Alcaraz-López, C., Muries, B., Mota-Cadenas, C. & Carvajal, M. Physiological aspects of rootstock-scion interactions. Sci. Hortic. 127, 112-118 (2010).

[11] Colla, G., Rouphael, Y., Leonardi, C. & Bie, Z. Role of grafting in vegetable crops grown under saline conditions. Sci. Hortic. 127, 147-155 (2010).

[12] Schwarz, D., Rouphael, Y., Colla, G. & Venema, J. H. Grafting as a tool to improve tolerance of vegetables to abiotic stresses: thermal stress, water stress and organic pollutants. Sci. Hortic. 127, 162-171 (2015).

[13] Li, H. et al. Microarray and genetic analysis reveals that csa-miR159b plays a critical role in abscisic acid-mediated heat tolerance in grafted cucumber plants. Plant Cell Environ. 39, 1790 (2016).

[14] Li, H. et al. The sub/supra-optimal temperature-induced inhibition of photosynthesis and oxidative damage in cucumber leaves are alleviated by grafting onto figleaf gourd/luffa rootstocks. Physiol. Plant. 152, 571-584 (2014).

[15] Wang, Z. Construction of genetic map and QTL analysis of important agronomic characters in Momordica charantia., 8-9 (2012).

[16] Arnon, D. I. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in beta vulgaris. Plant Physiol. 24, 1-15 (1949).

[17] Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408 (2001).

[18] Hurkman, W. J. & Tanaka, C. K. Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 81, 802 (1986).

[19] Yamori, W. et al. Enhanced leaf photosynthesis as a target to increase grain yield: insights from transgenic rice lines with variable Rieske FeS protein content in the cytochrome b6/f complex. Plant Cell Environ. 39, 80-87 (2016).

[20] Weis, E . Reversible heat-inactivation of the calvin cycle: a possible mechanism of the temperature regulation of photosynthesis. Planta 151, 33-39 (1981).

[21] Yang, B. et al. Genetic engineering of the Calvin cycle toward enhanced photosynthetic CO2 fixation in microalgae. Biotechnol. Biofuels. 10, 229 (2017).

[22] Craftsbrandner, S. J. & Salvucci, M. E. Rubisco activase constrains the photosynthetic potential of leaves at high temperature and CO2. Proc. Natl Acad. Sci. USA 97, 13430-13435 (2000).

[23] Deridder, B. P. & Salvucci, M. E. Modulation of Rubisco activase gene expression during heat stress in cotton (Gossypium hirsutum L.) involves post-transcriptional mechanisms. Plant Sci. 172, 246-254 (2007).

[24] Harrison, E. P., Willingham, N. M., Lloyd, J. C. & Raines, C. A. Reduced sedoheptulose-1,7-bisphosphatase levels in transgenic tobacco lead to decreased photosynthetic capacity and altered carbohydrate accumulation. Planta 204, 27-36 (1997).

[25] Olçer, H., Lloyd, J. C. & Raines, C. A. Photosynthetic capacity is differentially affected by reductions in sedoheptulose-1,7-bisphosphatase activity during leaf development in transgenic tobacco plants. Plant Physiol. 125, 982-989 (2001).

[26] Laisk, A. et al. A computer-operated routine of gas exchange and optical measurements to diagnose photosynthetic apparatus in leaves. Plant Cell Environ. 25, 923-943 (2002).

[27] Heide, H. & Kalisz, H. H. The oxygen evolving enhancer protein 1 (OEE) of photosystem II in green algae exhibits thioredoxin activity. J. Plant. Physiol. 161, 139-149 (2004).

[28] Mayfield, S. P., Bennoun, P. & Rochaix, J. D. Expression of the nuclear encoded OEE1 protein is required for oxygen evolution and stability of photosystem II particles in Chlamydomonas reinhardtii. EMBO J. 6, 313-318 (1987).

[29] Sang, Q. et al. Proteomic analysis reveals the positive effect of exogenous spermidine in tomato seedlings’ response to high-temperature stress. Front. Plant Sci. 8, 120 (2017).

[30] Albery, W. J. & Knowles, J. R. Free-energy profile of the reaction catalyzed by triosephosphate isomerase. Biochemistry 15, 5627-5631 (1976).

[31] Ma, W., Wei, L., Wang, Q., Shi, D. & Chen, H. Increased activity of the non-regulated enzymes fructose-1,6-bisphosphate aldolase and triosephosphate isomerase in Anabaena sp. strain PCC 7120 increases photosynthetic yield. J. Appl. Phycol. 19, 207-213 (2007).

[32] Hu, W. J. et al. Proteome and calcium-related gene expression in Pinus massoniana needles in response to acid rain under different calcium levels. Plant Soil 380, 285-303 (2014).

[33] Das, S. et al. Proteomic changes in rice leaves grown under open field high temperature stress conditions. Mol. Biol. Rep. 42, 1545-1558 (2015).

[34] Takabayashi, A., Niwata, A. & Tanaka, A. Direct interaction with ACR11 is necessary for post-transcriptional control of GLU1-encoded ferredoxin-dependent glutamate synthase in leaves. Sci. Rep. 6, 29668 (2016).

[35] Rolletschek, H. et al. Combined noninvasive imaging and modeling approaches reveal metabolic compartmentation in the barley endosperm. Plant Cell 23, 3041-3054 (2011).

[36] Deitzer, G. F., Kempf, O., Fischer, S. & Wagner, E. Endogenous rhythmicity and energy transduction: IV. Rhythmic control of enzymes involved in the tricarboxylic-acid cycle and the oxidative pentose-phosphate pathway in Chenopodium rubrum L.. Planta 117, 29-41 (1974).

