Functional Analysis In Hevea Brasiliensis Of Hberf-Ixc4 And Hberf-Ixc5 Genes, Two Potential Orthologs Of The Erf1 Gene From Arabidopsis

Abstract

Natural rubber (NR) (cis-1,4-polyisoprene) is the main production from Hevea brasiliensis (Willd. Ex A. Juss.) Müll. Arg. NR is a very important industrial material for transportation, consumer, and medical. The demand for NR is increasing from year to year. NR is obtained from latex. The latex flows out from laticifers after tapping the bark. Ethephon, an ethylene releaser, can be applied on clones to stimulate the latex production. Tapping and ethephon stimulation are sources of harvesting stresses conducing to the production of secondary metabolites and consequent rubber. Ethylene (ET) and jasmonic acid (JA) biosynthesis and signalling pathways play a crucial role in the response to latex harvesting stress. Two Hevea ethylene response factor genes, HbERF-IXc4 and HbERF-IXc5, were predicted to be orthologs to ERF1 from Arabidopsis. ERF1 was suggested to be a component of defence responses through the involvement of ethylene and jasmonic acid signalling pathways. Transcripts of HbERF-IXc4 and HbERF-IXc5 were dramatically accumulated by combining wounding, methyl jasmonate, and ethylene treatment. These factors were assumed to be a regulator at the crosstalk of ethylene and jasmonate signalling pathways in latex cells. HbERF-IXc4 and HbERF-IXc5 have several features of transcription factor revealed by transactivation experiment and subcellular localization, respectively: they can activate the GCC cis-acting element of promoters of target genes and are localized in nucleus, In this study, functional analysis of HbERF-IXc4 and HbERF-IXc5 genes have been carried out by overexpression of these genes under the control of 35S CaMV and HEV2.1 as a promoter in transgenic Hevea lines obtained by Agrobacterium tumefaciens-mediated genetic transformation. This overexpression of genes led to emphasize the effect of native HbERF-IXc4 and HbERF-IXc5 genes. Twenty-nine GFPpositive lines were established on paromomycin selection medium. Twelve lines regenerated plants but only ten led to produce a sufficient number of plants for further phenotyping with totally 1,622 transgenic plants in greenhouse. These ten lines were confirmed as transgenic by Southern blot hybridization. Observation of morphology until one year showed both genes (HbERF-IXc4 and HbERF-IXc5) promoted a better growth in terms of plant height, stem diameter, and weight of aerial and root system with higher vigour and better tolerance to some abiotic stresses. Plants overexpressing HbERF-IXc5 have also a better performance than HbERF-IXc4. Data also showed a vigorous root system well balanced with regard to the whole plant. Real-time RT-PCR analyses revealed that expression of HbERF-IXc4 and HbERF-IXc5 genes was higher in transgenic lines compared to wild-type. Analysis in details of HbERF-IXc5 lines also showed some changes in anatomy (cambium activity, number of latex cells, starch, and width of xylem). This work is the first successful functional analysis of transcription factors in Hevea. Some differences have been observed between HbERF-IXc4 and HbERF-IXc5. As ERF1, HbERF-IXc4 and HbERF-IXc5 proteins should drive the response to some stresses. HbERF-IXc5 protein might be a regulator of laticifer differentiation. This study could be completed with analysis of silenced transgenic lines, comparison of transcriptome, metabolome of wild-type and transgenic lines, and identification of target genes controlled by HbERF-IXc4 and HbERF-IXc5. These results could be applied through development of molecular genetic markers for tolerance to harvesting stress and development of new stimulants for agronomical application.