Won-Yong Song

Engineering tolerance and accumulation of lead and cadmium in transgenic plants

We have studied the utility of the yeast protein YCF1, which detoxifies cadmium by transporting it into vacuoles, for the remediation of lead and cadmium contamination. We found that the yeast YCF1-deletion mutant DTY167 was hypersensitive to Pb(II) as compared with wild-type yeast. DTY167 cells overexpressing YCF1 were more resistant to Pb(II) and Cd(II) than were wild-type cells, and accumulated more lead and cadmium. Analysis of transgenic Arabidopsis thaliana plants overexpressing YCF1 showed that YCF1 is functionally active and that the plants have enhanced tolerance of Pb(II) and Cd(II) and accumulated greater amounts of these metals. These results suggest that transgenic plants expressing YCF1 may be useful for phytoremediation of lead and cadmium.

Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters.

Arsenic is an extremely toxic metalloid causing serious health problems. In Southeast Asia, aquifers providing drinking and agricultural water for tens of millions of people are contaminated with arsenic. To reduce nutritional arsenic intake through the consumption of contaminated plants, identification of the mechanisms for arsenic accumulation and detoxification in plants is a prerequisite. Phytochelatins (PCs) are glutathione-derived peptides that chelate heavy metals and metalloids such as arsenic, thereby functioning as the first step in their detoxification. Plant vacuoles act as final detoxification stores for heavy metals and arsenic. The essential PC–metal(loid) transporters that sequester toxic metal(loid)s in plant vacuoles have long been sought but remain unidentified in plants. Here we show that in the absence of two ABCC-type transporters, AtABCC1 and AtABCC2, Arabidopsis thaliana is extremely sensitive to arsenic and arsenic-based herbicides. Heterologous expression of these ABCC transporters in phytochelatin-producing Saccharomyces cerevisiae enhanced arsenic tolerance and accumulation. Furthermore, membrane vesicles isolated from these yeasts exhibited a pronounced arsenite [As(III)]–PC2 transport activity. Vacuoles isolated from atabcc1 atabcc2 double knockout plants exhibited a very low residual As(III)–PC2 transport activity, and interestingly, less PC was produced in mutant plants when exposed to arsenic. Overexpression of AtPCS1 and AtABCC1 resulted in plants exhibiting increased arsenic tolerance. Our findings demonstrate that AtABCC1 and AtABCC2 are the long-sought and major vacuolar PC transporters. Modulation of vacuolar PC transporters in other plants may allow engineering of plants suited either for phytoremediation or reduced accumulation of arsenic in edible organs.

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 metallothioneins 2a and 3 enhance resistance to cadmium when expressed in Vicia faba guard cells

The Arabidopsis metallothionein genes AtMT1andAtMT2confer Cd(II) resistance to Cd(II)-sensitive yeast, but it has not been directly shown whether they or other metallothioneins provide the same protection to plants. We tested whether AtMT2aandAtMT3can confer Cd(II) resistance to plant cells by introducing GFP- or RFP-fused forms into guard cells of Vicia faba by biolistic bombardment. AtMT2a and AtMT3 protected guard cell chloroplasts from degradation upon exposure to Cd(II), an effect that was confirmed using an FDA assay to test the viability of the exposed guard cells. AtMT2a- and AtMT3-GFP were localized in the cytoplasm both before and after treatment of V. faba guard cells or Arabidopsis protoplasts with Cd(II), and the levels of reactive oxygen species were lower in transformed guard cells than in non-transformed cells after Cd(II)-treatment. These results suggest that the Cd(II)-detoxification mechanism of AtMT2a and AtMT3 may not include sequestration into vacuoles or other organelles, but does involve reduction of the level of reactive oxygen species in Cd(II)-treated cells. Increased expression of AtMT2a and AtMT3 was observed in Arabidopsis seedlings exposed to Cd(II). Together, these data support a role for the metallothioneins AtMT2a and AtMT3 in Cd(II) resistance in intact plant cells.

Arabidopsis ABCG14 is essential for the root-to-shoot translocation of cytokinin

Significance Roots and shoots communicate with each other to synchronize and optimize plant growth and respond to environmental changes. Shoots and roots exchange signals to sense the status and respond to the needs of the other organ. Cytokinins, which are phytohormones that regulate various aspects of growth and development, are recognized as the most important signal transmitted from roots to shoots. Whereas the enzymes underlying cytokinin biosynthesis and the corresponding receptors have been identified, our knowledge of cytokinin transport is limited. In this study, we identified the Arabidopsis ATP-binding cassette transporter subfamily G14 as a major component in the transfer of cytokinins from roots to shoots and hence as a regulator of shoot development. This finding represents a major breakthrough in the field. Cytokinins are phytohormones that induce cytokinesis and are essential for diverse developmental and physiological processes in plants. Cytokinins of the trans-zeatin type are mainly synthesized in root vasculature and transported to the shoot, where they regulate shoot growth. However, the mechanism of long-distance transport of cytokinin was hitherto unknown. Here, we report that the Arabidopsis ATP-binding cassette (ABC) transporter subfamily G14 (AtABCG14) is mainly expressed in roots and plays a major role in delivering cytokinins to the shoot. Loss of AtABCG14 expression resulted in severe shoot growth retardation, which was rescued by exogenous trans-zeatin application. Cytokinin content was decreased in the shoots of atabcg14 plants and increased in the roots, with consistent changes in the expression of cytokinin-responsive genes. Grafting of atabcg14 scions onto wild-type rootstocks restored shoot growth, whereas wild-type scions grafted onto atabcg14 rootstocks exhibited shoot growth retardation similar to that of atabcg14. Cytokinin concentrations in the xylem are reduced by ∼90% in the atabcg14 mutant. These results indicate that AtABCG14 is crucial for the translocation of cytokinin to the shoot. Our results provide molecular evidence for the long-distance transport of cytokinin and show that this transport is necessary for normal shoot development.

