LIU Xi,YI Xin,YANG Yan-rong,HUANG Qian-qian

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

Abstract Chloroplasts are essential for plant photosynthesis and growth. Many genes have been identified that regulate plant chloroplast development. However,it is not known at a molecular level how these genes regulate chloroplast biogenesis.In this study,we isolated a mutant ygl2 (yellow-green leaf2) that exhibited a pigment-defective phenotype. YGL2 encodes a geranylgeranyl reductase,and in mutant ygl2,there was a single base change (T1361G) located in the third exon of YGL2that resulted in a missense mutation (L454R) in the encoded product. Transmission electron microscopy revealed that chloroplast development was impaired in the ygl2 mutant. The expression levels of plastid-encoded genes were significantly altered in the ygl2 mutant. Furthermore,in a yeast two-hybrid assay,we found that YGL2 interacted with the RNA editing factor MORF8.

Keywords:rice (Oryza sativa L.),chloroplast,YGL2,geranylgeranyl reductase

In higher plants,RNA editing converts more than 400 cytidines (C) to uridines (U) in mitochondrial genes and another 30-40 cytidines in plastid genes (Corneilleet al.2000;Li and Mason 2014). Multiple organellar RNA editing factor (MORF) proteins affect RNA editing in plants. TheArabidopsisgenome encodes nine MORF proteins.ArabidopsisMORF2 and MORF9 are targeted exclusively to chloroplasts,whereas MORF8/RIP1 is targeted to both chloroplasts and mitochondria (Bentolilaet al.2012;

1.Introduction

Chloroplasts are semi-autonomous organelles,referred to as plastids;they have their own genome,which encodes 100 genes (Sakamotoet al.2008). Chloroplasts develop from proplastids,and,in higher plants,chloroplast development can be subdivided into three stages which are regulated by plastid and nuclear-encoded genes (Jarvis and Lopez-Juez 2013;Pogsonet al.2015). The coordinated expression Takenakaet al.2012). Only one rice MORF gene has been reported,WSP1 (Zhanget al.2017),which is localized to chloroplasts and affects the editing of multiple organellar RNA sites (rpoB,Rps14,ndhG,andndhD). However,the molecular mechanisms that regulate plant RNA editing remain unknown,particularly in rice.

Geranylgeranyl reductase (GGR) is essential for vitamin E and chlorophyll biosynthesis. GGR participates in the reduction of geranylgeranyl pyrophosphate (GGPP) to phytate pyrophosphate (PPP) and geranylgeranylchlorophyll to chlorophyll. There is only one GGR gene inArabidopsis thaliana. Two rice genes are homologous toArabidopsisGGR,namely,Os02g0744900(OsGGR1) andOs01g0265000(OsGGR2). OsGGR1 and OsGGR2 share 64 and 53% DNA and amino acid sequence identity,respectively (Kimuraet al.2018). Mutation ofLYL1/OsGGR1results in a yellow-green leaf phenotype and influences chloroplast development (Zhouet al.2013).However,it is not known at the molecular level howLYL1/OsGGR1regulates chloroplast activity.

In this study,we characterized a riceyellow-green leaf 2(ygl2) mutant that displayed yellow-green leaves throughout the entire growth period.YGL2encodes a geranylgeranyl reductase that is targeted to chloroplasts.Expression analyses suggested thatYGL2coordinates expression of many nuclear-and plastid-encoded genes involved in chloroplast development. Our work provides significant new understanding of the function ofYGL2in regulating rice chloroplast development.

2.Materials and methods

2.1.Plant materials and growth conditions

Theygl2mutant was identified from tissue cultures ofjaponicacultivar Dongjin. F2mapping populations were generated fromygl2×Nanjing 11 (indica). Plants were grown either in a paddy field in Huai’an,Jiangsu Province,China or in a growth chamber with a 14-h photoperiod/10 h dark cycle at 30°C/25°C.

