Abscisic acid (ABA) is a ubiquitous phytohormone involved in many developmental processes and stress responses of plants. ABA moves within the plant, and intracellular receptors for ABA have been recently identified; however, no ABA transporter has been described to date. Here, we report the identification of the ATP-binding cassette (ABC) transporter Arabidopsis thaliana Pleiotropic drug resistance transporter PDR12 (AtPDR12)/ABCG40 as a plasma membrane ABA uptake transporter. Uptake of ABA into yeast and BY2 cells expressing AtABCG40 was increased, whereas ABA uptake into protoplasts of atabcg40 plants was decreased compared with control cells. In response to exogenous ABA, the up-regulation of ABA responsive genes was strongly delayed in atabcg40 plants, indicating that ABCG40 is necessary for timely responses to ABA. Stomata of lossof-function atabcg40 mutants closed more slowly in response to ABA, resulting in reduced drought tolerance. Our results integrate ABAdependent signaling and transport processes and open another avenue for the engineering of drought-tolerant plants.abscisic acid transporter | drought resistance | guard cell

Carbon dioxide uptake and water vapour release in plants occur through stomata, which are formed by guard cells. These cells respond to light intensity, CO2 and water availability, and plant hormones. The predicted increase in the atmospheric concentration of CO2 is expected to have a profound effect on our ecosystem. However, many aspects of CO2-dependent stomatal movements are still not understood. Here we show that the ABC transporter AtABCB14 modulates stomatal closure on transition to elevated CO2. Stomatal closure induced by high CO2 levels was accelerated in plants lacking AtABCB14. Apoplastic malate has been suggested to be one of the factors mediating the stomatal response to CO2 (Refs 4,5) and indeed, exogenously applied malate induced a similar AtABCB14-dependent response as high CO2 levels. In isolated epidermal strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the AtABCB14 and slower in AtABCB14-overexpressing plants, than in wild-type plants, indicating that AtABCB14 catalyses the transport of malate from the apoplast into guard cells. Indeed, when AtABCB14 was heterologously expressed in Escherichia coli and HeLa cells, increases in malate transport activity were observed. We therefore suggest that AtABCB14 modulates stomatal movement by transporting malate from the apoplast into guard cells, thereby increasing their osmotic pressure. University of Science and Technology, Pohang, Korea; [10][11][12] and had a strongly reduced sensitivity to glibenclamide, ABA, calcium and auxin, which are well known to control stomatal movement. We therefore were interested whether AtABCB14 also exhibits a regulatory function in guard cell physiology.AtABCB14 expression, as visualized by the activity of an AtABCB14 promoter::GUS fusion construct, is not restricted to guard cells of leaves only, but is also found in guard cells of stems, flowers and siliques ( Fig. 1a-f). In leaves, GUS activity was also detected in epidermal and at very low levels in mesophyll cells (Fig. 1c). These promoter::GUS expressions corresponded to the transcript levels detected in mesophyll and guard cell protoplasts (Fig. 1g). Transient expression of an 35S::AtABCB14:GFP construct in Arabidopsis protoplasts revealed that AtABCB14 is targeted to the plasma membrane (Fig. 1h, i). AtABCB14:sGFP expressed under the control of the AtABCB14 native promoter was targeted to the plasma membrane of guard cells (Fig. 1n). Coexpression of AtABCB14 with AtAHA2:RFP, a fusion protein of a plasma membrane localized proton pump with a red fluorescent protein 13 , resulted in a perfect co-localization ( Fig. 1j-l). Fractionation of microsomes on a sucrose density gradient further confirmed that AtABCB14:HA protein was targeted to the plasma membrane: the distribution pattern of the protein crossreacting with the HA antibody corresponded to that of AtPDR8, a plasma membrane protein 14 and differed from the patterns of the ER (Bip) and vacuolar markers (γ-TIP) (Fig. 1m). These results indica...

