A metallothionein-like (rgMT) gene was isolated from a rice (Oryza sativa L.) root cDNA library that was prepared from plants grown under NaHCO3 stress. The rgMT gene expression was induced in rice leaves and roots under several abiotic stresses from salts (NaCl and NaHCO3), drought (PEG) and metals (CuCl2, ZnCl2, CdCl2). The results suggested that the rgMT gene was expressed in response to environmental stresses. The rgMT gene was expressed in Escherichia coli, and the final yield of the purified rgMT protein was 4.8 mg g(-1) dry cells. Tolerance of E. coli expressing GST-rgMT fusion protein to Cu2+, Zn2+ and Cd2+ was enhanced, and cells dry weight increased 0.04 mg, 0.17 mg and 0.07 mg in 1 ml culture treated with either CuCl2, ZnCl2 or CdCl2, respectively, compared with control after 6 h culture.

Polypeptide synthesis and accumulation were examined in the roots of tomato seedlings exposed to a polyethylene glycol-imposed water deficit stress. In these roots, the synthesis of a number of polypeptides was induced, while that of several others was enhanced or repressed. To examine the role played by abscisic acid (ABA) in co-ordinating the accumulation of these proteins, water-deficit-stressresponsive polypeptide synthesis was investigated in the roots of the ABA-deficient mutant flacca. In the roots of this mutant, the ability to accumulate a complete set of water-deficit-stress-responsive polypeptides was impaired, indicating that ABA is required for their synthesis. The role of ABA was further examined by exposing the roots of both genotypes to exogenous ABA, which, with one exception, elicited the accumulation of all water-deficit-stressresponsive proteins. Polyethylene glycol-induced polypeptide accumulation was accompanied by a 1·6-fold increase in the level of endogenous ABA in the roots of wild-type plants and a 5-fold increase in the roots of flc. Thus, although the absolute level was lower than that of the wildtype, flc has the capacity to accumulate ABA in its roots. When fluridone was used to prevent the biosynthesis of ABA, the accumulation of several water-deficit-stressresponsive polypeptides was reduced further. The synthesis of polypeptides was also examined in the roots of salttreated seedlings. Salt altered the accumulation of several polypeptides, all of which were previously observed in water-deficit-stressed roots, indicating that their synthesis was the result of the osmotic component of the salt stress. However, the accumulation of these polypeptides was not impaired in flc roots, indicating that the role played by ABA in regulating their accumulation in salt-and polyethylene glycol-treated roots differs. As such, salt-and waterdeficit-stress-induced changes in gene expression may be effected by different mechanisms, at least at the level of polypeptide accumulation.

A type 2 metallothionein gene, SsMT2, was cloned from Suaeda salsa, a salt- and alkali-tolerant plant, which is dominant species on the saline/alkali soil of northeast China. The SsMT2 gene was expressed in all organs except the flower and its expression was induced by various stresses such as CdCl2, NaCl, NaHCO3, and H2O2 treatments. SsMT2-transgenic yeast (Saccharomyces cerevisiae) and plants (Arabidopsis thaliana) showed significantly increased resistance to metal, salt and oxidant stresses. These transgenics accumulated more Cd2+, but less Na+ than their wild type counterparts. SsMT2 transgenic Arabidopsis maintained lower level of H2O2 than wild type plants did in response to the stress treatments. These results demonstrated that the SsMT2 gene plays an important role in reactive oxygen species scavenging and confers enhanced metal and oxidant tolerance to plants.

Metallothioneins (MTs) are cysteine-rich proteins of low molecular weight with many attributed functions, such as providing protection against metal toxicity, being involved in regulation of metal ions uptake that can impact plant physiology and providing protection against oxidative stress. However, the precise function of the metallothionein-like proteins such as the one coded for rgMT gene isolated from rice (Oryza sativa L.) is not completely understood. The whole genome analysis of rice (O. sativa) showed that the rgMT gene is homologue to the Os11g47809 on chromosome 11 of O. sativa sp. japonica genome. This study used the rgMT coding sequence to create transgenic lines to investigate the subcellular localization of the protein, as well as the impact of gene expression in yeast (Saccharomyces cerevisiae) and Arabidopsis thaliana under heavy metal ion, salt and oxidative stresses. The results indicate that the rgMT gene was expressed in the cytoplasm of transgenic cells. Yeast cells transgenic for rgMT showed vigorous growth compared to the nontransgenic controls when exposed to 7 mM CuCl2, 10 mM FeCl2, 1 M NaCl, 24 mM NaHCO3 and 3.2 mM H2O2, but there was no significant difference for other stresses tested. Similarly, Arabidopsis transgenic for rgMT displayed significantly improved seed germination rates over that of the control when the seeds were stressed with 100 μM CuCl2 or 1 mM H2O2. Increased biomass was observed in the presence of 100 μM CuCl2, 220 μM FeCl2, 3 mM Na2CO3, 5 mM NaHCO3 or 1 mM H2O2. These results indicate that the expression of the rice rgMT gene in transgenic yeast and Arabidopsis is implicated in improving their tolerance for certain salt and peroxide stressors.

High abundance proteins like ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) impose a consistent challenge for the whole proteome characterization using shot-gun proteomics. To address this challenge, we developed and evaluated Polyethyleneimine Assisted Rubisco Cleanup (PARC) as a new method by combining both abundant protein removal and fractionation. The new approach was applied to a plant insect interaction study to validate the platform and investigate mechanisms for plant defense against herbivorous insects. Our results indicated that PARC can effectively remove Rubisco, improve the protein identification, and discover almost three times more differentially regulated proteins. The significantly enhanced shotgun proteomics performance was translated into in-depth proteomic and molecular mechanisms for plant insect interaction, where carbon re-distribution was used to play an essential role. Moreover, the transcriptomic validation also confirmed the reliability of PARC analysis. Finally, functional studies were carried out for two differentially regulated genes as revealed by PARC analysis. One of the constant challenges for proteomics is inadequate protein identification because of the interference of high abundance proteins (1). The challenge is particularly critical for plant proteomics analysis because of the prevalence of Rubisco (Ribulose-1,5-bisphosphate carboxylase oxygenase) in green tissue. As a major enzyme involved in carbon fixation, Rubisco consists of 30 to 50% of total plant protein from green tissues and causes less sensitivity, dynamic range, and protein identification of plant proteomics (2-4). Influences of high abundance proteins like Rubisco affect both gel-based and shot-gun proteomics analysis. In one of the most popular shot-gun proteomics platforms with the data-dependent MS/MS acquisition, the peptides derived from the abundant proteins have more chance to be sampled by the MS instrument than the peptides from other functional proteins. Thus, the dynamic range and detection sensitivity will be sacrificed because of the prevalence of high abundance proteins (2). To address this challenge, we developed and evaluated a new method by combining PEI (Polyethyleneimine) 1 precipitation and protein sample fractionation to improve the performance of multidimensional protein identification technology (Mud-PIT)-based proteomics analysis.PEI is a positively charged polymer broadly used for removing nucleic acids from proteins (5). The compound can also be employed to remove acidic proteins like Rubisco from the total protein (6). PEI precipitation can be considered as a fractionation process to separate acidic proteins from the total protein, and thus can be used for both Rubisco removal as well as fractionation of plant proteins from green tissues. Despite the potential to be used for sample preparation, very few studies optimized the PEI precipitation for plant proteomics analysis and evaluated the effectiveness of the approach for enhancing proteomics performance. In this study, w...