HbMT2, an ethephon-induced metallothionein gene from Hevea brasiliensis responds to H2O2 stress

doi:10.1016/j.plaphy.2010.04.004

Plant Physiology and Biochemistry

Volume 48, Issue 8, August 2010, Pages 710-715

https://doi.org/10.1016/j.plaphy.2010.04.004 Get rights and content

Abstract

Metallothioneins (MTs) are the cysteine-rich proteins with low molecular weight, which play important roles in maintaining intracellular ion homeostasis, detoxification of heavy metal ions and protecting against intracellular oxidative damages. In this study a novel ethephon-induced metallothionein gene, designated as HbMT2, was isolated and characterized from Hevea brasiliensis. The HbMT2 cDNA contained a 237 bp open reading frame encoding 78 amino acids and the deduced protein showed high similarity to the type 2 MTs from other plant species. Expression analysis revealed more significant accumulation of HbMT2 transcripts in leaves and latex than in roots and barks. The transcription of HbMT2 in latex was strongly induced by ethephon and hydrogen peroxide (H₂O₂) stress. Overproduction of recombinant HbMT2 protein gave the Escherichia coli cells more tolerance on Cu²⁺ and Zn²⁺, and the recombinant HbMT2 could scavenge the reactive oxidant species (ROS) in vitro. All these results indicated that HbMT2 could respond to ethephon stimulation and H₂O₂ stress as a ROS scavenger in H. brasiliensis. It is also suggested that HbMT2 function in improving the tolerance of rubber trees to heavy metal ions, and repressing the ethephon-induced senilism and tapping panel dryness (TPD) development by ROS scavenge system in H. brasiliensis.

Introduction

Metallothioneins (MTs) are a group of low molecular and cysteine-rich metal-binding proteins widely found in diverse organisms including mammals, plants, fungi and some prokaryotes [1], [2]. The first plant MT, a wheat EcMT protein was identified in 1987 [3], and since then more than 140 MT sequences have been recorded from various species [2], [4]. Plant MTs have been organized into four types according to the distribution of cysteine residues in the amino- and carboxy-terminal regions [2] and different type of MT genes have different expression characterization. Preferential synthesis of type 1 seems located in the subterranean tissues [5] while that of type 2 in aerial organs. The transcripts of type 3 MT genes have been mainly detected from ripening fruits and developing embryos [6], [7] and the transcripts of type 4 genes are especially abundant in seeds [8]. Recent studies have demonstrated that MTs are not only involved in maintaining the homeostasis of essential metal ions, the detoxification of heavy metal ions and the storage of essential metal ions [9], [10], but also implicated in a range of physiological processes, including scavenging ROS [9], [11], the regulation of fruit ripeness and cell growth, the regulation of activities of metalloenzymes and transcription factors [12], and the responses to various stresses.

Natural rubber is synthesized in over 2000 plant species, representing about 300 genera from seven families [13]. The rubber tree (Hevea brasiliensis) has been established as a key commercial rubber source due to the good yield and excellent physical properties of its rubber products. Ethephon, a mimic of the plant hormone ethylene, is widely used on rubber tree to stimulate the production of latex in H. brasiliensis, but the underlying physiological and molecular processes are unclear [14]. In order to clarify the underlying mechanisms and to identify the related genes, we constructed an ethephon-induced latex suppression subtractive sybridization (SSH) cDNA library from latex in H. brasiliensis [15], in which HbSSH147, an expressed sequence tag (EST) with high homology to the type 2 MT genes was identified. Here, for the first time, we report on the isolation and characterization of an ethephon-induced metallothionein gene (HbMT2) from H. brasiliensis. The expression of HbMT2 gene was examined in H. brasiliensis organs and under various abiotic stresses, and the metal-binding properties and the ability of scavenging ROS of HbMT2 protein were also investigated.

