| Senescence in plants is the last stage of development,which ultimately leads to death of a cell,a tissue,an organ,and an organism.It determines the biomass,product quality of cereals in the field.Senescence is a controlled process that plants utilize to remobilize nutrients from source leaves to developing tissues.While during the past decades,molecular signal underlying the onset of senescence have been well studied,however knowledge remains scarce on the age dependent mechanisms that control the onset of senescence.Currently,S40 genes in barlay(HvS40)and Arabidopsis(Ats40-3)showed enhanced expression level during natural senescence as well as artificially induced senescence.The detail role of S40 gene family has not been illustrated.Herein,S40 gene family of rice,a homologue of AtS40 and HvS40 was functionally characterized in rice during leaf senescence and the main findings are as followings:1.S40 gene family belongs to DUF584 group members of the which share a highly conserved sequence in the C-terminal part as follows:GRXLKGR(D/E)(L/M)XXXR(D/N/T)X(I/V)XXXXG(F/I).A previous study has classified proteins based on similarities of their amino acid sequences into five groups.Four of the sixteen DUF584 proteins of rice belong to group Ⅰ to which also the HvS40 and AtS40 protein is assigned.From the further proteins of rice,two belong to group Ⅱ and Ⅲeach while only one protein belong to group Ⅳ.Group Ⅴ includes seven DUF584 proteins of rice.2.Chemical characteristics of rice S40 proteins coupled with gene structure analysis,motif determination and chromosomal location were identified using different software and databases.The rice S40 proteins were labeled as OsS40.The value of isoionic point(IP)varied from 5.36(OsS40-16)to 11.27(OsS40-11).The corresponding molecular weight varied from 14.14 kDa(OsS40-7)to 28.52 KDa(OsS40-10).The molecular weights ranged from with an average of 19.67 kDa.Among these proteins,all had an unstable structure except OsS40-13 with the instability index to be 27.77.OsS40-16 was the most instable one with an instability index of 89.11.All S40 proteins in rice were found to be hydrophilic proteins with Hydrophobicity score(arbitrary unit)below 0.S40 genes of rice showed high structural similarities as 14 out of 16 genes had 1 exon while 13 out of 16 genes has no intron.Structural detail of ten conserved motifs for 16 rice genes was provided to give further insight to understand S40 genes in rice.Furthermore,Chromosomal location of S40 genes in rice was also identified which revealed that 9 out of 16 S40 genes were distributed among three chromosomes(Chrl,Chr4,and Chr5).In contrast,chromosomes 7 and 10 have 2 genes while Chr3,Chr11 and Chr12 contained only one S40 gene each.3.To investigate the role of S40 genes in regulation of leaf senescence in rice,the expression pattern of rice S40 genes during natural senescence were examined:Six of the sixteen genes,OsS40-4,OsS40-5,OsS407,OsS40-9,OsS40-14 and OsS40-15 showed increased expression level at early stage of leaf senescence and decreased expression level at late senescent leaves,indicating the same expression pattern of OSH36,one of the senescence associated gene.It demonstrated that these six members of the OsS40 family might be involved in regulatory network at the onset of senescence during natural development of rice leaf.4.To explore the expression pattern of rice S40 candidate genes response to nitrogen treatment,rice plants were grown in the concentration of normal conc(1N),Half conc(1/2 of normal)and double conc(2N)for how many days.Semi quantitative RT-PCR results showed that 12 out of 16 S40 genes showed detectable expression level.Among them,only 6 genes,OsS40-1,OsS40-2,OsS40-4,OsS40-6,OsS40-10 and OsS40-12 showed differential expression in plant supplied with different concentration of nitrogen.The results of quantitative RT-PCR further confirm the enhanced transcript levels of these six genes as well as a stress related senescence associated gene OSH36 in plants under the same condition.5.To examine the effect of different phytohormones treatment including Abscisic acid(ABA),Jasmonic acid(JA)and Salicyclic acid(SA)on the expression pattern of rice S40 candidate genes.Semiquatitative RT-PCR results showed that eight of 16 OsS40 members enhanced their expression after treatment with