DREB2B Regulates Seed Longevity Negatively Through ABA Pathway In Plant

Seed longevity is an essential character for agronomy,ecology,and economy and varies with different plant species and environmental conditions,which is very important for plant development and crop yield.In the model plant species such as Arabidopsis thaliana,seed vigor and longevity regulatory have been studied for decades,but the underlying networks is yet unclear.Dehydration-Responsive Element-Binding Protein 2B(DREB2B)belongs to the DREB2 a subgroup of DREB transcription factor family,which is involved in drought response regulation;however,the role of DREB2B in the regulation of seed germination and vigor need to be considered.In this work,the dreb2b knockout mutant of Arabidopsis was identified and its phenotype in seed vigor and longevity was determined by examining the seed germination ratio of dreb2b seed after different weeks and months intervals of dry storage.After that dreb2b seed vigor was verified by examining the seed germination speed of dreb2b seed at different hour intervals after imbibition.Further,dreb2b seed vigor and viability was confirmed by calculating seed germination rate and viability ratio after 3 days Controlled Deterioration test(CDT)in freshly harvested seeds after two weeks of dry storage.All above researches revealed that dreb2b seeds displayed quick germination,higher germination rate after longer period of dry storage and reduced sensitivity to CDT treatment.DREB2B complementary lines were obtained by over-expressing the DREB2B gene into dreb2b and showed the similar phenotype to that of WT.It is well known that the phytohormones abscisic acid(ABA)mediates various plant growth and developmental processes containing seed dormancy,germination,and seed viability and longevity.Hyposensitivity to ABA in dreb2b seeds was observed by applying exogenous ABA.All these indicated that DREB2B is a negative regulator in seed germination and vigor dependent on ABA at least partially.To investigate the correlation between DREB2B and ABA in seed vigor regulation,some expression of ABA pathway genes were determined in dreb2b seeds.The key factor ABI3 was clearly down-regulated in dreb2b.Moreover,dreb2b/abi3-16 double mutant was generated to determine the clear pathway through which DREB2B is involved in seed vigor regulation.dreb2b/abi3-16 double mutant seeds showed more enhanced seed vigor and longevity and are more less affected by CDT compared to dreb2b and abi3-16 single mutant lines which indicated that DREB2B functions synergistically with ABI3 in seed vigor regulation by using different pathways.Then,DREB2B homolog gene(GhDREB2B-A09)was identified in cotton(G.hirsutum)and expression results showed that DREB2B and GhDREB2B-A09 mRNA levels were significant higher in mature dry seed.Furthermore,over-expression transgenic Arabidopsis of DREB2B and GhDREB2B-A09 genes,generated by flower dip method,exhibited slower seed germination speed and higher sensitivity to CDT compared to WT.Moreover,the biochemical analysis of DREB2Bs from Arabidopsis and cotton all displayed the clear transcriptional activity and cytoplasm and membrane subcellular localizations,indicating their specific functional mechanisms.So,DREB2B and GhDREB2B-A09 interaction proteins were identified through online prediction tool and confirmed by BiFC.The results showed that DREB2B and GhDREB2B-A09 interacted with RADICAL-INDUCED CELL DEATH1(RCD1,SRO1,and GhRCDlLs)proteins in and cotton.Furthermore,the role of rcdl-3,srol-1 and srol-2 mutants in seed vigor and longevity was determined and showed similar germination speed and sensitivity to CDT to that of dreb2b.Taken together,these results illustrated that DREB2B transcription factors interact with RCDls and partially control the expression of key ABA signaling factor ABI3 in Arabidopsis and cotton to regulate seed vigor negatively through an unidentified downstream factors or pathway.