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Mapping the leaf proteome of Miscanthus sinensis and its application to the identification of heat-responsive proteins

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Abstract

Miscanthus sinensis is a promising bioenergy crop; however, its genome is poorly represented in sequence databases. As an initial step in the comprehensive analysis of the M. sinensis proteome, we report a reference 2-DE protein map of the leaf. A total of 316 protein spots were excised from the gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) or MALDI-TOF/TOF MS. Two hundred and thirty-two protein spots were identified, which are involved in a variety of cellular functions through distinct metabolic pathways. Functional annotation of the proteins revealed a nearly complete C3 and C4 cycle, starch and sugar synthesis pathway, glycolysis pathway, a significant portion of the pentose phosphate pathway, and many enzymes involved in secondary metabolism such as flavonoid/isoflavonoid, kaurene, chalcone, sesquiterpene and lignin biosynthesis. Other proteins belong to primary metabolism, transcription, protein synthesis, protein destination/storage, disease/defense, cell growth/division, transportation and signal transduction. To test the applicability of the constructed map, we studied the effect of heat stress on M. sinensis leaf proteome. Twenty-five protein spots were upregulated, five were newly induced and twenty-five spots were downregulated by heat treatment. The differentially accumulated proteins were involved in photosynthesis, energy metabolism, gene transcription, protein kinases and phosphatases, signal transduction, protein synthesis and heat shock responses. C4-specific pyruvate orthophosphate dikinase, Rubisco large subunit, Rubisco activase and some associated proteins were upregulated during heat stress and tend to restore upon recovery. Identification of these proteins provides some important clues regarding the way M. sinensis copes with hot climate. This work represents the first extensive proteomic description of M. sinensis and provides a reference map and heat-responsive candidates for future molecular and physiological studies of this bioenergy crop.

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Abbreviations

2-D PAGE:

Two dimensional polyacrylamide gel electrophoresis

DTT:

Dithiothreitol

HSP:

Heat-shock protein

IPG:

Immobilized pH gradient

MALDI-TOF MS:

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry

NGS:

Next-generation sequencing

PCD:

Programmed cell death

PMF:

Peptide mass fingerprinting

PPDK:

Pyruvate phosphate dikinase

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Acknowledgments

We thank anonymous reviewers for their constructive suggestions. This work was supported by the National Research Foundation of Korea (NRF) Grant (NRF-2011-616-F00013). We thank Dr. Mitsue Miyao (National Institute of Agrobiological Sciences, Tsukuba, Japan) for providing PPDK antibody. S.A. Sharmin, K.H. Kim and Y.G. Kim are supported by scholarship, and I. Alam is supported by a postdoctoral grant from BK21 Program at the Gyeongsang National University, Republic of Korea.

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  1. Division of Applied Life Sciences (BK21 program), IALS, PMBBRC, Gyeongsang National University, Jinju, 660-701, Korea

    Shamima Akhtar Sharmin, Iftekhar Alam, Md. Atikur Rahman, Kyung-Hee Kim, Yong-Goo Kim & Byung-Hyun Lee

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  1. Shamima Akhtar Sharmin
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425_2013_1900_MOESM1_ESM.tif

Suppl. Fig. S1 Separation of M. sinensis leaf proteins by 2-DE showing that most proteins are located in a pI range of 4–7 (TIFF 2484 kb)

425_2013_1900_MOESM2_ESM.tif

Suppl. Fig. S2 2-DE images of the leaf proteome obtained using silver nitrate staining (a) and Coomassie Brilliant Blue G-250 staining (b). One hundred (silver) or 500 μg (CBB) proteins were separated by 2D PAGE as described in Materials and methods. Both staining methods result in very similar spot number and distribution, which facilitates the matching of spots between images (TIFF 1718 kb)

425_2013_1900_MOESM3_ESM.tif

Suppl. Fig. S3 The relative expression levels of the heat-induced proteins. Bars indicates the relative expression level of control (C) 24 h of heat treatment (H1), 48 h of heat treatment (H2) and a 48 h of recovery followed by a 48 h of heat treatment (R) of individual spots mentioned. Spot intensities were measured using a densitometer and compared to those of the controls. The average values of the relative increase levels of three replicate samples are shown in the histograms. The data represent the mean values and SE of three independent experiments. Different letters above the bars indicate statistically significant differences (P < 0.05) (TIFF 2705 kb)

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Sharmin, S.A., Alam, I., Rahman, M.A. et al. Mapping the leaf proteome of Miscanthus sinensis and its application to the identification of heat-responsive proteins. Planta 238, 459–474 (2013). https://doi.org/10.1007/s00425-013-1900-6

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