Expression of the Rice Arginase Gene OsARG in Cotton Influences the Morphology and Nitrogen Transition of Seedlings

RESEARCH ARTICLE Expression of the Rice Arginase Gene OsARG in Cotton Influences the Morphology and Nitrogen Transition of Seedlings Zhigang Meng1☯, Zhaohong Meng1☯, Rui Zhang1, Chengzhen Liang1, Jianmin Wan2, Yanling Wang1,3, Honghong Zhai1, Sandui Guo1* 1 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China, 2 Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China, 3 School of Life Science, Anhui Agricultural University, Anhui, China ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Meng Z, Meng Z, Zhang R, Liang C, Wan J, Wang Y, et al. (2015) Expression of the Rice Arginase Gene OsARG in Cotton Influences the Morphology and Nitrogen Transition of Seedlings. PLoS ONE 10 (11): e0141530. doi:10.1371/journal.pone.0141530 Editor: Xianlong Zhang, National Key Laboratory of Crop Genetic Improvement, CHINA Received: November 26, 2014 Accepted: October 9, 2015 Published: November 3, 2015 Copyright: © 2015 Meng 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. Arginase is the only enzyme capable of producing urea in plants. This enzyme also contributes to many important biological functions during plant growth and development, such as seed development, root development and plant nitrogen using. The unique rice arginase gene OsARG is known to affect nitrogen use efficiency and is also associated with higher yields in rice. In this study, we transformed OsARG into upland cotton R18 by Agrobacterium-mediated genetic transformation and analyzed the function of OsARG in transgenic cotton. Two independent OsARG expression transgenic cotton lines, ARG-26 and ARG-38, were obtained via transformation. Southern blot analysis indicated that two copies and one copy of the OsARG gene were integrated into the ARG-26 and ARG-38 genomes, respectively. Enzyme activity and RNA transcription analysis revealed that the OsARG gene is highly expressed in cotton. The nitric oxide content and the morphology of ARG-26 and ARG-38 seedlings were both affected by expression of the OsARG gene. Field experiments indicated that the polyamine and nitrogen content increased by more than two-fold in the T3 generation plants of the transgenic cotton lines ARG-26-2, ARG-26-7, ARG-38-8, and ARG-38-11, as compared with the control plants. After harvesting cotton fibers grown in field conditions, we analyzed the quality of fiber and found that the fiber length was increased in the transgenic lines. The average cotton fiber length for all of the transgenic cotton lines was two millimeters longer than the fibers of the control plants; the average cotton fiber lengths were 31.94 mm, 32.00 mm, 32.68 mm and 32.84 mm in the ARG-26ARG26-2, ARG-26-7, ARG-38-8 and ARG-38-11 lines, respectively, but the average fiber length of the control plants was 29.36mm. Our results indicate that the OsARG gene could potentially be used to improve cotton fiber length traits. Funding: This work was supported by the National Natural Science Foundation of China (grant no. 31301373). Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0141530 November 3, 2015 1 / 19 The Role of Rice Arginase in Cotton Introduction The application of nitrogen (N) is an important practice in cotton production. The quantity of nitrogen fertilizer applied and the nitrogen use efficiency of cotton plants both strongly influence cotton yield and fiber quality [1, 2], and these factors are also known to influence plant defense responses to biotic and abiotic stress [3]. Efforts to improve nitrogen utilization efficiency aimed at improving cotton yield and quality have been of particular significance for cotton production in nitrogen-limited conditions. Plants mainly acquire nitrogen via three steps: uptake, assimilation, and remobilization. Plant nitrogen uptake and assimilation determine external nitrogen use efficiency from soil, while nitrogen remobilization determines the in planta efficiency of nitrogen storage and reutilization. Nitrogen remobilization is an important factor influencing plant nitrogen use efficiency; high nitrogen remobilization efficiency can cause extra organic state nitrogen to be restored and reused in plant metabolism, a situation that prevents the conversion of this nitrogen into nitric oxide (NO) and other inorganic nitrogen forms that would be wasted. Examples of nitrogen remobilization include transamination metabolism processes that synthesize new amino acids and protein to achieve reuse of organic nitrogen and the synthesis of some transferable amino acids that are used in nitrogen translocation between old leaves and developing or photosynthetically-active leaves or seeds and other reproductive organs [4, 5]. In most plant species, nitrogen remobilization is mainly executed via arginine (Arg) metabolism. Arg metabolism has an important significance for processes including seed development, germination, and seedling growth and development. Arg is a primary storage form of nitrogen in seeds, often accounting for 50% of the content of nitrogen storage. After germination, Arg content can account for 90% of the soluble nitrogen in the tender tissues of seedlings [6, 7, 8]. Compared to amino acids generally, Arg, which contains a guanidine and an amino group, has a particularly high nitrogen content. Arg is also known to be an important nitrogen storage molecule and an important transport form for nitrogen translocation [9, 10]. In plant Arg metabolism, Arg is a precursor for the biosynthesis of some important functional molecules such as amino acids, polyamines, agmatine, and NO. Polyamines and agmatine are associated with plant resistance to environmental adversity. NO is involved in signal transduction in plants [11, 12]. Arginase can transform endogenous nitrogen into organic nitrogen to be reused in plant by hydrolyzing Arg to produce urea and ornithine (Orn). Urea can be utilized in the urea cycle to reuse nitrogen for the synthesis of amino acids and proteins; ornithine can be used as a precursor for the synthesis of both polyamines and proteins [13, 14, 15]. Arginase is involved in the production of many important molecules, and the biological functions of these molecules are receiving increasing amounts of research attention in a variety of plants [11, 16, 17, 18]. Ma et al. [16] found that, in the rice mutant nglf-1 for the arginase gene (OsARG), plant height, panicle number, seed size, and seed setting rate were all decreased compared to wild type plants, The seed setting rate phenotype was particularly pronounced in the mutant, with a more than 80% decrea (...truncated)