OsDXR interacts with OsMORF1 to regulate chloroplast development and the RNA editing of chloroplast genes in rice

CAO Peng-hui ,WANG Di ,GAO Su ,LlU Xi ,QlAO Zhong-ying ,XlE Yu-lin,DONG Ming-hui,DU Tan-xiaoZHANG XianZHANG RuiJl Jian-hui

1 Institute of Agricultural Sciences in Taihu Area of Jiangsu,Suzhou 215155,P.R.China

2 Huaiyin Institute of Agricultural Sciences of Xuhuai Region in Jiangsu,Huai’an 223001,P.R.China

3 School of Life Sciences,Huaiyin Normal University,Huai’an 223300,P.R.China

4 Key Laboratory of Eco-Agricultural Biotechnology around Hongze Lake,Regional Cooperative Innovation Center for Modern Agriculture and Environmental Protection,Huaiyin Normal University,Huai’an 223300,P.R.China

Abstract Plant chlorophyll biosynthesis and chloroplast development are two complex processes that are regulated by exogenous and endogenous factors. In this study,we identified OsDXR,a gene encoding a reductoisomerase that positively regulates chlorophyll biosynthesis and chloroplast development in rice. OsDXR knock-out lines displayed the albino phenotype and could not complete the whole life cycle process. OsDXR was highly expressed in rice leaves,and subcellular localization indicated that OsDXR is a chloroplast protein. Many genes involved in chlorophyll biosynthesis and chloroplast development were differentially expressed in the OsDXR knock-out lines compared to the wild type.Moreover,we found that the RNA editing efficiencies of ndhA-1019 and rpl2-1 were significantly reduced in the OsDXR knock-out lines. Furthermore,OsDXR interacted with the RNA editing factor OsMORF1 in a yeast two-hybrid screen and bimolecular fluorescence complementation assay. Finally,disruption of the plastidial 2-C-methyl-derythritol-4-phosphate pathway resulted in defects in chloroplast development and the RNA editing of chloroplast genes.

Keywords: rice,OsDXR,Chloroplast development,RNA editing,OsMORF1

1.lntroduction

Isoprenoids are essential for plant growth and development,and tens of thousands of these compounds have been isolated from archaea,bacteria and eukaryotes (Rodríguez-Concepción 2014;Tarkowská and Strnad 2018). Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are two basic fivecarbon molecules required for isoprenoid biosynthesis.In higher plants,two isoprenoid synthetic pathways are located in plastids and the cytoplasm,namely the cytoplasmic methyl valerate (MVA) pathway and the 2-C-methyl-derythritol-4-phosphate (MEP) pathway,respectively (Rohdichet al.2001). Chlorophylls,gibberellins,and abscisic acids are synthesizedviathe MEP pathway (Okadaet al.2002). Previous studies have shown that DOXP reductoisomerase (DXR) can catalyze the conversion of 1-deoxy-D-xylulose-5-phosphate (DOXP) into MEP (Carretero-Pauletet al.2002). In peppermint,overexpression ofDXRincreases the biosynthesis of essential oils (Mahmoudet al.2001),while inArabidopsis,the T-DNA insertion mutant ofDXRwas albino and dwarf,it could not complete seedling establishment (Xinget al.2010),the number of trichomes was reduced,and stomatal closure was affected (Xinget al.2010). However,the function of DXR has not been reported in crop plants,including rice.

