Characterization Of Genes Involved In Laticifer Differentiation Of Hevea Brasiliensis

Laticifer is a special tissue in Hevea brasiliensis where natural rubber is biosynthesized and accumulated.The number of laticifer is a key factor that influences the production of rubber.Previous work showed that secondary laticifer differentiation was induced from vascular cambia in young stems of epicormic shoots after mechanical wounding or treatment with exogenous jasmonic acid(JA).The exploited trees produced 2-3 times more laticifer rings and latex yield than that of unexploited trees,suggesting that JA-mediated wounding responses are involved in laticifer differentiation and latex biosynthesis.However,the underlying molecular mechanism is so far less known.Recent reports showed that the differentiation of secondary laticifers was prevented when the wounding site of epicormic shoots was wrapped immediately after wounding.In this work,the differentially expressed genes were screened by suppression subtractive hybridization(SSH)and macroarrays,leading to the identification of a NAC transcription factor(termed HbNAC1)and two dehydrins(termed HbDHN1 and HbDHN2)that might be involved in laticifer differentiation in H.brasiliensis.The induction of HbDHN1,HbDHN2 and HbNAC1 was proposed to be related with local tissue dehydration caused by wounding,and the local tissue dehydration might be an important signal that induced rubber laticifer differentiation.To verify this hypothesis,this work carried out the following researchs:(1)To further verify the differential expression pattern of HbDHN,HbDHN2 and HbNACl,quantitative real-time PCR(qRT-PCR)was used to monitor the gene expression in exposed or wrapped bark after wounding treatment.As revealed by macroarray analysis,the expression of HbDHNl,HbDHN2 and HbNAC1 in wrapped bark samples were higher than those in exposed bark samples 1 h after wounding treatment,which was verified by quantitative real-time PCR.However,the induction of HbNAC1 and HbDHN2 in the exposed samples was over a longer duration and displayed a shift in peak expression levels,compared to that in wrapped bark samples.The expression peak of exposed samples appeared at 6 h;in contrast,the peak expression of the wrapped samples appeared 1 h after wounding treatment.(2)It was therefore important to know whether local tissue dehydration is a key signal for wounding responses,such as laticifer induction,in rubber trees.In order to test this,the epidermis of epicormics shoots was wounded by slight scraping with a sharp razor blade,following which the wounding site was daubed with 10%PEG6000 and immediately wrapped with parafilm,the result indicates that local tissue dehydration is essential for laticifer induction by wounding in rubber trees.(3)In this work,the DHN genes(HbDHNl and HbDHN2)were further investigated.Multiple sequence alignment and phylogenetic analysis showed that two HbDHNs belonged to the SK2 subgroup.(4)Differential expression analysis:HbNAC1,HbDHN1 and HbDHN2 genes are expressed in the rubber tree different organizations.The expression level of HbNAC1 is little in latex,HbDHN1 and HbDHN2 genes are expressed predominantly in the stem tip.We analyzed the promoter fragment of the HbNAC1,HbDHNl and HbDHN2 for the presence of putative cis-acting regulatory elements.These include ABREs,TGACG-motifs CGTCA-motifs,and LTR motif etc.The qRT-PCR analysis showed that the expression levels of the HbNAC1,HbDHN1 and HbDHN2 could be markedly up-regulated by cold,salt,drought,wounding,ABA,ET and MeJA treatment.The results show that HbNACl,HbDHNl and HbDHN2 may participate in multiple abiotic stress responses.(5)In order to study whether HbNAC1 is a transcription factor,subcellular localization analysis results show that HbNACl is a nuclear localized protein.The transactivation ability of HbNACl was analyzed using a yeast assay system.These data confirmed that HbNAC1 exhibited transactivation activity at the C-terminus.(6)To further investigate whether HbNAC1 is involved in biosynthesis of rubber,Yeast hybrid and EMSA suggest that HbNACl bound to CACG and CATGTG binding sites,and we screen HbNAC1 associated with five promoters of rubber latex synthesis related genes by yeast one hybrid.The results show that only pHbSRPP had the potential to interact with HbNAC1.To investigate whether HbNAC1 could bind to the promoter of HbSRPP and regulate the expression in plants,the double transgenic line(pHbSRPP::GUS and 35S::HbNAC1)showed significantly higher GUS activity thanthe pHbSRPP::GUS line,suggesting that HbNAC1 might interact with the HbSRPP promoter and upregulate the expression of the reporter ?-glucuronidase in tobacco.(7)To investigate whether HbNAC1 is also responsive for drought tolerance in plants,transgenic lines overexpressing HbNAC1 were subjected to drought stress,compared with the wild-type,survival rate,relative water content,chlorophyll content,proline content and APX activity of the transgenic plants were significantly higher than that of the wild-type,whereas,electrical conductivity,O2-and H2O2 content is significantly lower than the wild-type.In addition,the expression of stress response related genes,such as ERD10C,LEA5,PP2C2 and NECD3 in transgenic tobacco,is significantly higher than the wild-type.These results suggest that HbNAC1 has pleiotropic effects on improving plant adaptation to abiotic stresses through regulation of the expression of downstream genes.(8)Overexpression of HbDHN1 and HbDHN2 in Arabidopsis thaliana increased tolerance to salt,osmotic and drought stresses.Compared with the wild-type,the transgenic plants accumulated more proline and suffered less membrane damage under salt,osmotic and drought stresses,the HbDHNs might be a ROS scavenger directly or indirectly regulating the redox status in vivo that affect the laticifer differentiation.Taken together,we may draw the conclusion that the HbDHN1,HbDHN2 and HbNAC1 genes are involved in dehydration and drought stress.The epidermis of epicormics shoots was wounded,the wound is exposed to the air can lead to local tissue dehydration,induced dehydration related genes,HbDHN1,HbDHN2 and HbNAC1 gene expression,and local tissue dehydration is an important signal that induced rubber laticifer differentiation.The HbDHN1,HbDHN2 and HbNAC1 gene are participate in multiple abiotic stress responses,the HbDHNs might be a ROS scavenger directly or indirectly regulating the redox status in vivo that affect the laticifer differentiation.In addition,HbNAC1 could bind to HbSRPP promoter of rubber synthesis related gene and regulate the expression of the gene,so is an adhesive related genes with dehydration induced laticifer differentiation and latex biosynthesis.