Section snippets

Plant materials

Rubber trees (H. brasiliensis clone RRIM 600) were planted at the experimental farm of Chinese Academy of Tropical Agricultural Sciences in Hainan Island in China. Six-year-old virgin trees were treated with 1.5% ethephon or 1.5% H₂O₂ according to the Hao’s method [16]. The concentrations of ethephon and H₂O₂ were expressed as weight percentages in the lanolin paste. The latex was respectively sampled at 0, 1, 3, 6, 12, 24 h after treatments. The latex was allowed to drop directly into liquid

Isolation and characterization of HbMT2

Among the identified 302 ESTs from the ethephon-induced latex SSH cDNA library in H. brasiliensis (data not shown), a 205 bp-length EST with a poly (A) tail of 28 bp, HbSSH147, showed high similarity to the type 2 MT genes from other plant species. With the help of 5′-RACE, RT-PCR techniques and some bioinformatic software, the 354 bp-length cDNA of corresponding gene in H. brasiliensis was isolated, in which a 237 bp-length ORF was flanked by an 85 bp 5′-UTR and a 32 bp 3′-UTR with a poly (A)

Discussion

Most plant MT genes have been detected and characterized in Arabidopsis, rice, cotton and other plant species [5], [8], [9], [26], [27]. In previous reports, some plant MT genes were found to be highly induced by some heavy metal ions such as Zn²⁺, Cd²⁺, Hg²⁺, and Cu²⁺ [28], and plant MTs were thought to play important roles as they did in animals and fungi. Moreover, an increasing number of observations indicate that plant MTs may be involved in the production and elimination of ROS [9], [11],

Acknowledgments

This work was supported by a grant of the National Natural Science Foundation of China (30760197) and National Nonprofit Institute Research Grant of CATAS-ITBB (ITBBZD0716).

References (35)

J.P. Bourdineaud et al.
Challenging the model for induction of metallothionein gene expression
Biochimie
(2006)
S.N.A. Abdullah et al.
Isolation and characterization of two divergent type 3 metallothioneins from oil palm, Elaeis guineensis
Plant Physiol. Biochem.
(2002)
F. Haq et al.
Signaling events for metallothionein induction
Mutat. Res.
(2003)
B.Z. Hao et al.
Laticifer differentiation in Hevea brasiliensis: induction by exogenous jasmonic acid and linolenic acid
Ann. Bot.
(2000)
C.R. Tang et al.
A convenient and efficient protocol for isolating high-quality RNA from latex of Hevea brasiliensis (para rubber tree)
J. Biochem. Biophys. Methods
(2007)
L.T. Zhu et al.
mRNA and 18S RNA coapplication-reverse transcription for quantitative gene expression analysis
Anal. Biochem.
(2005)
N. Smirnoff et al.
Hydroxyl radical scavenging activity of compatible solutes
Phytochem.
(1989)
S.V. Rana
Metals and apoptosis: recent developments
J. Trace. Elem. Med. Biol.
(2008)
K. Smeets et al.
Oxidative stress-related responses at transcriptional and enzymatic levels after exposure to Cd or Cu in a multipollution context
J. Plant Physiol.
(2009)
C. Cobbett et al.
Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis
Annu. Rev. Plant Biol.
(2002)

B. Lane et al.

The wheat-germ Ec protein is a zinc-containing metallothionein

Biochem. Cell Biol.

(1987)

G.K. Zhou et al.

Molecular analyses of the metallothionein gene family in rice (Oryza sativa L.)

J. Biochem. Mol. Biol.

(2006)

W.J. Guo et al.

Characterization of the Arabidopsis metallothionein gene family: tissue-specific expression and induction during senescence and in response to copper

New Phytol.

(2003)

V.N. Guyon et al.

Isolation and characterization of cDNAs expressed in the early stages of flavonol-induced pollen germination in Petunia

Plant Physiol.

(2000)

C.L. Chyan et al.

Cloning and expression of a seed-specific metallothionein-like protein from sesame

Biosci. Biotechnol. Biochem.

(2005)

T.T. Xue et al.