[37] Jung, Y. M., Lee, J. N., Shin, H. D. & Lee, Y. H. Role of tktA gene in pentose phosphate pathway on odd-ball biosynthesis of poly-P-hydroxybutyrate in transformant Escherichia coli HarboringphbCAB Operon. J. Biosci. Bioeng. 98, 224-227 (2004).

[38] Du, N. et al. Proteomic analysis reveals the positive roles of the plant-growth-promoting rhizobacterium NSY50 in the response of cucumber roots to Fusariumoxysporumf. sp.cucumerinumInoculation. Front. Plant Sci. 7, 1859 (2016).

[39] An, Y. et al. Root proteomics reveals cucumber 24-epibrassinolide responses under Ca(NO3)2 stress. Plant Cell Rep. 35, 1081-1101 (2016).

[40] Das, K. & Roychoudhury, A. Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants. Front. Environ. Sci. 2, 53 (2014).

[41] Jiang, L., Zhang, L., Shi, Y., Lu, Z. & Yu, Z. Proteomic analysis of peach fruit during ripening upon post-harvest heat combined with 1-MCP treatment. J. Proteom. 98, 31-43 (2014).

[42] Wang, C. Y., Guo, S. R. & Liu, C. J. Effects of calcium on expression of defense enzymes isoenzymes in roots of cucumber seedlings under root-zone hypoxic stress. Acta Bot. Boreal. Occident. Sin. 29, 1874-1880 (2009).

[43] Song, X. S. et al. Response of ascorbate peroxidase isoenzymes and ascorbate regeneration system to abiotic stresses in Cucumis sativus L.. Plant Physiol. Biochem. 43, 1082-1088 (2005).

[44] Weissbach, H., Resnick, L. & Brot, N. Methionine sulfoxide reductases: history and cellular role in protecting against oxidative damage. Biochim. Biophys. Acta 1703, 203-212 (2005).

[45] Capriotti, A. L. et al. Proteomic study of a tolerant genotype of durum wheat under salt-stress conditions. Anal. Bioanal. Chem. 406, 1423 (2014).

[46] Yuan Y, Min Z, Sheng S, et al. Proteomic and Physiological Analyses Reveal Putrescine Responses in Roots of Cucumber Stressed by NaCl. Front Plant Sci. 7, 2016,.

[47] Percipalle, P. & Visa, N. Molecular functions of nuclear actin in transcription. J. Cell Biol. 172, 967-971 (2006).

[48] Ahsan, N. et al. Analysis of arsenic stress-induced differentially expressed proteins in rice leaves by two-dimensional gel electrophoresis coupled with mass spectrometry. Chemosphere 78, 224-231 (2010).

[49] Filipowicz, W. & Pogačić, V. Biogenesis of small nucleolar ribonucleoproteins. Curr. Opin. Cell Biol. 14, 319 (2002).

[50] Polge, C. et al. Evidence for the existence in Arabidopsis thaliana of the proteasome proteolytic pathway: ACTIVATION IN RESPONSE TO CADMIUM. J. Biol. Chem. 284, 35412 (2009).

[51] Martin, J. & Hartl, F. U. Molecular chaperones in cellular protein folding. Nature 381, 571-579 (1996).

[52] Priya, S., Sharma, S. K. & Goloubinoff, P. Molecular chaperones as enzymes that catalytically unfold misfolded polypeptides. FEBS Lett. 587, 1981-1987 (2013).

[53] Charng, Y., Liu, H., Liu, N., Hsu, F. & Ko, S. Arabidopsis Hsa32, a novel heat shock protein, is essential for acquired thermotolerance during long recovery after acclimation. Plant Physiol. 140, 1297-1305 (2006).

[54] Muthusamy, S. K., Dalal, M., Chinnusamy, V. & Bansal, K. C. Genome-wide identification and analysis of biotic and abiotic stress regulation of small heat shock protein (HSP20) family genes in bread wheat. J. Plant. Physiol. 211, 100 (2017).

[55] Nakamura, K. et al. Functional specialization of calreticulin domains. J. Cell Biol. 154, 961 (2001).

[56] Kyriacou, M. C., Rouphael, Y., Colla, G., Zrenner, R. & Schwarz, D. Vegetable grafting: the implications of a growing agronomic imperative for vegetable fruit quality and nutritive value. Front. Plant Sci. 8, 741 (2017).

[57] Haroldsen, V. M. et al. Mobility of transgenic nucleic acids and proteins within grafted rootstocks for agricultural improvement. Front. Plant Sci. 3, 39 (2012).

[58] Takahashi, S. & Murata, N. How do environmental stresses accelerate photoinhibition?. Trends Plant. Sci. 13, 178 (2008).

[59] Yang, Y. et al. Proteomic study participating the enhancement of growth and salt tolerance of bottle gourd rootstock-grafted watermelon seedlings. Plant Physiol. Biochem. 58, 54-65 (2012).

[60] Zhou, Y. et al. Grafting of Cucumis sativus onto Cucurbita ficifolia leads to improved plant growth, increased light utilization and reduced accumulation of reactive oxygen species in chilled plants. J. Plant. Res. 122, 529-540 (2009).

[61] Zhang, S., Gu, X., Wang, Y. & Zhang, S. Effect of low temperature stress on the physiological and biochemical indexes in cucumber seedling grafted on wild cucumber ({/sl Sicyos angulatus}). Acta Bot. Boreal. Occident. Sin. 771, 243-247 (2005).

[62] He, Y., Zhu, Z., Yang, J., Ni, X. & Zhu, B. Grafting increases the salt tolerance of tomato by improvement of photosynthesis and enhancement of antioxidant enzymes activity. Environ. Exp. Bot. 66, 270-278 (2009).

[63] Jensen, P. J. et al. Rootstock-regulated gene expression patterns in apple tree scions. Tree Genet. Genomes 6, 57-72 (2010).