Single-point Mutation of an Histidine-aspartic Domain-containing Gene involving in Chloroplast Ribosome Biogenesis Leads to White Fine Stripe Leaf in Rice

Plant leaves are a crucial organ associated closely with chloroplast development, photosynthesis rate and crop productivity. In this study, a white fine stripe leaf 1 (wfsl1) mutant was isolated and characterized from the japonica rice Zhonghua11 (ZH11) after ethyl methanesulfonate mutagenesis. The wfsl1 displayed white fine stripe leaves since tillering stage and abnormal chloroplast structure. Map-based cloning and Bioinformatic analysis indicated that WFSL1 on chromosome 1 contains an “A” to “T” substitution in protein coding region, and encodes a putative metal-dependent phosphohydrolase with HD domain at the N-terminus. WFSL1 was targeted to the chloroplasts and had higher expression in mature leaves and sheaths. RNA-seq analysis revealed that chloroplast development and photosynthesis genes were significantly affected in wfsl1 plants. Levels of WFSL1 and chloroplast encoded proteins were decreased in wfsl1 mutants via western blot analysis. Compared with WT, wfsl1 exhibits lower Chl content and defective in biogenesis of chloroplast ribosomes, which resulted in reduced grain yield. Taken together, our results show that WFSL1 is critical for chloroplast development, ribosome biogenesis, and light energy utilization, finally affects grain yield.

assembly. The knock down and RNAi of ObgC result in chlorotic phenotype in rice 8 . Thus, 70 S ribosome biogenesis is essential for chloroplast development in higher plants.
Plant variegations are characterized by the presence of white sectors and green sectors. The white sectors contain defective chloroplasts and the green sectors contain normal chloroplasts 11 . Immutans (im) and var2 are two types variegation mutants in Arabidopsis. The im mutant is induced by a nuclear recessive gene and the extent of variegation can be modulated by light and temperature. IM protein is a plastid homologue of the mitochondrial alternative oxidase and functions as a redox component of the phytoene desaturation pathway 11 . Mutations in the VAR2 locus cause variegation due to loss of a chloroplast thylakoid membrane protein that is similar to the FtsH family of AAA protein. FtsH functions in a number of diverse membrane-associated events and VAR2 protein functions in thylakoid membrane biogenesis 12 . Both im and var2 mutants provide an excellent system to understand the molecular mechanism of nuclear-plastid interactions in Arabidopsis. In rice, many genes were also associated with nuclear-plastid interaction. V1 encodes a chloroplast-localized protein NUS1 regulating chloroplast RNA metabolism [13][14][15][16] . V2 encodes a plastids/mitochondria Guanylate kinase (pt/mtGK) which catalyzes GMP to GDP in guanylate biosynthesis and affects chloroplast development. It suggests that pt/mtGK is an important part for chloroplast development 17 . RNRL1 and RNRS1 encode the large and small subunits of rice ribonucleotide reductase, respectively. They are necessary for the DNA replication in chloroplast 18 . OsDVR plays certain roles in nuclear-cytoplasmic signal transduction by which nucleus directly regulates cytoplasm 19,20 . Leaf-color mutations are a crucial for dissection of regulation mechanism of with chloroplast development, photosynthesis and key agronomic traits.
Here, we isolated a white f ine stripe leaf (wfsl1) mutant in rice. The phenotype of mutant not very likely with im and var2 mutants, the green and white sectors longitudinal distributed on the blade of mutants. It's interesting that mutant phenotype mainly first occurs on the newly grown leaves and sheath at tillering stage, continued to the mature stage. Our study discovers that map-based cloning identifies WFSL1 on chromosome 1 encoding an HD domain containing gene. The WFSL1 protein is localized to chloroplast and was decreased in wfsl1 mutant leaves. The wfsl1 mutant was defective in biogenesis of chloroplast ribosomes, and affected photosynthesis rate and grain yield. Our results suggest that WFSL1 is critical for chloroplast development and regulating chloroplast ribosome biogenesis.

