Introgression of opaque2 into Waxy Maize Causes Extensive Biochemical and Proteomic Changes in Endosperm

RESEARCH ARTICLE Introgression of opaque2 into Waxy Maize Causes Extensive Biochemical and Proteomic Changes in Endosperm Zhiqiang Zhou1☯, Liya Song2☯, Xiaoxing Zhang1, Xinhai Li1, Na Yan1, Renpei Xia1, Hui Zhu1, Jianfeng Weng1, Zhuanfang Hao1, Degui Zhang1, Hongjun Yong1, Mingshun Li1*, Shihuang Zhang1* 1 Department of Crop Genetics and Breeding, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China, 2 Beijing Key Lab of Plant Resource Research and Development, Beijing Technology and Business University, Beijing, China a11111 ☯ These authors contributed equally to this work. * (ML); (SZ) Abstract OPEN ACCESS Citation: Zhou Z, Song L, Zhang X, Li X, Yan N, Xia R, et al. (2016) Introgression of opaque2 into Waxy Maize Causes Extensive Biochemical and Proteomic Changes in Endosperm. PLoS ONE 11(7): e0158971. doi:10.1371/journal.pone.0158971 Editor: Guangyuan He, Huazhong University of Science and Technology, CHINA Received: April 11, 2016 Accepted: June 26, 2016 Published: July 8, 2016 Copyright: © 2016 Zhou 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 China Agriculture Research System (CARS-02-01) and the funding was received by MSL. The Beijing Municipal Natural Science Foundation (31401390) and the funding was received by LYS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Waxy maize is prevalently grown in China and other countries due to the excellent characters and economic value. However, its low content of lysine can’t meet the nutritional requirements of humans and livestock. In the present study, we introgressed the opaque2 (o2) allele into waxy maize line Zhao OP-6/O2O2 by using marker-assisted selection (MAS) technique and successfully improved the lysine content and quality of waxy maize. Transcript abundance analysis indicated that the wx1 expression levels had no difference between Zhao OP-6/o2o2 and Zhao OP-6/O2O2. However, Zhao OP-6/o2o2 was characterized by a phenotype of hard and vitreous kernels and accumulation of protein bodies at smaller size (one third of that of parents) but in larger numbers. Biochemical analyses showed that Zhao OP-6/o2o2 had 16.7% less free amino acids than Zhao OP-6/O2O2, especially those derived from glycolytic intermediates, but its content of lysine was increased by 51.6% (0.47% vs. 0.31%). The content of amylopectin was 98.5% in Zhao OP6/o2o2, significantly higher than that in Zhao OP-6/O2O2 (97.7%). Proteomic analyses indicated that o2 introgression not only decreased the accumulation of various zein proteins except for 27-kDa γ-zein, but also affected other endosperm proteins related to amino acid biosynthesis, starch-protein balance, stress response and signal transduction. This study gives us an intriguing insight into the metabolism changes in endosperm of waxy maize introgressed with opaque2. Introduction Waxy maize (Zea mays L. sinensis Kulesh), also known as sticky maize, is one sub-type of maize that was first discovered in Southwestern China and then prevalently grown in other Asian countries [1–3]. The endosperm of waxy maize has a high content of amylopectin (nearly 100%), and is thus characterized by high viscosity, easy digestion, and good light PLOS ONE | DOI:10.1371/journal.pone.0158971 July 8, 2016 1 / 16 Waxy Maize Introgressed with o2 transmittance [4]. These excellent characters and fresh harvest make waxy maize widely used in frozen food processing, paper-making and livestock feeding industries. However, due to the limited levels and types of essential amino acids, especially lysine, the nutritional value of waxy maize is relatively low. Generally, the lysine content in maize grain should be more than 0.5% (>51 mg per gram of protein) to meet human and livestock requirements [5], but waxy maize has a lysine content of only 0.24–0.34%. By introgression of opaque2 (o2) and opaque2 modifier (o2m) alleles into elite maize inbred lines with marker-assisted selection (MAS) technique, the genetically modified opaque2 maize, also known as quality protein maize (QPM), shows an improved lysine content of approximately 0.4% [6–9]. Therefore, it is of importance to breed a novel waxy maize line with high lysine content by introgressing the o2 and o2m traits with MAS. Accumulation of starch and storage proteins occurs in the developing endosperm of maize, the quality of which is contributed to by the action of the Waxy1 (Wx1) and Opaque2 (O2) genes [10]. The single copy 3.8-kb Wx1 contains 14 exons and is mapped on the short arm of chromosome 9 [11,12]. Previous studies have shown that transposable elements Ac/Ds and En/Spm, deletion mutation and mutagenic ethylmethane sulfonate (EMS) mutagenesis account for the pre-mRNA splicing or translation errors and result in a low expression level of Wx1 [13]. As a result, the granule-bound starch synthase I (GBSS I) activity of the wx1 mutant has a decreased activity (5–95%) in amylose synthesis, leading to the low level of amylose but high level of amylopectin in maize endosperm and pollen [14,15]. O2 is a transcriptional factor that contains a basic leucine-zipper (bZIP) motif. It is specifically expressed in the developing endosperm and directly regulates the expression of 22-kDa α-zeins [16–18]. The substantial reduction of α-zeins is concomitant with increased accumulation of non-zeins, consequently accounting for the increased contents of lysine and tryptophan in maize mutants [19, 20]. In addition, a large number of studies have shown that O2 also has pleiotropic effects on the expression of non-storage proteins including ribosome-inactivating protein b-32 (RIP), cytosolic pyruvate phosphate dikinase 1 (cyPPDK1), lysine ketoglutarate reductase-saccaropine dehydrogenase (LKR-SDH), acetohydroxyacid synthase (AHAS) and Opaque2 heterodimerizing protein 1 (OHP1) [21–23]. Thus O2 as a regulator plays a crucial role in maize endosperm development by influencing the storage protein and nitrogen/carbon metabolism. Although the individual functions of Wx1 and O2 are well known, how these genes interact to maintain the starch-protein balance is yet unknown. It has been reported that o2 mutation can alter the transcriptional patterns of Wx1 in varying degrees [24–26], but no evidence revealed the regulatory effect of o2 on the expression of wx1. By backcrossing of o2 and o16 traits with MAS, the quality and lysine content of waxy maize have been successfully improved [27,28]. However, the molecular mechanism underlying the ameliorated amin (...truncated)