Analyses Of Desiccation Sensitivity Of Quercus Variabilis Seeds

Quercus variabilis is a widely distributed deciduous broad-leaved tree specie,which has high ecological and economic value.The propagation of Q.variabilis seedlings mainly by sowing,therefore the quality of seed determines quality of the seedlings.However,the seeds of Q.variabilis are typically recalcitrant.Recalcitrant seeds die rapidly when treated under the conventional method used to store orthodox seeds.The desiccation sensitivity of recalcitrant seeds is considered as a functional and secondary evolutionary characteristic.The analysis of desiccation sensitivity molecular regulation mechanism has great significance for seed storage and long-term preservation of germplasm resources.In this study,on the basis of continuous field investigation of Q.variabilis,the seeds collected from one selected tree were used as the test material.The critical periods of Q.variabilis seed desiccation sensitivity were confirmed by desiccation and germination experiment.We performed physiological indices determination,transcriptome and proteome analyses of the critical periods seeds.The candidate genes and proteins related to desiccation sensitivity were identified.The research can provide theoretical basis for analyzing the possible molecular regulation mechanism of the desiccation sensitivity of Q.variabilis seeds.It can also provide reference for the seedling breeding,seed storage and long-term preservation of germplasm resources of Q.variabilis.The main findings are as follows:(1)The moisture content and germination percentage of Q.variabilis seeds continued to decrease with the extension of desiccation time.A significant positive correlation was observed between the moisture content and germination percentage.The germination percentage and moisture content of seeds without desiccation treatment were 84.00% and 34.27%,respectively.When the moisture content decreased from 34.27% to 28.20%,nearly half of the seeds lost vitality;when the moisture content reached 24.79%(T4),the seed germination percentage declined rapidly again;when the seed moisture content reached 17.79%,almost all seeds lost vitality.It indicates that Q.variabilis seeds are highly sensitive to water loss.The possible critical and lethal water content are 28.20% and 17.79%,respectively.Based on the desiccation and germination experiments,three critical periods of Q.variabilis seeds desiccation sensitivity were determined for subsequent experimental analysis.(2)During desiccation of Q.variabilis seeds,the content of soluble sugars showed a rising trend,while the soluble protein showed a rise-decline-rise trend.At the same time,the superoxide anion showed a rise-decline-rise trend,and the content of malondialdehyde showed a rising trend.During the desiccation,superoxide dismutase and peroxidase activities of seeds showed a decline-rise-decline trend.The activity of ascorbate peroxidase showed a declining trend,while the activity of glutathione reductase first decreased and then increased.Furthermore,the content of abscisic acid,salicylic acid and cytokinin first reduced and then increased.The content of auxin increased all the time.The jasmonic acid content first increased,and then reduced before increasing again.(3)Transcriptome analysis was performed on Q.variabilis seeds that were not desiccated(CK),desiccated for 2 days(T2),4 days(T4)and 11 days(T11).A total of 596,166,538 clean reads were obtained.Through differential expression analysis,4405,4891,5907,1975,4066 and898 differentially expressed genes were obtained in the comparison groups T2 vs CK,T4 vs CK,T11 vs CK,T11 vs T4,T11 vs T2 and T4 vs T2,respectively.There are 2219 overlapped differentially expressed genes in T2 vs CK,T4 vs CK and T11 vs CK.Further analysis revealed that many genes related to plant hormones,late embryogenesis abundant proteins,heat stress proteins and glycerophospholipid metabolism are differentially expressed during seed desiccation.In addition,multiple transcription factors such as ABI5 and ERF1 B might play important roles in the desiccation sensitivity of recalcitrant Q.variabilis seeds.(4)Proteomics studies were performed on the non-desiccated(CK),desiccated for 2 days(T2),4 days(T4)and 11 days(T11)of Q.variabilis seeds.A total of 6386 proteins were obtained.Through differential expression analysis,332,281,656,239,156 and 44 differentially expressed proteins were identified in the comparison groups T2 vs CK,T4 vs CK,T11 vs CK,T11 vs T4,T11 vs T2 and T4 vs T2,respectively.And 133 proteins are differentially expressed in T2 vs CK,T4 vs CK and T11 vs CK.Further analysis revealed that many proteins related to protein synthesis,SNARE interaction in vesicle transport,linolenic acid metabolism and late embryogenesis abundant proteins were differentially expressed during seed desiccation.In addition,12 differentially expressed proteins with unknown functions were found,which may play important roles in the regulation of the desiccation sensitivity of recalcitrant Q.variabilis seeds.(5)In this study,we combined the transcriptome and proteome analyses to expound the mechanism of seed desiccation sensitivity.Finally,254,98,253,29,71 and 6 genes are differentially expressed at the transcription and protein levels,which were identified in the comparison groups T2 vs CK,T4 vs CK,T11 vs CK,T11 vs T4,T11 vs T2 and T4 vs T2,respectively.Based on this,9 all up-regulated,11 all down-regulated,8 down-regulated genes at the transcription level and up-regulated at the protein level were found in T2 vs CK,T4 vs CK and T11 vs CK,respectively.Among them,genes encoding formate dehydrogenase,11 k Da late embryogenesis abundant proteins and U-box domain-containing protein 7 may play important roles in the regulation of the desiccation sensitivity of recalcitrant Q.variabilis seeds.