2.2.Map-based cloning of YGL2

Sequence polymorphisms between Nipponbare (japonica) and 93-11 (indica) were identified from the National Center for Biotechnology Information (NCBI;http://www.ncbi.nlm.nih.gov/). We used the Primer Premier 5.0 Software to develop new molecular markers for mapping. The primers used in this study are listed in Appendix A. The PCR procedure was as follows:94°C for 3 min,followed by 30 cycles of 94°C for 30 s,annealing at 55°C for 30 s,72°C for 1 min,and a final elongation step at 72°C for 5 min.

2.3.Chlorophyll measurement and transmission electron microscopy (TEM) analysis

Fresh leaf tissues (～200 mg) fromygl2and WT were immersed in 10 mL extract solution for 48 h in the dark. The absorbance of the supernatant was measured with a dual-beam ultraviolet spectrophotometer (TU1901,Beijing) at 645 and 663 nm. Chlaand Chlbcontents were measured according to Porraet al.(1989). The data are expressed as the mean±SD of three biological replicates.

For TEM analysis,flag leaves sampled from the 3-leafstage WT andygl2seedlings were fixed in a solution of 2.5% glutaraldehyde. Samples were prepared as described (Liuet al.2018) and observations were performed with a JEOL JEM-1230 EX (Tokyo,Japan) TEM.

2.4.Structure prediction and sequence alignment analysis

Homologous protein sequences of YGL2 were identified by NCBI BLAST. The structure analysis was performed with the I-TASSER algorithm (https://zhanglab.ccmb.med.umich.edu/I-TASSE/). Protein sequences were aligned using BioXM version 2.6 Software.

2.5.Subcellular localization analysis

The full-length coding sequence ofYGL2was PCR-amplified from the wild type and introduced into vector pAN580-GFP to form the YGL2-GFP and GFP-YGL2 constructs. The transient expression constructs were transformed into rice protoplasts and incubated in darkness according to published protocols (Wanget al.2017). GFP fluorescence was visualized with a confocal laser scanning microscope (Zeiss,LSM700).

2.6.RT-PCR and quantitative real-time PCR (qRTPCR) analysis

Total RNA was extracted from different rice tissues using an RNA Prep Pure Plant Kit (CWBIO Co.,Beijing,China). First strand cDNAs were synthesized from 2 μg total RNA using a PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa,Japan).qRT-PCR was performed with a CFX96 Touch Real-time PCR Detection System using the SYBR PremixEx TaqTMKit (TaKaRa). The riceUBQgene was used as an internal control. The primers are listed in Appendix A. The data are expressed as the mean±SD of three biological replicates.

2.7.RNA editing analysis

Specific cDNA fragments were generated by RT-PCR amplification. The cDNA sequences were compared to identify C-to-T changes resulting from RNA editing.All primers for RNA editing were obtained as reported (Wanget al.2017;Zhanget al.2017).

2.8.Yeast two-hybrid analysis

The full-length coding sequences of YGL2 and MORF8 were amplified and inserted into the bait (pGBKT7) and prey (pGADT7) vectorsviathe ClonExpress®II One Step Cloning Kit (C112-02,Vazyme,Nanjing,China). Yeast transformation and screening procedures were performed according to the Clontech protocol. Primers are listed in Appendix A.

3.Results

3.1.Characterization of the ygl2mutant

We isolated a mutant with a pigment-defective phenotype from tissue cultures ofjaponicacultivar Dongjin. The mutant was designated asyellow-green leaf2(ygl2). The yellow-leaf phenotype ofygl2appeared at the seedling stage and persisted to plant maturity (Fig.1-A;Appendix B). Compared with the wild type,chlorophyllaandbcontents were greatly reduced in theygl2mutant at the seedling,tillering and heading stages (Fig.1-B-D). Four agronomic traits (plant height,tiller number,panicle length,and spikelets per panicle) were markedly reduced inygl2(Appendix C). These results suggested that theYGL2mutation impaired normal chlorophyll synthesis and affected plant growth.

