An integrated molecular and physiological investigation of the fundamental mechanisms of heavy metal accumulation was conducted in Thlaspi caerulescens, a Zn͞Cd-hyperaccumulating plant species. A heavy metal transporter cDNA, ZNT1, was cloned from T. caerulescens through functional complementation in yeast and was shown to mediate high-affinity Zn 2؉ uptake as well as lowaffinity Cd 2؉ uptake. It was found that this transporter is expressed at very high levels in roots and shoots of the hyperaccumulator. A study of ZNT1 expression and high-affinity Zn 2؉ uptake in roots of T. caerulescens and in a related nonaccumulator, Thlaspi arvense, showed that alteration in the regulation of ZNT1 gene expression by plant Zn status results in the overexpression of this transporter and in increased Zn influx in roots of the hyperaccumulating Thlaspi species. These findings yield insights into the molecular regulation and control of plant heavy metal and micronutrient accumulation and homeostasis, as well as provide information that will contribute to the advancement of phytoremediation by the future engineering of plants with improved heavy metal uptake and tolerance. R ecently, there has been considerable interest in the use of terrestrial plants as a green technology for the remediation of surface soils contaminated with toxic heavy metals. This technology, termed phytoremediation, uses plants to extract heavy metals from the soil and to concentrate them in the harvestable shoot tissue (1, 2). A major factor behind the interest in phytoremediation of metal-polluted soils has been the growing awareness of the existence of a number of metalaccumulating plant species. These plant species, called hyperaccumulators, are endemic to metalliferous soils and can accumulate and tolerate high levels of heavy metals in the shoot (3, 4). Among the best known hyperaccumulators is Thlaspi caerulescens. This member of the Brassicaceae family has attracted the interest of plant biologists for over a century because of its ability to colonize calamine and serpentine soils containing naturally elevated levels of heavy metals such as Zn, Pb, Cd, Ni, Cr, and Co.

In Arabidopsis thaliana, signal transduction of the hormone ethylene involves at least two receptors, ETR1 and ERS, both of which are members of the two-component histidine protein kinase family that is prevalent in prokaryotes. The pathway also contains a negative regulator of ethylene responses, CTR1, which closely resembles members of the Raf protein kinase family. CTR1 is thought to act at or downstream of ETR1 and ERS based on double mutant analysis; however, the signaling mechanisms leading from ethylene perception to the regulation of CTR1 are unknown. By using the yeast two-hybrid assay, we detected a specific interaction between the CTR1 amino-terminal domain and the predicted histidine kinase domain of ETR1 and ERS. We subsequently verified these interactions by using an in vitro protein association assay(s). In addition, we determined that the amino-terminal domain of CTR1 can associate with the predicted receiver domain of ETR1 in vitro. Based on deletion analysis, the portion of CTR1 that interacts with ETR1 roughly aligns with the regulatory region of Raf kinases. These physical associations support the genetic evidence that CTR1 acts in the pathway of ETR1 and ERS and suggest that these interactions could be involved in the regulation of CTR1 activity.Ethylene has numerous effects on plant growth and development, such as fruit-ripening, organ abscission, seed germination, senescence, and the induction of certain defense responses (1). The cloning of genes corresponding to several ethylene-response mutants in Arabidopsis has begun to provide us with insight into the molecular basis of ethylene signal transduction (2-6). In Arabidopsis, there are at least two ethylene receptors, ETR1 and ERS, that are similar to each other. Plants appear to have multiple ethylene receptors; several different ETR1 and ERS homologs have been cloned recently from Arabidopsis (7) and tomato (8, 9). The ETR1 and ERS gene products are predicted to function as histidine protein kinases based on their sequence similarities with the two-component regulator family. The two-component regulators are prevalent in prokaryotes (10) and are starting to be identified in various eukaryotes, including fungi, slime mold, and higher plants (11,12); one of these, the Arabidopsis CKI1 protein, is potentially a receptor for cytokinin (13). The basic two-component system consists of a histidine autokinase sensor component that directs the activity of a cognate response regulator, which in turn controls downstream signaling (10). After autophosphorylation of the sensor kinase, the phosphoryl group is transferred from the histidine of the sensor kinase to an aspartic acid residue in the receiver domain of the response regulator (10). The ETR1 protein is an example of a hybrid histidine kinase because it contains both a histidine kinase domain and a receiver domain (2). ERS, in contrast, contains only a histidine kinase domain (3). The significance of having an attached receiver domain is unknown, and whether there are separate cognate...

