Main conclusions on the effects of promoters and candidate genes

influenced by drought and salt stress has special characteristic. The size of stomatal aperture becomes smaller, so it will reduce transpirational water loss Cheng et al. 2013. Duan 2011 has been reported that HbERF-IXc5 functions like the ERF1. The response of HbERF-IXc5 to the combined treatment of wounding, MeJA and ET was significantly multiplied Duan 2011. Unfortunately, histological analyses were carried only for HbERF-IXc5 gene, so no comparison was possible for the two candidate genes at this level. The effect of HbERF-IXc4 and HbERF-IXc5 genes in stress response ability of transgenic plants was successfully conducted on several environmental stress treatments. The ability of plants species to tolerate cold stress was varying; it depends on gene expression to modify their physiology, metabolism, and growth Chinnusamy et al. 2010. In cold stress response, HbERF-IXc5 performed a better response than HbERF-IXc4 under control HEV2.1 promoter in parameters: FvFm value, P. Index, and SPAD value. The maximum photochemical efficiency of the photosystem II FvFm indicated particular responses of the genotypes. However, both of genes HbERF-IXc4 and HbERF-IXc5 had good ability to face senescence than control. It was described with the leaflets number, all transgenic lines showed more leaflets compared to the control. Similar with ERF1 function, HbERF-IXc5 might play a role to enhance the plants ability to cold stress response. ERF1 was directly regulates abiotic stress response genes cold, salinity, and water deficit in Arabidopsis Cheng et al. 2013; Hao et al. 2002; Sakuma et al. 2002. With regard to FvFm and SPAD value, both of genes had a same capability in salinity stress tolerance, but HbERF-IXc5 performed a better P. Index than HbERF-IXc4. The ratio of v m is a direct measure of the “optimal uantum efficiency” of the plant Genty et al. 1989. P.Index reflects the functionality of both photosystems I and II and gives quantitative information on the current state of plant performance under stress conditions Strasser et al. 2004. In the other hand, the SPAD value can be used as an indicator of chlorophyll content that measure greenness based on optical responses Kariya et al. 1982. However, number of leaflets analysis revealed that transgenic plant with construct of HEV2.1::HbERF-IXc5 also showed a better performance to adapt with salinity stress compared to control by growing a new shoot. Leaf abscission was reflecting the effect of ethephon treatment. Ethephon or native ethylene was promoted in abscission by induction of hydrolytic enzyme synthesis and secretion of this enzyme into cell wall Abeles 1969; Abeles and Leather 1971; Horton and Osborne 1967. Analysis of leaflets number and leaves morphology revealed that HbERF- IXc4 gene had a great ability to face senescence. It might suggest that this gene could raise the ethephon tolerance in Hevea transgenic plants.

4.2.3 Main conclusions on the effects of promoters and candidate genes

The previous research showed that the promoter 35S CaMV drives a strong GUS activity in embryogenic callus and plant vessels Lagier 2004, but these results showed contrary result. This suggests that HbERF-IXc4 has a negative effect on callus proliferation and plant regeneration when driven by this strong promoter. HEV2.1::HbERF-IXc4 and HEV2.1::HbERF-IXc5 lines can regenerate a large number of plantlets with normal phenotype but with higher performance in terms of growth for root and aerial parts. Similarly, no obvious changes could be observed in plants from 35S::HbERF-IXc5 or HEV2.1::HbERF-IXc5 lines. These observations led to conclude that there is no deleterious effect of this gene whatever the stages of development. HbERF-IXc4 and HbERF-IXc5 genes likely play different role especially in the control of different target genes. Indeed, HbERF-IXc4 only activates the promoter of the SUT3 gene. HbERF-IXc4 has been identified as a binding factor to the promoter of the gene HbSUT3 by simple hybrid technique Zhang et al. 2014. This suggests involvement of HbERF-IXc4 in activating the sucrose loading in laticifers, which is a carbon source for the biosynthesis of polyisoprene chains. Table 57 Summary effect of lines on morphology of plants. Red highlight is significant difference and blue highlight is not significant difference. R is total root weight and R1 is taproot Construct Line Height Stem diameter Leaves and leaflets Total weight Leaf weight Stem weight Root weight Ratio Rtot plant Ratio R1tot R Wild-type CI07060 35S::HbERF-IXc4 TS17A61 TS17A79 HEV2.1::HbERF-IXc4 TS18A37 TS18A09 TS18A13 TS18A20 TS18A69 35S::HbERF-IXc5 TS19A46 TS19A90 HEV2.1::HbERF-IXc5 TS20A47 TS20A69 TS20A75 Table 58 Summary effect of construct on abiotic and biotic stress based on average value. Red highlight is significant difference and blue highlight is not significant difference Construct Abiotic stress Biotic stress Cold Salinity Ethephon Water FvFm P. Index SPAD Leaflets FvFm P. Index SPAD Leaflets Wild-type HEV2.1::HbERF-IXc4 35S::HbERF-IXc5 HEV2.1::HbERF-IXc5 4.3 HbERF-IXc4 and HbERF-IXc5 are putatively orthologs to ERF1 according to the phylogenetic analysis but might have additional functions

4.3.1 ERF1 phenotype in other species