Clonging And Functional Analysis Of Leaf Senescence-associated Receptor-like Kinase Gene SARK1 In Arabidopsis

Receptor-like protein kinases?RLKs?as an important class of cell surface receptors,are present in many plant species,playing critical roles in multiple biological processes including cell–cell communications,plan development and stress responses?There are more than 600 and 100 RLKs in model plants Arabidopsis and rice,respectively.Except a few genes including CLAVATA1?HAESA?BAK7?FLS2?RPK2?BAM1/BAM2/BAM3?OsSIK1?OsLRK2?OsNPR1?OsBRI1,function of most RLK genes are still unknown.In this study,we adopted the method of reverse genetics to identify a leaf senescence-associated receptor-like gene,designated as SARK1.The main results are as follows:?1?We have analyzed the expression profile of SARK1 in young leaf,mature leaf,early senescen-ce leaf and later senescence leaf by using qRT-PCR.The results showed that the SARK1 expression level was lower in non-senescence and increased in late senescence leaves.In a given leaf,senescence starts from the leaf tip and progresses towards the leaf base.The yellow tip showed stronger SARK1 expression than the proximal part of a leaf.These results indicated that SARK1 is upregulated during leaf senescence and might be involved in leaf senescence regulation in Arabidopsis.?2?Using RT-PCR to detect SARK1 expression level of T-DNA insertion homozygous line,RT-PCR analysis indicated that SARK1 was not completely knocked out,with a small amount of expression remained.Compared with wild-type,there were no visible differences in growth and development,except for the earlier leaf senescence phenotype in the sark1 knockdown plants?Determination of chlorophyll content,F_v/F_m of sark1 mutant and wild type rosette leaves suggested that the chlorophyll content and of sark1 leaves were lower than that of the wild type,F_v/F_m of sark1 is also lower than wild type.The above results exhibited that the loss of SARK1 cause precocious senescence of Arabidopsis leaf.?3?We have also compared overexpression lines with wild-type,and find that SARK1 overexpre-ssing plants showed delayed leaf senescence.Consistent with a retarded visible yellowing phenotype,chlorophyll levels in rosett leaves of the overexpression line was generally higher than in counterpart leaves of the age-matched wild-type plants.The Fv/Fm ratios in leaves of the overexpression line were also higher than in counterpart leaves of the age-matched wildtype plants.To confirm that the loss of SARK1 is responsible for the precocious senescence phenotype,we performed a complementation test experiment.The wild-type copy of SARK1,actived by native promoter,was introduced into the sark1knockdown plants,finding that SARK1 can restores the sark1 knockdown mutant plants to wild type.These data confirmed that loss of SARK1 expression in the sark1 knockdown was the only cause of the premature senescence phenotype.?4?When treating the detached wild-type arabidopsis leaves with 50?M strigolactone,theexpression level of SARK1 was upregulated within 6 hours.The result demonstrated that SARK1expression might be induced by strigolactone.?5?70 RLP family genes were identified in Nicotiana tabacum by bioinformatics,and NtRLPfamily members could be classified into 6 subfamilies.We also found that subfamily 5 was distinct from others.Transcriptome analysis revealed that the expression level of some genes,including NtRLP05?NtRLP06?NtRLP50 and NtRLP53,were higher in senscing leaf than non-senescent leaf.So,we could spectulate that NtRLP05?NtRLP06?Nt RLP50 and NtRLP53 are probably involved in regulating maturity of tobacco leaves.