Posttranscriptional processing,including RNA editing,RNA splicing,RNA cleavage,and RNA stability,plays an important role in regulating plant chloroplast development and chlorophyll biosynthesis (Barkan and Goldschmidt-Clermont 2000). RNA editing converts cytidine nucleotides (C) to uridine (U) in the transcripts of plastidial and mitochondrial genes,and is affected by temperature,fungal infection,and stresses (Karcher and Bock 2002;García-Andradeet al.2013;Rodrigueset al.2017). Many studies have shown that pentatricopeptide repeat (PPR) proteins,multiple organellar RNA editing factors (MORFs),and thioredoxin z play important roles in plant RNA editing (Takenakaet al.2012;Huanget al.2020;Wanget al.2021).OsPGL1encodes a dual-localized PPR protein that affects chloroplast and mitochondrial RNA editing (Xiaoet al.2018).WP2encodes a thioredoxin z protein that affects the efficiency of RNA editing in many plastidial-encoded genes (Wanget al.2021). Recently,the MEP pathway geneOsHMBPP/OsHDRhas been shown to influence plastidic RNA editing and interact with OsMORF8 (Liuet al.2020),suggesting that the MEP pathway might affect plastidic RNA editing.

There is only one DXR in rice,OsDXR. In this study,OsDXR was identified and shown to be involved in regulating rice chlorophyll biosynthesis and chloroplast development. To elucidate the mechanism,we demonstrated that OsDXR interacts with the RNA editing factor,OsMORF1,resulting in reduced RNA editing of two chloroplast genes.

2.Materials and methods

2.1.Plant materials

Nipponbare,ajaponicavariety of rice (O.sativa),was used as the wild-type (WT) plant. We developed twoOsDXRknock-out lines through the CRISPR/Cas9 system (Luet al.2017). The mutation target(5′-TTCCTCGACTCCAACAG-3′) was constructed in a 1305-CRISPR plasmid vector using theAarI enzyme,and introduced into Nipponbare byA.tumefaciens-mediated transformation. Fragments containing the target were PCR amplified (5′-GAGTCTCAGATTCCCATCTCGTC-3′and 5′-CTGCGGATTATCTTGAAACAGG-3′) and the sequence was verified. All plant seedlings were grown in a growth chamber with a photoperiod of 14 h light and 10 h dark at 30/25°C,respectively.

2.2.Chlorophyll content analysis

Chlorophylls were extracted from 10-day-old WT andOsDXRknock-out leaves as described previously (Porraet al.1989) with some modifications. Briefly,～0.2 g of leaves harvested from WT andOsDXRknock-out plants were placed in 15-mL centrifugal tubes in 5 mL of extraction buffer (95% ethanol),and stored in the dark for 48 h. All pigmented solutions were combined and centrifuged for 2 min. The light absorbance of three biological replicates was measured at 663 and 647 nm.

2.3.Transmission electron microscopy (TEM)

Leaf samples of 10-day-old WT andOsDXRknock-out seedlings were collected and fixed in 4% glutaraldehyde,and then vacuum-treated for 1 h. The samples were dehydrated through a series of alcohol solutions,and imaged by TEM (Hitachi,Tokyo,Japan) as described previously (Liuet al.2020).

2.4.Sequence analysis

Protein sequences homologous to OsDXR were searched using the BLAST Ssearch Program (www.ncbi.nlm.nih.gov/BLAST/),and aligned using the DNAMAN Software.

2.5.Subcellular localization

To determine the subcellular localization of OsDXR,the full-length ORF ofOsDXRwas amplified with the primers 5′-CGGAGCTAGCTCTAGAATGGCGCTCAAGGTCGTC TC-3′ and 5′-TGCTCACCATGGATCCACAGGTACAGGG CTGA-3′ and introduced into pAN580-GFP at theXbaI andBamHI sites. Transformation was performed as described previously (Liuet al.2020). The GFP fluorescence of rice protoplasts was observed by confocal laser scanning microscopy (LSM700;Zeiss),and chlorophyll autofluorescence was used as a control.

