Effect Of Grafting And Stress On MiRNAs Expression In Citrus

Plant growth and development is affected by biotic and abiotic stresses.miRNAs play the important roles in regulation of plant growth and development,responding to biotic and abiotic stresses.In this study,the seedlings of ‘Jincheng'orange?Citrus sinensis Osbeck?,‘Ziyang Xiangcheng'?C.junos Sied.Ex Tanaka?,and Flying Dragon?Poncirus trifoliata Raf.?,and the grafted plants of ‘Jincheng/Ziyang Xiangcheng',‘Jincheng/Flying Dragon' were used as materials.In order to analyze the expression profiles of miRNAs and their target genes before and after grafting and identify the difference on miRNA expression between scion and rootstock,the expression of sixteen miRNAs and their target genes which might respond to grafting were examined by fluorescence quantitative PCRs with the leaf and root samples.Small RNA sequencing was performed with the materails from phloems of seedlings,grafted rootstocks and scions.By means of bioinformatics and molecular biology analysises to identify differentially and specifically expressed miRNAs and the miRNAs that could be transported in a long distance.After alkali stress treament,the contents of proline?Pro?and malondialdehyde?POD?,the activities of superoxide dismutase?SOD?and catalase?CAT?,as well as the expression of miRNA and their target genes were detected in citrus leaves.The aims of this study are to explore the molecular mechanism of interaction between citrus rootstock and scion,and clarify the genetic mechanism of alkaline resistance in miRNA level.The main results are as follows:1.Total RNAs were extracted from the materials collected from the leaves and roots of seedling and grafted trees four months after grafting.The results of luorescence quantitative PCRs indicated that the grafting significantly affected the expression of miRNAs and their target genes in both scions and rootstocks.In the leaves of scion,effect of grafting more likely prompted the expression of miRNAs and suppressed their target genes expression involved in the regulation of plant growth and development,stress response and hormone signal transduction.In the roots of rootstock,effect of grafting tended to suppress the expression of miRNAs related to the regulation of plant growth and development,and stress response.The varieties of miRNAs and the level of expression were significantly different between the two rootstocks.2.Identification of miRNAs in the phloem.By high-throughput sequencing and bioinformatics analysis,59 known miRNAs and 118 novel miRNAs belonged to 47 miRNA families were identified.The target genes of these miRNAs were predicted.3.Identification of miRNA possibly transfer between rootstock and scion was conducted by analysis of the sequence data of specifically expressed miRNAs respondiong to grafting.The results indicated that in the scions,8 miRNAs were up-regulated and 5 were down-regulated in the combination of C.sinensis/C.junos after grafting.In the combination of C.sinensis/ P.trifoliata,there were 9 miRNAs up-regulated and 14 miRNAs down-regulated.Seven common differentially expressed genes were found in two different scion combinations.In rootstocks,5 miRNAs were up-regulated and 4 down-regulated in the combinations of C.sinensis/ C.junos after grafting.In the combinations of C.sinensis/ P.trifoliata,there were 19 miRNAs up-regulated and 3 down-regulated.There were two common differential genes shared in two different scion combinations.Further analysis revealed that compared to the seedling of scion variety,in the scion of C.sinensis/C.junos,expression of 7 miRNAs were found highly up-regulated and corresponded to the high expression of those miRNAs in the rootstock with or without grafting.Eight similar miRNAs were also found in the scion of C.sinensis/ P.trifoliata.Except the grafting induced differentially expression of those miRNAs,higher level of expression in some of them might be due to the miRNA transferred from rootstock to scion.Similarly,by compared to the seedlings of rootstock varieties,3 and 6 miRNAs were found in the rootstocks of C.junos and P.trifoliata,respectively,which were highly up-regulated and corresponded to the high expression of those miRNAs in the scion variety with or without grafting.The high expression of those 9 miRNAs also could be due to the transfer of miRNA from scion to rootstocks.The realtime qRT-PCR was performed to validate the expression of those miRNAs.The results showed that in above miRNAs,only two miRNAs,miR395 and chrUn₃5093 demonstrated their high expression in grafted scion or rootstock correlated to the low expression or no expression in the seedlings of same scion or rootstock varieties.This suggested that those two miRNAs might be the kind of miRNA which possibly transfer in a long distance between rootstock and scion.4.The pH value of 9.0 was used for alkaline stress treatment with pH 6.0 as control.Leaves were collected from the plants at 0 days,5 days,10 days,20 days and 30 days of treatment for analysis.The contents of Malondialdehyde?MDA?and Proline,activities of POD,SOD and CAT,and the expression of 16 miRNAs and mRNAs were investigated.The results of physiological and biochemical indexes showed various patterns of change with prolonged time of stress treatment.Pro content decreased in early phase and then increased afterwards.MDA content increased at first and then decreased.POD and SOD activity also increased in early phase and decreased afterwards.The activity of CAT was increasing at first and then declining and then increasing again later.The results of principal component and membership function analysis indicated that the alkaline tolerance of scion variety was improved by grafting.The results of fluorescence quantitative PCRs showed that compared with the control,the majority of miRNAs in the leaves of P.trifoliata seedlings,grafted C.sinensis/C.junos and C.sinensis/P.trifoliata were down-regularly expressed at 5d,10 d and 30 d after alkaline stress treatment.The expression of some of miRNAs were up-regulated in leaves of C.junos and C.sinensis,while some miRNAs were down-regulated.The expressions of most target genes were negatively correlated with the miRNAs,except some target genes did not show negative correlation with miRNAs.Under alkaline stress,the expression of miRNA and their target genes involved in plant development and growth,stress response and signal transduction were significantly changed compared to the control.Those changes were correlated to the alkaline tolerances of different genetic materials.The results of this study are helpful for clarifying the effects of grafting on expression of miRNA and the effects of miRNAs on plant growth and development,as well as alkaline stress tolerance in citrus.This study also laid a foundation for further study of miRNAs that can transfer in a long distance between rootstock and scion.