Results
Phenotypic characterization of wfsl1 mutant. The wfsl1 mutant was isolated from an M 2 population of the japonica rice Zhonghua11 (ZH11) after Ethyl methanesulfonate mutagenesis, and was designated according to the chromosome location and white fine stripe leaf phenotype. At seedlings wfsl1 showed normal phenotype ( Fig. 1a) but it emerged white fine stripe leaf at tillering stage and continued to the mature stage ( Fig. 1b-e). Cross-section observation showed content of chlorophyll decreased in wfsl1 mutants (Fig. 1f,i). The chlorophyll and carotenoid levels were nearly the same at seedling stage (Fig. 1j), but they remarkably decreased in wfsl1 mutants at tillering stage (Fig. 1k). Transmission electron microscopy (TEM) indicated that wfsl1 had green, green-white and white types of cells ( Fig. 2a-d). The green cells have more chloroplast and well-developed thylakoid membrane systems, whereas the chloroplast were small with no thylakoid membranes in wfsl1 white cells ( Fig. 2e-j). So the cells are heteroplastidic in mutants.
The wfsl1 plants reduced grain yield. Agronomic trait analysis indicated that the plant height, panicle length, setting rate, and thousand seed weight were remarkably reduced in wfsl1, compared with WT (Fig. 3a). The length of topmost and second topmost interondes was significantly decreased in wfsl1 (Fig. 3a). The setting rate was 70.1% of wfsl1 while 89% of wild type (Fig. 3a). The thousand seed weight reduced to 22.48 g of wfsl1 where wild type was 25.98 g (Fig. 3a). The photosynthesis rate reduced in wfsl1 (Fig. 3b). However, no difference of tiller number between wild type and wfsl1 was found (Fig. 3a). TEM suggested chloroplasts were small and thylakoid membrane impaired in wfsl1 which may reduce photosynthesis. In summary, the wfsl1 mutation resulted in the reduction of grain yield.
Map-based cloning and functional confirmation of WFSL1. The WFSL1 was cloned in an F 2 population derived from the cross between the wfsl1 and an indica cultivar NJ06. The segregation ratio of wild type to white fine stripe leaf phenotype among the F 2 population was 3:1 (365:107, χ 2 = 1.3672 < χ 2 0.05, 1 ; P = 0.2423 > 0.05), suggesting that the phenotype of wfsl1 mutant is controlled by a single recessive gene (Supplementary Table S2). The WFSL1 gene was primarily mapped to the region between the markers RM3252 and RM5336 on chromosome 1 (Fig. 4a). Then eight insertion-deletion polymorphism (InDel) markers were developed for further fine-mapping (Supplementary Table S1). The location of WFSL1 was narrowed down to a 50.5 kb region which includes 12 ORFs (open reading frame). The predicted region was sequenced to detect the mutation and discovered that the target ORF (LOC_Os01g01920) had an "A" to "T" substitution in protein coding region (Fig. 4a). The gene includes eighteen exons and seventeen introns. The mutation site on the seventeenth exon, with the amino acid Asn changed into Tyr (Fig. 4b). Gene prediction (Rice Genome Annotation Project) indicates that it encodes a putative metal-dependent phosphohydrolase. Sequencing alignment indicates that WFSL1 has a conserved HD domain motif ( Supplementary Fig S1). To confirm whether the phenotype of wfsl1 mutant is caused by the loss function of WFSL1, complementation test was conducted. The genomic DNA fragment containing WFSL1 sequence, the upstream and downstream sequences were inserted into a binary vector to construct pCAMBIA1300-WFSL1 vector. This vector was introduced into the wfsl1 mutants by Agrobacterium-mediated transformation. More than eight transgenic lines exhibited normal green leaves (Fig. 4c), and the content of chlorophyll had no difference contrast to wild type (Fig. 4d). Besides, sequencing results confirmed the complementation plants (COM) (Fig. 4e). And a 1,743-bp fragment of full-length cDNA from the cDNA library of wild type was inserted into a binary overexpression vector to construct Scientific RepoRts | 7: 3298 | DOI:10.1038/s41598-017-03327-2 pCAMBIA1300S-WFSL1. The vector was transformed into wfsl1 mutants to generate overexpression plants (OE). The overexpression plants exhibited normal green leaves and relative expression increased significantly compared with wild type and mutants (Fig. 4f). Thus, the white fine stripe leaf phenotype was rescued. These results suggest that the single base substitution in LOC_Os01g01920 (WFSL1) is responsible for the phenotype of wfsl1 mutant.