2.6.RT-PCR and qPCR analysis

Total RNA was extracted from roots,stems,leaves,and panicles of WT plants with an RNA Prep Pure Plant Kit (Tiangen,Beijing,China). First-strand cDNA was reverse transcribed with an RT primer mix. Real-time PCR was performed using a SYBR Premix Ex TaqTMKit (TaKaRa,Japan) on a CFX96 Touch Real-Time PCR Detection System with three biological replicates.The primers used to analyze the expression level ofOsDXRwere 5′-AAACGAGGGACAGAAGAGCA-3′and 5′-GAACCGGTTGAGCCAACAAT-3′. The primers for chlorophyll biosynthesis (Zenget al.2020),chloroplast development-related genes (Wanget al.2017),and the MEP pathway genes (Liuet al.2020)were obtained as reported previously. The riceUBQgene was used as an internal control. The primers used forUBQwere 5′-GCTCCGTGGCGGTATCAT-3′ and 5′-CGGCAGTTGACAGCCCTAG-3′.

2.7.RNA editing assay

To analyze RNA editing,total RNA from 10-day-old WT andOsDXRknock-out plants were isolated and treated with DNaseI (CWBIO,Jiangsu,China). The RNAs were reverse transcribed with random primers. cDNA fragments containing RNA editing sites were amplified by RT-PCR. Primers were used to detect the RNA editing sites of chloroplast genes as described previously (Wanget al.2017). The RT-PCR products were sequenced directly,and C to T changes were compared using the BioXM 2.6 Software.

2.8.Yeast two-hybrid (Y2H) screen and bimolecular fluorescence complementation (BlFC) assay

The full-length cDNAs ofOsDXR(5′-CATGGAGGCCGAA TTCATGGCGCTCAAGGTCGTCTC-3′ and 5′-GCAGGTC GACGGATCCCTAGACAGGTACAGGGCTGA-3′) and seven MORF genes were amplified and cloned into theEcoRI andBamHI sites of pGBKT7 and pGADT7 vectors with a ClonExpress II One Step Cloning Kit (Nanjing Vazyme Biotech Co.,Ltd.,China),respectively. Different combinations of plasmids were introduced into the yeast strain,AH109,following the manufacturer’s protocol(Clontech,PT1172-1). The primers for the pGADT7 vector were obtained as reported previously (Liuet al.2021). For the BiFC assay,OsDXR(5′-CATTTACGAACG ATAGTTAATTAAATGGCGCTCAAGGTCGTCTC-3′ and 5′-CACTGCCACCTCCTCCACTAGTGACAGGTACAGG GCTGA-3′) andLOC_Os11g11020were cloned into pVYNE and pVYCE,respectively. VYL interacts with OsClpP4 in rice chloroplasts (Donget al.2013). YNOsDXR/YC-VYL and YN-OsClpP4/YC-OsMORF1 were used as the negative controls. Recombinant green fluorescence signals fromNicotianabenthamianawere examined as described previously (Waadtet al.2008).

2.9.Statistical analysis

In this study,all experiments were performed with three biological replicates. The results are presented as mean±SD in the figures,while＊and＊＊indicate significant differences atP＜0.05 andP＜0.01,respectively.

3.Results

3.1.Characterization of the OsDXR protein in rice

TheOsDXRgene consists of a 5 989-bp open-reading frame,comprising 12 exons and 11 introns. The OsDXR protein has 473 amino acids and a calculated molecular weight of 51 kD. The first 49 amino acids were predicted to be a chloroplast transit peptide by ChloroP (http://www.cbs.dtu.dk/services/ChloroP/). Multiple amino acid sequence alignments by DNAMAN Software indicated that OsDXR has similarities among many species (Fig.1),includingArabidopsisAt5g62790,with 80.1% sequence homology (Xinget al.2010). The results indicate that the OsDXR protein is highly conserved in plants.

Fig.1 Comparison of the amino acid sequences of five DXR homologs. The following sequences were compared: Oryza sativa LOC_ Os01g01710 (OsDXR),Setaria italica XP_004967950.1,Zea mays XP_008655547.1,Arabidopsis thaliana NP_201085.1,and Sorghum bicolor XP_021311303.1. The amino acids in dark blue are conserved.

