| dc.description.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. | id |
