Overexpression of Hevea brasiliensis ethylene response factor HbERF-IXc5 enhances growth and tolerance to abiotic stress and affects laticifer differentiation

Abstract

Ethylene response factor 1 (ERF1) is an essential integrator of the jasmonate and ethylene signalling pathways coordinating a large number of genes involved in plant defences. Its orthologue in Hevea brasiliensis, HbERF-IXc5, has been assumed to play a major role in laticifer metabolism and tolerance to harvesting stress for better latex production. This study sets out to establish and characterize rubber transgenic lines overexpressing HbERF-IXc5. Overexpression of HbERF-IXc5 dramatically enhanced plant growth and enabled plants to maintain some ecophysiological parameters in response to abiotic stress such as water deficit, cold and salt treatments. This study revealed that HbERF-IXc5 has rubber-specific functions compared to Arabidopsis ERF1 as transgenic plants overexpressing HbERF-IXc5 accumulated more starch and differentiated more latex cells at the histological level. The role of HbERF-IXc5 in driving the expression of some target genes involved in laticifer differentiation is discussed.

Keywords: genetic modification; latex; plant hormone; rubber; transcription factor.

Figure 1

Description of plant regeneration from friable callus lines of wild‐type and HbERF‐IXc5 transgenic lines. Callus of wild‐type (a), and transgenic lines TS19A90 (b), TS20A75 (c). View of RITA system with brown callus and somatic embryo of wild‐type (d), and transgenic lines TS19A90 (e), TS20A75 (f); 12‐month‐old plant of wild‐type (g), and transgenic lines TS19A90 (h), TS20A75 (i). Root system of wild‐type (j), and transgenic lines TS19A90 (k), TS20A75 (l)

Figure 2

Determination of copy number by Southern blot hybridization. Genomic DNA of leaves were digested with Eco RI. The blot was hybridized with a ³²P radio‐labelled probe corresponding to NPTII gene

Figure 3

Comparison of photosynthetic nitrogen use efficiency (PNUE, A₄₀₀/Na) between wild‐type and several transgenic lines under optimum growth conditions without water stress. Statistical analysis was performed with an ANOVA followed by the Newman and Keuls test. Values with the same letter were not significantly different at the 0.05 probability level

Figure 4

Histological analysis revealing the accumulation of starch reserves. (a) Leaves of wild‐type, (b) Leaves of HbERF‐IXc5 transgenic lines, (c) Green stem of wild type, (d) Green stem of HbERF‐IXc5 transgenic lines, (e) Lignified stem of wild type, (f) Lignified stem of HbERF‐IXc5 transgenic lines, (g) Taproot of wild type, (h) Taproot of HbERF‐IXc5 transgenic lines. The histological sections were stained with Schiff Naphthol Blue Black. Histological sections were annotated by arrows as follows: MR, midrib; P, pith; X, xylem; Ca, cambium; St, starch

Figure 5

Presence of latex cells in leaves, stem and root of 1‐year‐old plants from wild‐type and HbERF‐IXc5 transgenic lines. (a) Latex cell in leaves of wild type; (b) Latex cell in leaves of HbERF‐IXc5 transgenic lines; (c) Latex cell in green stem of wild type; (d) Latex cell in green stem of HbERF‐IXc5 transgenic lines; (e) Latex cell in lignified stem of wild‐type; (f) Latex cell in lignified stem of HbERF‐IXc5 transgenic lines; (g) Latex cell in taproot of wild type; (h) Latex cell in taproot of HbERF‐IXc5 transgenic lines. The histological sections were stained with Oil Red O. Histological sections were annotated by arrows as follows: B, bark; Ca, cambium; PLC, primary latex cells; SLC, secondary latex cell; X, xylem

Figure 6

Antioxidant contents in leaves of 1‐year‐old plants from wild‐type (WT) and transgenic lines. (a) Total glutathione. (b) Ascorbic acid content (ASA)

Figure 7

Effect of water deficit on 8‐month‐old plants from wild‐type and transgenic lines and their FTSW. (a) Plants before water stress on day 0 (FTSW about 1). (b) Plants after 8 days of water deficit (FTSW ≥ 0.2). (c) Plants after 14 days of water deficit (FTSW < 0.2). (d) Daily change in FTSW value after water deficit treatment. Highlighting in green (1 > FTSW > 0.4), in orange (0.4 > FTSW > 0.2), in red (FTSW < 0.2). nd: nondetermined

Figure 8

Effect of cold and salinity stress on various ecophysiological parameters for wild‐type and several transgenic lines. (a) Effect of cold stress on Fv/Fm value. (b) Effect of salinity stress on Fv/Fm value. (c) Effect of cold stress on P. Index value. (d) Effect of salinity stress on P. Index value. (e) Effect of cold stress on SPAD value. (f) Effect of salinity stress on SPAD value. Statistical analysis was performed with an ANOVA followed by the Tukey test. Values with the same letter were not significantly different at the 0.05 probability level