Cloning And Function Analysis Of PEBP Family From Rubber Tree(Hevea Brasiliensis Muell.Arg.)

Flowering is a very important process of reproduction in higher plant species.It has been confirmed that phosphatidylethanolamine-binding protein(PEBP)family played a critical role in regulating the flowering transition.Rubber tree(Hevea brasiliensis Muell.Arg.)which belongs to woody perennial species is nowadays the most important plant among 7500 plant species for producing natural rubber.The new cultivars or clones characterized by higher quantity and quality,disease and stress resistance are always selected mainly from traditional breeding practice.However,the breeding processes take a very long time since rubber tree grows mature for several years from the post-embryo development to flowering,and some special agronomic traits only occur in mature tree.It is of great significance to shorten the breeding processes via molecular assistance to improve the breeding program.In this context the PEBP family members are hence cloned from rubber tree for function analysis to elucidate their biological roles during flowering transition,which will facilitate improvement of the breeding program as combined with the transgenic method.Our study results are summarized as follows.1.Eight PEBP family members were cloned from rubber tree and were designated as HbFTI HbFT2,HbTFLl-1,HbTFL1-2,HbTFLI-3,HbCEN1,HbCEN2 and HbMFT1,respectively.All of the members,except HbCEN1 which contained 4 exons and 4 introns,conserved the same genomic structure,including 4 exons and 3 introns.Our phylogenetic analysis showed that the eight proteins fall into three clades,HbFT1 and HbFT2 into the FT clade,HbTFL1-1,HbTFL1-2,HbTFL1-3,HbCEN1 and HbCEN2 into the TFL1/CEN clade,and HbMFT1 into the MFT clade.2.qRT-PCR results revealed that the eight members showed divergent expression profiles.HbMFT1 was mainly expressed in embryos.HbFT1 mRNA was abundant in mature leaves of a mature tree.The quantitatively preferential expression of HbFT1 was in reproductive organs.HbTFL1-1 was expressed predominantly in roots of three-month-old seedlings and the mature leaves of trees at different years.HbTFLl-2 was primarily detected in the roots of the three-month-old seedlings.HbTFL1-3 was most highly expressed in the shoot apex meristems(SAM)of the three-month-old seedlings and open male flowers.HbCEN1 and HbCEN2 showed a predominant expression in the stems and SAM of rubber trees at different ages.The mRNA expression levels of HbTFL1-1,HbTFL1-2 and HbTFLl-3 increased whereas those of HbCENl and HbCEN2 tended to decrease as inflorescences developed,especially the mRNA level of HbCEN2 which was under the detection limit in open inflorescences.3.qRT-PCR of FT-like genes of the mature leaves in different months showed that the same gene displayed similar expression pattern between rubber trees at different ages,and that the mRNA accumulation was increased with tree age,but HbFT1 differed from HbFT2 in the expression pattern.HbFTl mRNA was mostly detected in September and October,but weakly detected from March to May,which is the floral initiation period,whereas HbFT2 transcript was abundant in March,June,July and November.4.qRT-PCR revealed that HbMFTl and FT-like genes were strongly induced in mature leaves of the three-month-old seedlings under short-day conditions,but exhibited different expression profiles,in which the expression of HbMFT1 peaked at the beginning of the light conditions(8 am),whereas two peaks of HbFTl mRNA were at 12 pm and 6 am and HbFT2 transcript was detected highest at 10 pm.On the contrary,quite weak expressions could be detected for three of them under long-day conditions.Additionally,three of them exhibited the highest expression levels at 28?.5.In order to investigate the biological functions of these members of PEBP family from rubber tree,we constructed several recombinant constructs carrying the 35S promoter which was fused respectively with them and transformed into the wild-type Arabidopsis plants,respectively.As a result,the transgenic plant transformed with HbMFTl delayed germination,growth and development,and the aerial tissues and flowering transition.The transformants with TFL1/CEN-like genes were affected in plant architecture,showing a prolonged vegetative phase and delayed flowering,with floral and silique abnormalities to some different degrees.Moreover,a few tranformants with each of TFL1/CEN-like genes did not flower until death.For both of the FT-like genes the transformants could trigger the appearance of premature flowering,as seen in the phenotypes of transgenic Arabidopsis plants,which bore terminal flowers.6.These recombinant constructs carrying the 35S promoter fused respectively with PEBP family members were further transformed into tfl-1 and ft-10 mutants,respectively.As a result,some of the transgenic plant carrying each of TFL1/CEN-like genes exhibited a normal phenotype similar to wild-type plant,whereas fewer severely delayed flowering and very few showed non-flowering phenotype.By the same way,some of transformants with FT-like genes also showed similar patterns to wt in phenotype and some other ones showed earlier flowering even than wt.7.Eight promoters were respectively cloned and fused with GUS gene(except HbCEN2 promoter),and then transformed into Arabidopsis,respectively.As a result,the transgenic plants with each promoter showed that GUS activity was in more organs relative to the original tissue-specific expression in rubber tree,largely consistent with the tissue-specific cis-elements existing in the promoter.Accordingly,it was speculated that the activities of some of cis-elements in each of promoters may somehow be inhibited by the up-stream regulated elements in rubber tree.In addition,the GUS activity of HbMFTl promoter in the transgenic Arabidopsis plants was induced by ABA.On the basis of all the results mentioned above,we supposed that HbMFTl was mainly involved in the embryos developmental process,but inhibited seed germination,pant growth and development and the initiation of flowering.Five TFL1/CEN-like genes may be implicated in maintenance of juvenility or vegetative growth.The expression pattern of HbFT1 hinted at roles of development in other aspects,such as leaf senescence and shoot dormancy,rather than flowering transition,even though it could advance flowering as ectopically over-expressed in Arabidopsis,whereas HbFT2 may be a flowering promoter in rubber tree,favourable for formation and development of floral organs.