OsCHR4

=Brief introduction Mi-2 protein, the central component of the NuRD nucleosome remodeling and histone deacetylase complex, plays a role in transcriptional repression in animals. Mi-2-like genes have been reported in Arabidopsis, though their function in monocots remains largely unknown. In the present study, a rice Mi-2-like gene, OsCHR4 (Oryza sativa Chromatin Remodeling 4, LOC_Os07g03450), was cloned from a rice mutant with adaxial albino leaves. The Oschr4 mutant exhibited defective chloroplasts in adaxial mesophyll, but not in abaxial mesophyll. Ultrastructural observations indicated that proplastid growth and/or thylakoid membrane formation in adaxial mesophyll cells was blocked in the Oschr4 mutant. Subcellular localization revealed that OsCHR4::GFP fusion protein was targeted to the nuclei. OsCHR4 was mainly expressed in the root meristem, flower, vascular bundle, and mesophyll cells by promoter::GUS analysis in transgenic rice. The transcripts of some nuclear- and plastid-encoded genes required for early chloroplast development and photosynthesis were decreased in the adaxial albino mesophyll of the Oschr4 mutant. These observations provide evidence that OsCHR4, the rice Mi-2-like protein, plays an important role in early chloroplast development in adaxial mesophyll cells. The results increase our understanding of the molecular mechanism underlying tissue-specific chloroplast development in plants. =

=Gene function The chloroplast is the important organ of plant photosynthesis, and is closely related with carbon assimilation. The chloroplasts development has a direct impact on photosynthesis abilities and yields, thus screening abnormal leaf mutants can be used to study chloroplast development as good materials. In this study, we isolated a rice （Oryza sativa L.） mutant showing leaf albino only in the adaxial side, from an EMS genetic mutant library. By transmission electron microscopic images of chloroplasts, the adaxial albino phenotype of the mutant leaves is caused by abnormal formation of chloroplasts. However, the mesophyll cells in abaxial side contained morphologically normal chloroplasts. The mutated gene 0sCHR4 was cloned using the map-based cloning strategy and its gene locus is LOC07g31450. This gene was shown to encode a chromatin remodeling factor 4. Transgenic complementation of the mutant verified that OsCHR4 is involved in adaxial chloroplast development. OsCHR4 belongs to the highly conserved CHD family of proteins and its homologous proteins have been identified in vertebrates, Drosophila, yeast, C. elegans and plants.OsCHR4 is targeted to the nucleus and constitutively expressed in all tissues tested. Histochemical staining for GUS activity showed that OsCHR4 is expressed in the active root tissues of mitosis, flowers, all mesophyll cells and vascular bundles of stems. In the end, we further investigated the effect of OsCHR4 mutation on chloroplast development, and the result showed that the transcripts of eight genes associated with chloroplast development were significantly reduced in the adaxial mesophyll of the mutant. On the other hand, KNOX homeobox genes were significantly increased in the mutant which indicates OsCHR4 probably related to the AS1/AS2 pathway for repression of KNOX genes expression. The present results reveal that OsCHR4 is essential for chloroplast biogenesis at the early stage in adaxial mesophyll; this is the first report on the function of a chromatin remodeling factor involved in chloroplast development in plants. =