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.

Tonoplast-localized Abc2 Transporter Mediates Phytochelatin Accumulation in Vacuoles and Confers Cadmium Tolerance

Phytochelatins mediate tolerance to heavy metals in plants and some fungi by sequestering phytochelatin-metal complexes into vacuoles. To date, only Schizosaccharomyces pombe Hmt1 has been described as a phytochelatin transporter and attempts to identify orthologous phytochelatin transporters in plants and other organisms have failed. Furthermore, recent data indicate that the hmt1 mutant accumulates significant phytochelatin levels in vacuoles, suggesting that unidentified phytochelatin transporters exist in fungi. Here, we show that deletion of all vacuolar ABC transporters abolishes phytochelatin accumulation in S. pombe vacuoles and abrogates 35S-PC2 uptake into S. pombe microsomal vesicles. Systematic analysis of the entire S. pombe ABC transporter family identified Abc2 as a full-size ABC transporter (ABCC-type) that mediates phytochelatin transport into vacuoles. The S. pombe abc1 abc2 abc3 abc4 hmt1 quintuple and abc2 hmt1 double mutant show no detectable phytochelatins in vacuoles. Abc2 expression restores phytochelatin accumulation into vacuoles and suppresses the cadmium sensitivity of the abc quintuple mutant. A novel, unexpected, function of Hmt1 in GS-conjugate transport is also shown. In contrast to Hmt1, Abc2 orthologs are widely distributed among kingdoms and are proposed as the long-sought vacuolar phytochelatin transporters in plants and other organisms.

Plant ABC Transporters Enable Many Unique Aspects of a Terrestrial Plant's Lifestyle.

Terrestrial plants have two to four times more ATP-binding cassette (ABC) transporter genes than other organisms, including their ancestral microalgae. Recent studies found that plants harboring mutations in these transporters exhibit dramatic phenotypes, many of which are related to developmental processes and functions necessary for life on dry land. These results suggest that ABC transporters multiplied during evolution and assumed novel functions that allowed plants to adapt to terrestrial environmental conditions. Examining the literature on plant ABC transporters from this viewpoint led us to propose that diverse ABC transporters enabled many unique and essential aspects of a terrestrial plants lifestyle, by transporting various compounds across specific membranes of the plant.

Phytochelatin–metal(loid) transport into vacuoles shows different substrate preferences in barley and Arabidopsis

Cadmium (Cd) and arsenic (As) are toxic to all living organisms, including plants and humans. In plants, Cd and As are detoxified by phytochelatins (PCs) and metal(loid)-chelating peptides and by sequestering PC-metal(loid) complexes in vacuoles. Consistent differences have been observed between As and Cd detoxification. Whereas chelation of Cd by PCs is largely sufficient to detoxify Cd, As-PC complexes must be sequestered into vacuoles to be fully detoxified. It is not clear whether this difference in detoxification pathways is ubiquitous among plants or varies across species. Here, we have conducted a PC transport study using vacuoles isolated from Arabidopsis and barley. Arabidopsis vacuoles accumulated low levels of PC2 -Cd, and vesicles from yeast cells expressing either AtABCC1 or AtABCC2 exhibited negligible PC2 -Cd transport activity compared with PC2 -As. In contrast, barley vacuoles readily accumulated comparable levels of PC2 -Cd and PC2 -As. PC transport in barley vacuoles was inhibited by vanadate, but not by ammonium, suggesting the involvement of ABC-type transporters. Interestingly, barley vacuoles exhibited enhanced PC2 transport activity when essential metal ions, such as Zn(II), Cu(II) and Mn(II), were added to the transport assay, suggesting that PCs might contribute to the homeostasis of essential metals and detoxification of non-essential toxic metal(loid)s.

The Arabidopsis Small G Protein ROP2 Is Activated by Light in Guard Cells and Inhibits Light-Induced Stomatal Opening

ROP small G proteins function as molecular switches in diverse signaling processes. Here, we investigated signals that activate ROP2 in guard cells. In guard cells of Vicia faba expressing Arabidopsis thaliana constitutively active (CA) ROP2 fused to red fluorescent protein (RFP-CA-ROP2), fluorescence localized exclusively at the plasma membrane, whereas a dominant negative version of RFP-ROP2 (DN-ROP2) localized in the cytoplasm. In guard cells expressing green fluorescent protein–ROP2, the relative fluorescence intensity at the plasma membrane increased upon illumination, suggesting that light activates ROP2. Unlike previously reported light-activated factors, light-activated ROP2 inhibits rather than accelerates light-induced stomatal opening; stomata bordered by guard cells transformed with CA-rop2 opened less than controls upon light irradiation. When introduced into guard cells together with CA-ROP2, At RhoGDI1, which encodes a guanine nucleotide dissociation inhibitor, inhibited plasma membrane localization of CA-ROP2 and abolished the inhibitory effect of CA-ROP2 on light-induced stomatal opening, supporting the negative effect of active ROP2 on stomatal opening. Mutant rop2 Arabidopsis guard cells showed phenotypes similar to those of transformed V. faba guard cells; CA-rop2 stomata opened more slowly and to a lesser extent, and DN-rop2 stomata opened faster than wild-type stomata in response to light. Moreover, in rop2 knockout plants, stomata opened faster and to a greater extent than wild-type stomata in response to light. Thus, ROP2 is a light-activated negative factor that attenuates the extent of light-induced changes in stomatal aperture. The inhibition of light-induced stomatal opening by light-activated ROP2 suggests the existence of feedback regulatory mechanisms through which stomatal apertures may be finely controlled.