| Trehalose is a stable non-reducing disaccharide that enhances the ability of resurrection plants to resist abiotic stress.However,in most plants,the concentration of trehalose is maintained at a very low level that makes it impossible to play the roles of osmotic adjustment and stress tolerance.In stead,its biosynthesis intermediate,trehalose-6-phosphate(T6P),was found to act as an important signaling molecule,which together with the energy-metabolizing control switch,SnRKl(sucrose non-fermenting-1-related protein kinase-1),participates extensively in plant growth and development,and stress response.In Hevea brasiliensis(para rubber tree),rubber biosynthesis that occurs in specialized laticiferous cells uses sucrose as the precursor molecule.The regulation of the sucrose metabolism in laticifers becomes a key factor affeecting rubber production.To understand the physiological functions of T6P synthase genes in H.brasiliensis(HbTPSs),especially whether this type of protein and its catalytic product T6P are involved in the regulation of sucrose metabolism and latex regeneration in laticifers,we cloned and functionally characterized the TPS gene family in this species.The main results obtained are as follows:1.By searching the Hevea genome database,we identified a total of 14 putative HbTPS genes,named HbTPS1 to 14,and submitted to GenBank under accession numbers of KT313591 to KT313604,respectively.Of the 14 HbTPS genes,four(HbTPS1 to 4)were grouped in class Ⅰ,with each having 17 exons and 16 introns,and the remaining ten(HbTPS5 to 14)in class Ⅱ with 3 exons and 2 introns.We successfully cloned the full-length cDNAs of 12 HbTPS genes,with the exception of two ones.HbTPS3 and HbTPS4.2.RNA-seq analysis was conducted to explore the tissue expression patterns of HbTPS genes.It turned out that HbTPS5 was the most abundant isoform in latex,Hb TPS1 being the predominant in trunk bark,Hb TPS6 and Hb TPS9 the major in root,HbTPS7 and HbTPS13 the most abundant in mature seeds,HbTPS11 and HbTPS12 the highest in mature leaf and female flower,respectively,and HbTPS1,HbTPS5,HbTPS6,HbTPS11 and HhTPS13 genes all exhibiting higher expressions in male flowers.However,the expressions of HbTPS3 and 4 were not detected or at very low levels in any of the seven tissues(latex,mature leaf,trunk bark,mature seed,male flower,female flower and root)examined,and they were thought to be pseudogenes.3.Functional complementation experiments using yeast mutants demonstrated that of the 12 cloned HbTPS genes,only the two Class I TPS genes(HbTPS1 and 2)encode proteins with TPS activity,and their overexpression in the yeast mutants showed enhanced resistance to salt stress.4.Immunofluorescence histological localization showed that HbTPS5 protein was mainly located in Hevea laticifers,but the failure of its direct interaction with HbTPS1 or 2 as revealed by a yeast two-hybrid assay suggested the requirement of other factors for the participation of HbTPS5 in regulating TPS enzyme activity.5.In Hevea seedlings,the expressions of HbTPS1 and 2 genes were significantly affected by temperature and drought stresses.Although their expressional response to the treatments of low temperature(4℃)and high temperature(40 ℃)was similar,but a significant difference was observed in their response to drought stress.In the latex of regularly tapped Hevea trees,the ethylene treatment induced the expressions of HbTPS1 but inhibited those of HbTPS2.In comparison,the treatments of 2,4-D,MeJA(methyl jasmonate),SA(salicylic acid)and wounding all significantly up-regulated the expressions of both Hb TPS genes.6.The promoter sequence of HbTPS1 gene was obtained by the genome-walking method and predicted to harbor multiple SA-,MeJA-,light and heat-stress responsive cis-elements,as well as endosperm-specific elements,suggesting that HbTPS1 gene is involved in a variety of biological processes.The studies of HbTPS1 promoter::GUS transgenic Arabidopsis showed that HbTPS1 gene was widely expressed in different Arabidopsis stages,and in different organs including leaves,young siliques and flowers.7.In virgin Hevea trees,the expression of HbTPS1 in latex was induced by tapping,while those of HbTPS2,HbTPS5,HbSnRK1-1 and HbSnRK1-2 were inhibited.With the increasing number of tapping,the TPS activity in latex was enhanced,the T6P content increased,but the SnRK1 kinase activity decreased.In addition,the SnRK1 activity in latex was significantly inhibited by the addition of exogenous T6P.These results suggested that tapping might have enhanced the synthesis of T6P through regulating the expression of TPS genes in laticifers,which thereby inhibiting the activity of SnRK1 protein kinases,and then activating sucrose metabolism to promote latex regeneration.8.The expressions of HbTPS and HbSnRK1 genes,TPS activity,sucrose and T6P contents were analyzed in the leaves at different developmental stages of Hevea tree.It was found that the T6P-SnRK1 signaling pathway was also involved in the regulation of sucrose metabolism and development in Hevea leaves.9.Transgenic Arabidopsis plants overexpressing HbTPS1 and 2 were generated,and their phenotypes were studied.Transgenic plants overexpressing HbTPS1 exhibited improved resistance to freezing,heat and drought stresses,but not to the salt stress.Transgenic Arabidopsis plants overexpressing HbTPS2 showed an accelerated growth,an early flowering,as well as an increase in leaf area,leaf number,and hundred-seed weight compared with the wild-type plants.To conclude,based on the assays of gene expression,enzyme activity and metabolite content as well as transgenic research,the TPS gene family that consists of multiple transcription-active isoforms was suggested to extensively participate in sucrose metabolism,latex regeneration,abiotic stress response and plant growth and development in Hevea tree.The results of this paper have not only Iaied a good foundation for the studies into the physiological functions of TPS genes in Hevea tree,but provided useful hints into the relevant studies in other plant species. |
PDF Full Text Request