Cloning and characterization of HbMT2a, a metallothionein gene from Hevea brasiliensis Muell. Arg differently responds to abiotic stress and heavy metals.

Metallothioneins (MTs) are of low molecular mass, cysteine-rich proteins. They play an important role in the detoxification of heavy metals and homeostasis of intracellular metal ions, and protecting against intracellular oxidative damages. In this study a full-length cDNA of type 2 plant metallothioneins, HbMT2a, was isolated from 25 mM Polyethyleneglycol (PEG) stressed leaves of Hevea brasiliensis by RACE. The HbMT2a was 372bp in length and had a 237bp open reading frame (ORF) encoding for a protein of 78 amino acid residues with molecular mass of 7.772 kDa. The expression of HbMT2a in the detached leaves of rubber tree clone RY7-33-97 was up-regulated by Me-JA, ABA, PEG, H2O2, Cu(2+) and Zn(2+), but down-regulated by water. The role of HbMT2a protein in protecting against metal toxicity was demonstrated in vitro. PET-28a-HbMT2-beared Escherichia coli. Differential expression of HbMT2a upon treatment with 10 °C was observed in the detached leaves of rubber tree clone 93-114 which is cold-resistant and Reken501 which is cold-sensitive. The expression patterns of HbMT2a in the two rubber tree clones may be ascribed to a change in the level of endogenous H2O2.

Molecular and biochemical characterization of a cyanogenic ß-glucosidase in the inner bark tissues of rubber tree (Hevea brasiliensis Muell. Arg.).

Tapping causes the loss of large amounts of latex from laticifers and subsequently enhances latex regeneration, a high carbon- and nitrogen-cost activity in rubber tree. It is suggested that a 67 kDa protein associated with protein-storing cells in the inner bark tissues of rubber tree plays an important role in meeting the nitrogen demand for latex regeneration. Here, the 67 kDa protein was further characterized by a combination of cell biological, molecular biological and biochemical techniques. Immunogold labeling showed that the 67 kDa protein was specifically localized in the central vacuole of protein-storing cells. A full-length cDNA, referred to as HbVSP1, was cloned. The HbVSP1 contained a 1584 bp open reading frame encoding a protein of 527 amino acids. The putative protein HbVSP1 shared high identity with the P66 protein from rubber tree and proteins of the linamarase, and bg1A from cassava (Manihot esculenta). HbVSP1 contained the active site sequences of ß-glucosidase, TFNEP and I/VTENG. In vitro analysis showed that the 67 kDa protein exhibited the activity of both ß-glucosidase and linamarase and was thus characterized as a cyanogenic ß-glucosidase. Proteins immuno-related to the 67 kDa protein were present in leaves and lutoids of laticifers. Tapping down-regulated the expression of HbVSP1, but up-regulated the expression of genes encoding the key enzymes for rubber biosynthesis, while the effect of resting from tapping was the reverse. Taken together, the results suggest that the 67 kDa protein is a vacuole-localized cyanogenic ß-glucosidase encoded by HbVSP1 and may have a role in nitrogen storage in inner bark tissues of trunk during the leafless periods when rubber tree is rested from tapping.

Characterization of HbEREBP1, a wound-responsive transcription factor gene in laticifers of Hevea brasiliensis Muell. Arg.

AP2/ERF transcription factors play an important role in regulation of the cross-talk between ethylene and jasmonate signaling pathways mediating defense responses of plants to biotic and abiotic stresses. In this study, an AP2/ERF transcription factor gene was isolated and characterized from laticifers of rubber tree by using RACE and real time PCR. The full length cDNA, referred to as HbEREBP1, was 1,095 bp in length and contained a 732 bp open reading frame encoding a putative protein of 243 amino acid residues. The molecular mass of the putative protein is 26.4 kDa with a pI of 9.46. The deduced amino acid sequence had a specific domain of AP2 superfamily and an ethylene-responsive element binding factor-associated amphiphilic repression motif, sharing 42.4, 39.1, and 38.0% identity with that of AtERF11, AtERF4, and AtERF8 in Arabidopsis, respectively. HbEREBP1 expression was down-regulated by tapping and mechanical wounding in the laticifers of adult trees. It was also down-regulated at early stage while up-regulated at late stage upon treatment with exogenous ethephon or methyl jasmonate, which was reverse to the case of defense genes in laticifers of epicormic shoots of rubber tree. Our results suggest that HbEREBP1 may be a negative regulator of defense genes in laticifers.

MYC genes with differential responses to tapping, mechanical wounding, ethrel and methyl jasmonate in laticifers of rubber tree (Hevea brasiliensis Muell. Arg.).

MYC2 transcription factor is a key component of the core module COI1-JAZ-MYC2 of jasmonate signaling in Arabidopsis, but the MYC transcription factor (s) associated with jasmonate signaling in jasmonate-responsive laticifer cells remains to be identified. Two full-length cDNAs, designated HblMYC1 and HblMYC2, were isolated from laticifer cells in Hevea brasiliensis by the method of RACE. HblMYC1 contained 1431bp ORF encoding a putative protein of 476 amino acids while HblMYC2 contained 1428bp ORF encoding a putative protein of 475 amino acids. Bioinformatic analysis showed that the putative proteins, HblMYC1 and HblMYC2, possessed a bHLH domain and were most related to the MYC2 among the selected 27 MYC members with identified functions in Arabidopsis. In addition to the presence of cis-regulatory elements involving jasmonate responsiveness in the promoter regions of HblMYC1 and HblMYC2, the abscisic acid-, salicylic acid- and gibberellin-responsive elements were found in the promoter region of HblMYC1. Transcripts of HblMYC1 and HblMYC2 were most abundant in latex, relatively low in male flowers and nearly undetected in bark tissues and roots by real-time RT-PCR analysis. Regular tapping, mechanical wounding, and ethrel remarkably up-regulated HblMYC1 expression, but had little effect on the expression of HblMYC2 in laticifer cells. Successive tapping, however, significantly down-regulated the expression of HblMYC2 while up-regulating the expression of HblMYC1. The HblMYC2 expression took a mutual ebb and flow relationship with the HblMYC1 expression upon treatment with methyl jasmonate. Characterization of HblMYC1 and HblMYC2 will contribute to the understanding of jasmonate signaling in laticifiers, a kind of specialized tissue for natural rubber biosynthesis in Hevea brasiliensis.