Aluminum (Al) toxicity in acid soils is a worldwide agricultural problem that severely limits crop productivity through inhibition of root growth. Previously, Arabidopsis mutants with increased Al sensitivity were isolated in order to identify genes important for Al tolerance in plants. One mutant, als3, exhibited extreme root growth inhibition in the presence of Al, suggesting that this mutation negatively impacts a gene required for Al tolerance. Map-based cloning of the als3-1 mutation resulted in the isolation of a novel gene that encodes a previously undescribed ABC transporter-like protein, which is highly homologous to a putative bacterial metal resistance protein, ybbM. Northern analysis for ALS3 expression revealed that it is found in all organs examined, which is consistent with the global nature of Al sensitivity displayed by als3, and that expression increases in roots following Al treatment. Based on GUS fusion and in situ hybridization analyses, ALS3 is primarily expressed in leaf hydathodes and the phloem throughout the plant, along with the root cortex following Al treatment. Immunolocalization indicates that ALS3 predominantly accumulates in the plasma membrane of cells that express ALS3. From our results, it appears that ALS3 encodes an ABC transporter-like protein that is required for Al resistance/tolerance and may function to redistribute accumulated Al away from sensitive tissues in order to protect the growing root from the toxic effects of Al.

Ethylene signaling is a complex pathway that has been intensively analyzed partly due to its importance to the manifestation of horticultural phenomena, including fruit ripening and tissue senescence. In order to further our understanding of how this pathway is regulated, a screen for Arabidopsis mutants with increased ethylene response was conducted. From this, a mutant was identified as having a dark-grown hypocotyl that is indistinguishable from Col-0 wt in the presence of the ethylene perception inhibitor AgNO₃, yet has extreme responsiveness to even low levels of ethylene. Map-based cloning of the mutation revealed a T-DNA insertion in the coding sequence of the receptor-like kinase FERONIA, which is required for normal pollen tube reception and cell elongation in a currently unknown capacity. In contrast to a previous report, analysis of our feronia knockout mutant shows it also has altered responsiveness to brassinosteroids, with etiolated fer-2 seedlings being partially brassinosteroid insensitive with regard to promotion of hypocotyl elongation. Our results indicate that FERONIA-dependent brassinosteroid response serves to antagonize the effect of ethylene on hypocotyl growth of etiolated seedlings, with loss of proper brassinosteroid signaling disrupting this balance and leading to a greater impact of ethylene on hypocotyl shortening.

Ethylene signaling in Arabidopsis begins at a family of five ethylene receptors that regulate activity of a downstream mitogen-activated protein kinase kinase kinase, CTR1. Triple and quadruple loss-of-function ethylene receptor mutants display a constitutive ethylene response phenotype, indicating they function as negative regulators in this pathway. No ethylene-related phenotype has been described for single loss-of-function receptor mutants, although it was reported that etr1 loss-of-function mutants display a growth defect limiting plant size. In actuality, this apparent growth defect results from enhanced responsiveness to ethylene; a phenotype manifested in all tissues tested. The phenotype displayed by etr1 loss-of-function mutants was rescued by treatment with an inhibitor of ethylene perception, indicating that it is ethylene dependent. Identification of an ethylene-dependent phenotype for a loss-of-function receptor mutant gave a unique opportunity for genetic and biochemical analysis of upstream events in ethylene signaling, including demonstration that the dominant ethylene-insensitive phenotype of etr2-1 is partially dependent on ETR1. This work demonstrates that mutational loss of the ethylene receptor ETR1 alters responsiveness to ethylene in Arabidopsis and that enhanced ethylene response in Arabidopsis not only results in increased sensitivity but exaggeration of response.Ethylene is a simple gaseous molecule that is one of five classic plant hormones, being critical for the control of physiological processes at all stages of plant growth and development. Example processes include seed germination, response to pathogen attack, tissue senescence, and fruit ripening (Abeles et al., 1992). Work to understand the molecular mechanisms of ethylene signaling has utilized Arabidopsis as a model system through mutagenesis and screening for seedlings that display an aberrant ethylene phenotype, resulting in the elucidation of a linear signaling pathway (Kieber, 1997;Johnson and Ecker, 1998;Chang and Shockey, 1999;Bleecker and Kende, 2000;Stepanova and Ecker, 2000).In Arabidopsis, ethylene perception initiates with binding of ethylene to a family of five receptors (ETR1, ERS1, ETR2, EIN4, and ERS2). Ethylene binding is mediated by a copper cofactor (Rodriguez et al., 1999) that is provided to the receptors by the copper transporter RAN1 (Hirayama et al., 1999;Woeste and Kieber, 2000). The ethylene receptors are structurally similar to a family of proteins from bacteria, collectively known as two-component regulators, which are responsible for sensing changes in the growth environment (Chang and Shockey, 1999;Bleecker and Kende, 2000). As with two-component regulators, the ethylene receptors can be divided into multiple functional domains including a sensor domain that consists of a transmembrane region responsible for ethylene binding (Schaller and Bleecker, 1995;Hall et al., 2000); a GAF domain of unknown function (Aravind and Ponting, 1997); a His kinase domain, of which only ETR1 and ERS1 contain all o...