=Mutants Phenotypic characterization of the Oschr4 mutant One mutant exhibiting albino leaves on the adaxial side was isolated by screening an EMS-generated mutant，japonica rice, cv. Ishikari-shiroge library (Fig. 1a–c). The mutant was designated Oschr4 according to the annotation after map-based cloning. Cross-sections of fresh leaves from WT and Oschr4 plants revealed that nearly all cells in the adaxial mesophyll of the Oschr4 mutant lacked chlorenchyma, aside from a few cells on both sides of the large vascular bundles. Transverse Spurr’s resin sections of leaves showed that viable chloroplasts were absent from adaxial mesophyll cells in the Oschr4 mutant, but present in WT (Fig. 1f), as indicated by methylene blue staining. At the mature stage, the mutant plants were shorter (WT, 96.5 ± 2.9 cm; Oschr4,84.3 ± 5.7 cm) and had a lower tiller number (WT,10.3 ± 1.7; Oschr4, 2.7 ± 1.5), smaller panicle size (WT,19.9 ± 1.1 cm; Oschr4, 17.3 ± 1.2 cm), and fewer grains per panicle (WT, 147.2 ± 16.6; Oschr4, 73.8 ± 9.2; all values ± SD, n = 8) than WT plants . The chlorophyll content of leaves from Oschr4 mutant plants was decreased approximately 50 % compared with WT。To examine the morphological defects of chloroplasts in the adaxial albino cells of Oschr4 mutant, the ultrastructure of chloroplasts was examined by TEM. The unexpanded third and fully expanded second leaves were sampled from 8-day-old WT and Oschr4 seedlings. In the third leaf, the Oschr4 mutant had fewer chloroplasts and lighter-dyed adaxial mesophyll cells (Fig. 2a) compared with WT plants, whereas the development of chloroplasts in abaxial mesophyll cells was similar among WT and Oschr4 plants (Fig. 2b, c). Two types of cells were observed in the adaxial mesophyll of Oschr4 mutant: no chloroplastcontaining cells (designated as NCC cells) in which no chloroplast-like organelles were observed, and chloroplastcontaining cells (designated as CC cells) in which chloroplast-like organelles were visible but lighter-dyed .The lighter-dyed chloroplasts in CC cells from the Oschr4 mutant lacked thylakoid membranes and were smaller than those in WT plants. In contrast, chloroplast structure and thylakoid membrane organization were well developed in the abaxial mesophyll cells from the Oschr4 mutant (Fig. 2f). Consistent with the third leaf, chloroplasts in adaxial mesophyll cells from the second leaf of the Oschr4 mutant were lighter-dyed (Fig. 2g–i) than WT . These chloroplasts exhibited an irregular shape and fuzzy two-layer membrane structures, lacked thylakoid membrane organization, or contained fewer fragmentary thylakoid membranes. Clustered, dark plastoglobuli were also present, suggesting degradation. Nevertheless,mitochondrial and nuclear structures were visible。cloned into the vector pCAMBIA1300::GFP. The resulting plasmid, OsCHR4p::OsCHR4::GFP, was then transformed into calli from the Oschr4 mutant using Agrobacterium tumefaciens-mediated transformation. More than 15 transgenic lines exhibiting normal green leaves were obtained. Two independent transgenic lines are listed. Subsequent phenotypic observations and chlorophyll analyses confirmed that the mutated traits were rescued in the transgenic lines. Based on BLASTp searching, we analyzed the phylogenetic relationships between OsCHR4 and its related proteins from a variety of eukaryotes. Alignment analysis of OsCHR4 homologs showed a high similarity in the amino acid sequences between Chromo domain and DNAbinding domain, especially in SNF2_N and Helicase C domains (Supplemental Fig. S1). Neighbor-joining tree constructed using this alignment demonstrated that all these proteins can be separated into animal (clade I) and plant (clade II and III) groups. OsCHR4 is evolutionarily close to the clade III Arabidopsis CHR4. =

=Subcellular and tissue localization To examine the subcellular localization of OsCHR4, the protein was fused in-frame to the amino-terminus of GFP driven by the CaMV35S promoter and transiently expressed in onion epidermal cells. The green fluorescence signal of OsCHR4::GFP was detected exclusively in nuclei (Fig. 4a). Nuclear localization was also observed by green fluorescence in the root meristem of OsCHR4p::Os-CHR4::GFP transgenic plants (Fig. 4b). To determine the tissue localization of OsCHR4, the 3,096-bp OsCHR4p was amplified and inserted into the 5 end of the GUS reporter gene in the vector pBI101.3. The resulting construct was transformed into WT calli. Histochemical staining for GUS activity in transgenic plants revealed that the gene was expressed in meristem zones of primary and lateral roots (Fig. 5a, b), lateral root primordial (Fig. 5c), leaf blade (Fig. 5d), and flowers (Fig. 5e, f). Cross-sections revealed that OsCHR4 was expressed in the vascular bundles of the stem Longitudinal section of the stem base indicated that OsCHR4 is expressed in leaf primordia, but not in shoot apical meristem (Fig. 5k). The results of the RT-PCR analysis were consistent with those of GUS staining analysis (Fig. 5l). To continue investigating the expression of OsCHR4 in mesophyll tissues, adaxial and abaxial mesophyll cells from WT plants were obtained using LCM (Fig. 6a). qRTPCR analysis indicated that OsCHR4 is expressed at a similar level in adaxial and abaxial mesophyll (Fig. 5m). =

=Differential expression of chloroplast genes To examine whether the expression pattern of genes associated with chloroplast development is different in the Oschr4 mutant, adaxial and abaxial mesophyll cells from WT and Oschr4 plants were sampled from the basal portions of the unexpanded third leaves using LCM (Fig. 6a). Three types of genes expressed during the course of chloroplast development were analyzed by qRT-PCR: NEP-dependent plastid genes (RpoA, B, and Rps7) (Inada et al. 1996; Hedtke et al. 1997), PEP-dependent plastid gene (PsbA) (Liere et al. 1995), and PEP-dependent nuclear genes (RbcS and Cab1R) (Leutwiler et al. 1986; Hanley-Bowdoin and Chua 1989). Similar levels of RpoB were transcribed in adaxial and abaxial mesophyll from Oschr4 and WT plants (Fig. 6b). RpoA and Rps7 transcripts were decreased significantly in the adaxial mesophyll from the Oschr4 mutant compared with WT, but their expression in the abaxial mesophyll was similar for both plants (Fig. 6b). As expected, the expression of three photosynthetic genes (RbcS, Cab1R, and PsbA) was downregulated in the adaxial mesophyll from the Oschr4 mutant (Fig. 6b). = [1] [2] [3] [http://www.medsci.cn/sci/show_paper.asp?id=41473319274