Location of WFSL1 to chloroplast. To investigate the subcellular localization of WFSL1, the full-length WFSL1 cDNA was amplified from the cDNA library of wild type. The WFSL1 cDNA was ligated to GFP sequence, and injected into rice protoplasts. Its expression was under the control of 35 S promoter. The GFP fluorescence in transformed protoplasts was examined using confocal fluorescence microscope, which showed that WFSL1 was located to chloroplast (Fig. 5a,b).
Analysis of WFSL1 and plastid-encoded genes expression pattern. qRT-PCR was used to examine the tissue-specific expression pattern of WFSL1. RNA was extracted from young leaves and young roots (three leaf stage), mature leaves and mature roots (heading stage), stems, sheaths and panicles, respectively. WFSL1 was highly expressed in sheaths and mature leaves, but low expression in young leaves, roots, stems and panicles (Fig. 5c). Next, we investigated the transcription levels of the genes associated with chlorophyll   and OsChlH 26 . UBQ5 (Actin) was used as control 27 (Supplementary Table S3). qRT-PCR analysis showed that the transcriptions of OsPORB, NOL, V1, OsDVR, OsChlH were significantly suppressed in wfsl1 mutant at seedling stage. The expression level of OsPORA, OsPORB, SGR were increased and OsCAO1, OsCAO2, V1, V2, OsDVR, OsChlH were decreased in mutant at tillering stage (Fig. 5d,e). The relative expression levels of plastid-encoded genes PsaA, PsbA, AtpB and Rubisco large subunit (RbcL), Rubisco activase (RCA) were all decreased in wfsl1. while the expression levels of nuclear-encoded genes RpoA, RpoB, RpoC1, RpoC2 increased in wfsl1. These results suggest wfsl1 is defective in biogenesis of plastid-encoded genes and affects chloroplast development.
Expression of photosynthesis related genes is repressed in wfsl1. RNA-seq was performed to analyze the effect of the wfsl1 mutation on gene expression. More than 40 million reliable clean reads were obtained from wild type and wfsl1. About 355 up regulated genes while 536 repressed genes in wfsl1  Table S4). We randomly selected 10 down-regulated and 10 up-regulated genes, and using qRT-PCR methods to verify the results of RNA-seq. The qRT-PCR results were consistent with the RNA-seq (Fig. 6e). Go and KEGG enrichment analysis indicated that genes encoding photosynthesis, light reaction, chloroplast envelope, PSI and PSII, chlorophyll binding, carbon fixation were remarkably reduced in wfsl1 (Supplementary Figs S2 and S3).

Defects of WFSL1 and plastid proteins in wfsl1.
We tested the accumulation of WFSL1 protein in wild type and wfsl1 mutants using western-blot analysis. However, the accumulation of WFSL1 protein was decreased in wfsl1 mutant at tillering stage (Fig. 7a). SDS-PAGE gel and western-blot suggest the protein levels of the large subunit of Rubisco (RbcL) and Rubisco activase (RCA) were decreased in wfsl1 (Fig. 7b,c).Other plastidic proteins including ATP synthase subunit beta (AtpB), A1 of PSI, D1 of PSII, "alpha and beta" subunits of RNA polymerase were also tested. The results showed that the levels of plastid-encoded proteins were significantly decreased in wfsl1 (Fig. 7c). qRT-PCR results suggest the expression levels of class I genes RbcL, PsbA, AtpB were remarkably decreased, while class III genes including RpoA, RpoC2 increased (Fig. 7d). RNA-seq results indicated that expression levels of many plastidic genes changed between wild type and wfsl1. Class I, II and III genes are three types of plastidic genes in plant. Class I genes are transcribed by PEP, class II genes are transcribed by NEP and PEP, and class III genes are mainly transcribed by NEP. The expression levels of class III genes were increased while class I genes decreased (Fig. 8d). These results indicate that wfsl1 was defective in PEP activity and chloroplast protein biosynthesis.

Reduction of chloroplast ribosome activity in wfsl1.
