Identification Of Mutants For Pseudouridine Synthase Family And Functional Analysis Of Genes In Arabidopsis

There are many posttranscriptional modifications in RNA,such as methyladenosine,methylcytosine,inosine,pseudouridine and so on in living organisms.Pseudouridine is the most abundant RNA modification known to date,and ubiquitously exists in noncoding RNAs including rRNA,tRNA,snRNA and snoRNA.In recent years,pseudouridine is also found in mRNA in yeast and mammals.Pseudouridine synthase catalyzes the isomerization of uridine in RNA to pseudouridine.All pseudouridine synthases contain a conserved core folding region and an active site,they can catalyze their substrates without the help of any cofactors.The functions of pseudouridine modification and pseudouridine synthase have been studied in E.coli,yeast,human and mammals.However,little is known about them in plants.In order to investigate the functions of pseudouridine synthases in plants,we ordered 46 T-DNA insertion mutants from 22 pseudouridine synthase family genes,and finally obtained 36 T-DNA insertion homozygous mutants.These mutants were treated by four different stresses and their phenotypes were identified to study their function.Our results showed that germination of seeds from the mutants and wild type(WT)plants was delayed for 1-2 d under 120 mM NaCl stress compared with normal growth condition.However,the germination rates between mutants and WT were not has no significant difference at both conditions.We found that gene AtCYG1(Cotyledons green,AtCYG1)may play a role in salt stress response.The green cotyledons rates of atcyg1-1 and atcyg1-2 were significantly higher than that of WT after treatment with 120 mM NaCl.However,there were no remarkably difference under normal condition.In the presence of 300 mM Mannitol,the seed germination of all the mutants and WT was delayed for approximately 2 d.But there was no significant difference in seed germination rates.Under 0.05 ?M IAA,the seed germination rates of the mutants were similar to those of WT.After exposure to 0.5 ?M ABA,the seed germination of the mutants and WT was delayed for about 1 d,and their cotyledons appeared later;however,no significant difference in germination rates was observed between various mutants and WT.Our results revealed that deletion in AtFCS1(Leaf curly and small,AtFCS1)gene led to growth defect.The atfcs1 mutant has a weak and small phenotype with curled leaves,less seeds and prolonged growth period.Moreover,the Fv/Fm value of atfcs1-1 was prominently smaller than WT,indicating that the photosynthesis efficiency is lower in the mutant than in WT.The water loss rate of the mutant was faster than WT,and the surface temperature of the leaf was lower in the mutant than in WT,and the mutant was insensitive to ABA-induced stomatal clousure.These findings suggest that AtFCS1 plays an important role in adaptation to drought stress in plants.To further study the functions of AtFCS1,we constructed the fusion expression vectors such as proAtFCS1::AtFCS1,proSuper::AtFCS1-GFP and proAtFCS1::GUS,and obtained transgenic plants.We found that the phenotype of atfcs1-1 could be recovered by expression of proAtFCS1::AtFCS1 in the mutant,indicating that the phenotype is caused by the function loss of AtFCS1.We also demonstrated that AtFCS1 was localized in the mitochondria,and highly expressed in guard cells.Together,these results will lay foundations for further study on the functional mechanisms of AtFCS1 and roles of pseudourdine synthases in plants.