Aspartic proteases gene family in rice: Gene structure and expression, predicted protein features and phylogenetic relation Jiongjiong Chen 1 , Yidan Ouyang 1 , Lei Wang, Weibo Xie, Qifa Zhang ⁎ National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China abstract article info Article history: Received 10 December 2008 Received in revised form 22 April 2009 Accepted 23 April 2009 Available online 3 May 2009 Received by J.G. Zhang Keywords: Aspartic protease Rice Microarray Expression profile Phytohormone Aspartic proteases constitute a large family in eukaryotes, which play fundamental roles in protein processing, maturation and degradation. In this study, we identified 96 OsAP genes in rice (Oryza sativa L.), the model plant for monocots, by a reiterative database search. The analysis of the complete set of OsAP genes is presented, including chromosomal location, phylogenetic relationships, classification and gene structure. Moreover, a comprehensive expression analysis of OsAP family genes was performed using 24 tissues during the plant life cycle of two rice cultivars. Sixty-six OsAP genes were found to be expressed in at least one of the examined developmental stages, which were divided into 3 classes based on their transcript levels. OsAP genes were also found to be differentially up- or down-regulated in rice seedlings in response to treatments with phytohormones, as well as in plumules/radicles under light/dark conditions. The comprehensive annotation and expression profiling undertaken in this research add to our understanding of OsAP family genes in rice growth and development. Our results also provide a basis for selection of candidate genes for functional validation in future studies. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Aspartic proteases (APs; EC 3.4.23) are widely distributed in all living organisms, constituting one of the four superfamilies of proteolytic enzymes (Davies, 1990; Barrett, 1992; Rawlings and Barrett, 1999). Most of the APs contain two aspartic acid residues at the active sites. They are active in acidic pH and are specifically inhibited by pepstatin A. The catalytic Asp residues are located within the conserved Asp-Thr/Ser-Gly (DT/SG) motif. In most of the known APs, a pair of aspartic residues act together to bind and activate the catalytic water molecule. However, in some APs, residues of other amino acids replace the second Asp (Barrett et al., 2004). APs are synthesized as single chain preproenzymes and converted to mature two-chain enzymes during activation. Plant APs, especially Arabidopsis APs, are divided into three categories, including typical APs, nucellin-like APs and atypical APs (Faro and Gal, 2005). The characteristic of typical plant AP precursors is the presence of an extra protein domain of approximately 50–100 amino acids known as the plant specific insert (PSI). This segment, inserted into the C-terminal domain of typical plant AP precursors, is usually removed during protein maturation. The sequence, structure and function of the PSI domain show no homology with animal or microbial APs, but it is highly similar to that of saposin-like proteins (Mutlu and Gal, 1999). The nucellin-like APs encode proteins similar to nucellin first detected in barley nucellar cells (Chen and Foolad, 1997). Atypical APs display intermediate features between the typical and nucellin-like sequences (Faro and Gal, 2005). APs have been detected or purified from both monocotyledons and dicotyledons as well as from gymnosperms. They are expressed in different plant organs, such as seed, grain, tuber, leaf, flower, petal, root and pollen, as well as in the digestive fluids of carnivorous plants. Plant APs have been implicated in protein processing and/or degradation in different plant organs, as well as in plant senescence, stress response, programmed cell death, and reproduction (Simões and Faro, 2004). In rice (Oryza sativa L.), a typical aspartic protease, oryzasin, was purified from seed and had been characterized in details (Asakura et al., 1995, 1997, 2000). Recently, a rice atypical aspartic protease was identified as playing a major role in regulating indica– japonica hybrid sterility and wide compatibility by conditioning embryo-sac fertility (Chen et al., 2008). Genome-wide identification and phylogenetic analyses of AP genes had been reported in Arabidopsis (Beers et al., 2004; Faro and Gal, 2005; Takahashi et al., 2008). In addition, the phylogenetic relationships of different eukaryotic AP genes had also been investigated (Hughes et al., 2003; Pimentel et al., 2007). However a complete analysis of the AP content of the O. sativa genome is still lacking. Gene 442 (2009) 108–118 Abbreviations: OsAP, Oryza sativa aspartic protease; KOME, Knowledge-based Oryza Molecular biological Encyclopedia; NCBI, National Center for Biotechnology Informa- tion; TIGR, The Institute for Genomic Research; NAA, naphthalene acetic acid; GA3, gibberellic acid; KT, kinetin. ⁎ Corresponding author. Tel.: +86 27 87282429; fax: +86 27 87287092. E-mail address: qifazh@mail.hzau.edu.cn (Q. Zhang). 1 These authors contributed equally to this work. 0378-1119/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2009.04.021 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene