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 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.

SummaryEthylene signaling in Arabidopsis begins with a family of ®ve ethylene receptors that regulate the activity of the Raf-like kinase, CTR1. Recent work to identify novel factors required for modulating ethylene signaling resulted in the isolation of enhanced ethylene response 1 (eer1), a mutant that displays both increased sensitivity and increased amplitude of response to ethylene. Molecular cloning of eer1 reveals that its mutant phenotype results from a loss-of-function mutation in the previously characterized RCN1, one of three PP2A A regulatory subunits in Arabidopsis. Our analysis shows that neither RCN1 expression nor PP2A activity is regulated by ethylene. Instead, we found that Arabidopsis PP2A-1C, a PP2A catalytic subunit previously characterized as interacting with RCN1, associates strongly with the kinase domain of CTR1 in vitro. This likely represents a role for PP2A in modulation of CTR1 activity because an in vitro kinase assay did not reveal phosphorylation of either RCN1 or PP2A-1C by CTR1, indicating that neither of them is a substrate for CTR1. PP2A activity is required for Ras-dependent activation of mammalian Raf, with reductions in PP2A activity signi®cantly compromising the effectiveness of this mechanism. Our genetic and biochemical results suggest that a similar requirement for PP2A activity exists for ethylene signaling, with loss-of-function mutations affecting PP2A activity possibly reducing the effectiveness of CTR1 activation, thus lowering the threshold required for manifestation of ethylene response.

SummaryBy screening etiolated Arabidopsis seedlings for mutants with aberrant ethylene-related phenotypes, we identi®ed a mutant that displays features of the ethylene-mediated triple response even in the absence of ethylene. Further characterization showed that the phenotype observed for the dark-grown seedlings of this mutant is reversible by prevention of ethylene perception and is dependent on a modest increase in ethylene production correlated with an increase in 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) activity in the hypocotyl. Molecular characterization of leaves of the mutant revealed severely impaired induction of basic chitinase (chiB) and plant defensin (PDF)1.2 following treatment with jasmonic acid and/or ethylene. Positional cloning of the mutation resulted in identi®cation of a 49-bp deletion in RCE1 (related to ubiquitin 1 (RUB1)-conjugating enzyme), which has been demonstrated to be responsible for covalent attachment of RUB1 to the SCF (Skpl Cdc 53 F-box) ubiquitin ligase complex to modify its activity. Our analyses with rce1-2 demonstrate a previously unknown requirement for RUB1 modi®cation for regulation of ethylene biosynthesis and proper induction of defense-related genes in Arabidopsis.