Chloroplast ribosome is composed of 30 S small subunit and 50 S large subunit. The 30 S and 50 S subunits are mainly comprised of 16 S and 23 S rRNAs and ribosomal genes 9 . We found that the 16 S and 23 S rRNAs were decreased in wfsl1 (Fig. 8a). We used an Agilent 2100 to analyze the composition and content of rRNAs from wild type and wfsl1 at tillering stage. We found that the 16 S and 23 S rRNAs were reduced to one half of wild type levels (Fig. 8b-d). RNA-seq analysis indicates that the expression levels of some ribosomes 30 S genes RPS1, RPS5, RPS17 and 50 S genes RPL5, RPL11, RPL13, RPL18 were all decreased in wfsl1 (Fig. 8e). These results indicated that wfsl1 mutant was defective in biogenesis of chloroplast ribosomes.

Discussion
Rice leaf-color mutants are key for investigation of chloroplast development, light energy utilization rate and reproduction. In this study, we isolated the wfsl1 mutant displayed white fine stripe leaves at tillering stage (Fig. 1b,c). Compared with the wild type, wfsl1 mutant exhibited pleiotropic phenotypes including reduction of plant height, panicle length, seed setting rate, and thousand seed weight. Also the chlorophyll content decreased in wfsl1 plants since tillering stage compared with WT (Fig. 1f,i,j,k). wfsl1 has three types of cells (green, green and white, white) and its chloroplasts were defective in tillering stage (Fig. 2e-j). TEM results identified the pattern of the white fine stripe phenotype, suggesting that wfsl1 cells are heteroplastidic containing white cell sector Cluster analysis of differently expressed genes in wild type and wfsl1 Red represents high expression genes. Blue represents low expression genes. (e) qRT-PCR analysis differently expression genes of RNA-seq. 20 up-regulated or down-regulated genes were tested. Error bars represent the SD from three independent experiments (Student's t-test, *P < 0.05; **P < 0.01) with abnormal chloroplasts (Fig. 2a-d). The wfsl1 mutant only has single-base mutation and leads to a series of phenotypic changes. These phenotypic differences between the wfsl1 and previously reported mutants may be due to the different genetic backgrounds and the different mutation sites in the target genes [28][29][30] . So determination of the pattern of white fine stripe leaf using the wfsl1 mutant is an important to understand the mechanism of white fine stripe and the function of WFSL1 protein. The reduction of the content of chlorophyll in wfsl1 is useful to study the relationship among WFSL1 and chloroplast development, light energy utilization and grain yield.
We cloned WFSL1 using map-based cloning method and confirmed its function in transgenic plants (Fig. 4). WFSL1 has a HD domain motif 31 (Supplementary Fig. S1) with a H…HD…H…H…D sequence motif. The histidines or aspartates in HD domain are highly conserved and coordinate with the metal ion to regulate the activity of protein. HD domain is globular in dGTPases 32 and has the (Guanosine 5′-triphosphate 3′-diphosphate) ppGpp hydrolase activity of SpoT protein in E.coli, which supports that the protein containing HD-domain is a phosphohydrolase 33 . Cyclic nucleotide Phosphodiesterase (PDEs) has a HD domain, and is regulated by Zn 2+ coordination. Other metals, such as Mn 2+ , Co 2+ and Mg 2+ , also have catalytic activity 34 . RelA and SpoT regulate ppGpp levels in E.coli 35 . In SpoT protein, HD domain has ppGpp ase activity 33 , and the His-Asp doublet is involved in the hydrolysis for ppGpp 36 . Arabidopsis has four RelA/SpoT homologues AtRSH1, AtRSH2, AtRSH3 and AtCRSH. They are all targeted to plastids and expressed in green tissues and flowers which have important functions in chloroplast development and reproduction via ppGpp synthetase activity 37,38 . The transcripts encoding the RC and LHC subunits of PSI and PSII, the small and large subunits of Rubisco was decreased in OX:RSH2 and OX:RSH3 plants. Plastid-encoded genes were affected in OX:RSH2 and OX:RSH3 plants 39 .
