| In the present work,the biological functions of the alternative splicing variants generated by Arabidopsis and woad SEP3 orthologs and the E-class floral homeotic genes IiSEP1 and IiSEP2 in woad were studied.At SEP3 of Arabidopsis and IiSEP3 of woad show the identical splicing patterns,and can produce the full-length transcript(At SEP3-2 and IiSEP3-2)and the alternative splicing variant lacking the sixth exon(At SEP3-3 and IiSEP3-3).This conservative way in splicing may possess important regulatory function in the process of plant growth.In Nterminus of IiSEP3-2,IiSEP3-3,IiSEP1 and IiSEP2,a nuclear localization signal sequence KRIENKINRQVTFAKRR could be found,indicating these proteins are all located in the nucleus.Analysis of the amino acid sequences revealed that there are three conserved motifs in the C-terminus of IiSEP3-2,IiSEP3-3,IiSEP1 and IiSEP2,including SEP motif I(FFQPLE),SEP/AGL6 motif(PILQIGY)and SEP motif II(NNYMLGWLP).It suggests that these proteins may have redundant functions.qRT-PCR was used to analyze the expression patterns of At SEP3-2,At SEP3-3 IiSEP3-2,IiSEP3-3,IiSEP1 and IiSEP2 in transcriptional level.The results showed that Ii AP1 and At AP1 exhibited similar expression patterns.They are not expressed in vegetative growth stage,but are expressed in inflorescences,flowers,sepals and petals,and have the highest expression level in inflorescences.IiSEP1,IiSEP2,At SEP1 and At SEP2 are not expressed in vegetative growth stage,but are expressed at high levels in reproductive growth stage.IiSEP1 showed the highest expression level in silicles,whereas the expression level of At SEP1 peaked in the pistils.The transcript of IiSEP2 displayed the highest abundance in petals,whereas the number of At SEP2 transcript peaked in stamens.IiSEP3-2 and At SEP3-2 were not expressed in roots and stems,and their expression levels in leaves were very low.In floral organs,the expression levels of IiSEP3-2 and At SEP3-2 were significantly increased.Petals displayed the peak value of IiSEP3-2 expression,while the highest abundance of At SEP3-2 was observed in siliques.The transcriptional signals of IiSEP3-3 and At SEP3-3 were barely detected in roots and stems,but began to accumulate in leaves.IiSEP3-3 peaked in petals,whereas At SEP3-3 peaked in pistils.At the same time,the accumulation amounts of At SEP3-2,At SEP3-3,IiSEP3-2 and IiSEP3-3 in different tissues and organs were analyzed in the protein level.Western blotting analysis showed that IiSEP3-2 and IiSEP3-3 signals could not be detected in roots and stems,whereas IiSEP3-2 could be detected in rosette leaves and flowers,and IiSEP3-3 could be detected in rosette leaves,cauline leaves and flowers.In Arabidopsis,At SEP3-2 was only accumulated in flowers,stamens and pistils,while At SEP3-3 could be accumulated in all the tissues and organs except roots and stems.These results showed that At SEP3-2,At SEP3-3,IiSEP3-2 and IiSEP3-3 displayed different expression patterns,indicating the alternative splicing variants might possess distinct functions in regulation of floral transition and floral organ formation.On this basis,At SEP3-2 and At SEP3-3 splicing variants were silenced by ami RNA(artificial mi RNA)technology and their functions in growth and development of Arabidopsis plants were analyzed.Compared with the wild-type Arabidopsis plants,the growth states of mi R-sep3-2 and mi R-sep3-3 transgenic Arabidopsis plants in vegetative stage are more vigorous,leaf area became larger and the margin of the leaves showed an obvious serrated phenotype.In addition,the arrangement mode of the sepals and the petals in mi R-sep3-2 transgenic Arabidopsis plants was also changed.Transcriptome sequencing results revealed that the expression levels of 1116 genes were altered in mi R-sep3-2 transgenic Arabidopsis lines,including 559 up-regulated genes and 557 down-regulated genes.In like manner,the m RNA accumulation levels of 1347 genes were changed in mi R-sep3-3 transgenic Arabidopsis lines,including 879 up-regulated genes and 468 down-regulated genes.Subsequently,the