The Mechanisms Of Regulating Flowering And Senescence By CCCH Zinc Finger Domain And KH Domain Proteins In Arabidopsis

CCCH zinc-finger proteins and K-homolog proteins are widely present in plants and play important roles in plant growth and stress responses.There are 68 CCCH zinc finger proteins and 26 KH proteins in Arabidopsis.KHZ1 and KHZ2 are two proteins which contain both CCCH zinc finger domain and KH domain,and they belong to the ? subfamily of the CCCH protein family.Both KHZ1 and KHZ2 contain two non-tandem CCCH zinc finger domains,one at the N-terminal region(C-X8-C-X5-C-X3-H)and the other at the C-terminal region(C-X7-C-X5-C-X3-H),between them is an I-type KH domain.However,the functions of the KHZ proteins remain uncharacterized so far.Previously,our laboratory have obtained the T-DNA insertion single mutant khz1,as well as the overexpression lines of KHZ1 and KHZ2.In order to study the functions of KHZ1 and KHZ2,the single mutant khz2 and double mutant khzl khz2 were obtained by the CRISPR/Cas9 technology.Then the phenotype was observed,and the single mutant khz1 had a slight late-flowering phenotype,khz2 was similar to WT,while the double mutant khz1 khz2 delayed the flowering time seriously.In contrast,the overexpression lines showed early flowering phenotype.These results indicate that KHZ1 and KHZ2 positively regulate the floral transition of Arabidopsis.To study the floral transition mechanisms of KHZ proteins,the key genes of photoperiod,vernalization,and GA pathways were examined,and their expression had no significant differences in khzl khz2 when compared with WT.In addition,khzl khz2 showed a late-flowering phenotype both under long-day and short-day conditions.After the vernalization and GA treatment,the khzl khz2 still displayed a late-flowering phenotype,but the flowering time was earlier than that of the untreated khz1 khz2 plants.These results indicate that khz1 khz2 responds normally to the different conditions.KHZ1 and KHZ2 might not be involved in the photoperiod,vernalization or GA pathways.The assays of flowering integrator and floral meristem identity genes revealed that the expression of FLC in khz1 khz2 was significantly increased,while the expression of FT,SOC1,AGL24,API and FUL were significantly decreased.These results were consistent with the late-flowering phenotype of khz1 khz2.If the FLC gene mutation in khzl khz2 was applied,the flowering time of the triple mutant khz1 khz2 flc was basically the same as WT.In addition,overexpression of FT in khz1 khz2 also restored the late-flowering phenotype of khz1 khz2.These results suggest that KHZ1 and KHZ2 are new members of the autonomous pathway and promote the floral transition of Arabidopsis by inhibiting FLC and further promoting the expression of F1.KHZ1 and KHZ2 also positively regulate the senescence of Arabidopsis,including the leaf senescence and the whole plant senescence.Under the normal growth conditions,khz1 khz2 showed delayed leaf senescence and whole plant senescence significantly,khz1 could delay senescence of the whole plant,while khz2 only delayed the death time of rosette leaves compared with the WT.In contrast with the mutants,premature senescence of leaf and the whole plant were observed in overexpression lines.Under the treatment with ABA,the single mutants khz1 and khz2 and the double mutant khz1 khz2 showed delayed leaf senescence.Therefore,KHZ1 and KHZ2 might play roles in the ABA-mediated leaf senescence signaling pathway.These results indicate that KHZ1 and KHZ2 have an important roles redundantly on the floral transition and senescence of Arabidopsis.The analysis of KHZ1 and KHZ2 expression patterns showed that they were ubiquitously expressed throughout the tissues of Arabidopsis,such as seedlings,roots,stems,leaves and flowers,and preferentially express in flowers and seedlings.The overall level of KHZ1 mRNA was higher than KHZ2,which indicated that KHZ1 might be more predominant than KHZ2 in regulation of the development of Arabidopsis.Subcellular localization analysis of KHZ 1 and KHZ2 found that they were localized to nuclear dots.KHZ1 and KHZ2 had transcriptional activities in Arabidopsis protoplasts,whereas no transcriptional activities were detected in yeast.This suggests that the transcriptional activation of KHZ1 and KHZ2 might require cofactors or some modifications.In addition,KHZ1 and KHZ2 also had RNA binding activities,suggesting that they might be involved in RNA processing such as RNA splicing or polyadelation.Further experimental results showed that KHZ1 and KHZ2 repressed the splicing efficiency of FLC pre-mRNA.In summary,KHZI and KHZ2 positively regulate floral transition and senescence of Arabidopsis,and they are new members of the autonomous pathway.KHZ1 and KHZ2 have RNA binding activities and they negatively regulate the expression of FLC by inhibiting the splicing of FLC pre-mRNA,thereby promoting floral transition in Arabidopsis.KHZ1 and KHZ2 also have transcriptional activities,but their functions at the transcriptional level remain unclear.