3.2.Characterization of OsDXR knock-out mutants

To investigate the functions ofOsDXRin rice development,we developed twoOsDXRknock-out lines,dxr-1anddxr-2,by CRISPR/Cas9-targeted mutagenesis. Two and four bases were deleted indxr-1anddxr-2,respectively,leading to a frameshift mutation and premature termination (Fig.2-A;Appendix A).Subsequent analysis was performed usingdxr-2since it is the shortest mutant protein. In addition,we detected the potential off-target sites and did not find any mutations in any of the potential off-target sites (Appendix B). Bothdxr-1anddxr-2displayed an albino phenotype and ultimately died (Fig.2-B).OsDXRexpression was remarkably reduced in bothdxr-1anddxr-2(Fig.2-C). Consistent with the albino leaves,the contents of chlorophyllaandbin the mutants were significantly reduced compared with the WT (Appendix C).

Fig.2 Disruption of the OsDXR gene results in chlorophyll biosynthesis defects in rice. A,sketch map of the target gene,OsDXR,and the mutations of the knockout lines. The 19-bp gene-specific target site and protospacer adjacent motif (PAM)are underlined and shaded red,respectively. Red lines represent the missing bases. B,the phenotypes of dxr-1 and dxr-2 at the seedling stage (bar=5 cm). C,the expression levels of OsDXR in the dxr mutants. Bars mean SD (n=3). ＊＊ indicates significant differences at P＜0.01.

In order to determine whether the structure of chloroplasts was affected in thedxr-2mutant,we used TEM to observe the structures of chloroplasts in the WT anddxr-2seedlings. Chloroplast morphology of the WT seedlings was normal,and the thylakoid lamellae showed an orderly arrangement (Fig.3-A and B). However,the chloroplasts in thedxr-2mutant were abnormal,and completely lacking thylakoid lamellae(Fig.3-C and D). These results suggest thatOsDXRis essential for chloroplast development in rice.

Fig.3 Chloroplast ultrastructure of wild-type (WT) (A and B) and dxr (C and D) leaves. A and C,bars=2 μm;B and D,bars=500 nm.

3.3.Expression pattern and subcellular localization of OsDXR

To examine the expression pattern ofOsDXR,we first analyzed the expression ofOsDXRin the rice expression database (http://bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi). As shown in Appendix D,OsDXRis expressed in various organs,including leaves,panicles,and seeds.To verify this result,we extracted RNA from the panicles,stems,leaves,and roots,and used quantitative RT-PCR to analyze the expression ofOsDXRin each tissue.OsDXRwas highly expressed in leaves and stems (Fig.4-A).To determine the subcellular localization of the OsDXR protein,we generated a transient expression system in rice protoplasts. Strong green fluorescence signals from OsDXR were co-localized with the chloroplast (Fig.4-B),indicating thatOsDXRis constitutively expressed in various tissues and that the OsDXR protein localizes to chloroplasts,providing additional evidence for its role in chloroplast development.

Fig.4 Expression analysis and subcellular localization of OsDXR. A,expression levels of OsDXR in various rice organs. Bars mean SD (n=3). B,localization of the OsDXR-GFP protein in rice protoplasts. Bar=10 μm.

3.4.Expression levels of chlorophyll biosynthesis and plastid development-related genes were altered in the dxr mutant

Genes expressed in plastids by the nucleus and plastid coordinate together to produce normal chloroplasts and synthesize chlorophyll. To examine whether the loss of function ofOsDXRaffects the expression of chlorophyll biosynthesis and chloroplast developmentrelated genes,we performed qRT-PCR to analyze the expression levels of these genes in WT anddxrplants.The qRT-PCR analysis indicated that the expression levels of genes related to chlorophyll biosynthesis and chloroplast development were significantly altered in thedxrmutant. For instance,the expression levels of several chlorophyll biosynthetic genes,includingHEMA,DVR,CHLMandPORA,were significantly reduced indxrcompared to WT (Fig.5). Additionally,the expression levels of several plastid development-related genes,includingrbcL,NDHB,andV1,were significantly reduced indxrcompared to WT. However,the expression of some chlorophyll biosynthesis genes,such asCRDandCHLG,and the plastid development-related gene,psaA,increased relative to the WT (Fig.5).