Fig.1 Phenotypic characteristics of ygl2. A,phenotypes of 2-wk-old wild type (WT) and ygl2 seedlings. Scale bar,1 cm. B,pigment contents of 2-wk-old WT and ygl2 seedlings. C,pigment contents of WT and ygl2 at the tillering stage. D,pigment contents of WT and ygl2 at the heading stage. Chl,chlorophyll. Error bars represent the SD from three independent biological replicates.

We used TEM to examine the chloroplast ultrastructure of WT andygl2at the three-leaf stage. WT plants had mature chloroplasts with a normal morphology and well-developed thylakoid structure (Fig.2-A and B). The chloroplasts ofygl2showed an abnormal morphology,with deformed thylakoid membranes (Fig.2-C and D). These observations indicated thatYGL2has an important function in rice chloroplast formation.

Fig.2 Ultrastructure of chloroplasts from wild type (WT) and ygl2 leaves.A and B,chloroplasts from WT leaves. C and D,chloroplasts from ygl2 leaves. Scale bars,5 μm in A and C; 1 μm in B and D.

3.2.Map-based cloning and expression analysis of YGL2

To isolate theYGL2gene,we generated an F2population fromygl2×Nanjing 11 (indica). We initially mappedYGL2to the long arm of chromosome 2,between the simple sequence repeat (SSR) markers RM13915 and RM14001 (Fig.3-A). Ultimately,we located theYGL2locus to between RM13952 and insertion-deletion marker Y-1,which had a 284-kb interval.Sequencing analysis in the target region revealed a single nucleotide substitution in the third exon ofLOC_Os02g51080(Fig.3-A). This polymorphism changed a leucine residue to arginine (Fig.3-A). To verify that the nucleotide substitution was responsible for theygl2mutant phenotype,the full-length coding sequence ofYGL2plus approximately 2.5 kb of 5´ sequence was inserted into the pCUbi1390 vector and transformed into theygl2callus byAgrobacterium. Phenotypic observations showed that transgenic lines rescued the mutant phenotype (Fig.3-B).These findings suggested that the substitution inLOC_Os02g51080was responsible for the pigment-defective phenotype ofygl2.

Fig.3 Map-based cloning of YGL2. A,the YGL2locus was mapped to a 284-kb region between markers RM13952 and Y-1,on chromosome 2L. Candidate gene YGL2(LOC_Os02g51080) is shown by the red arrow. ATG and TGA represent the start and stop codons,respectively. Black boxes indicate the exons. An SNP in the third exon in YGL2 causes a leucine-to-arginine amino acid substitution. B,complementation of ygl2by transformation. WT,wild type;Com,complementation.

The expression profile ofYGL2was predicted using the Rice eFP Browser (http://bar.utoronto.ca/efprice/;Appendix D).YGL2was expressed in various organs at different growth stages,with higher levels in leaves and panicles. To investigate the expression pattern ofYGL2in rice,we detected theYGL2transcript by qRT-PCR of RNA from roots,leaf sheaths,stems,young leaves,panicles,and seeds.YGL2transcript abundance was high in young leaves,leaf sheaths and stems,and low in panicles,roots and seeds (Fig.4-A). Also,we found that the peak ofYGL2expression appeared during the alternation of the light and dark periods (Fig.4-B).

Fig.4 Expression pattern analysis of YGL2. A,qRT-PCR analyses of YGL2 in various tissues of wild type (WT). YL,young leaf;LS,leaf sheath. B,diurnal expression of YGL2in leaves. White box,light;black box,darkness. Bars mean SD (n=3).

3.3. YGL2encodes a chloroplast-targeted geranylgeranyl reductase

To identify an effect of the single nucleotide substitution on ygl2 protein structure,we used the I-TASSER algorithm (https://zhanglab.ccmb.med.umich.edu/I-TASSER/).Compared with the wild type,the three-dimensional structure of ygl2 was altered (Fig.5-A).YGL2was isolated previously as a nuclear-encoded protein that controls rice leaf color (namedLYL1;Zhouet al.2013). Multiple protein sequence alignment analysis indicated that YGL2 is conserved betweenZea mays,Arabidopsis thalianaandSorghum bicolor(Fig.5-B).