Cotton metallothionein GhMT3a, a reactive oxygen species scavenger, increased tolerance against abiotic stress in transgenic tobacco and yeast

J. Exp. Bot.

(2009)

V.H. Hassinen et al.

Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens

J. Exp. Bot.

(2009)

Cited by (34)

Role of transcriptomics in countering the effect of abiotic stresses in wheat
2023, Abiotic Stresses in Wheat: Unfolding the Challenges
Unfavorable environmental circumstances have an impact on plant growth and development. Advancement in the development of omics technologies has steadily increased in importance as a subject of academic investigation due to the need to unravel the molecular mechanism concerning adverse abiotic stresses. Transcriptomics is a dynamic process representing the expression level of all genes in a single cell, tissue, or organ of an organism at a given stage of growth and development. In this chapter, we investigate the use of transcriptomics to understand the wheat plant response to abiotic stressors such as salt, drought, high and low temperature, and their response under abiotic stress conditions to provide a framework for future study. This chapter is aimed to evaluate current advancements in transcriptome studies for wheat and changes in differentially expressed genes (DEGs) and their involvement in metabolic pathways under abiotic stress environmental conditions. It also provides a roadmap for introducing these transcripts for future studies and improving the wheat genome to withstand abiotic stress conditions.
SaMT3 in Sedum alfredii drives Cd detoxification by chelation and ROS-scavenging via Cys residues
2022, Environmental Pollution
Metallothioneins (MTs), a group of cysteine-rich proteins, are effective chelators of cadmium (Cd) and play a key role in plant Cd detoxification. However, little is known about the role of single cysteine (Cys) residues in the MTs involved in the adaptation of plants to Cd stress, especially, in hyperaccumulators. In the present study, we functionally characterised SaMT3 in S. alfredii, a Cd/Zn hyperaccumulator native to China. Our results showed that the C- and N- terminal regions of SaMT3 had differential functional natures in S. alfredii and determined its Cd hypertolerance and detoxification. Two CXC motifs within the C-terminal region were revealed to play a crucial role in Cd sensing and binding, whereas the four Cys-residues within the N-terminal region were involved in scavenging reactive oxygen species (ROS). An S. alfredii transgenic system based on callus transformation was developed to further investigate the in-planta gene function. The SaMT3-overexpressing transgenic plant roots were more tolerant to Cd than those of wild-type plants. Knockout of SaMT3 resulted in significantly decreased Cd concentrations and increased ROS levels after exposure to Cd stress. We demonstrated the SaMT3-mediated adaptation strategy in S. alfredii, which uses metal chelation and ROS scavenging in response to Cd stress. Our results further reveal the molecular mechanisms underlying Cd detoxification in hyperaccumulating plants, as well as the relation between Cys-related motifs and the metal binding properties of MTs. This research provides valuable insights into the functions of SaMT3 in S. alfredii, and improves our understanding of Cd hyperaccumulation in plants.
ScMT10, a metallothionein-like gene from sugarcane, enhances freezing tolerance in Nicotiana tabacum transgenic plants
2022, Environmental and Experimental Botany
Citation Excerpt :
Two MT2 genes from Arabidopsis, AtMT2a and AtMT2b, were expressed predominantly in leaf tissues compared to root tissues (Guo and Goldsbrough, 2003). A similar situation was observed in MT2 genes from O. sativa (Zhou et al., 2006), Sedum alfredii (Zhang et al., 2014), Hevea brasiliensis (Zhu et al., 2010). Some MT2 gene members from sugarcane showed different tissue-specific expression patterns, e.g., ScMT2–1–3 was highly expressed in root and bud but lowly expressed in stem and leaf (Guo et al., 2013).
Sugarcane (Saccharum spp. hybrid) is of tropical origin, yet generally cultivated in subtropical areas. Cold stress is a major environmental factor that impairs the production of sugarcane in that area. Metallothioneins, a family of small, cysteine-rich proteins, are proved to be involved in metal homeostasis and detoxification and thus alleviate stress-induced ROS accumulation. Here, a cDNA clone deducing a single Cys-rich domain type 2 metallothionein-like protein, designated ScMT10, was isolated from sugarcane. The level of ScMT10 expression was up-regulated by salicylic acid (SA), cold, and oxidative stress (H₂O₂) in sugarcane seedlings, but it was down-regulated in response to abscisic acid (ABA), methyl jasmonate (MeJA), heavy metal ions (Cu²⁺, Cd²⁺), and salinity. Transgenic tobacco (Nicotiana tabacum) plants (ScMT10-OE) showed a remarkable enhancement in freezing tolerance compared with wild-type plants (WT). Furthermore, ScMT10-OE tobacco showed a lower electrolyte leakage, reduced malondialdehyde contents, and slower ROS (including H₂O₂) accumulation under freezing stress and retained higher proline, soluble sugar, and relative chlorophyll content compared to WT plants. The expression profiles of ScMT10 in the transgenic tobacco were similar to those in sugarcane seedlings under different stresses. Besides, the level of H₂O₂ in the ScMT10-OE plants was comparable to that observed in WT plants under heavy metal, salt, and drought stress. Therefore, the transgenic plants did not perform better than WT plants under those stresses. Taken together, we present a hypothesis that ScMT10 was exclusively and positively involved in the cold stress-induced accumulation of reactive oxygen species (ROS). The over-expression of ScMT10 in tobacco decreased the contents of endogenous ROS, MDA, and relative electric conductivity. It increased the accumulation of proline, soluble sugar, and relative chlorophyll under freezing stress, which alleviated the oxidative damage to the cell membrane during freezing exposure. ScMT10 can be considered as a candidate gene for cold-tolerance trait improvement.
