Mechanism Of SlCycB2 Regulating Multicellular Trichome Formation And Flower Organ Development In Plant

Plant trichomes play important roles in response of biological and abiotic stresses and provide an excellent model for studying the molecular mechanisms underlying cell fate determination.Trichomes in Arabidopsis are typically unicellular structures,which are controlled by a WD40-bHLH-MYB complex.The active complex triggers trichome formation by directly inducing the expression of downstream genes.However,the regulatory pathway of multicellular trichomes in tomato and tobacco is different from that in Arabidopsis.Until now,the regulatory mechanism of tobacco and tomato multicellular trichome formation is unknown.Cyclins that determine the transition between the phases of the cell cycle in eukaryotes,function in many development processes,play important roles in trichome formation.Floral organ development is of great importance in biology.Although the ABCDE model is constructed to study the developmental regulation of flowers,the regulation of B-type cyclin on flower development is still limited.In this study,the putative homologs of SlCycB2 were identified in tomato,tobacco and Arabidopsis and the function of these genes were analyzed in tomato and tobacco.The main results are as follows:1.Amino acid sequence analysis demonstrated that seven homologs of SlCycB2 were identified in tomato,tobacco and Arabidopsis,and these genes were named as SlCycB3(Solyc06g073990),SlCycB4(Solyc01g007870),AtCycB2(AT5G06270)and AtCycB3(AT3G11600),NtCycB2(gnl|UG|Nta#S57566025),NtCycB3(gnl|UG|Nta#S45437178)and NtCycB4(gnl|UG|Nta#S33541254),respectively.Structural analysis revealed that these genes contained one exon,which encode about 100 amino acids with three conserved motifs.Phylogenetic analysis revealed that SlCycB2 was highly homology with SlCycB3,SlCycB4,AtCycB2 and AtCycB3,all of which belongs to a very conservative B type cyclin family.2.In order to identify homologs of SlCycB2 in different species,we also conducted pBLAST with the amino acids of SlCycB2-type cyclins in tomato,tobacco and Arabidopsis at NCBI database,the results showed that SlCycB2-type cyclins are wide existed in different species and the function of which are unknown,indicating that these cyclins must have important biological functions.3.Tissue expression profiles analysis of SlCycB2 and SlCycB3 genes revealed that SlCycB2 and SlCycB3 are expressed in all tested tissues,being higher in young leaves and flowers than other tissues.Transient expression of CaMV35S::SlCycB2-EGFP fusionconstruct in onion epidermal cells showed that SlCycB2 localized in the nucleus.SlCycB2 promoter sequence analysis showed that several light response cis-elements were found.The transcripts levels of SlCycB2 and SlCycB3 were determined in AC plants which treated by light or dark.The results exhibited that transcripts accumulation of SlCycB2 and SlCycB3 was significantly suppressed by dark but induced by light.Promoter analysis demonstrated that GUS activity was detected in several tissues,including trichomes of hypocotyledonary axis,shoot and leaves,stigma and immature fruit,especially very high expressed in glands.4.Function analysis of SlCycB2 demonstrated that SlCycB2 regulates tomato multicellular trichomes formation.Over-expression vector and RNAi vector of SlCycB2 were transformed to Ailsa Craig mediated by Agrobacterium.qRT-PCR analysis showed that SlCycB2 was higher expressed in over-expression plants than that in AC plants and was lower expressed in RNAi plants than that in AC plants.Compared with AC plants,the trichome density of SlCycB2 over-expression transgenic plants decreased significantly,type I,type III and type V trichomes are disappeared in the leaves and stems and the density of type VI,type VII is also significantly decreased.In contrast,the trichome density,especially type III and type V trichome of SlCycB2 RNAi plants dramatically increased obviously.Furthermore,a novel type III-like trichome increased in the stems of SlCycB2 RNAi plants.5.Monoterpenes and sesquiterpenes were detected in the leaf extracts of AC plants and SlCycB2-OE lines.The results showed the synthesis of these monoterpenes and sesquiterpenes were dramatically inhibited in transgenic plants compared with the AC plants.Terpenoids have an important effect on defense against insect herbivores.Thus,we assess the effect of transgenic leaves on defense against insect herbivores,the results indicated that SlCyc B2 over-expression transgenic plants were more susceptible to insects.These results suggest that the glandular trichomes including type VI play important roles in herbivore defense in tomato.6.Function analysis of SlCycB2 also demonstrated that SlCycB2 regulated tomato flower organs development.SlCycB2 over-expression transgenic plants showed that style length is significantly shorter than that in AC plants,stamens in SlCycB2 over-expression transgenic plants are unclosed and fruits cannot finish the processes of enlargement.SEM observation indicated that the decreased style resulted from the shorter cell length of the styles.More interestingly,almost all the stigma papilla cells are collapsed and the size of stigma becomes much smaller due to the overexpression of SlCycB2 gene.Furthermore,many pollen grains were aberrant and uninflated in these transformants.These results suggest that SlCycB2 participate in the processes of stamen,style and pollen development.7.RNA-seq analysis illustrated that there were 241 differentially expressed genes(DEGs)between SlCycB2 over-expression transgenic plants and AC plants.There were84 DEGs between SlCycB2 RNAi transgenic plants and AC plants.There were 834 DEGs between SlCycB2 over-expression and RNAi transgenic plants.The putative biological functions of these DEGs using the KEGG pathway were analyzed.The results demonstrated that the expression of the genes involved in cutin,suberine and wax biosynthesis,steroid biosynthesis,diterpenoid biosynthesis,peroxisome,biosynthesis of secondary metabolites were obviously repressed in SlCycB2-OE plants.These results indicated the decrease of terpenes led to weakness of the defenses against insect herbivores in SlCycB2-OE plants.8.Function analysis of SlCycB2-type genes declared that this type genes have the same functions in regulating multi-cellular trichome formation in tomato.We construct constructed SlCycB3,SlCycB4,NtCycB2,NtCycB3,NtCycB4,AtCycB2 and AtCycB2over-expression constructs using pMV2 vector and SlCycB3 and SlCycB4 RNAi suppression constructs using the pHELLSGATE2 vector.These vectors were transformed to Ailsa Craig mediated by Agrobacterium.qRT-PCR and RT-PCR analysis showed that all these genes were higher expressed in their over-expression plants than that in AC plants and SlCycB3 and SlCycB4 were lower expressed in their RNAi plants than that in AC plants.Compared with AC plants,the trichome density of SlCycB3,Nt CycB2 and AtCycB2 over-expression transgenic plants was decreased significantly and the trichome density of SlCycB3 RNAi plants was increased.These results demonstrated that these small cyclins had same function in tomato multicellular trichome formation.9.The transcription levels of SlCycB2 and SlCycB3 in SlCycB2,SlCycB3,NtCycB2 and AtCycB2 over-expression transgenic plants were also determined,respectively.SlCycB2 as well as SlCycB3 were lower expressed in NtCycB2 and AtCycB2over-expression transgenic plants than that in AC plants.Furthermore,SlCycB2 was lower expressed in SlCycB3 over-expression transgenic plants,as the same results,SlCycB3 was lower expressed in SlCycB2 over-expression transgenic plants.The results showed that the expression of this type of cyclins could be repressed by overexpression of its homologs.10.SlCycB2,SlCycB3,SlCycB4,NtCycB2,NtCycB3,NtCycB4,AtCycB2 andAtCycB2 over-expression vectors were transformed to tobacco mediated by Agrobacterium.qRT-PCR and RT-PCR analysis showed that all these genes were higher expressed in their over-expression transgenic plants than that in transgenic backgound tobacco plants(WT).Compared with WT,the trichome density of SlCycB2,SlCycB3,AtCycB2 and NtCycB2 over-expression transgenic plants was obviously decreased.Two type trichomes were reduced in these transgenic plants through SEM observation.The similar roles of its homologs in multicellular trichome formation suggest that Solanaceous species may share common multicellular trichome formation regulatory pathway.