Fig.5 Expression levels of chlorophyll biosynthesis and plastid development-related genes in wild type (WT) and dxr-2 leaves.Total RNA was extracted from the leaves of WT and dxr-2 mutant plants. Values are presented as the mean±SD of three biological replicates. ＊ and ＊＊ indicate significant differences at P＜0.05 and P＜0.01,respectively.

To investigate whether the expression levels of the MEP pathway genes were affected by theOsDXRmutation,we used qRT-PCR to measure the expression levels of the other genes of the MEP pathway. Compared with the wild type,the expression levels ofCMK,DXS,HDS,CMS,andMCSwere significantly down-regulated(Appendix E).

3.5.RNA editing of rpl2-1 and ndhA-1019 were impaired in the dxr mutants

Previous studies have shown that RNA editing and other posttranscriptional modifications are involved in the regulation of plant chlorophyll synthesis and chloroplast development (Tanget al.2017;Cuiet al.2019). To determine whether RNA editing is altered in theOsDXRmutants,we examined the editing efficiency of 18 editing sites in the rice chloroplast genome in WT,dxr-1,anddxr-2seedlings. The editing efficiencies ofrpl2-1 andndhA-1019 were greatly reduced (Fig.6). Chloroplastrpl2andndhAencode ribosomal protein subunit L2 and NADPH dehydrogenase,respectively.rpl2participates in the peptidyl-transferase reaction in theEscherichiacoliribosome,andndhAinfluences NADH dehydrogenase(NDH) activity (Nierhaus 1982;Linet al.2017). The other 16 editing sites displayed normal editing in the WT anddxrmutants (Appendix F).

Fig.6 RNA editing analyses of rpl2-1 and ndhA-1019 in wild type (WT),dxr-1,and dxr-2. Nucleotides shown in red letters indicate the editing sites.

3.6.OsDXR interacted with the multiple organellar RNA editing factor OsMORF1

To explore the function of OsDXR,we performed a Y2H screen to identify OsDXR-interacting proteins.We constructed a yeast cDNA library from Nipponbare seedlings and screened the yeast using OsDXR as a target. We obtained 58 colonies that grew well on media lacking Leu/Trp/His/Ade,and found that five of those colonies corresponded to rice LOC_Os11g11020.Additionally,we found an orthologous gene ofLOC_Os11g11020inArabidopsisnamed multiple organellar RNA editing factor 1 (MORF1). Hence,we referred to the rice LOC_Os11g11020 as OsMORF1. Since there are seven MORF genes in the rice genome,we used the Y2H assay to assess the interactions between OsDXR and the other six rice MORF proteins,and only observed an interaction between OsDXR and OsMORF1 in the Y2H assay (Fig.7-A). Moreover,using the BiFC assay withN.benthamiana,a green fluorescence signal was only observed in the protein group of OsDXR/OsMORF1,compared with the YN-OsDXR/YC-VYL and YN-OsClpP4/YC-OsMORF1 combinations (Fig.7-B). Therefore,the Y2H and BiFC assays revealed that OsDXR specifically interacts with OsMORF1.

Fig.7 Interaction identification of OsDXR with OsMORF1. A,yeast two-hybrid assay of the interaction between OsDXR and OsMORF1.DDO and QDO indicate SD-Leu/-Trp dropout plates and SD-Leu/-Trp/-His/-Ade dropout plates,respectively. QDO contains 40 μg mL-1 X-α-Gal. B,BiFC assays of the interaction between OsDXR and OsMORF1 in Nicotiana benthamiana leaves. Bar=10 μm.