Also WFSL1 was located to chloroplast (Fig. 5a,b), and the same that the HD domain containing proteins OsCRSH1, OsCRSH2 and OsCRSH3 were located to chloroplast 38 . Go term analysis found that genes of photosynthesis, photosystem II assembly, and plastid were highly expressed in wfsl1 (Supplementary Fig. S1). KEGG analysis found that photosynthesis, and carbon fixation metabolism were significantly difference in WT and wfsl1 (Supplementary Fig. S2). These suggest WFSL1 was essential in chloroplast development. The expression of chlorophyll synthesis genes OsCAO1 and OsCAO2, chloroplast development genes V1, V2, OsDVR and OsChlH and plastid encoded genes PsaA, PsbA, AtpB, RCA were all decreased in wfsl1 at tillering stage. The expression level of nuclear-encoded genes RpoA, RpoB, RpoC1, RpoC2 was increased in wfsl1 (Fig. 7d). These suggest plastid encoded genes were defective in wfsl1. WFSL1 is highly expressed in mature leaves and sheaths, indicating that it functions at tillering and mature stages (Fig. 5d). The expression class I genes decreased and class III genes increased in wfsl1. It is similar to these PEP-related mutants obgc 8 , rh3 40 , and PPR proteins mutants 41, 42 . So we further conducted western blot analysis, and found WFSL1 protein levels were decreased in wfsl1 at tillering stage (Fig. 7a). These results in decreased expression levels of WFSL1 at tillering stage (Fig. 5h,i). The rubisco large subunit, plastid-encoded proteins and Rubisco activase (RCA) were decreased in wfsl1. Nuclear-encoded protein RpoB increased in wfsl1. qRT-PCR results indicate that the expression levels of plastid-encoded genes decreased and nuclear-encoded genes increased in wfsl1. These are very likely previously reported mutant wp1 9 . The wp1 mutant decreased plastid-encoded proteins and defective in chloroplast developmental. Since levels of plastid-encoded proteins decreased in wfsl1. We analyzed the content and composition of rRNA using an Agilent 2100. The results showed little difference between 18 S and 25 S rRNA levels in wild type and wfsl1. However, we found that wfsl1 16 S and 23 S rRNAs contents were dropped one half that of wild type (Fig. 8a-d). RNA-seq analysis suggest expression levels of ribosomal genes, including 50 S ribosomal genes RPL5, RPL10, RPL18, RPL21 and 30 S ribosomal genes RPS1, RPS5, RPS9, RPS17 were all decreased in wfsl1 (Fig. 8e). These results indicated that wfsl1 was defective in chloroplast ribosome biogenesis Chloroplast plays an important role in light reception in higher plants 1 . In this study, the wfsl1 displayed white fine stripe leaves and reduced Chl content at tillering stage (Fig. 1f,i,k). The thylakoid was abnormal in wfsl1 ( Fig. 2e-j), and leads to the reduced of photosynthesis rate. Some plastid-encoded proteins A1 of PSI (PsaA), D1 of PSII (PsbA), Rubisco large subunit and Rubisco activase (RCA) were decreased in wfsl1 (Fig. 7a-c). PSI and PSII are two pathways that responsible for electron transfer during photosynthesis. Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco), which constitute of RbcL and RbcS subunit that catalyzes the first step in net photosynthetic CO 2 assimilation and photorespiratory carbon oxidation 43,44 . PSI and PSII may inhibit protein levels of RbcL in wfsl1, suggesting that the photosynthesis was affected in wfsl1 plants. The decreased setting rate and thousand seed weight (Fig. 3a) were associated with decreased photosynthesis in wfsl1 plants. These results showed that defective developmental of chloroplast affected the photosynthesis rate and grain yield in wfsl1.
In conclusion, this study suggested that WFSL1, which is essential in expression of plastid genes and plastid ribosome biogenesis, is important for chloroplast development.

Materials and Methods
Plant materials and growth conditions. The wfsl1 mutant was isolated from a M 2 population of the japonica rice ZH11 (wild type) after Ethyl methanesulfonate mutagenesis. The japonica rice ZH11 and wfsl1 mutant were grown in the paddy fields of Zhejiang (30°03′N, summer season, temperate climate) and Hainan (18°48′N, winter season, subtropical climate) provinces in China under local growing conditions. Chlorophyll analysis. The leaves from different stages of wild type and wfsl1 were collected and weighted.
