Molecular Mechanisms Regulation Of Alternative Splicing Of FT Related To Differences In Flowering Time Between Tall And Drawf Coconut

Coconuts can be divided into two distinct categories according to morphological characteristics and breeding habits: tall and dwarf coconut.Tall coconut is slow to mature(flowering 8-10 years after plants).Cn.dwarf is early-flowering(4-6 years after planting).The enormous divergence of flowering time between tall and dwarf coconut supports experimental materials for our study.The flowering time is an important agronomic trait and their molecular mechanism had been full documented in modern plants and some crops.There are generally five signal transduction pathway regulating plant flowering time,including vernalization pathway,photoperiod pathway,autonomous pathway,hormone pathway,and age pathway.Coconut is a tropical oil and fruit crop no study was reported about the molecular mechanism of flowering time in it.Some documents had reported that the flowering time of tropcial woody crop can be regulated by photoperiod pathway.Short sunshine conditions can accelerate the transformation from vegetative growth to reproductive growth.Our experimental data had showed that candidate genes Suppressor of Overexpression of Constans1(SOC1,CCG008883)、FT(CCG011565)and Apetala 1(AP1,CCG027514)can be induced at the stage of reproductive growth,indicating that photoperiod pathway may be play a important role in coconut flowering time.Meanwhile,Flowering Locus T(FT,florigen)were detected to have two different pre-m RNA splicing style between tall and dwarf coconut and the alternative splicing is significantly associated with the divergence of fowering time between tall and dwarf coconut among coconut population comprised of 28 tall coconut andd84 dwarf coconut.Candidate genes involved in flowering-time pathways in coconut were identified by aligning with sequences of arabidopsis genes involving in flowering time.Expression profiles and alternative splicing analysis showed that higher expression was detected for genes involved in photoperiod pathway in mature trees compared to in seedlings of tall and dwarf coconut.Among them,different transcripts of Cn FT6 were detected between tall and dwarf coconut and result in one six-nucleotide indel.The relationship between the six-nucleotide indel and flowering time were validated in 112 coconuts.Yeast two-hybrid were used to demonstrate that Cn FT6 a and Cn FT6 b can interact with Cn FD.In orther to,overexpression vector of two types of Cn FT6 were constructed and transformed into wild-type arabidopsis.The detailed results are as follows:Firstly,CDS sequences of 174 arabidopsis genes involving in flowering time were downloaded from the National Center for Biotechnology information(NCBI),and were aligned with the CDS(coding sequences)database of coconut.A total of 148 candidate genes involving in flowering-time pathways were identified,Among them,74 candidate genes were classified into photoperiod pathway,11 candidate genes were classified into gibberellin(GA)pathway and 63 candidate genes were classified into autonomous pathway and vernalization pathway.Secondly,the expression profiles analysis of 148 candidate genes involved in flowering-time pathways showed that SOC1(CCG008883)had RPKM values of 0.22 and0.34 in tall and dwarf coconut seedlings respectively,but RPKM values of 55.16 and 50.12 in mature trees of tall and dwarf coconut respectively,FT(CCG011565)had RPKM values of8.87 and 8.1 in tall and dwarf coconut seedlings respectively,but RPKM values of 38.18 and48.14 in mature trees of tall and dwarf coconut respectively,AP1(CCG027514)had RPKM values of 0.74 and 0.15 in tall and dwarf coconut seedlings respectively,but RPKM values of56.6 and 51.41 in mature trees of tall and dwarf coconut respectively.These results indicate that Photoperiod Pathway may be play a important role in coconut flowering time.Alternative splicing analysis in tall and dwarf coconut showed that due to alternatice 3’ splicing site(A3SS)in the fourth exon of Cn FT6,one six-nucleotide indel was detected between tall and dwarf coconut.Meanwhile,the relationship between the six-nucleotide indel and flowering time were validated in 112 coconuts.These results indicate that alternative splicing of Cn FT6 may be play a important role in coconut flowering time.Thirdly,in order to verify the interaction between Cn FT6 and Cn FD,yeast two-hybrid were used to demonstrate that Cn FT6 a can interact with Cn FD,as well as Cn FT6 b and Cn FD.These results indicate that this alternative splice variant of Cn FT6 do not affect the interaction with Cn FD.The further investigation and validation is to verify whether this alternative splice variant of Cn FT6 play roles in the transcription of Cn AP1.Fourthly,overexpression vector of Cn FT6 a and Cn FT6 b were constructed,and transformed into wild-type arabidopsis thaliana.By comparing the flowering time of two transgenic arabidopsis and the wild-type arabidopsis,functions of Cn FT6 a and Cn FT6 b were identified preliminarily.