Diversity and versatile functions of metallothioneins produced by plants: A review
2020, Pedosphere
Metal ions are essential for plant growth and development, but in excess, these compounds can become highly toxic. Plants have adopted numerous ways to maintain metal homeostasis while mitigating adverse effects of excess metal ions, including phytochelatin and the metal-chelating proteins metallothioneins (MTs). A family of cysteine (Cys)-rich, intracellular, and low-molecular-weight (4–8 kDa) MTs are proteins found in nearly all phyla including plants, animals, and fungi, and they have the potential to scavenge reactive oxygen species and detoxify toxic metals including copper, cadmium, and zinc. Based on their Cys numbers and residues, MTs have been categorized into three major classes. Class I MTs, which have highly conserved Cys residues, are found in animals, while class II MTs, with less conserved Cys residues, are present in plants and are classified further into four groups. Class III MTs include phytochelatins, a group of enzymatically synthesized Cys-rich proteins. The MTs have been an area of interest for five decades with extensive studies, which have been facilitated by advancements in instrumental techniques, protein science, and molecular biology tools. Here, we reviewed current advances in our understanding of the regulation of MT biosynthesis, their expression, and their potential roles in the alleviation of abiotic stresses (i.e., drought, salinity, and oxidative stresses) and heavy metal detoxification and homeostasis.
Cyamopsis tetragonoloba type 1 metallothionein (CtMT1) gene is upregulated under drought stress and its protein product has an additional C-X-C motif and unique metal binding pattern
2018, International Journal of Biological Macromolecules
Citation Excerpt :
Hydroxyl radical scavenging activity of purified CtMT1 protein was determined by assessing its ability to compete with salicylate for hydroxyl radicals by using hydroxyl radical generating and detecting system. The assay was performed as described previously by Smirnoff and Cumbes [31] and Zhu et al. [32] with the following minor modifications. In our experiment, the final reaction mixture volume of 3 ml contained 150 mM KH2PO4-KOH buffer (pH 7.4), 0.15 mM Fe-EDTA, 0.6 mM H2O2, 0.26 mM ascorbic acid, 2 mM sodium salicylate and varying concentrations (10–50 μM) of CtMT1.
Metallothioneins (MTs) are involved in cellular homeostasis of essential metal ions and detoxification of nonessential metal ions. We report here the identification of four MT genes, CtMT1, CtMT2, CtMT3 and CtMT4, encoding CtMT1, CtMT2, CtMT3 and CtMT4 proteins, respectively, from the industrial guar crop. The primary structures of last three proteins were similar to those of respective MT proteins of other plants but the CtMT1 protein primary structure was different from the other plant MT1 proteins in having an additional C-X-C motif. The four MT genes showed tissue specific expression patterns suggesting their specific roles in different tissues. High expression of CtMT1 gene was observed in roots and nodules whereas CtMT2 and CtMT3 genes showed high expression in leaves. The expression of CtMT4 gene was high in seeds. The qRT-PCR studies revealed upregulation in expression of CtMT1 gene under drought stress. Recombinant CtMT1 protein was produced in E. coli Rosetta cells and purified by metal affinity chromatography. The purified protein showed antioxidant property and the order of its metal ion binding affinities was Cu²⁺ > Zn²⁺ > Fe²⁺ > Cd²⁺. This information about CtMT1 protein is expected to be useful in understanding its role in drought tolerance and other physiological processes of guar.
Responses of Phytochelatins and Metallothioneins in Alleviation of Heavy Metal Stress in Plants: An Overview
2015, Plant Metal Interaction: Emerging Remediation Techniques
Heavy metal detoxification in plants is a phenomenon resulting from complex interactions among interconnected physiological pathways and defense shunts leading to reactive oxygen species scavenging and subsequent protection of cellular vitals. These signaling pathways involve cross-talk between a number of antioxidant compounds including two main groups of amino acid rich metal chelators, namely the phytochelatins (PCs) and metallothioneins (MTs). This book chapter traces the mechanism of metal tolerance and detoxification strategies possessed by these biological molecules in addition to their biosynthesis, roles played and genetic aspects involved in their course of action. The isolation, characterization of PC and MT genes involved in metal compartmentalization and their successful induction in other plants is a much more recent application because this is of immense importance to the world of agronomics. Genetic validation and success for the same has been reported widely in this decade and many prominent reports have been included in the text to highlight this. Extending this vast information about the PC and MT gene pool at the proteomic level is gaining a lot of momentum currently and shall remain the future line of investigation for understanding metal resistance pathways at the cellular as well as subcellular level.

