Comparative Study On B-class MADS-box Genes Response To Photoperiods In Three Viola Species

Viola philippica,Viola prionantha,and Viola tricolor are all species of the genus Viola.Previous studies in our laboratory have found that photoperiod is the main ecological factor affecting the development of the two types of flowers for V.philippica and V.prionantha.Both of them form chasmogamous(CH)flowers under short daylight and cleistogamous(CL)flowers under long daylight.Howere,floral morphology of V.tricolor is not regulated by photoperiod.All flowers of V.tricolor are normal CH flowers.Previous analysis of the two types flowers of V.philippica and V.prionantha in our laboratory showed that the difference between CH and CL flowers lies in the size and quantity of petals and stamens.According to the "ABC model" of flower development,class B MADS-box genes act as floral organ identity-determining genes to regulate development of stamens and petals.Therefore,we speculate that class B MADS-box genes influence dimorphic flower development by responding to different photoperiod.However,the flower morphology of V.tricolor does not change with the change of photoperiod.Thus,V.tricolor was introduced as a relative control species.Based on the above research basis,in this study,the V.philippica,V.prionantha and V.tricolor were used as experimental materials to study the expression characteristics of class B MADS-box genes in three species of Viola.To explore the effects of photoperiod on class B MADS-box genes in three species of Viola,we used chromosome walking,quantitative real-time PCR and yeast two-hybrid technology to provide more theoretical basis for the molecular mechanism of dimorphic flowers’ development.The main findings of this study are as follows:(1)The open reading frame sequences of PI(PISTILLATA)and TM6-1(Tomato MADS box gene 6-1)genes in three species of the Viola were cloned.Sequence analysis showed that PI and TM6-1 genes were highly conserved in three species of the Viola.Compared with V.tricolor,the PI and TM6-1 genes in V.philippica and V.prionantha have higher similarity.(2)The prey vector and bait vector of AP3 gene,PI gene and TM6-1 gene were constructed for the yeast two-hybrid system of V.philippica,V.prionantha and V.tricolor.The interactions between AP3 protein and PI protein,PI protein and TM6-1protein were found by yeast two-hybrid experiment,and AP3 protein and PI protein,PI protein and TM6-1 protein between V.tricolor and V.philippica,V.prionantha,respectively.It was further demonstrated that the ORF sequences of the B-class MADS-box genes were highly conserved in the Viola.(3)The gDNA sequences of PI and TM6-1 genes in three species of the Viola were cloned.The sequence analysis showed that the gDNA sequences of PI and TM6-1 genes in three species of the Viola were very similar in exon number and sequence,and the differences were mainly reflected in intron sequence.(4)The upstream promoter sequences of PI and TM6-1 genes in three species of the Viola were cloned by chromosome walking.Based on the shortest PI and TM6-1promoter sequence length of three species,the response elements of PI and TM6-1promoter sequences in three species of the Viola were compared and analyzed comprehensively.The results showed that the light response elements on the promoters of PI and TM6-1 gene were similar in type and quantity to those of V.philippica and V.prionantha.However,compared with V.philippica and V.prionantha,PI and TM6-1 in V.tricolor have great differences in the types and quantities of photoresponsive elements.(5)The expression of PI and TM6-1 genes in three species of the Viola were detected by real-time quantitative PCR at 5 different flower development stages.The results showed that the expression of PI and TM6-1 genes in three species of the Viola showed an increasing trend with the increasing maturity of flower development.With the prolongation of photoperiod,the expression of PI and TM6-1 genes in the flower buds of V.philippica and V.prionantha showed a decreasing trend at 5 stages.In addition,the expression levels of PI and TM6-1 genes were lowest in the long daylight(16h photoperiod).The expression levels of PI and TM6-1 genes in V.tricolor showed no significant difference or increased trend with the prolongation of photoperiod.Thus,the photoperiod effecting the expression levels of PI and TM6-1 genes in V.philippica,V.prionantha and V.tricolor plants was different.The generation of CL flowers in V.philippica and V.prionantha may be the result of down-regulation of the class B-class MADS-box genes expression by long daylight.The only CH flowers in V.tricolor plants may be because they are insensitive to photoperiod.The change of photoperiod does not change the floral type.The expression of PI and TM6-1 genes in newly induced flower budsd of V.philippica and V.prionantha decreased with the increase of conversion days when the plants grew in short daylight were switched to long daylight.However,the expression of PI and TM6-1 genes in the two plants increased with the increase of Conversion days after switching from long daylight to short daylight.The results reveal that the formation of dimorphic flowers in V.philippica and V.prionantha may be the results of the B-class MADS-box genes expression responsing on different photoperiod.This study showed that PI and TM6-1 genes had good conservation in three species of Viola by gene cloning and yeast two-hybrid experiments.The photoperiodic characteristics of PI and TM6-1 gene expression in these three plants were studied by chromosome walking and qRT-PCR technique.The results showed that V.tricolor buds induced by long daylight were normal CH flowers,but not CL flowers,which may be related to the insensitivity of PI and TM6-1 gene expression to photoperiod response,while V.philippica and V.prionantha buds induced by long daylight were CL flowers,which may be related to the down-regulation expression of class B MADS-box genes.In conclusion,this study provides new insights into the evolution and development of the CH-CL flowers in Viola.