Cloning And Functional Analysis Of Var2 Genetic Modifier In Arabidopsis

In higher plants,chloroplasts are the site for photosynthesis and produce an array of fundamental molecules.The biogenesis and proper development of chloroplast is pivotal for the plant growth and development.The biogenesis and development of chloroplast is regulated by a number of biological processes.The Arabidopsis var2 mutant exhibits a unique leaf variegation phenotype because of lacking FtsH2.Variegation mutants have used as an ideal model to elucidate the mechanism of chloroplast development since the severity of variegation depends on developmental and environmental cues.Our laboratory are interesting in characterizing second-site var2 modifier including (?)UPPRESSOR OF (?)A(?)IEGATION(SVR)and (?)NHANCER OF (?)A(?)IEGATION(EVR)to further understand the mechanism of the chloroplast development regulation.In this study,we report the cloning two genetic enhancer genes,EVR1 and EVR2 using activation tagging screening.We performed to investigate the functions of EVR1 and EVR2 in the enhancing of var2-mediated leaf variegation,all these works in this study was aim to provide insightful information regarding VAR2 role on the chloroplast development regulation.(1)In our large-scale screens for var2-5 modifier,two genetic enhancer,evr1-1 and evr2-1 were isolated.evr1-1 displayed delayed growth and pointed leaf and evr2-1 exhibited pale green phenotype.Genetic analysis showed that both mutants were able to enhance the var2-4 leaf variegation.(2)Thermal asymmetric interlaced PCR was used to identify of the EVR1/At3g53890.Semi-quantitative RT-PCR showed that the expression of At3g53890 declined in evr1-1 and enhancer line,098-004.The transformation of homozygous evr1-1 plants and enhancer line 098-004 with P35S:At3g53890g completely restored the wild type phenotype and the phenotype of leaf variegation similar to var2-5,respectively.These results confirmed that At3g53890 is EVR1 that reduction of At3g53890 expression is the cause for the enhancement of var2 leaf variegation in 098-004,and the leaf developmental phenotypes of evr1-1.(3)The At3g53890 gene was annotated as the cytosolic RPS21 B,a protein of the small 40 S ribosomal complex subunit.Arabidopsis ribosomal proteins(r-proteins)are were encoded by the gene families,and RPS21 family comprises two functional members,including RPS21B/EVR1 and RPS21C/EVR1L1.Bioinformatics analysis indicated that these two members shared a high degree of amino acid identity,and EVR1 and EVR1L1 protein were localized to cytoplasm and nucleus using protein fusions to green fluorescent protein(GFP)transient assay analysis.(4)The results using semi-quantitative RT-PCR and histochemical staining showed that EVR1 and EVR1L1 were ubiquitously expressed in tissue examined and demonstrated a higher expressed quantity in the meristem especially.And EVR1 expression pattern in the leaves was regulated by the development time.(5)The homozygous evr1l1-1 displayed growth retardation and pointed leaf similar to evr1-1,but the phenotypes of evr1l1-1 single mutant were less severe than evr1-1.evr1l1-1 was also able to enhance the var2-5 or var2-4 leaf variegation.Consistent with the weaker overall developmental phenotype of evr1l1-1 compared to evr1-1,the enhancement of var2 variegation by evr1l1-1 was also less pronounced than that of evr1-1.Transgenic lines overexpression of EVR1L1 in evr1l1-1 backgrounds showed wild type phenotype.(6)EVR1 and EVR1L1 are functionally redundant.Genetic analysis showed that nonallelic non-complementation was observed in both evr1-1 and evr1l1-1 mutant.The result of genetic analysis between evr1-1 and evr1l1-1 suggests that the RPS21 family requires at least two active gene copied for plant viability.Over-expressing EVR1L1 in evr1-1 or 098-004 can complemented the developmental phenotypes of single mutant or leaf variegation phenotype of double mutant,respectively.Transgenic lines overexpression of EVR1 in evr1l1-1 and evr1l1-1 var2-5 backgrounds showed wild type and var2-5 phenotypes,respectively.Additionally,epidermal specific over-expression of EVR1 or EVR1L1 in evr1-1 and 098-004 also complemented the developmental phenotypes or leaf variegation phenotype,respectively.(7)The result using semi-quantitative RT-PCR showed that four FtsHs genes had no discernible alteration of the transcripts level in enhancer line and evr1-1 mutant compared to wild type and var2-5.The result of immunoblots analysis showed that EVR2 and chloroplast protein encoded by nuclear genome had no discernible alteration,whereas that encoded by plastid genome were decreased in 098-004.(8)EVR1 regulates many auxin-related developmental phenotype.evr1-1 and evr1l1-1 mutant exhibited pointed first leaf and retarded growth in early development stage,abnormal cotyledon veins,reduced root and hypocotyl length.(9)Genetic analysis showed that the enhancing effect observed in var2-5 for RPS21 family were a common feature of different ribosomal protein mutant.The enhancing effect was different between 40 S and 60 S subunit mutants.The small subunit ribosomal protein mutants showed dramatic stronger leaf variegation enhancement than large subunit ribosomal protein mutants and the degree of variegation enhancement was positively related with the severity of 40 S subunit single mutants.Cycloheximide did not result in significant enhancement of variegation in var2,suggesting that activities mediated by 40 S subunit probably play more important roles in VAR2-mediated chloroplast biogenesis pathway.(10)evr1-1 showed weak enhancement of var1-1 and thf1-2 leaf variegation.(11)Genetic analysis showed that evr1-1 mutant have no effect on color phenotype of var2 suppressors that defective in chloroplast translation,such as svr8,svr9,and prpl11-1.But the leaf variegation phenotype of triple mutant is associated with the severity of the var2 suppressors mutant when evr1-1 combined var2-5 or var2-4 and var2 suppressors,indicating that the enhancement of var2 leaf variegation by cytosolic ribosomal protein mutants is dependent on chloroplast translation.(12)EVR2/At2g35260 was identified using positional cloning.Sequence analysis indicated that the EVR2 deletion in evr2-1 mutant caused by T-DNA insertion.The result of semi-quantitative RT-PCR showed that EVR2 transcript was not detected in evr2-1 and 085-004.The transformation of homozygous evr2-1 plants and enhancer line 085-004 with P35S:At2g35260g completely restored the wild type phenotype and the phenotype of leaf variegation was phenotype similar to var2-5,respectively.These results confirmed that At2g35260 is EVR2 that the deletion of At2g35260 is the cause for the enhancement of var2 leaf variegation in 085-004,and the leaf color phenotypes of evr2-1.(13)Confocal microscopy revealed that EVR2-GFP were detected in the chloroplast,when we expressed 35 S promoter driven EVR2-GFP fusion genes in Arabidopsis leaf protoplasts.The result of semi-quantitative RT-PCR showed EVR2 was ubiquitously expressed in tissue examined.(14)evr2-1 dramatically enhanced var1-1 leaf variegation,but had no effect on thf1-2 leaf variegation.In conclusion,the 40 S ribosomal subunit play more important roles in VAR2-mediated chloroplast biogenesis and development pathway.Moreover,the balance between cytosolic and chloroplast translation controls the extent of var2 variegation.