Kwan Sam Choi

A Novel Family of Cys-Rich Membrane Proteins Mediates Cadmium Resistance in Arabidopsis

Cadmium (Cd) is a widespread pollutant that is toxic to plant growth. However, only a few genes that contribute to Cd resistance in plants have been identified. To identify additional Cd(II) resistance genes, we screened an Arabidopsis cDNA library using a yeast (Saccharomyces cerevisiae) expression system employing the Cd(II)-sensitive yeast mutant ycf1. This screening process yielded a small Cys-rich membrane protein (Arabidopsis plant cadmium resistance, AtPcrs). Database searches revealed that there are nine close homologs in Arabidopsis. Homologs were also found in other plants. Four of the five homologs that were tested also increased resistance to Cd(II) when expressed in ycf1. AtPcr1 localizes at the plasma membrane in both yeast and Arabidopsis. Arabidopsis plants overexpressing AtPcr1 exhibited increased Cd(II) resistance, whereas antisense plants that showed reduced AtPcr1 expression were more sensitive to Cd(II). AtPcr1 overexpression reduced Cd uptake by yeast cells and also reduced the Cd contents of both yeast and Arabidopsis protoplasts treated with Cd. Thus, it appears that the Pcr family members may play an important role in the Cd resistance of plants.

Arabidopsis PCR2 Is a Zinc Exporter Involved in Both Zinc Extrusion and Long-Distance Zinc Transport

This work shows that PCR2 is a membrane protein implicated in two processes, namely, the detoxification of zinc in the presence of high concentrations of zinc and the transfer of zinc from the root to the shoot. This dual role is likely made possible by PCR2’s expression pattern that differs in different parts of the root. Plants strictly regulate the uptake and distribution of Zn, which is essential for plant growth and development. Here, we show that Arabidopsis thaliana PCR2 is essential for Zn redistribution and Zn detoxification. The pcr2 loss-of-function mutant was compromised in growth, both in Zn-excessive and -deficient conditions. The roots of pcr2 accumulated more Zn than did control plants, whereas the roots of plants overexpressing PCR2 contained less Zn, indicating that PCR2 removes Zn from the roots. Consistent with a role for PCR2 as a Zn-efflux transporter, PCR2 reduced the intracellular concentration of Zn when expressed in yeast cells. PCR2 is located mainly in epidermal cells and in the xylem of young roots, while it is expressed in epidermal cells in fully developed roots. Zn accumulated in the epidermis of the roots of pcr2 grown under Zn-limiting conditions, whereas it was found in the stele of wild-type roots. The transport pathway mediated by PCR2 does not seem to overlap with that mediated by the described Zn translocators (HMA2 and HMA4) since the growth of pcr2 hma4 double and pcr2 hma2 hma4 triple loss-of-function mutants was more severely inhibited than the individual single knockout mutants, both under conditions of excess or deficient Zn. We propose that PCR2 functions as a Zn transporter essential for maintaining an optimal Zn level in Arabidopsis.

Overexpression of a yeast cadmium factor 1 (YCF1) enhances heavy metal tolerance and accumulation in Brassica juncea

A yeast cadmium factor 1 (YCF1) is a member of the ATP-binding cassette (ABC) transporter family associated with multi-drug resistance, and it is localized at the vacuolar membrane in Saccharomyces cerevisiae. To determine ability to increase heavy metal tolerance and accumulation, YCF1 was introduced into Brassica juncea plants by Agrobacterium-mediated genetic transformation. YCF1 gene presence in transgenic plants was demonstrated by polymerase chain reaction (PCR). Reverse transcriptase-PCR analysis confirmed YCF1 gene expression in the transgenic plants, but the degree of YCF1 expression varied among the lines. YCF1 overexpression in B. juncea conferred enhanced tolerance to cadmium (Cd[II]) and lead (Pb[II]) stress. Transgenic B. juncea seedlings showed 1.3- to 1.6-fold tolerance to Cd stress and 1.2- to 1.4-fold tolerance to Pb stress compared to wild type (WT) plants (per gram fresh weight). Most importantly, the shoot tissues of transgenic seedlings contained about 1.5- to 2-fold higher Cd(II) and Pb(II) levels than those of WT, demonstrating significantly increased accumulation of both Cd(II) and Pb(II) in transgenic plants.

Overexpression of AtATM3 in Brassica juncea confers enhanced heavy metal tolerance and accumulation

AtATM3, a member of the ATP-binding cassette transporter family, is localized at the mitochondrial membrane of Arabidopsis thaliana and is involved in the biogenesis of Fe–S clusters and iron homeostasis in plants. Through Agrobacterium-mediated genetic transformation, the AtATM3 gene driven by the cauliflower mosaic virus 35S promoter (CaMV35S) was introduced into Brassica juncea (Indian mustard), a plant species suitable for phytoremediation, with the aim of improving heavy metal tolerance and accumulation in plants. The presence of the AtATM3 gene in transgenic plants was confirmed by polymerase chain reaction (PCR). Reverse transcriptase-PCR analysis confirmed AtATM3 expression in transgenic plants, but the level of AtATM3 expression varied between lines. AtATM3 overexpression in B. juncea conferred enhanced tolerance to cadmium [Cd(II)] and lead [Pb(II)] stresses. Importantly, the shoot tissues of transgenic seedlings contained about 1.5- to 2.5-fold higher Cd(II) and Pb(II) levels than wild type (WT) seedlings, demonstrating significantly-increased accumulation of both Cd(II) and Pb(II) in transgenic plants. The enhanced capacity of heavy metal tolerance and accumulation by AtATM3 transgenic plants was attributed to higher BjGSHII (B. juncea glutathione synthetase II) and BjPCS1 (phytochelatin synthase 1) expression levels induced by AtATM3 overexpression. In addition, AtATM3 overexpression regulated the expression of several metal transporters in the transgenic B. juncea plants under heavy metal stress conditions. Therefore, AtATM3 transgenic plants are more tolerant of and can accumulate more heavy metals to enhance phytoremediation of contaminated soils.