Fig.5 Evolutionary analysis of YGL2 protein. A,structure prediction of YGL2 and ygl2 by the I-TASSER algorithm. B,amino acid sequence alignment of YGL2 homologs from Arabidopsis thaliana(NP_177587),Sorghum bicolor(XP_002454287) and Zea mays(NP_001149382). Amino acids fully or partially conserved are shaded black and pink,respectively. The amino acid difference between YGL2 and ygl2 is marked with a red arrow. The numbers indicate the amino acid positions.

ChloroP analysis suggested that YGL2 has a 37 amino acid chloroplast transit peptide at the N-terminal (http://www.cbs.dtu.dk/services/ChloroP/;Appendix E). To determine the subcellular localization of YGL2,we performed transient expression assays of a YGL2-GFP and GFP-YGL2 fusion in rice protoplasts. The YGL2-GFP signal merged fully with chlorophyll autofluorescence,and the GFP fluorescence of GFP-YGL2 was mostly focused in the cytoplasm,as was the free GFP,indicating that YGL2 localizes to chloroplasts (Fig.6).

Fig.6 Subcellular localization of YGL2. Green fluorescence shows GFP,red fluorescence shows chlorophyll and orange indicates the two types of fluorescence combined.

3.4.Mutation in YGL2 affects plastid gene expression

To investigate whether the mutation inYGL2affected plastid gene expression,we used qRT-PCR to measure the transcript levels of plastid genes inygl2. As shown in Fig.7,the expression levels of plastid genes were significantly altered between the wild type andygl2. Compared with the wild type,the transcript levels of plastid genes that are transcribed by plastid-encoded RNA polymerase (PEP),such aspsaA,ndhAandrbcL,were greatly reduced inygl2.However,the transcript levels of plastid genes transcribed by NEP (nuclear-encoded polymerase),such asrps8,rpoA,rpoB,rpoCandrpoC2,were increased inygl2(Fig.7).These results suggested thatYGL2is essential for proper expression of plastid genes.

Fig.7 Expression of plastid genes in wild type and ygl2by qRT-PCR. Three independent experiments were analyzed. Bars mean SD (n=3).

3.5.YGL2 interacts with MORF8 in yeasts

To understand the molecular function of YGL2,we performed yeast two-hybrid screening of a rice cDNA library prepared from young rice leaves. We found that MORF8 strongly interacted with YGL2 (Fig.8). Also,we found that ygl2 interacted with MORF8 in yeasts (Fig.8). MORF proteins are involved in the RNA editing of plant chloroplast and mitochondrial genes (Hammaniet al.2009;Takenakaet al.2010;Zhanget al.2017). Thus,we speculated that the mutation in YGL2 affected chloroplast RNA editing. To date,21 rice chloroplast RNA editing sites have been reported (Corneilleet al.2000). Among them,20 sites were located in gene coding regions and one was found in an untranslated region. Editing is non-synonymous at all the protein-coding regions. There were no differences in chloroplast RNA editing between the wild type andygl2(Appendix F). These results suggested that theYGL2was not required for RNA editing of rice chloroplast genes.

Fig.8 Yeast two-hybrid assay of YGL2 and MORF8. YGL2 interacts with MORF8 in yeasts. DDO,control medium (SDLeu/-Trp);QDO,selective medium (SD-Leu/-Trp/-His/-Ade). Empty pGBKT7 and pGADT7 vectors served as negative controls.

