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A Nuclear-Encoded Chloroplast Protein Harboring a Single CRM Domain

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A Nuclear-Encoded Chloroplast Protein Harboring a Single CRM Domain Lee et al. BMC Plant Biology 2014, 14:98 http://www.biomedcentral.com/1471-2229/14/98 RESEARCH ARTICLE Open Access A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response Kwanuk Lee1, Hwa Jung Lee1, Dong Hyun Kim1, Young Jeon2, Hyun-Sook Pai2 and Hunseung Kang1* Abstract Background: Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Results: Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. Conclusions: These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts. Keywords: Arabidopsis thaliana, Chloroplast, CRM domain, RNA-binding protein, RNA metabolism Background regulatory process between chloroplasts and nuclei is Chloroplasts are derived from cyanobacteria through endo- required to fine-tune chloroplast gene expression, and symbiosis, and massive gene transfer from the plastid to many nuclear-encoded RNA-binding proteins (RBPs) the nucleus occurred during evolution [1]. Chloroplasts have been recently regarded as the primary elements possess approximately 100-150 genes in their own circular that modulate posttranscriptional steps in chloroplasts genome that encodes messenger RNAs, ribosome RNAs, [2,4,6,7]. Although chloroplasts share some features of and transfer RNAs. In addition to its own gene products, RNA metabolism with their bacterial ancestors, chlo- functional communication between chloroplasts and nu- roplasts require a more complicated mechanism of cleus is required, and many nuclear-encoded proteins are RNA metabolism compared to their ancestor, which targeted to chloroplasts and play fundamental roles in the have both prokaryotic and eukaryotic characteristics [8,9]. regulation of chloroplast gene expression. Expression of One particular example is the splicing of group I and group chloroplast genes is commonly regulated at posttranscrip- II introns. In contrast to self-splicing of prokaryotic introns, tional level, including mRNA processing, splicing, editing, chloroplasts have lost their capacity to self-splice, and the decay, and translational control [2-5]. A sophisticated splicing of group I and group II introns in chloroplasts re- quires many nuclear-encoded proteins that form protein * Correspondence: [email protected] complexes similar to spliceosomal complexes found in 1Department of Plant Biotechnology, College of Agriculture and Life eukaryotes [10-14]. Therefore, involvement of nuclear- Sciences, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Korea encoded RBPs is indispensible for posttranscriptional Full list of author information is available at the end of the article © 2014 Lee et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee et al. BMC Plant Biology 2014, 14:98 Page 2 of 11 http://www.biomedcentral.com/1471-2229/14/98 regulation of RNA metabolism and gene expression in protein (Figure 1A and Additional file 1). The two sin- chloroplasts. gle CRM domain-containing proteins (At4g39040 and CRM (chloroplast RNA splicing and ribosome matur- At2g21350) share approximately 56% amino acid se- ation) domain-containing proteins were first found in ar- quence homology with each other. To examine whether chaea and bacteria. Based on their structural data and the single CRM domain proteins are conserved in dicoty- predicted domain structures, CRM domain proteins were ledonous and monocotyledonous plants, the amino acid suggested to have RNA-binding activity [12]. Prokaryotes sequences of single CRM domain proteins in diverse plant contain proteins harboring only a single CRM domain, species, including Arabidopsis, Zea mays, Medicago trun- whereas land plants contain proteins harboring multiple catula, Vitis vinifera, Hordeum vulgare, Sorghum bicolor, CRM domains. Splicing of group I and group II introns and Oryza sativa, were compared. The results showed and tRNAs and processing of rRNAs in chloroplasts re- that CFM4 family proteins share 35-50% amino acid quire different complexes of CRSs (for chloroplast RNA sequence homology among dicot and monocot plants splicing), CAFs (for CRS2-associated factors), and CFMs and share > 70% amino acid sequence homology among (for CRM family members) that harbor multiple CRM do- monocot plants (Additional file 1), suggesting that the mains [12,13,15-19]. It has also been demonstrated that single CRM domain-containing proteins are function- mutation in CRM domain-containing protein genes re- ally conserved in dicots and monocots. sults in pale-green phenotypes, delayed development, and The CRM proteins in Arabidopsis and rice have been aborted seed production in plants, indicating the import- predicted to be targeted mainly to chloroplasts or mito- ant roles of CRM domain proteins in plant growth and de- chondria. To determine the subcellular localization of velopment [11,12,17,20,21]. CFM4, the cDNA encoding CFM4 was ligated in front Despite an increased understanding of the roles of CRM of the green fluorescence protein (GFP) gene, and ex- domain-containing proteins in chloroplast RNA metabol- pression of the CFM4-GFP fusion protein was investi- ism and plant growth and development, the functional gated in transgenic Arabidopsis plants. Strong GFP signals roles of most of the CRM domain-containing proteins were observed in chloroplasts (Figure 1B). To examine have not been demonstrated experimentally. The Arabi- whether CFM4 is also localized to mitochondria, Arabi- dopsis (Arabidopsis thaliana)andrice(Oryza sativa) dopsis mitochondria were stained with Mito-tracker that genomes harbor the genes encoding 16 and 14 CRM is a red-fluorescent dye and stains mitochondria in live domain-containing proteins, respectively [13]. Among cells,andthesignalsfromplastidsinrootsandchloro- the 16 Arabidopsis CRM domain-containing protein plasts in leaves were examined. The results showed genes, two genes (At4g39040 and At2g21350) encode that the signals from mitochondria did not overlap the smallest proteins harboring a single CRM domain with the signals from chloroplasts, and GFP signals [13]. However, the role of single CRM domain-containing were observed exclusively in chloroplasts (Figure 1C proteins has not been demonstrated in plants. Here, we and 1D). These results clearly indicate that CFM4 is determined the developmental and stress response roles localized to chloroplasts. of a single CRM domain-containing protein (At4g39040). Because this protein belongs to subfamily group 4 among CFM4 plays a role in Arabidopsis growth and senescence CRM domain proteins [13], we designated it as CRM fam- To determine the role of CFM4 during plant growth and ily member subfamily4 (CFM4). We show that CFM4 pos- development, the T-DNA insertion mutant lines in CFM4 sesses RNA chaperone activity and is involved in rRNA (SALK_076439 and SALK_126978) were obtained, and processing, which is important for normal growth, devel- their phenotypes were analyzed under normal and stress opment, and the stress response in plants. conditions. The absence of CFM4 expression in the knockout mutant lines was confirmed by RT-PCR ana- Results lysis (Additional file 2). The wild-type and cfm4 mu- Structural features and characterization of CFM4 in tantplantsweregrowninMSmediumorsoil,and Arabidopsis their phenotypes were observed during the entire life Sixteen predicted CRM domain family members occur in cycle (from germination to senescence) of the plants. the Arabidopsis genome, and they are classified into four Growth of the wild-type and mutant plants was not groups, such as CRS1 subfamily, CAF subfamily, subfamily significantly different at 7 days after germination (DAG) 3, and subfamily 4. Among the 16 CRM domain-containing (Additional file 3). However, growth of the plants was protein genes, two genes (At4g39040 and At2g21350) en- markedly different at
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