Chloroplast-targeted BrMT1 (Brassica rapa type-1 metallothionein) enhances resistance to cadmium and ros in transgenicatabidopsis plants

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that bind to heavy metals. Type-1 MTs function under various abiotic stresses, including exposure to the cadmium ion. We have now isolated theBrassica rapa type-1 metallothioneirt gene (BrMT1)using yeast systems, and have found that it confers resistance to Cd in otherwise Cd-sensitive yeast. Using a constitutive CaMV35S promoter and an RbsS transit peptide, we successfully targeted BrMT1 to the chloroplastsof Arabidopsis. Overexpression in either the chloroplasts or the cytosol effectively detoxified cadmium and H2O2 stresses in transgenicArabidopsis. in particular, the chloropfast-targeted BrMTl was associated with a significant reduction in paraquat-induced chlorosis and the accumulation of H2O2. This is the first report regarding the effects of type-1 MT1 targeted to chloroplasts. Our results suggest that this may be applicable to the development of plants with enhanced tolerance against environmental stresses.

An efficient and novel plant selectable marker based on organomercurial resistance

A selectable marker gene facilitates the detection of genetically modified plant cells during transformation experiments. So far, these marker genes are almost exclusively of two types, conferring either antibiotic resistance or herbicide tolerance. However, more selectable markers must be developed as additional transgenic traits continue to be incorporated into transgenic plants. Here, we used mercury resistance, conferred by the organomercurial lyase gene, as a selectable marker for transformation. The merB gene fromStreptococcus aureus was modified for plant expression and transferred to a hybrid poplar(Populus alba xPopulus glandulosa), using the stem segment-agrobacteria co-cultivation method. The transformed cells were selected on a callus-inducing medium containing as little as 1 µM methylmercury. Subsequent plant regeneration was done in the presence of methylmercury. Resistance to Hg was stably maintained in mature plants after two years of growth in the nursery. We suggest that this gene could serve as an excellent selectable marker for plant transformation.

Mercury-tolerant Transgenic Poplars Expressing Two Bacterial Mercury-metabolizing Genes

Mercury is one of the most toxic metals to various organisms, including humans. Genes involved in mercury metabolism have been cloned fromStaphylococcus aureus, and were modified here to be expressed in plants. Transgenic poplars containing both chimeric genes (p35S-merA andp35S-merB) were developed via two rounds of transformation usingnos-nptll andnos-hpt genes as selectable markers. Although expression levels varied among transgenic lines, tolerance to either ionic mercury or organic mercury matched well with the degree of expression revealed by northern hybridization. In culture, these trees were tolerant to 50 μM HgCl2 and 2 μM CH3HgCI. Variations in mercury tolerance among the transgenic lines indicates that vigorous selection is required to select the best clones for use in phytoremediation.

Preferential spreading of RNA silencing into the 3’ downstream region of the transgene in tobacco

The amplification mechanism of short interfering RNAs (siRNAs) along the transgene sequence exists in RNA interference (RNAi). The RNA-dependent RNA polymerase synthesizes complementary RNAs by using the transgene mRNA as a template, and the secondary siRNAs are generated from the outside of primary RNAi target. Four independent RNAi vectors which produced primary siRNAs against distinct regions of the tobacco endoplasmic reticulum ω-3 fatty acid desaturase gene (NtFAD3) were transiently expressed in leaves of theNtF4D3-overexpressed transgenic plants. Regardless of the RNAi vector used, the secondaryNtFAD3 siRNAs were generated preferentially from the 3’ downstream region of the transgene. Secondary siRNAs from the 5’ upstream region adjacent to the annealing site of primary siRNAs accumulated under the detection level. Our results suggest that different regulatory mechanisms are involved in the spreading of RNA silencing into 5’ upstream and 3’ downstream regions of the target sequence, respectively.

Factors for high frequency plant regeneration in tissue cultures of Indian mustard (Brassica juncea L.)

An efficient system for high frequency plant regeneration was established through investigating various factors such as plant growth regulator combinations, explant types and ages, and addition of influenced on shoot regeneration in Brassica juncea L. cv. BARI sarisha-10. Murashige and Skoog (MS) medium supplemented with 0.1 mg/L NAA (1-naphthaleneacetic acid) and 1 mg/L BA (6-benzyladenine) showed the maximum shoot regeneration frequency (56.67%) among the different combinations of NAA and BA. Explant type, explant age, and addition of also significantly affected shoot regeneration. Of the four type of explants (cotyledon, hypocotyl, root, and leaf explants)- cotyledon explants produced the highest shoot regeneration frequency and hypocotyls explants produced the highest number of shoots per explant, whereas root explants did not produce any shoot. The cotyledonary explants from Four-day-old seedlings showed the maximum shoot regeneration frequency and number of shoots per explant. Shoot regeneration frequency increased significantly by adding to the medium. Two mg/L appeared to be the best for shoot regeneration with the highest shoot regeneration frequency (86.67%) and number of shoots per explant (7.5 shoots). Considerable variation in shoot regeneration from cotyledonay explants was observed within the B. juncea L. genotypes. The shoot regeneration frequency ranged from 47.78% for cv. Shambol to 91.11% for cv. Rai-5. In terms of the number of shoots produced per explant, B. juncea L. cv. Daulot showed the maximum efficiency. MS medium supplemented with 0.1 mg/L NAA showed the highest frequency of rooting. The regenerated plantlets were transferred to pot soil and grown to maturity in the greenhouse. All plants were fertile and morphologically identical with the source plants.