downstream genes controlled by the encoding products of the alternative At SEP3 splicing variants were analyzed by chromatin immunoprecipitation(Ch IP)method.The results showed that At SEP3-2 could bind directly to the CAr G elements in the promoter regions of AGL17?AGL20?AGL42?ARF17?CCA1?FT?GI?GRF8?LFY?SEP3?SHP1?STM?TCP3?TCP12 and TT16,and indirectly control the transcription of AGL15,AGL24,TCP3,TCP12 and TCP23.At SEP3-3 can directly regulate the expression activity of AGL15,AGL24,ARF17,CUC2,GI,GRF8,MAF5,SEP3,SHP1,STM and TT16,and indirectly control the transcription of AGL17,AGL20,AGL42,FT and LHY.These results illustrated that there are differences in function between two splicing variants of At SEP3.Constitutive expression of IiSEP3-2,IiSEP3-3,IiSEP1 and IiSEP2 in Arabidopsis all could result in early flowering and abnormal development of floral organs.Compared to wild-type Arabidopsis plants,35S::IiSEP3-2,35S::IiSEP3-3,35S::IiSEP1 and 35S::IiSEP2 transgenic Arabidopsis lines also exhibited an obviously dwarf phenotype.The rosette leaves of 35S::IiSEP3-2,35S::IiSEP3-3 and 35S::IiSEP1 transgenic Arabidopsis plants curled inwardly,while the rosette leaves of 35S::IiSEP2 transgenic plants did not change significantly.In IiSEP3-2 transgenic Arabidopsis plants,the number of sepals,petals and stamens was reduced,and cauline leaves and sepals were converted into carpelloid structures.In 35S::IiSEP3-3 transgenic Arabidopsis plants,the number of sepals,petals and stamens was reduced or even missing,the pollens were not fully developed,the four whirls of floral organs would be wilted prematurely,and the whole flowers showed an abortive state.The morphogenesis of the flowers in 35S::IiSEP1 transgenic Arabidopsis lines was also abnormal and homeotic conversion could be observed in the floral organs.In 35S::IiSEP2 transgenic Arabidopsis lines,the petals were abnormal in development,the number of the sepals and the stamens was reduced,accompanied by the conversion of the sepals into carpelloid structures.Silencing of IiSEP3-2 or IiSEP3-3 by VIGS(virus-induced gene silencing)technology based on the tobacco rattle virus(TRV)could delay the flowering time and perturb the normal development process of floral organs in woad.Compared with the control woad plants,the vegetative growth time of the woad plants treated with TRV-IiSEP3-2 and TRV-IiSEP3-3 was extended significantly,the number of the petals and the stamens was reduced,and the anther development was abnormal.Moreover,the number of the sepals in woad plants treated with TRV-IiSEP3-3 was also reduced,and the shape of the petals was changed obviously.At the transcriptome level,the expression levels of 1861 genes were significantly changed in woad plants treated with TRV-IiSEP3-2,including 1035 up-regulated genes and 826 down-regulated genes.Similar to this,the expression levels of 2063 genes were changed in woad plants treated with TRV-IiSEP3-3,of which the expression of 1289 genes was promoted and the transcription of 774 genes was inhibited.The above results showed that IiSEP3 was associated with the floral transition,and woad plants would not be able to enter the reproductive growth stage normally from the vegetative growth stage when the alternative IiSEP3 splicing variants were silenced.The results of yeast two-hybrid experiments showed that IiSEP3-2 could interact with IiSEP1,IiSEP2,IiSEP4,Ii SVP and Ii SHP2,but could not interact with Ii FUL.IiSEP3-3 could form heterodimers with IiSEP1,IiSEP4 and Ii SVP,but could not form heterodimers with IiSEP2,Ii SHP2 and Ii FUL.These results showed that IiSEP3-2 and IiSEP3-3 possessed different interacting proteins and had discrepant functions in growth regulation of woad plants.In conclusion,At SEP3-2 and At SEP3-3 participate in leaf morphogenesis and floral organ development by regulation of different downstream genes in Arabidopsis.IiSEP3-2 and IiSEP3-3 can interact with different proteins and influence the floral transition and the floral organ development in woad. |
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