Function Analysis Of Senescence-associated Genes PheORE1s And PheNAPs In Phyllostachys Edulis

Moso bamboo(Phyllostachys edulis)is an important biological resource in China,for food as juvenile shoots and industrial production as timber.It is widely distributing in China,accounting for more than 70% of the national bamboo forest area,and has important economic and ecological value.As a perennial monocotyledonous plant,moso bamboo start aging process after once-in-a-lifetime flowering process.The flowering may result in the decreasing of regeneration ability,the accumulational senescence leaves,and even,the death of stock bamboo and forest aging.Flower senescence and degraded senescence cause a huge economic and ecological loss.NAC protein is a kind of plant-specific transcription factor.Among them ORE1 and NAP not only participate in plant development and stress response,but also regulate plant senescence process.Most of researches about NAC family concentrate on model plants and crop such as Arabidopsis,rice and cotton,but comprehensive studies about ORE1 and NAP members in moso bamboo are lacking.Thus,the regulatory function of NACs in the bamboo senescence process,which may provide a sight into the molecular mechanism of bamboo senescence.Firstly,genome-wide analysis of moso bamboo NACs(Phe NACs)were performed to obtain the essential information of Phe NACs.Evolutionary analysis and expression pattern analysis indicated the potential function of NACs.Several putative senescence-associated NACs were identified by analyzing their expression pattern during leaf senescence process and post-harvest shoot senescence process.The putative senescence-associated NACs were conducted into yeast-hybrid system and model plant Arabidopsis for functional analysis.1.ORE1 and NAP subfamily members of the moso bamboo were identified as candidate genes related to senescence.Based on phylogenetic analysis and conserved motifs enrichment,a total of 112 Phe NACs were identified in moso bamboo genome.Then,the expression patterns of Phe NACs were studied during flower development,shoot development and post-harvest senescence process.Stress-related NAP subfamily related to stress and NAM subfamily members related to growthwere strongly induced by the flower development and the post-harvest senescence of bamboo shoots.In addition,the members of NAP subfamily were significantly up-regulated after drought stress and salt stress treatment,indicating that these genes might also involve in the stress-induced senescence process.2.ORE1 and NAP subfamily members were involved in the senescence process of bamboo leaves and bamboo shoots after harvestA total of 7 Phe NACs genes and one alternative splicing transcript were obtained by cloning,named Phe NAC5-11 and Phe NAC6 S,respectively.Three Phe NACs(Phe NAC5-7)were homologous to Os NAC2 of rice(belongs to ORE1 subfamily),and four Phe NACs(Phe NAC8-11)were homologous to At NAP of Arabidopsis and Os NAP of rice.Phe NAC5-10 were induced significantly during leaf natural aging,phytohormone-induced leaf senescence(incudling ABA,ACC ? Me JA and SA),dark-induced leaf senescence,and post-harvest senescence of shoots.Phe NAC11 was also induced significantly in these different senescence process,except for bamboo leaf natural aging process.3.PheORE1 s and PheNAPs promote/delay the leaf senescence of ArabidopsisPhe NAC6,Phe NAC6 S and Phe NAC7 were overexpressed in wild-type Arabidopsis thaliana Col-0 and Arabidopsis nac2-1 mutants,and the results showed no delayed or senescent phenotype after natural conditions and ACC-induced Phe NAC6 transgenic plants.After ACC treatment,the detached leaves of Phe NAC6 S transgenic plants showed a delayed chlorotic phenotype,indicating that Phe NAC6 S plays a negative regulation during ACC-induced senescence.After ACC treatment,the chlorosis process of detached leaves of Phe NAC7 transgenic plants was earlier than that of nac2-1 mutants and Col-0,indicating that Phe NAC7 participates in the process of ACC-induced senescence.The overexpression of Phe NAC10 and Phe NAC11 in Arabidopsis nap mutation(35S:Phe NAC10/nap and 35S:Phe NAC11/nap)could complement function and restore the Col-0 phenotype.Overexpression of Phe NAC10 in Col-0(35S:Phe NAC10/WT)leads to premature leaf senescence prior to flowering.The leaves of 35S:Phe NAC10/nap and35S:Phe NAC11/nap turned yellow under ABA treatment,and was the same with Col-0,excluding the nap mutant.This demonstrates that ABA induces leaf senescence of Arabidopsis,but requires the presence of Phe NAC10 and Phe NAC11 to elicit the interaction.All of these verified the positive regulation of Phe NAC10 and Phe NAC11 during ABA-induced leaf senescence in Arabidopsis.The overexpression of Phe NAC9 in Arabidopsis nap mutation(35S:Phe NAC9/nap)and col(35S:Phe NAC9/WT)delayed the senescence of Arabidopsis comparing to nap mutation and col,respectively.Moreover,the expression of Phe NAC9 also remited the process of ABA-induced leaf senescence,which suggested Phe NAC9 was a negative regulator during leaf senescence of Arabidopsis.4.PheORE1 s and PheNAPs directly bind to the promoter of Phe ABCG40-1,Phe SAG12,Phe NYC3 associated with senescence.Phe NAC5-11 showed nuclear localization protein by transient transformation of Phe NAC-e GFP fusion gene in protoplasts of Arabidopsis leaves and tobacco leaves.However,Phe NAC6 S,as the translation product of splicing production of Phe NAC6,showed whole-cell localization that showed no difference with e GFP protein.Additionally,Phe NAC5-GAL4,Phe NAC6-GAL4,Phe NAC7-GAL4,Phe NAC8-GAL4,Phe NAC10-GAL4 and Phe NAC11-GAL4 could bind and activate the expression of His3 and Lac Z in yeast,except Phe NAC6S-GAL4 and Phe NAC9-GAL4.Based on the yeast single-hybrid assay,Phe NAC9 was target genes of Phe NAC6 S,Phe NAC7 and Phe NAC10.Besides,Phe NAC10 could bind to the promoter of Phe SAG12 associated with senescence.Phe NAC9 could also regulate the expression of Phe NYC3.Yeast two-hybrid assay revealed the interaction of Phe NAC9 and Phe NAC8.Above the results,ORE1 s and NAPs formed a complex regulatory network working for bamboo senescence.5.PheORE1 s and PheNAPs related to senescence were involved in various regulatory processesYeast single-hybrid assay and multi-sequence comparison of candidate NAC binding elements revealed that PheORE1 s and PheNAPs NAC binded conservative TACG[G/T][T/G]or GTCA[A/C] core sequences,which is different from NAC binding elements containing CGT[G/A] core sequences.Based on the NAC binding element,the candidate downstreamgenes were screened at the genome-wide level,and functional annotation results of the candidate downstream genes showed that they may be related to flowering time regulation,hormone response,and biotic and abiotic stress responses.ORE1 s and NAPs associated with senescence may be involved in various regulatory processes.Overall,based on the evolution,structure and expression pattern analysis of the NAC family of the moso bamboo,ORE1 s and NAPs subfamily members were selected as candidate senescence-related genes.Seven Phe NACs genes and one alternative splicing transcript were obtained.Eight genes were involved in the senescence process of different tissues of moso bamboo and could positively/negatively regulate the senescence of Arabidopsis leaves.Candidate genes could regulate ABA pathway-related genes or chloroplast degradation-related genes,and Phe NAC9 were node genes of ORE1 and NAP subfamilies which could regulate senescence.These results presented here opened a new avenue for the researchers to further investigate the mechanism of leaf senescence in moso bamboo.