4.Discussion

OsDXRgenes have been isolated from many plants,includingArabidopsis,peppermint,and mint,through T-DNA insertion mutants,homologous cloning,and transgene overexpression (Lange and Croteau 1999;Mahmoud and Croteau 2001;Xinget al.2010). So far,only three genes involved in the MEP pathway,IspE,IspFandOsHMBPP,have been cloned,but noOsDXRgene has yet been identified in rice (Chenet al.2018;Huanget al.2018;Liuet al.2020). In rice,numerous genes responsible for an albino phenotype have been isolated,such asOsCAF1,YSA,OsPPR16,andRA1(Suet al.2012;Zhanget al.2019;Zhenget al.2019;Huanget al.2020).In this study,we constructed twoOsDXRknock-out mutants which exhibited the albino phenotype and had abnormal chloroplasts. In the two mutants,deletions of two and four bases in theOsDXRgene resulted in a frameshift mutation and premature termination.

InArabidopsis,the T-DNA insertionDXRmutant(Xinget al.2010) displays an albino phenotype,grows purple cotyledons,and exhibits impaired chloroplast development. Mutations in several genes of the MEP pathway,includingDXS,IspD,IspE,IspF,IspGandIspH,also show the albino phenotype (Hsieh and Goodman 2005,2006;Hsiehet al.2008;García-Alcázaret al.2017). These studies indicated that cytoplasmic isoprenoids from the MVA pathway could not effectively compensate for the lack of plastid isoprenoids inArabidopsis. In rice,theIspEmutant,gry340,showed a green-revertible phenotype,while theIspFmutant,505ys,exhibited a yellow-green phenotype during the whole growth period (Chenet al.2018;Huanget al.2018). RiceIspEandIspFmutants could set seeds at maturity,whereas theArabidopsisIspEandIspFmutants died at the seedling stage. The riceIspHmutant,las1,had an albino phenotype,which was consistent with the phenotype of theArabidopsisIspHmutant (Hsieh and Goodman 2005;Liuet al.2020). In rice,theDXRmutants died at the seedling stage and could not complete the whole life cycle,which was different from theArabidopsisDXRmutants. These results suggest a divergence in the functions of the MEP pathway genes in the dicotyledonArabidopsis thalianaand the monocot rice. In addition,we found that there are 112 SNPs and 9 InDel variants in the genome ofOsDXRwith the software RiceVarMap V2.0. Based on the SNP variants ofOsDXR,15 subhaplotypes ofOsDXRwere found (Appendix G),suggesting that the genetic diversity ofOsDXRis rich.

TheIspH/LAS1mutation affects plastdic RNA editing,and IspH/LAS1 interacts with the MORF family protein Os09g33480 (Liuet al.2020). In this study,we found that the RNA editing efficiencies ofrpl2-1 andndhA-1019 in thedxrmutants were significantly reduced compared with the WT. In addition,OsDXR interacted with OsMORF1invivo,suggesting that OsDXR may be a component of an RNA editing complex. These results indicate that proteins in the MEP pathway might regulate rice chlorophyll biosynthesis and chloroplast developmentviatheir interactions with the MORF family of proteins,which will be the focus of our future research.

5.Conclusion

We characterized two rice OsDXR mutants and confirmed thatOsDXRpositively regulates chloroplast development in rice. The results suggest thatOsDXRmay be involved in regulating the expression of chlorophyll biosynthesis and plastid development-related genes,and that it interacts with the RNA editing factor OsMORF1.

AcknowledgementsThis study was supported by the Program for Subsidized Project of Suzhou Academy of Agricultural Sciences,China (20028),the Science and Technology Foundation of Suzhou (SNG2020048),the Huaishang Talents,China,the National Natural Science Foundation of China(32070345),the Huai’an Academy of Agricultural Sciences Initiation and Development of Scientific Research Fund for High-level Introduced Talents,China (0062019016B),the Six Talents Summit Project of Jiangsu Province,China(NY-129),and the Natural Science Foundation of Jiangsu Province,China (BK20190239 and BK20180107).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm