Functional And Evolutional Analysis Of Flowering Regulatory Genes In Platanus Acerifolia

Intensive studies of flowering regulation have been carried out in herbaceous model species(i.e.Arabidopsis and rice)and some core eudicots and monocots,but little is known in woody plants and basal eudicots.London plane(Platanus acerifolia Willd)is a basal eudicot tree belonging to the Platanaceae family of the order Proteales,which is also an excellent landscaping plant praised as 'the king of street trees' and is widely planted in roadside and courtyard for shading.However,countless flowers and fruits with pollens and seed hairs,respectively,scatter in spring and early summer from the adult trees,which is an increasingly serious problem that need to be resolved urgently.Understanding the mechanisms of flowering regulation in Platanus is helpful for breeding non-flowering or fruitless varieties.Based on the achievements in model species and the transcriptome data during the flowering initiation phase of London plane,a series of genes related to flowering regulation,including the members of FT/TFL1 and MADS-box gene family,and LFY/UFO homologs,were isolated and characterized in this study,which could not only shed light on the understanding of flowering regulatory mechanism in Platanus and the functional evolution of these genes,but also provide scientific basis for genetic improvement of the species.The main results are as following:1.Five FT/TFL1 family of genes were cloned and identified from London plane according to the homologous cloning and transcriptome sequencing data combined with RACE and Tail-PCR methods.Sequence alignment and phylogenetic analysis suggested that three of them(PlacTFLla,PlacTFLlb and PlacBFT)belong to TFL1-like clade,while the other two(PlacMFT1 and PlacMFT2)were grouped into the MFT-like clade.qRT-PCR analysis showed that three TFL1-like genes primarily expressed in vegetative phase.The expression of PlacTFL1a was much higher and wider than that of PlacTFL1b.PlacTFL1b was only detected in apical and lateral buds in April.The expression of PlacBFT peaked in young stem of adult trees and next in vegetative organs of juvenile plants.Two MFT-like genes were expressed mainly in fruits,and PlacMFT2 was limited expressed in fruits.Ectopic expression in Arabidopsis and petunia showed that three TFL1-like genes repressed flowering while two MFT-like genes promoted flowering.Yeast two-hybrid analysis indicated that only weak interactions were detected between PlacTFL1a and PlacFDL1,which demonstrated that PlacTFL1a might have more conserved functions.2.Five(SOC1-like genes were obtained according to the homologous cloning and transcriptome data combined with RACE method,named as PlacSOC1a/b/c/d/e.They show similar spatiotemporal expression pattern:primarily expressed in vegetative phase except PlacSOCla that was also obviously expressed in reproductive organs.Overexpression of PlacSOCls in Arabidopsis showed that PlacSOCla/b/e evidently promoted flowering but PlacSOC1c/d didn't advance flowering time and even had signs of late flowering.Yeast two-hybrid analysis indicated that only PlacSOC1a/b/e interact with PlacSVP1,which is in accordance with the phenotypic changes of transgenic plants.It's worthy to mention that 35S:PlacSOC1c transgenic Arabidopsis plants showed affected floral organ development.In addition,PlacSOC1c can interact with PlacAGL6a/b,indicating the functions of PlacSOClc may related with the E-class genes.3.Three SVP-like genes were isolated and characterized in London plane,named as PlacSVP 1/2/3,qPCR analysis indicated that they were expressed principally in vegetative phase.PlacSVP1 and PlacSVP3 had a gradually increased expression level in subpetioilar and apical buds during the early stage of flower bud differentiation,whereas PlacSVP2 expression was declined during the same development stages.We speculated that PlacSVP2 may maintain vegetative meristem identity and suppress flower bud differentiation,and PlacSVP1 and PlacSVP3 may have functions opposite to PlacSVP2.Ectopic expression in Arabidopsis showed that SVP-like genes from Platanus delayed flowering and changed floral organ development.However,overexpression of PlacSVP1 in petunia only influenced the development of floral organs.4.Three FUL-like genes designated as PlacFL1/2/3 were cloned and functional analyzed.The expression of PlacFL2 was much higher and wider than that of PlacFL1 and PlacFL3.PlacFL2 was also detected in vegetative organs in addition to male and female inflorescences development.Overexpression of PlacFL1/2/3 in Arabidopsis resulted in early flowering.PlacFLl and PlacFL3 may play roles in floral transition and inflorescence development.PlacFL1 may also have functions in leaf and vegetative bud development.5.Five SEP-like genes were cloned and identified from London plane.Sequence alignment and phylogenetic analysis indicated that three genes belong to the SEP1/2/4 clade,while the other two genes are grouped into the SEP3 clade.qRT-PCR analysis showed that all PlacSEPs,except PlacSEP1.1 and PlacSEP1.2,were expressed during the male and female inflorescence initiation,and throughout the flower and fruit development process.PlacSEP1.2 gene expression was only detected clearly in female inflorescence at April.PlacSEP1.3 and PlacSEP3.1 were also expressed,although relatively weak,in vegetative buds of adult trees.Overexpression of PlacSEPs in Arabidopsis plants resulted in different phenotypic alterations.35S:PlacSEP1.1.35S:PlacSEP1.3 and 35S:PlacSEP3.2 transgenic Arabidopsis plants showed evident early flowering,with less rosette leaves but more cauline leaves,while 35S:PlacSEP1.2 and 35S:PlacSEP3.1 transgenic plants showed no visible phenotypic changes.35S:PlacSEP1.1 and 35S:PlacSEP3.2 transgenic Arabidopsis plants also produced smaller and curled leaves.Yeast two-hybrid analysis indicated that PlacSEPs proteins can form homo-or hetero-dimers with the Platanus API/FUL,B-,C-and D-class MADS-box proteins in different interacting pattern and intensity.6.Two AGL6-like genes,PlacAGL6a and PlacAGL6b,were identified from Platanus.The spatiotemporal expression pattern of PlacAGL6a/b were similar,with high expression in inflorescence differentiation and development and relative lower expression in fruit development.No expression was detected in vegetative organs.Ectopic expression of PlacAGL6a/b in Arabidopsis showed precocious flowering,curled leaves and more lateral branches,however,only 35S:PlacAGL6a transgenic plants showed altered floral organ development.In addition,PlacAGL6a can form homodimer and interact with C-and D-class MADS-box proteins,but PlacAGL6b can't,suggesting that PlacAGL6a and PlacAGL6b may have redundant and distinct functions.7.The UFO homolog of Platanus(PlacUFO)and its 3.3 kb promoter sequence containing 5' UTR(pPlacUFO)were cloned and characterized.The results suggested that only one homolog of both UFO and LFY exists in Plastanus genome.PlacUFO exclusively expressed with low level in subpetiolar buds of juvenile,apical and subpetiolar buds during floral initiation and mix buds in June.Overexpression of PlacUFO in Arabidopsis and petunia plants didn't affect flowering time,but changed floral organ and leaf development.Ectopic expression of PlacLFY in Arabidopsis showed precocious flowering and more axillary branches,with lateral inflorescences transformed into single flowers.Yeast two-hybrid analysis indicated that PlacUFO had no interaction with PlacLFY.Histochemical staining of pPlacUFO:GUS and pPlacLFY.GUS transgenic Arabidopsis showed that pPlacUFO drives GUS expression mainly in petals,stamens and stigmas,and the vascular tissues of roots,stems and leaves of seedlings,while pPlacLFY showed a wider range of expression.