ABA.The quantitative RT-PCR results further showed the fast enhanced expression of OsS40-13 after only 24hrs of ABA treatment,the enhanced expression of OsS40-4,OsS40-5,OsS40-6 and OsS40-14 after 48hrs,while the slowly enhanced expression of OsS40-1,OsS40-2 and OsS40-12 after 72hrs,but the expression level of OsS40-1 and OsS40-4 didn’t show any change.After treatment with MeJa,OsS40-1 and OsS40-12 showed enhanced expression after 72hrs,OsS40-2,OsS40-6 and OsS40-14 showed enhanced expression after 48hrs,while OsS40-4,OsS40-5 and OsS40-13 didn’t show any change.There was no impact of IAA on the expression of genes at any time point.Expression of SAGs such as OsNAP,SGR,NYC1 and Os157 which were widely used as leaf senescence markers in rice,were used as positive controls for the efficiency of treatment by ABA,JA,SA,and IAA.6.To explore the expression of the S40 candidate genes of rice upon infection with pathogens,rice seedlings(Oryza sativa CO39)at 3-4 leaf stage of were inoculated by spraying fungal(Magnaporthe oryzae)spores.The total RNA were isolated from the leaf material after 24,48,72,96,and 108 hours treatment,the semi qRT-PCR results of them showed that 10 genes,OsS40-1,OsS40-2,OsS40-4,OsS40-6,OsS40-8,OsS40-11,OsS40-12,OsS40-13,OsS40-14 and OsS40-15 altered gene expression after treatment of the plants with the fungus.The qRT-PCR results confirm the enhanced transcript levels of these genes at the different responsive rate(time).NAC4 and WRKY45,pathogen related senescence marker genes,were used as positive controls for the efficiency of treatment with fungus,showing fast enhanced expression after 24 hours treatment.7.GFP fusion method was employed to test the subcellular localization of S40 genes in rice.In protoplasts expressing the OsS40-1GFP,OsS40-13-GFP and OsS40-14-GFP fusion protein,the green fluorescence of the GFP clearly demonstrates that these proteins were efficiently targeted to the nucleus while OsS40-16-GFP was targeted to cytoplasm.On the other hand,OsS40-3-GFP OsS40-6-GFP OsS40-7GFP OsS40-9-GFP and OsS40-15-GFP fluorescence were distributed as several speckles in cytoplasm.8.By mass spectrometry-based proteomics approaches,we have identified 68 proteins that interact with OsS40-14.These proteins,distributed in the cell,have numerous functions ranging from RNA binding,nucleic acid binding and structural constituent of ribosome to translation,ATP synthesis,ATP metabolic process,photosynthesis and many more.Because no work is done about the function of OsS40-14,therefore these findings suggest that the interaction between these proteins and OsS40-14 is plausible and may have functional implications.Moreover,four genes from the interacting proteins,IP-16,IP-28,IP-29 and IP-52 showed enhanced expression pattern upon exposure to darkness,hormones and pathogen induced senescence insinuates that OsS40-14 may interact with and regulate these proteins in a similar manner.However,our current study opens up avenues of research related to novel functions of the OsS40-14.Taken together,the expression of some of the members of OsS40 family was induced during age dependent as well as artificially induced senescence.The expression of OsS40-2 and OsS40-12 were induced by hormones induced senescence including ABA,SA,JA,as well as pathogen induced senescence and nitrogen deficient induced senescence.OsS40-1 expression was also induced by similar artificially induced senescence except SA.OsS40-14 showed enhanced expression during natural senescence,pathogen induced senescence and hormones including SA and JA induced senescence.Conclusively,OsS40-1,OsS40-2,OsS4012 and OsS40-14 were found to show enhanced expression level during natural and artificially induced senescence that shows their role in regulation of leaf senescence in rice.At the same time,four interacting protein candidates of OsS40 protein were identified by Mass Spectrometry and showed similar expression profile with OsS40 under stress condition.It indicates that OsS40 and its interacting protein together play a potential regulatory function during rice leaf senescence.The detail insight of the function of OsS40-1,OsS40-2,OsS40-12 and OsS40-14 will addressed the mechanism of crosstalk of natural senescence and stress induced senescence pathway in future. |
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