Identification and Functional Analysis of Light- Responsive Unique Genes and Gene Family Members in Rice Ki-Hong Jung1, Jinwon Lee2, Chris Dardick3, Young-Su Seo 1 , Peijian Cao 1 , Patrick Canlas1, Jirapa Phetsom1, Xia Xu1, Shu Ouyang 4 , Kyungsook An 2 , Yun-Ja Cho 2 , Geun-Cheol Lee5, Yoosook Lee6, Gynheung An 2 , Pamela C. Ronald1* 1 Department of Plant Pathology, University of California Davis, Davis, California, United States of America, 2 Functional Genomic Center, Pohang University of Science and Technology, Pohang, Republic of Korea, 3 The Appalachian Fruit Research Station, USDA-ARS, Kearneysville, West Virginia, United States of America, 4 J. Craig Venter Institute, Rockville, Maryland, United States of America, 5 College of Business Administration, Konkuk University, Gwangjin-gu, Seoul, Republic of Korea, 6 School of Veterinary Medicine, Department of Pathology, Immunology and Microbiology, University of California Davis, Davis, California, United States of America Abstract Functional redundancy limits detailed analysis of genes in many organisms. Here, we report a method to efficiently overcome this obstacle by combining gene expression data with analysis of gene-indexed mutants. Using a rice NSF45K oligo-microarray to compare 2-week-old light- and dark-grown rice leaf tissue, we identified 365 genes that showed significant 8-fold or greater induction in the light relative to dark conditions. We then screened collections of rice T-DNA insertional mutants to identify rice lines with mutations in the strongly light-induced genes. From this analysis, we identified 74 different lines comprising two independent mutant lines for each of 37 light-induced genes. This list was further refined by mining gene expression data to exclude genes that had potential functional redundancy due to co-expressed family members (12 genes) and genes that had inconsistent light responses across other publicly available microarray datasets (five genes). We next characterized the phenotypes of rice lines carrying mutations in ten of the remaining candidate genes and then carried out co-expression analysis associated with these genes. This analysis effectively provided candidate functions for two genes of previously unknown function and for one gene not directly linked to the tested biochemical pathways. These data demonstrate the efficiency of combining gene family-based expression profiles with analyses of insertional mutants to identify novel genes and their functions, even among members of multi-gene families. Citation: Jung K-H, Lee J, Dardick C, Seo Y-S, Cao P, et al. (2008) Identification and Functional Analysis of Light-Responsive Unique Genes and Gene Family Members in Rice. PLoS Genet 4(8): e1000164. doi:10.1371/journal.pgen.1000164 Editor: Gregory S. Barsh, Stanford University School of Medicine, United States of America Received March 5, 2008; Accepted July 15, 2008; Published August 22, 2008 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This research was funded in part by a competitive grants from the National Science Foundation Plant Genome Program (award: #DBI0313887), the USDA (# 2004-35604-14226) and the NIH (#5R01GM055962-0) to PCR, a grant from the Biogreen 21 Program (20070401-034-001-007-03-00) to GA, and a grant from the National Research Laboratory Program funded by the Ministry of Science and Technology (M10600000270-06J0000-27010) to GA. KHJ was supported by a Korea Research Foundation Grant (KRF-2005-C00155). Support was also provided by the Hatch Act and State of Iowa funds. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pcronald@ucdavis.edu Introduction Gene inactivation by the insertion of T-DNA is a common tool used for functional studies of genes in model plants such as Arabidopsis or rice. T-DNA insertional mutants have been generated for virtually all of the annotated genes in Arabidopsis thaliana. Recently, researchers working with Arabidopsis have combined the use of microarray technology with screens of T- DNA insertional mutant libraries to identify and characterize genes of unknown function. This strategy has successfully been used to characterize genes related to the formation of the secondary cell wall, those differentially regulated in phyto- chrome-mediated light signals, and those involved in host responses to pathogens [1,2,3]. These studies did not address the issue of multi-gene families, which limits functional analysis of genes in many plant species. Rice is a staple crop for more than half of the world’s population and a model for other cereal crops. Therefore, studies of the rice genome are expected to help elucidate the function of genes in other major cereal crops, all of which have much larger genomes than rice. So far the functions of only a handful of rice genes have been characterized. The rice research community has recently generated a large collection of rice insertional mutant lines and thus far 27,551 gene loci with knockout mutations have been collected and 172,500 flanking sequences tagging those mutations have been sequenced (http://signal.salk.edu/RiceGE/RiceGE_ Data_Source.html) [4]. Four microarray platforms covering nearly the entire rice transcriptome have also been developed [4,5,6,7]. Accordingly, studies similar to those carried out in Arabidopsis, combining microarray-derived expression data with reverse genetics to address gene function, can now be carried out in rice. Plants are continuously exposed to biotic and abiotic factors, light being one of the most important. In addition to providing the source of energy, light is also involved in regulating growth and development throughout the plant life cycle [8]. Genome-wide gene expression profiles of various light responses can help reveal PLoS Genetics | www.plosgenetics.org 1 August 2008 | Volume 4 | Issue 8 | e1000164