Differential Proteomic Analysis Using iTRAQ Reveals Alterations in Hull Development in Rice (Oryza sativa L.)

RESEARCH ARTICLE Differential Proteomic Analysis Using iTRAQ Reveals Alterations in Hull Development in Rice (Oryza sativa L.) Shuzhen Wang1,2,3☯, Wenyue Chen1☯, Wenfei Xiao1, Changdeng Yang2, Ya Xin1, Jieren Qiu1, Weimin Hu3, Wu Ying1, Yaping Fu2, Jianxin Tong1, Guocheng Hu2, Zhongzhong Chen1, Xianping Fang1, Hong Yu1, Wenguo Lai1, Songlin Ruan1*, Huasheng Ma1* 1 Laboratory of Plant Molecular Biology & Proteomics, Institute of Biotechnology, Hangzhou Academy of Agricultural Sciences, Hangzhou 310024, China, 2 State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China, 3 Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310012, China ☯ These authors contributed equally to this work. * (SR); (HM) OPEN ACCESS Citation: Wang S, Chen W, Xiao W, Yang C, Xin Y, Qiu J, et al. (2015) Differential Proteomic Analysis Using iTRAQ Reveals Alterations in Hull Development in Rice (Oryza sativa L.). PLoS ONE 10(7): e0133696. doi:10.1371/journal.pone.0133696 Editor: Shantanu Sengupta, CSIR-Institute of Genomics and Integrative Biology, INDIA Received: September 6, 2014 Accepted: July 1, 2015 Published: July 31, 2015 Copyright: © 2015 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Great Project of Science and Technology of Hangzhou City (Research Grant #20131812A02 to SLR), Zhejiang Provincial Natural Science Foundation of China (Research Grant # LR12C13001 to SLR) and the Open Project of State Key Laboratory of Rice Biology (Research Grant #12020313 to SZW and SLR). Competing Interests: The authors have declared that no competing interests exist. Abstract Rice hull, the outer cover of the rice grain, determines grain shape and size. Changes in the rice hull proteome in different growth stages may reflect the underlying mechanisms involved in grain development. To better understand these changes, isobaric tags for relative and absolute quantitative (iTRAQ) MS/MS was used to detect statistically significant changes in the rice hull proteome in the booting, flowering, and milk-ripe growth stages. Differentially expressed proteins were analyzed to predict their potential functions during development. Gene ontology (GO) terms and pathways were used to evaluate the biological mechanisms involved in rice hull at the three growth stages. In total, 5,268 proteins were detected and characterized, of which 563 were differentially expressed across the development stages. The results showed that the flowering and milk-ripe stage proteomes were more similar to each other (r=0.61) than either was to the booting stage proteome. A GO enrichment analysis of the differentially expressed proteins was used to predict their roles during rice hull development. The potential functions of 25 significantly differentially expressed proteins were used to evaluate their possible roles at various growth stages. Among these proteins, an unannotated protein (Q7X8A1) was found to be overexpressed especially in the flowering stage, while a putative uncharacterized protein (B8BF94) and an aldehyde dehydrogenase (Q9FPK6) were overexpressed only in the milk-ripe stage. Pathways regulated by differentially expressed proteins were also analyzed. Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase (Q9SDJ2), and two magnesiumchelatase subunits, ChlD (Q6ATS0), and ChlI (Q53RM0), were associated with chlorophyll biosynthesis at different developmental stages. The expression of Q9SDJ2 in the flowering and milk-ripe stages was validated by qRT-PCR. The 25 candidate proteins may be pivotal markers for controlling rice hull development at various growth stages and chlorophyll biosynthesis pathway related proteins, especially magnesium-protoporphyrin IX monomethyl PLOS ONE | DOI:10.1371/journal.pone.0133696 July 31, 2015 1 / 16 Differential Proteomic Analysis of Hull Development in Rice ester [oxidative] cyclase (Q9SDJ2), may provide new insights into the molecular mechanisms of rice hull development and chlorophyll associated regulation. Introduction Rice (Oryza sativa L.) supports nearly half the world population and is one of the world’s most important grain crops [1–4]. The mature rice seed consists of caryopsis and hull. The rice caryopsis contains the caryopsis coat, aleurone, embryo and starchy endosperm. The aleurone layer is the outermost layer of the endosperm, followed by the inner starchy endosperm [5]. The embryo consists of the scutellum, embryonic axis and various sheathing structures. The starchy endosperm, accounting for over 80% of the caryopsis, contains parenchyma cells filled with reserves [6]. The rice hull (husk), the outer cover of the rice grain, is an important floral organ comprised of lemma and palea. The lemma covers two-thirds of the seed, while the palea is the lining that hugs the seed. The hull is indigestible because of the presence of opaline silica (a hydrated form of silica) and lignin, a complex component that helps to hydrate the rice grain. Rice hull not only acts as a mechanical barrier to prevent damage and maintain humidity for the developing seed, but is also a critical factor that affects yield and milling quality of the grain in terms of its volume, shape, and size [7–12]. Genetic studies and gene cloning have helped understand the functions of the large numbers of genes involved in rice hull development. For example, a rice mutant with twisted hull (twh) derived from a breeding population of rice was reported to exhibit reduced grain weight compared with the wild type parent [13]. Similarly, a rice floral organ mutant (bh1) has a beakshaped hull, which was found to have a negative effect on grain yield [14]. The stunted lemma palea 1 rice mutant (slp1) displays severely degenerated lemmas/paleae, and SLP1 was reported to be localized in a 46.4-kb genomic region that contained three putative genes, OsSPL16, OsMADS45, and OsMADS37 [15]. A palea formation controlling gene, depressed palea 1 (dp1), encodes a nuclear-localized AT-hook DNA-binding protein that causes a primary defect in the main structure of the palea, which is required for palea formation and floral organ number control [16]. The rice MADS-box factor (OsMADS1) is an early-acting regulator of inner floral organs that controls the differentiation of specific cell types in the lemma and palea [17]. Li and coworkers found that TH1 accumulated mainly in young inflorescence and was important in controlling lemma and palea development in rice [8]. Other genes that have been reported to be associated with rice hull development include frizzy panicle (FZP) [18], abnormal hull (ah) (...truncated)