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STRIPE2 Encodes a Putative Dcmp Deaminase That Plays an Important Role in Chloroplast Development in Rice

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STRIPE2 Encodes a Putative Dcmp Deaminase That Plays an Important Role in Chloroplast Development in Rice Available online at www.sciencedirect.com ScienceDirect JGG Journal of Genetics and Genomics 41 (2014) 539e548 ORIGINAL RESEARCH STRIPE2 Encodes a Putative dCMP Deaminase that Plays an Important Role in Chloroplast Development in Rice Jing Xu a, Yiwen Deng a, Qun Li a, Xudong Zhu b,*, Zuhua He a,* a National Key Laboratory of Plant Molecular Genetics and National Center of Plant Gene Research, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China b China National Rice Research Institute, Hangzhou 31006, China Received 31 March 2014; revised 8 May 2014; accepted 9 May 2014 Available online 19 June 2014 ABSTRACT Mutants with abnormal leaf coloration are good genetic materials for understanding the mechanism of chloroplast development and chlorophyll biosynthesis. In this study, a rice mutant st2 (stripe2) with stripe leaves was identified from the g-ray irradiated mutant pool. The st2 mutant exhibited decreased accumulation of chlorophyll and aberrant chloroplasts. Genetic analysis indicated that the st2 mutant was controlled by a single recessive locus. The ST2 gene was finely confined to a 27-kb region on chromosome 1 by the map-based cloning strategy and a 5-bp deletion in Os01g0765000 was identified by sequence analysis. The deletion happened in the joint of exon 3 and intron 3 and led to new spliced products of mRNA. Genetic complementation confirmed that Os01g0765000 is the ST2 gene. We found that the ST2 gene was expressed ubiquitously. Subcellular localization assay showed that the ST2 protein was located in mitochondria. ST2 belongs to the cytidine deaminase-like family and possibly functions as the dCMP deaminase, which catalyzes the formation of dUMP from dCMP by deamination. Additionally, exogenous application of dUMP could partially rescue the st2 phenotype. Therefore, our study identified a putative dCMP deaminase as a novel regulator in chloroplast development for the first time. KEYWORDS: stripe2; Chloroplast development; dCMP deaminase; Oryza sativa INTRODUCTION materials to investigate regulation mechanisms of chlorophyll biosynthesis and chloroplast development in rice. The chloroplast is the crucial organelle for plant photosyn- Screening for chloroplast development mutants has identi- thesis and essential for the production of hormones and me- fied most steps in these biological processes, such as yellow- tabolites (Pogson and Albrecht, 2011). About 3000 proteins in green leaf1 ( ygl1) and faded green leaf ( fgl ), which result the chloroplast participate in transition from proplastids to from lesions of chlorophyll synthase that catalyzes esterifica- mature chloroplasts, and this process is coordinated by both tion of chlorophyllide in the last step of chlorophyll biosyn- nuclear and plastid genome involved in synthesis of chloro- thesis (Wu et al., 2007) and NADPH:protochlorophyllide plast DNA, the plastidic transcription/translation apparatus oxidoreductase that catalyzes the photoreduction of proto- and the photosynthetic system (Sakamoto et al., 2008). chlorophyllide (pchlide) to chlorophyllide (chlide) (Sakuraba Numerous chlorophyll-deficient or abnormal chloroplast mu- et al., 2013). Both mutations led to reduced contents of tants have been identified, and they provide ideal genetic chlorophyll and undeveloped chloroplasts. The magnesium chelatase, catalyzing the chelation of Mg2þ into proto IX to produce Mg-Proto IX, comprises three subunits including * Corresponding authors. Tel: þ86 21 5492 4121, fax: þ86 21 5492 4123 (Z. He); Tel: þ86 571 6337 0327, fax: þ86 571 6337 0389 (X. Zhu). ChlH, ChlD and ChlI (Jung et al., 2003; Zhang et al., 2006). E-mail addresses: [email protected] (X. Zhu); [email protected] (Z. He). Mutations of the chlorina-1 and chlorina-9 led to deficiency in http://dx.doi.org/10.1016/j.jgg.2014.05.008 1673-8527/Copyright Ó 2014, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. 540 J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548 chlorophyll content and incomplete development of chloro- ribonucleotide reductase (RNR), respectively, which regulates plasts, due to the disruption of ChlD and ChlI subunits, the rate of deoxyribonucleotide production for DNA synthesis respectively. Moreover, an insert mutation in ygl2 ( yellow- and repair. Yoo et al. (2009) speculated that, upon insufficient green leaf 2), encoding heme oxygenase (HO) that catalyzes activity of RNR, plastid DNA synthesis is preferentially the degradation of heme to synthesize phytochrome precursor, arrested to allow nuclear genome replication in developing results in significantly reduced content of chlorophyll and leaves to sustain the continuous plant growth. tetrapyrrole intermediates (Chen et al., 2013). In this study, we identified a stripe variegated mutant As a semiautonomous organelle, chloroplast genome only named st2 (stripe2). The st2 plants develop chlorotic leaves encodes about 100 genes (Delannoy et al., 2009). Most of the caused by low content of chlorophylls. Examination of the proteins essential for chloroplast development and function are ultrastructure showed that the thylakoid membranes are nuclear-encoded (Chen et al., 2010). It is well documented that extremely disturbed in the mutants. Map-based cloning and the coordination of nuclear and plastid genes is crucial for genetic complementation indicated that ST2 encodes a cyti- chloroplast biogenesis (Mullet, 1988). Under illumination, dine deaminase-like protein, which most likely functions as one-third of the nuclear genes change expression, including the dCMP deaminase. many transcription factors such as PIFs (phytochrome inter- acting factors). Either pif1 or pif3 mutant showed delayed RESULTS development of chloroplast (Moon et al., 2008; Stephenson et al., 2009). Gene transcription, RNA maturation, and pro- Phenotype characterization of stripe2 mutant tein translation in the chloroplasts also have impact on chlo- roplast biogenesis and development. There are two types of The rice (Oryza sativa L.) st2 mutant was isolated from g-ray- plastid RNA polymerases: plastid-encoded RNA polymerase induced mutations of an indica cultivar (Longtepu, LTP). The (PEP) and nucleus-encoded RNA polymerase (NEP) respon- mutant leaves were virescent with stripes (Fig. 1A and E). To sible for the transcription of the plastome. In Arabidopsis, determine its effect on chlorophyll formation, the leaves of mutations in SIG6 (sigma factor 6) cause a weakly virescent 3-week seedling were analyzed for chlorophyll contents. phenotype and transcripts of several PEP-dependent plastid Compared with the wild type, the chlorophyll contents in st2 genes are specifically reduced (Loschelder et al., 2006). were reduced by 30% (Fig. 1B), and autofluorescence in the Decreased expression of AtRpoTp, one of the NEP genes, st2 leaves also decreased (Fig. 1C and D). The ultrastructure of leads to a typical virescent phenotype which can be recovered the wild-type chloroplasts was crescent-shaped and contained after two weeks of growth (Swiatecka-Hagenbruch et al., well-formed thylakoid structure including stroma thylakoids 2008). PPR proteins (pentatricopeptide repeat proteins), and grana thylakoids (Fig. 2A). In contrast, the mutant chlo- characterized by tandem arrays of a 35 amino acid motif, have roplasts were small and thylakoid membrane was disturbed, been demonstrated to be critical for RNA processing, splicing, some with less thylakoid structure (Fig. 2B and C). Some editing, stability, maturation and translation in the chloroplast chloroplasts formed rudimentary thylakoids consisting of only (Takenaka et al., 2013). YSA (young seedling albino) encodes grana lamellae without formation of stroma lamellae a PPR protein and the disruption of its function causes a (Fig. 2D), while some chloroplasts displayed well-developed seedling stage-specific albino phenotype in rice. The mutant lamellar structures equipped with normally stacked grana but plants can recover and develop normal green leaves after the no starch grains in the st2 leaves (Fig. 2E and F). These results four-leaf stage. Interestingly, the ysa mutant has been used as a indicated the st2 phenotype is caused by the underdevelop- marker for efficient identification and elimination of false ment of the chloroplast. hybrids in commercial hybrid rice production (Su et al., 2012). The Arabidopsis mutant sel1 (seedling lethal 1) exhibited a Fine mapping of the ST2 Gene pigment-defective and seedling-lethal phenotype with a dis- rupted PPR gene. In the sel1 plants, RNA editing of acetyl- For genetic analysis of the st2 mutant, we firstly crossed st2 CoA carboxylase b subunit transcripts was disrupted (Pyo mutant with a japonica cultivar Zhonghua11 (ZH11), and the et al., 2013). The virescent rice mutants v1, v2 and v3 are F1 generation exhibited normal green leaves. Among the 762 temperature-conditional, which produce chlorotic leaves at a F2 individuals, 183 were virescent and 579 were green. The restrictive temperature (20 C) but develop nearly green segregation ratio of the F2 population accorded with 3:1 2 2 leaves at a permissive temperature (30 C). V1 encodes a (c ¼ 0.39 < c0.05 ¼ 3.81; P > 0.05), suggesting that the st2 chloroplast-localized protein NUS1 which is involved in the phenotype was controlled by a single recessive gene. Genetic regulation of chloroplast rRNA metabolism during early mapping was performed using the same population. The chloroplast
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