4.Discussion

4.1. YGL2 is essential for rice chloroplast development

Most leaf color mutants are defective in chloroplast development,and their photosynthetic efficiencies are greatly reduced,suggesting that chloroplast function is coordinated with normal plant growth and yield (Wanget al.2016;Niuet al.2017;Zhanget al.2017;Chenet al.2018). Theygl2mutant showed a yellow-green leaf phenotype at the seedling stage that persisted to the mature stage (Fig.1-A). Chlorophyll content in the mutant was greatly reduced compared with the wild type (Fig.1-B).Complementation analysis indicated that disruption ofYGL2causes the pigment-defective phenotype. The N-terminal fused YGL2-GFP was located in chloroplasts,while the empty GFP and C-terminal fused GFP-YGL2 were both located in the cytoplasm (Fig.6),indicating that the protein signal of YGL2 was located in the N-terminal and the YGL2 protein was targeted to chloroplasts. These findings indicated thatYGL2has important functions in rice chlorophyll biosynthesis and chloroplast development;this conclusion was also supported by the tissue-specific expression and subcellular localization of YGL2.

Zhouet al.(2013) reported a yellow-leaf mutantlyl1-1,an allele ofygl2. Consistent with our observations,thelyl1-1mutant also exhibited a yellow-green phenotype (Zhouet al.2013). However,ygl2had some molecular features and phenotypes distinct fromlyl1-1. The yellow-green phenotype ofygl2persisted throughout the entire growth period and some agronomic traits were changed. The young leaves from leaf sheaths oflyl1-1were green andlyl1-1was hypersensitive to strong light. Compared with the wild type,the expression levels of plastid-encoded genes were altered inygl2. The phenotype and molecular feature differences betweenygl2andlyl1-1might have been due to amino acid substitutions at different sites.

4.2.YGL2 is not required for chloroplast RNA editing in rice

Chloroplast and mitochondrial RNA editing events are essential for plant growth and development. Also,the editing events may be an indication of evolutionary adaptiveness (Chu and Wei 2019). Many PPR and MORF proteins are involved in chloroplast and mitochondrial RNA editing,especially inArabidopsis thaliana,maize and rice (Wanget al.2017;Zhanget al.2017;Cuiet al.2018;Liet al.2018).Our yeast two-hybrid assay demonstrated that YGL2 and ygl2 interacted with MORF8 (Fig.8),indicating that the SNP substitution mutation did not influence the interaction between YGL2 and MORF8. The SNP substitution mutation might have influenced the stability and protein level of the interacting proteins. However,we found thatygl2was not defective in chloroplast RNA editing,when compared with the wild type (Appendix F). Further studies should focus on the biological significance of the interaction between YGL2 and MORF8.

4.3.YGL2 has an important function in regulating plastid gene transcript abundance

We found by plastid gene expression analysis ofygl2that the transcript levels of PEP-dependent genes were dramatically reduced,whereas levels of NEP-dependent genes were increased (Fig.7). This finding indicated thatYGL2has an important function in regulating plastid gene expression. The opposite effects of reduced PEP transcript levels and increased NEP transcript levels have also been reported in PEP-related mutants (Pfalzet al.2006;Zhouet al.2009;Steineret al.2011). Therefore,the impaired chloroplast development inygl2may have been due to reduced PEP activity. TheArabidopsisortholog of YGL2 interacts with two light-harvesting-like proteins,LIL3:1 and LIL3:2,which regulate chlorophyll and tocopherol biosynthesis (Tanakaet al.2010). Thus,it is possible that YGL2 interacts with the corresponding rice light-harvesting-like proteins to regulate chloroplast development.

5.Conclusion

We have isolated a novel pigment-defective rice mutant,ygl2,that resulted from a mutation in the gene encoding a geranylgeranyl reductase. Our results demonstrated thatYGL2is required for plastid gene expression and normal chloroplast development. Further,we found that YGL2 interacted with MORF8 in yeasts,but the mutation did not affect plastid RNA editing. Thus,YGL2is essential for rice plastid gene expression and chloroplast development.

Acknowledgements

The authors would like to thank AiMi (www.aimieditor.com) for providing linguistic assistance. This work was supported by the National Natural Science Foundation of China (31900388),the Natural Science Foundation of Jiangsu Province,China (BK20191055) and the Natural Science Foundation of Colleges of Jiangsu Province,China (19KJB180009).

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