P 1B -type heavy-metal ATPases (HMAs) are transmembrane metal-transporting proteins that play a key role in metal homeostasis. Despite their importance, very little is known about their functions in monocot species. We report the characterization of rice (Oryza sativa) OsHMA9, a member of the P 1B -type ATPase family. Semiquantitative reverse transcription-polymerase chain reaction analyses of seedlings showed that OsHMA9 expression was induced by a high concentration of copper (Cu), zinc (Zn), and cadmium. We also determined, through promoterTb-glucuronidase analysis, that the main expression was in the vascular bundles and anthers. The OsHMA9:green fluorescence protein fusion was localized to the plasma membrane. Heterologous expression of OsHMA9 partially rescued the Cu sensitivity of the Escherichia coli copA mutant, which is defective in Cu-transporting ATPases. It did not rescue the Zn sensitivity of the zntA mutant, which is defective in Zn-transporting ATPase. To further elucidate the functional roles of OsHMA9, we isolated two independent null alleles, oshma9-1 and oshma9-2, from the T-DNA insertion population. Mutant plants exhibited the phenotype of increased sensitivity to elevated levels of Cu, Zn, and lead. These results support a role for OsHMA9 in Cu, Zn, and lead efflux from the cells. This article is the first report on the functional characterization of a P 1B -type metal efflux transporter in monocots.A number of heavy metals, including copper (Cu), zinc (Zn), manganese, and iron, are essential micronutrients for a wide variety of physical processes. These micronutrients can serve structural roles in proteins, act as enzyme cofactors, and function in cellular redox reactions . However, when present in excess, they can have deleterious effects because of their reactive nature (Schutzendubel and Polle, 2002). For example, Cu is an essential element for plant growth and is important in various biochemical reactions but, at toxic levels, it interferes with numerous physiological processes (Fernandes and Henriques, 1991). Likewise, Zn acts as a nutrient, but can be very toxic at higher concentrations (Rout and Das, 2003). Furthermore, some metals, such as cadmium (Cd), mercury, silver, and lead (Pb), are generally considered nonessential to plants and are potentially highly toxic because of their reactivity with sulfur and nitrogen in amino acid side chains (Clemens, 2001).Accumulations of these heavy metals in plants can occur following their uptake from contaminated soil, which can then lead to toxic levels in animals feeding on them . To maintain the concentration of essential metals within physiological limits and to minimize the detrimental effects of nonessential metals, plants, like other organisms, trigger a complex network of homeostatic mechanisms to control their uptake, accumulation, trafficking, and detoxification (Clemens, 2001). Specialized transport proteins, in the form of channels, carriers, or pumps, mediate the movement of heavy metals through membranes (Williams et al., 2000)....

SUMMARYAtHMA1 is a member of the heavy metal-transporting ATPase family. It exhibits amino acid sequence similarity to two other Zn(II) transporters, AtHMA2 and AtHMA4, and contains poly-His motifs that are commonly found in Zn(II)-binding proteins, but lacks some amino acids that are typical for this class of transporters. AtHMA1 localizes to the chloroplast envelope. In comparison with wild-type plants, we observed a more pronounced sensitivity in the presence of high Zn(II) concentrations, and increased accumulation of Zn in the chloroplast of T-DNA insertional mutants in AtHMA1. The Zn(II)-sensitive phenotype of AtHMA1 knock-out plants was complemented by the expression of AtHMA1 under the control of its own promoter. The Zn(II)-transporting activity of AtHMA1 was confirmed in a heterologous expression system, Saccharomyces cerevisiae. The sensitivity of yeast to high concentrations of Zn(II) was altered by the expression of AtHMA1 lacking its Nterminal chloroplast-targeting signal. Taken together, these results suggest that under conditions of excess Zn(II), AtHMA1 contributes to Zn(II) detoxification by reducing the Zn content of Arabidopsis thaliana plastids.

SUMMARYThe exine of the pollen wall shows an intricate pattern, primarily comprising sporopollenin, a polymer of fatty acids and phenolic compounds. A series of enzymes synthesize sporopollenin precursors in tapetal cells, and the precursors are transported from the tapetum to the pollen surface. However, the mechanisms underlying the transport of sporopollenin precursors remain elusive. Here, we provide evidence that strongly suggests that the Arabidopsis ABC transporter ABCG26/WBC27 is involved in the transport of sporopollenin precursors. Two independent mutations at ABCG26 coding region caused drastic decrease in seed production. This defect was complemented by expression of ABCG26 driven by its native promoter. The severely reduced fertility of the abcg26 mutants was caused by a failure to produce mature pollen, observed initially as a defect in pollenwall development. The reticulate pattern of the exine of wild-type microspores was absent in abcg26 microspores at the vacuolate stage, and the vast majority of the mutant pollen degenerated thereafter. ABCG26 was expressed specifically in tapetal cells at the early vacuolate stage of pollen development. It showed high co-expression with genes encoding enzymes required for sporopollenin precursor synthesis, i.e. CYP704B1, ACOS5, MS2 and CYP703A2. Similar to two other mutants with defects in pollen-wall deposition, abcg26 tapetal cells accumulated numerous vesicles and granules. Taken together, these results suggest that ABCG26 plays a crucial role in the transfer of sporopollenin lipid precursors from tapetal cells to anther locules, facilitating exine formation on the pollen surface.