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Dynamic changes in the leaf proteome of a C3 xerophyte, Citrullus lanatus (wild watermelon), in response to water deficit

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Abstract

Wild watermelon (Citrullus lanatus) is a xerophyte native to the Kalahari Desert, Africa. To better understand the molecular mechanisms of drought resistance in this plant, we examined changes in the proteome in response to water deficit. Wild watermelon leaves showed decreased transpiration and a concomitant increase in leaf temperature under water deficit conditions. Comparison of the proteome of stressed plants with that of unstressed plants by two-dimensional gel electrophoresis revealed that the intensity of 40 spots increased in response to the stress, and the intensity of 11 spots decreased. We positively identified 23 stress-induced and 6 stress-repressed proteins by mass spectrometry and database analyses. Interestingly, 15 out of the 23 up-regulated proteins (65% of annotated up-regulated proteins) were heat shock proteins (HSPs). Especially, 10 out of the 15 up-regulated HSPs belonged to the small heat shock protein (sHSP) family. Other stress-induced proteins included those related to antioxidative defense and carbohydrate metabolism. Fifteen distinct cDNA sequences encoding the sHSP were characterized from wild watermelon. Quantitative real-time PCR analysis of the representative sHSP genes revealed strong transcriptional up-regulation in the leaves under water deficit. Moreover, immunoblot analysis confirmed that protein abundance of sHSPs was massively increased under water deficit. Overall, these observations suggest that the defense response of wild watermelon may involve orchestrated regulation of a diverse array of functional proteins related to cellular defense and metabolism, of which HSPs may play a pivotal role on the protection of the plant under water deficit in the presence of strong light.

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Abbreviations

2DE:

Two-dimensional gel electrophoresis

CLsHSP:

Citrullus lanatus small heat shock protein

HSP:

Heat shock protein

LC/MS/MS:

Liquid chromatography–tandem mass spectrometry

MG:

Methylglyoxal

MS:

Mass spectrometry

PGM:

Phosphoglucomutase

PHGPX:

Phospholipid hydroperoxide glutathione peroxidase

RFO:

Raffinose family oligosaccharide

sHSP:

Small heat shock protein

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Acknowledgments

We thank Drs Taise Shimaoka and Kaori Kohzuma for helpful technical assistance on the proteome analysis. This research was supported partly by a grant from the Japan Society for the Promotion of Science, and by funds from the Nara Institute of Science and Technology (NAIST), both awarded to K. Akashi. This research was also supported partly by the Asia-Africa Science and Technology Strategic Cooperation Promotion Program from the Japan Science and Technology Agency, the NAIST Global COE program, and the Nissan Science Foundation.

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Author notes

  1. Kazuo Yoshida

    Present address: Central Research Laboratory, Daiwahouse Industry Co. Ltd., 6-6-2 Sakyo, Nara, 631-0801, Japan

  2. Masayoshi Kuwano

    Present address: Joint Research Division, Institute of Low Temperature Science, Hokkaido University, N19, W8, Kita-ku, Sapporo, 060-0819, Japan

  3. Kazuya Yoshimura

    Present address: Department of Food and Nutritional Science, College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi, 487-8501, Japan

Authors and Affiliations

  1. Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Ikoma, Nara, 630-0192, Japan

    Kinya Akashi, Kazuo Yoshida, Masayoshi Kuwano, Masataka Kajikawa, Kazuya Yoshimura, Saki Hoshiyasu, Naoyuki Inagaki & Akiho Yokota

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  1. Kinya Akashi
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Correspondence to Kinya Akashi.

Electronic supplementary material

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Supplementary Fig. 1 Three replicates of 2DE images of proteome in the leaf soluble fraction of wild watermelon. Leaf soluble proteins from three unstressed plants (a-c), and those from three plants under water deficit for three days (d-f), were extracted individually, and separated by 2DE with the isoelectric focusing in the first horizontal dimension, and SDS/PAGE in the second vertical dimension. These three sets of 2DE images, together with the data presented in Fig. 2, were used for the quantitative analysis of the spot intensity, and for the LC/MS/MS analyses for protein identification.

Supplementary Fig. 2 Amino acid alignment of CLsHSPs. Alignment was generated by ClustalW program, and visually inspected and edited. In each block of the alignments, amino acid positions counted from their N-terminus were presented at the rightmost columns

425_2010_1341_MOESM1_ESM.pdf

Supplementary Fig. 1 Three replicates of 2DE images of proteome in the leaf soluble fraction of wild watermelon. Leaf soluble proteins from three unstressed plants (a–c), and those from three plants under water deficit for three days (d–f), were extracted individually, and separated by 2DE with the isoelectric focusing in the first horizontal dimension, and SDS/PAGE in the second vertical dimension. These three sets of 2DE images, together with the data presented in Fig. 2, were used for the quantitative analysis of the spot intensity, and for the LC/MS/MS analyses for protein identification (PDF 682 kb)

425_2010_1341_MOESM2_ESM.pdf

Supplementary Fig. 2 Amino acid alignment of CLsHSPs. Alignment was generated by ClustalW program, and visually inspected and edited. In each block of the alignments, amino acid positions counted from their N-terminus were presented at the rightmost columns. (PDF 191 kb)

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Akashi, K., Yoshida, K., Kuwano, M. et al. Dynamic changes in the leaf proteome of a C3 xerophyte, Citrullus lanatus (wild watermelon), in response to water deficit. Planta 233, 947–960 (2011). https://doi.org/10.1007/s00425-010-1341-4

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