Aluminum toxicity in acid soils severely limits crop productivity through inhibition of root growth and, consequently, shoot development. Several Arabidopsis mutants were previously identified as having roots with Al hypersensitivity, suggesting that these represent deleterious mutations affecting genes required for either Al tolerance or resistance mechanisms. For this report, the als1-1 mutant was chosen for further characterization. The phenotype of als1-1 is most obviously presented in Al challenged roots, as evidenced by exaggerated root growth inhibition in conjunction with increased expression of Al-responsive genes compared to wt. Using a map-based cloning approach, the als1-1 mutation was isolated and found to represent a deleterious amino acid substitution in a previously uncharacterized half type ABC transporter, At5g39040, which is expressed in a non-Al dependent manner in all organs tested. GUS-dependent analyses revealed that ALS1 expression is primarily localized to the root tip and the vasculature throughout the plant. Concomitant with this, an ALS1: GFP fusion accumulates at the vacuolar membrane of root cells, indicating that ALS1 may be important for intracellular movement of some substrate, possibly chelated Al, as part of a mechanism of Al sequestration.

We have examined the expression of mRNAs for S-adenosylmethionine synthetase (EC 2.5.1.6), 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14), and the ethylene-forming enzyme (EFE) in various floral organs of carnation (Dianthus caryophyllus) during the increase in ethylene biosynthesis associated with petal senescence. The abundance of ACC synthase and EFE mRNAs increased and S-adenosylmethionine synthetase transcripts decreased concomitantly with the ethylene climacteric in senescing petals. The increase in abundance of ACC synthase and EFE mRNAs in aging flowers was prevented by treatment with the ethylene action inhibitor 2,5-norbornadiene. Furthermore, an increase in ACC synthase and EFE transcripts was detected in petals from presenescent flowers within 3 to 6 hours of exposure to 2 microliters per liter of ethylene. The increase in ethylene production by senescing petals was associated with a concomitant increase in ethylene biosynthesis in styles, ovary, and receptacle tissues. In all tissues, this increase was associated with increased activities of ACC synthase and EFE. The increase in EFE activities by all floral organs examined was correlated with increased abundance of EFE transcripts. In contrast, the level of ACC synthase mRNA, as detected by the cDNA probe pCARACC3, did not always reflect enzyme activity. The combined tissues of the pistil exhibited high rates of ACC synthase activity but contained low levels of ACC synthase mRNAs homologous to pCARACC3. In addition, pollinated styles exhibited a rapid increase in ethylene production and ACC synthase activity but did not accumulate detectable levels of ACC synthase mRNA until several hours after the initiation of ethylene production. These results suggest that transcripts for ACC synthase leading to the early postpollination increase in ACC synthase activity and ethylene production are substantially different from the mRNA for the ethylene-responsive gene represented by pCARACC3.Ethylene biosynthesis in plant tissues is under strict metabolic regulation and subject to induction by a variety ofsignals including mechanical wounding, auxin, and endogenous developmental factors in senescing flowers and ripening fruit (30). The ethylene biosynthetic pathway was elucidated by Adams and Yang (1) and is Met--SAM2--ACC--ethylene.

By screening for enhanced ethylene-response (eer) mutants in Arabidopsis, we isolated a novel recessive mutant, eer1, which displays increased ethylene sensitivity in the hypocotyl and stem. Dark-grown eer1 seedlings have short and thick hypocotyls even in the absence of added ethylene. This phenotype is suppressed, however, by the ethylene biosynthesis inhibitor 1-aminoethoxyvinyl-glycine. Following ethylene treatment, the dark-grown eer1 hypocotyl response is greatly exaggerated in comparison with the wild type, indicating that the eer1 phenotype is not simply due to ethylene overproduction. eer1 seedlings have significantly elevated levels of basic-chitinase expression, suggesting that eer1 may be highly sensitive to low levels of endogenous ethylene. Adult eer1 plants display exaggerated ethylene-dependent stem thickening, which is an ethylene response previously unreported in Arabidopsis. eer1 also has enhanced responsiveness to the ethylene agonists propylene and 2,5-norbornadiene. The eer1 phenotype is completely suppressed by the ethylene-insensitive mutation etr1-1, and is additive with the constitutive ethylene-response mutation ctr1-3. Our findings suggest that the wild-type EER1 product acts to oppose ethylene responses in the hypocotyl and stem.