iTRAQ Proteomic Analysis of Wheat (Triticum aestivum L.) Genotypes Differing in Waterlogging Tolerance

Abstract

Transient and chronic waterlogging constrains crop production in many regions of the world. Here, we invoke a novel iTRAQ-based proteomic strategy to elicit protein synthesis and regulation responses to waterlogging in tolerant (XM 55) and sensitive genotypes (YM 158). Of the 7,710 proteins identified, 16 were distinct between the two genotypes under waterlogging, partially defining a proteomic basis for waterlogging tolerance (and sensitivity). We found that 11 proteins were up-regulated and 5 proteins were down-regulated; the former included an Fe-S cluster assembly factor, heat shock cognate 70, GTP-binding protein SAR1A-like and CBS domain-containing protein. Down-regulated proteins contained photosystem II reaction center protein H, carotenoid 9, 10 (9', 10')-cleavage dioxygenase-like, psbP-like protein 1 and mitochondrial ATPase inhibitor. We showed that nine proteins responded to waterlogging with non-cultivar specificity: these included 3-isopropylmalate dehydratase large subunit, solanesyl-diphosphate synthase 2, DEAD-box ATP-dependent RNA helicase 3, and 3 predicted or uncharacterized proteins. Sixteen of the 28 selected proteins showed consistent expression patterns between mRNA and protein levels. We conclude that waterlogging stress may redirect protein synthesis, reduce chlorophyll synthesis and enzyme abundance involved in photorespiration, thus influencing synthesis of other metabolic enzymes. Collectively, these factors accelerate the accumulation of harmful metabolites in leaves in waterlogging-susceptible genotypes. The differentially expressed proteins enumerated here could be used as biological markers for enhancing waterlogging tolerance as part of future crop breeding programs.

Keywords: abiotic stress; anthesis; crop adaptation; iTRAQ; proteomics; waterlogging; wheat.

FIGURE 1

Phenotypes of XM 55 and YM 158 under waterlogging and control conditions. (A) Dynamic changes of chlorophyll concentration (SPAD unit) of the last expanded leaf after 7 days waterlogging at anthesis. (B) Dynamic changes of water content of leaf after waterlogging at anthesis between different varieties. (C) Dynamic changes of ear water content after waterlogging at anthesis between different varieties. (D) Dynamic changes of water content of stem and sheath after waterlogging at anthesis between different varieties. Vertical bars indicate standard error of the mean (n = 3).

FIGURE 2

Mass spectrometry analysis and protein identification. (A) The number of spectra and proteins. (B) The number of proteins with different unique peptides.

FIGURE 3

Quantitative and Venn analysis of the proteome of two wheat cultivars under different treatments. (A) Quantitative analysis of the proteome between the waterlogging treated and control samples. (B) Venn analysis of two wheat cultivars under different treatments. (C) Venn analysis of different treatments in different wheat cultivars; XM 55 and YM 158 are the two cultivars; CK, control; WL, waterlogging.

FIGURE 4

GO annotation of differentially expressed proteins between XM 55 and YM 158 under WL.

FIGURE 5

Correlation of differentially expressed protein at transcript and translation level. Differences in protein expression and qRT-PCR between XM 55 and YM 158 under waterlogging stress. (A) Differences in protein expression measured by iTRAQ and quantitative real-time reverse transcription-PCR (qRT-PCR) in XM 55 (B) and YM 158 (C) under WL and CK. Log2-RT-PCR represents RNA expression level; Log2-FC represents the differences in protein expression level.