Then, the leaves were soaked in an acetone and ethanol mixture solution at 26 °C in dark for 24 h. The content of chl a, chl b and carotenoids were calculated as described previously 45 . Transmission electron microscopy (TEM) analysis. The leaves of wild type and wfsl1 were selected at tillering stages and cut into small pieces. They were fixed by 2.5% glutaraldehyde (PH 7.2) and vacuumed until fully sinking to the bottom. Subsequently, samples were successively washed three times with 0.2 mol/L sodium cacodylate buffer for 30 min, fixed in 10% osmic acid for 1 h, distilled three times with deionized water for 45 min, dehydrated with ethanol, treated with acetone and embedded in epoxy resins and polymerized at 70 °C. The samples were then cut into about 500-800 Ǻ thick with a slicer and stained by the mixture of uranyl acetate dihydrate and lead citrate. The sections were washed with deionized water and visualized using HITACHI Transmission Electron Microscope (HT7700).

Genetic analysis and map-based cloning. For genetic analysis, we constructed the crosses between wfsl1
and the indica cultivars TN1, 93-11, SH527 (Shuhui527) to analyze whether a dominant/recessive single/multiple gene control(s) the wfsl1 phenotype. The segregation population of F 2 was examined by χ 2 test (Additional file 1: Table S2).
To map the genomic location of WFSL1, 1,900 mutants were selected from the F 2 population which was derived from the cross between wfsl1 and the indica cultivar NJ06 (Nanjing06). A total of 117 pairs of rice chromosome markers were used for primary mapping, and the sequence-tagged-site (STS) markers for fine mapping were developed based on the gap difference between the contig sequences of the japonica cultivar Nipponbare and the indica cultivar 93-11 (http://ensemblgenomes.org). WFSL1 was ultimately mapped to a 50.5 kb region on chromosome 1. The predicted region was PCR-amplified and sequenced to detect the mutation.

Sequence alignment of WFSL1 protein.
Using blastp program to search the protein sequence database at the NCBI with an E-value cut-off of 0.001. Using the Clustal Omega (http://www.ebi.ac.uk/Tools/msa/ clustalo/) to identify motif that are conserved in the alignment sequence.
Identifying the subcellular location of WFSL1. To investigate the subcellular location of WFSL1, a 1,743-bp fragment of full-length cDNA was amplified by PCR and ligated into pCAMBIA1300-GFP (containing 35 s promoter and GFP reporter protein) vector to generate the pCAMBIA1300-WFSL1-GFP construct. The PCR primers for WFSL1 were: 5′-gctctagaatgaaacatccctcccgcattaaattggc-3′ and 5′-cgggatcccggttgtaggttctcgaaggcttctg-3′. The pCAMBIA1300-WFSL1-GFP vector, as well as the control were transformed into rice protoplasts according to the protocol described previously 46 . The transformed rice protoplasts cells were incubated for 16 h at 28 °C under dark conditions. The GFP fluorescence in transformed protoplasts was examined by confocal fluorescence microscope (Carl Zeiss, LSM 780).
RNA extraction and qRT-PCR. Total RNA was extracted from the seedling and tillering stage of wild type and wfsl1 mutants using a Total RNA Extraction Kit (Axygene, cat No, AP-MN-MS-RNA-250) according to the manufacturer's instructions. The complementary DNA was synthesized using a ReverTra Ace qPCR-RT Kit (TOYOBA, Japan). RT-PCR was run in Applied Biosystems 7900HT Real-time System using 2 × SYBR Green PCR Master Mix (Applied Biosystems). The RT-PCR program was as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 1 min. The analysis of each sample was based on three technical replicates and biological replicates. The relative expression level of each transcript was compared with that of UBQ5 and quantified with the 2 −ΔΔC T method 47 . The primers for the genes are listed (Supplementary Table S3).
RNA-seq analysis. Total RNA was extracted from wild type and wfsl1 at tillering stage. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. cDNA was synthesized using random hexamer primers. The library was constructed and sequenced using an Illumina Hisequation 2000 (Novogene). A total of 45 million reads genes from wild type and 40 million from wfsl1 were obtained. The significance of differentially expressed genes (DEGs) were using log 2 (fold change) > 1 and q values < 0.05. Gene ontology analysis was performed on GOseq 48 . Pathway enrichment analysis was using the Kyoto Encyclopedia of Genes and Genomes database 49 .