Loose Panicle1 encoding a novel WRKY transcription factor, regulates panicle development, stem elongation, and seed size in foxtail millet [Setaria italica (L.) P. Beauv.]

RESEARCH ARTICLE Loose Panicle1 encoding a novel WRKY transcription factor, regulates panicle development, stem elongation, and seed size in foxtail millet [Setaria italica (L.) P. Beauv.] Jishan Xiang1,2,3☯, Sha Tang2☯, Hui Zhi2, Guanqing Jia2, Huajun Wang1*, Xianmin Diao2* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Gansu Provincial Key Lab of Aridland Crop Science/Gansu Key Lab of Crop Improvement & Germplasm Enhancement/College of Agronomy, Gansu Agricultural University, Lanzhou, People’s Republic of China, 2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, People’s Republic of China, 3 Chifeng University, Chifeng, People’s Republic of China ☯ These authors contributed equally to this work. * (HJW); (XMD) Abstract OPEN ACCESS Citation: Xiang J, Tang S, Zhi H, Jia G, Wang H, Diao X (2017) Loose Panicle1 encoding a novel WRKY transcription factor, regulates panicle development, stem elongation, and seed size in foxtail millet [Setaria italica (L.) P. Beauv.]. PLoS ONE 12(6): e0178730. https://doi.org/10.1371/ journal.pone.0178730 Editor: Hector Candela, Universidad Miguel Hernández de Elche, SPAIN Received: December 31, 2016 Accepted: May 19, 2017 Published: June 1, 2017 Copyright: © 2017 Xiang 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 National High Technology Research and Development Program of China (863 Program) (2013AA102603), the National Natural Science Foundation of China (31660423 and 31501324), China Agricultural Research System (CARS0713.5-A02), and the Agricultural Science and Panicle development is an important agronomic trait that aids in determining crop productivity. Foxtail millet and its wild ancestor green foxtail have recently been used as model systems to dissect gene functions. Here, we characterized a recessive mutant of foxtail millet, loose-panicle 1 (lp1), which showed pleiotropic phenotypes, such as a lax primary branching pattern, aberrant branch morphology, semi-dwarfism, and enlarged seed size. The loose panicle phenotype was attributed to increased panicle lengths and decreased primary branch numbers. Map-based cloning, combined with high-throughput sequencing, revealed that LP1, which encodes a novel WRKY transcription factor, is responsible for the mutant phenotype. A phylogenetic analysis revealed that LP1 belongs to the Group I WRKY subfamily, which possesses two WRKY domains (WRKY I and II). A single G-to-A transition in the fifth intron of LP1 resulted in three disorganized splicing events in mutant plants. For each of these aberrant splice variants, the normal C2H2 motif in the WRKY II domain was completely disrupted, resulting in a loss-of-function mutation. LP1 mRNA was expressed in all of the tissues examined, with higher expression levels observed in inflorescences, roots, and seeds at the grain-filling stage. A subcellular localization analysis showed that LP1 predominantly accumulated in the nucleus, which confirmed its role as a transcriptional regulator. This study provides novel insights into the roles of WRKY proteins in regulating reproductive organ development in plants and may help to develop molecular markers associated with crop yields. Introduction Foxtail millet [Setaria italica (L.) P. Beauv.] and green foxtail (S. viridis; the wild ancestor of foxtail millet), owing to their unique growing characteristics and small genomes, have emerged PLOS ONE | https://doi.org/10.1371/journal.pone.0178730 June 1, 2017 1 / 16 Loose panicle1 regulates panicle development in Setaria Technology Innovation Program of the Chinese Academy of Agricultural Sciences. Competing interests: The authors have declared that no competing interests exist. as model systems for studying genomics and genetics, C4 photosynthesis and stress biology [1– 4]. In recent years, our group has used S. italica as a model for gene mapping and functional genomic studies [5–7]. We used an S. italica variety with an available genome sequence, ‘Yugu1’, as the material for a large-scale ethyl methylsulfone (EMS)-induced mutant library. We identified an improved variety, ‘SSR41’, which has a similar flowering time and a high level of genetic polymorphism with ‘Yugu1’ [5]. ‘SSR41’ and a mutant originating from ‘Yugu1’ were used as pollen parents to construct mapping populations. Several functional genes, including SiYGL1 [5], SiDWARF2 [6], and SiAGO1b [7], were discovered recently through map-based cloning. These studies confirmed the potential of Setaria spp. to serve as promising models for gene discovery and pathway engineering. Panicle development is a major component that helps determine crop yield [8]. The molecular mechanisms related to panicle development have aroused wide attention [9]. Approximately 46 genes associated with inflorescence morphogenesis have been cloned and functionally characterized in Oryza sativa (http://www.ricedata.cn/). These genes act in various genetic pathways and are mainly involved in regulating the following biological processes: transcriptional regulation (e.g., LAX1/LAX2 [10, 11], FZP [12], and OsMADS15/34/50 [13]), photoperiods and flowering regulation (e.g., Ehd1 [14] and DTH8 [15]), heterotrimeric G proteins (e.g., DEP1 [16]), and plant hormone regulation (e.g., GNP1 [17] and TOB1 [18]). Of these genes, LAX1, LAX2, MOC1 [19], and FZP, which encode transcription factors and affect patterns of panicle branching, are similar to those investigated in our present study. Most of these transcriptional regulators are highly expressed in axillary meristems and directly regulate their formation, suggesting that transcription factors have extensive and conserved functions in regulating panicle development [11]. The WRKY transcription factor gene family is one of the largest families of transcriptional regulators in plants [20]. The name is derived from its most prominent functional domain, which contains a highly conserved amino acid signature ‘WRKYGQK’ (some specific members have ‘WKKYGNK’ instead). Approximately 74 and 102 WRKY family members have been identified in the model plants Arabidopsis thaliana and O. sativa [20]. Most of the reported studies on WRKY proteins address their involvement in biotic/abiotic stress responses [20, 21], and few characterize their roles in plant growth and seed development. TTG2 was the first WRKY transcription factor identified as controlling organ development in plant. It is strongly expressed in young leaves, trichomes, and seed coats, and it, together with TGG1 and GLABRA2, controls seed coat morphogenesis and trichome outgrowth [22]. OsWRKY78 is another example of a WRKY that functions in r (...truncated)