Alternative Splicing And Functional Analysis Of VvPMA1 Proton Pump Under Salt Alkaline Stress

As an important economic fruit,sustainable development of grape is restricted due to soil alkalized.However,currently limited information is available on this particular from stress.Usually,alkali stress involves salt and pH mixed stress.It is generally accepted that sustaining pH homeostasis is important to plant cell under alkalinity,in which plasma membrane H⁺-ATPase play a key role.Based on previous research,PM-H⁺-ATPase are uesd as target in alkalinity-insensitive Crimson seedless（vitis vinifera）.It will be ascertained whether or how regulatory pattern is changed in PM-H⁺-ATPase with increase of stress time by PM-H⁺-ATPase activity with protein amounts and expression profiles of PM-H⁺-ATPase.PM-H⁺-ATPase proteins responding to alkali stress VvPMA1 was cloned.Surprisingly,the sequence analysis in grape genomic data and further assay showed that the isolated VvPMA1 had two undiscovered splicing variants,VvPMA1αand VvPMA1β.The function of VvPMA1αand VvPMA1βwere investigated to explore its abilities in resisting salt stress.The results are as follows:1.The transcriptional amounts of PM-H⁺-ATPase subfamily isoforms in different tissue of grape seedling was investigated.The results showed that VvPMA1 was expressed mainly in root.2.And the full-length cDNA of root main PM-H⁺-ATPase VvPMA1 was isolated from alkalinity-insensitive Crimson seedless.Surprisingly,the sequence analysis in grape genomic data and further assay showed that the isolated VvPMA1 had two undiscovered splicing variants,VvPMA1αand VvPMA1β.VvPMA1αhas 2862 bp and encodes 954 amino acids,VvPMA1βhas 2757 bp and encodes 919 amino acids.Surprisingly,the sequence of VvPMA1βis 105 bp less than VvPMA1α.Comparing the two VvPMA1 cDNA sequences with the genomic sequence revealed that the second intron was retained in the VvPMA1βcDNA.This intron retention was predicted to generate a novel VvPMA1 through N-terminal truncation because of a 5’-terminal frame shift.A bioinformatics analysis of cis-acting elements of VvPMA1 promoter revealed that ABRE,CGTCA-motif,WUN-motif and light elements were presented.3.Yeast function complementation assay showed that the isolated VvPMA1βhad powerful function.4.The qPCR results showed that the VvPMA1βtranscriptional amount of increment was highest in alkali stress 3 hours,however,VvPMA1αtranscriptional amount decreased obviously compared with that of control.So,the proportion of the VvPMA1βtranscriptional amount in total VvPMA1αtranscriptional amount was enhanced,which was benefit to the total activity of PM-H⁺-ATPase in grape root at early stage of alkali stress.After that VvPMA1αtranscriptional amount increased accompanying by VvPMA1βtranscriptional amount decrease,which caused the proportion of the VvPMA1βtranscriptional amount in total VvPMA1 transcriptional amount was reduced and the total activity of PM-H⁺-ATPase changed accordingly.5.VvPMA1 was inserted into the vector of PRI-GFP,subcellar localization was performed and results showed that VvPMA1 expressed in plasma membrane.6.Phenotypes of wild type Arabidopsis and overexpression lines were observed.In T₀ generation,compared to wild type Arabidopsis,transgenic plants have slowly growth rate,multiple lateral root and inducing cluster buds,but the overexpression plants have high lethality rate.7.Compared to wild type,VvPMA1αhad no significant difference on growth rate,on the contrary,VvPMA1βhad lower growth rate than VvPMA1αand wild type,while the VvPMA1βplants showed abnormal inclination of leaves and twisted stems,at the same time,VvPMA1βplants had high lethality rate.8.VvPMA1αfirst generation in different lines showed abnormal inclination leaves and had less lateral branches compared to wild plants.To monitor the root exteral pH,the root systems from plants growed in MS culture medium containing the pH-sensitive dye,bromocresol purple.After 12 h,VvPMA1αhad a much larger change than wild plants and showed that VvPMA1αplants result in a lowering of the root exteral pH.Q-PCR showed that in transgenic plants VvPMA1 transcript levels were higher than in untransformed plants but LePMA showed the contrary results.After irragation saline solution 20 days,wild plants showed yellow leaves and resulted plants wilting while VvPMA1αtransgenic plants did not show any developmental abnormalities.In transformed plants,Na⁺/K⁺and MAD content were lower than wild plants and chlorophyII content and APX activity were significantly higher than wild plants.The results showed that the salt resistance of transgenic lines was stronger than wild plants.Furthermore,oxlic acid content in VvPMA1αoverexpression plants were higher than wild type and indirectly showed that H⁺-ATPase activities were higher.In this study,the alternative splicing（AS）of root VvPMA1 was discovered during RNA processing in alkali stress.Splice variant VvPMA1βappeared by intron retention.Yeast complementation assays of the two splice variants showed that compared to the constitutive variant,VvPMA1βcould enhance the ability to complement Saccharomyces cerevisiae deficient in PM H⁺-ATPase activity due to releasing N-terminal autoinhibition.In addition,the amount of VvPMA1 AS was enhanced in grape cultivars with high alkali resistance under alkaline.Our data suggests that through AS,the N-terminal length of VvPMA1 may be regulated to accurately modulate PM H⁺-ATPase activity of grape root in alkalic stress.Furthermore,overexpression of VvPMA1βin tomato seriously harmful to transgenic tomato growth under normal condition.We suggested that high activity of VvPMA1βexpends too much more ATP,which is disadvantageous to plant growth.Therefore,it is necessary for plant alkalic resistance that the suitable proportion of the splice variant is acquired by alternative splicing of plasma membrane H⁺-ATPases.