View all citing articles on Scopus

¹: Equally contributed.

View full text

Research articleHbMT2, an ethephon-induced metallothionein gene from Hevea brasiliensis responds to H2O2 stress

Abstract

Introduction

Section snippets

Plant materials

Isolation and characterization of HbMT2

Discussion

Acknowledgments

Biochimie

Plant Physiol. Biochem.

Mutat. Res.

Ann. Bot.

J. Biochem. Biophys. Methods

Anal. Biochem.

Phytochem.

J. Trace. Elem. Med. Biol.

J. Plant Physiol.

Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis

Annu. Rev. Plant Biol.

The wheat-germ Ec protein is a zinc-containing metallothionein

Biochem. Cell Biol.

Molecular analyses of the metallothionein gene family in rice (Oryza sativa L.)

J. Biochem. Mol. Biol.

Characterization of the Arabidopsis metallothionein gene family: tissue-specific expression and induction during senescence and in response to copper

New Phytol.

Isolation and characterization of cDNAs expressed in the early stages of flavonol-induced pollen germination in Petunia

Plant Physiol.

Cloning and expression of a seed-specific metallothionein-like protein from sesame

Biosci. Biotechnol. Biochem.

Cotton metallothionein GhMT3a, a reactive oxygen species scavenger, increased tolerance against abiotic stress in transgenic tobacco and yeast

J. Exp. Bot.

Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens

J. Exp. Bot.

Research article
HbMT2, an ethephon-induced metallothionein gene from Hevea brasiliensis responds to H₂O₂ stress