title: Analysis of weighted co-regulatory networks in maize provides insights into new genes and regulatory mechanisms related to inositol phosphate metabolism.
abstract: BACKGROUND
  D-myo-inositol phosphates (IPs) are a series of phosphate esters. Myo-inositol hexakisphosphate (phytic acid, IP6) is the most abundant IP and has negative effects on animal and human nutrition. IPs play important roles in plant development, stress responses, and signal transduction. However, the metabolic pathways and possible regulatory mechanisms of IPs in maize are unclear. In this study, the B73 (high in phytic acid) and Qi319 (low in phytic acid) lines were selected for RNA-Seq analysis from 427 inbred lines based on a screening of IP levels. By integrating the metabolite data with the RNA-Seq data at three different kernel developmental stages (12, 21 and 30 days after pollination), co-regulatory networks were constructed to explore IP metabolism and its interactions with other pathways.
  
  
  RESULTS
  Differentially expressed gene analyses showed that the expression of MIPS and ITPK was related to differences in IP metabolism in Qi319 and B73. Moreover, WRKY and ethylene-responsive transcription factors (TFs) were common among the differentially expressed TFs, and are likely to be involved in the regulation of IP metabolism. Six co-regulatory networks were constructed, and three were chosen for further analysis. Based on network analyses, we proposed that the GA pathway interacts with the IP pathway through the ubiquitination pathway, and that Ca(2+) signaling functions as a bridge between IPs and other pathways. IP pools were found to be transported by specific ATP-binding cassette (ABC) transporters. Finally, three candidate genes (Mf3, DH2 and CB5) were identified and validated using Arabidopsis lines with mutations in orthologous genes or RNA interference (RNAi)-transgenic maize lines. Some mutant or RNAi lines exhibited seeds with a low-phytic-acid phenotype, indicating perturbation of IP metabolism. Mf3 likely encodes an enzyme involved in IP synthesis, DH2 encodes a transporter responsible for IP transport across organs and CB5 encodes a transporter involved in IP co-transport into vesicles.
  
  
  CONCLUSIONS
  This study provides new insights into IP metabolism and regulation, and facilitates our development of a better understanding of the functions of IPs and how they interact with other pathways involved in plant development and stress responses. Three new genes were discovered and preliminarily validated, thereby increasing our knowledge of IP metabolism.
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. connshaojun.zhang@hotmail.com.|National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), 100081, Beijing, China. connshaojun.zhang@hotmail.com.
authors.forename: Shaojun
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. yangwenzhu@caas.cn.|National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), 100081, Beijing, China. yangwenzhu@caas.cn.
authors.forename: Wenzhu
authors.initials: W
authors.lastname: Yang
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. zhaoqianqian@yeah.net.|National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), 100081, Beijing, China. zhaoqianqian@yeah.net.
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. zhouxiaojin@caas.cn.|National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), 100081, Beijing, China. zhouxiaojin@caas.cn.
authors.forename: Xiaojin
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. jiangling@caas.cn.|National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), 100081, Beijing, China. jiangling@caas.cn.
authors.forename: Ling
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. mscclg@163.com.
authors.forename: Shuai
authors.initials: S
authors.lastname: Ma
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. qing0225@126.com.|National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), 100081, Beijing, China. qing0225@126.com.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Liu
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. liye20086@163.com.|National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), 100081, Beijing, China. liye20086@163.com.
authors.forename: Ye
authors.initials: Y
authors.lastname: Li
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. zhangchunyi@caas.cn.|National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), 100081, Beijing, China. zhangchunyi@caas.cn.
authors.forename: Chunyi
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. fanyunliu@caas.cn.|National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), 100081, Beijing, China. fanyunliu@caas.cn.
authors.forename: Yunliu
authors.initials: Y
authors.lastname: Fan
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. chenrumei@caas.cn.|National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), 100081, Beijing, China. chenrumei@caas.cn.
authors.forename: Rumei
authors.initials: R
authors.lastname: Chen
mesh_terms: D017398:Alternative Splicing; D017360:Arabidopsis / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D053263:Gene Regulatory Networks; D017343:Genes, Plant; D007295:Inositol Phosphates / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics; D010641:Phenotype; D010833:Phytic Acid / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D012639:Seeds / Q000737:chemistry / Q000235:genetics; D017423:Sequence Analysis, RNA; D014157:Transcription Factors / Q000235:genetics; D059467:Transcriptome; D003313:Zea mays / Q000235:genetics*
chemical_list: D007295:Inositol Phosphates; D035683:MicroRNAs; D018749:RNA, Plant; D014157:Transcription Factors; D010833:Phytic Acid