Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the genome of A. tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome. We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants.

Supplementary data are available at Bioinformatics online.

The Arabidopsis thaliana cDNA, KAT1, encodes a hyperpolarization-activated K ؉ channel. In the present study, we utilized a combination of random site-directed mutagenesis, genetic screening in a potassium uptakedeficient yeast strain, and electrophysiological analysis in Xenopus oocytes to identify strong modifications in cation selectivity of the inward rectifying K ؉ channel KAT1. Threonine at position 256 was replaced by 11 other amino acid residues. Six of these mutated KAT1 cDNAs complemented a K ؉ uptake-deficient yeast strain at low concentrations of potassium. Among these, two mutants (T256D and T256G) showed a sensitivity of yeast growth toward high ammonium concentrations and a dramatic increase in current amplitudes of rubidium and ammonium ions relative to K ؉ by 39 -72-fold. These single site mutations gave rise to Rb ؉ -and NH 4 ؉ -selective channels with Rb ؉ and NH 4 ؉ currents that were approximately 10 -13-fold greater in amplitude than K ؉ currents, whereas the NH 4 ؉ to K ؉ current amplitude ratio of wild type KAT1 was 0.28. This strong conversion in cation specificity without loss of general selectivity exceeds those reported for other mutations in the pore domain of voltage-dependent K ؉ channels. Yeast growth was greatly impaired by sodium in two other mutants at this site (T256E and T256Q), which were blocked by millimolar sodium (K1 ⁄2 ‫؍‬ 1.1 mM for T256E), although the wild type channel was not blocked by 110 mM sodium. Interestingly, the ability of yeast to grow in the presence of toxic cations correlated to biophysical properties of KAT1 mutants, illustrating the potential for qualitative K ؉ channel mutant selection in yeast. These data suggest that the size of the side chain of the amino acid at position 256 in KAT1 is important for enabling cation permeation and that this site plays a crucial role in determining the cation selectivity of hyperpolarizationactivated potassium channels.

K+ channels play diverse roles in mediating K+ transport and in modulating the membrane potential in higher plant cells during growth and development. Some of the diversity in K+ channel functions may arise from the regulated expression of multiple genes encoding different K+ channel polypeptides. Here we report the isolation of a nove1 Arabidopsis fhaliana cDNA (AKTZ) that is highly homologous t o the two previously identified K+ channel genes, KATl and AKT7. This cDNA mapped t o the center of chromosome 4 by restriction fragment length polymorphism analysis and was highly expressed i n leaves, whereas AKT7 was mainly expressed in roots. I n addition, we show that diversity in K+ channel function may be attributable t o differences i n expression levels. lncreasing KAT7 expression i n Xenopus oocytes by polyadenylation of the KATl mRNA increased the current amplitude and led t o higher levels of KATl protein, as assayed i n western blots. The increase in KATl expression in oocytes produced shifts in the threshold potentia1 for activation to more positive membrane potentials and decreased half-activation times. These results suggest that different levels of expression and tissue-specific expression of different K+ channel isoforms can contribute to the functional diversity of plant K+ channels. The identification of a highly expressed, leaf-specific K+ channel homolog in plants should allow further molecular characterization of K+ channel functions for physiological K+ transport processes in leaves.

A nove1 effect of ammonium ions on root growth was investigated to understand how environmental signals affect organ development. Ammonium ions (3-12 mM) were found to dramatically inhibit Arabidopsis thaliana seedling root growth in the absence of potassium even if nitrate was present. This inhibition could be reversed by including in the growth medium low levels (20-100 PM) of potassium or alkali ions Rb+ and Cs+ but not alkali ions Na' and Li+. The protective effect of low concentrations of potassium is not due to an inhibition of ammonium uptake. Ammonium inhibition is reversible, because root growth was restored in ammonium-treated seedlings if they were subsequently transferred to medium containing potassium. It is known that plant hormones can inhibit root growth. We found that mutants of Arabidopsis resistant to high levels of auxin and other hormones (auxl, axrl, and axr2) are also resistant to the ammonium inhibition and produce roots in the absence of potassium. Thus, the mechanisms that mediate the ammonium inhibition of root development are linked to hormone metabolic or signaling pathways. These findings have important implications for understanding how environmental signals, especially mineral nutrients, affect plant root development.

However, the conducting properties of transporters go well beyond this simple picture (Sonders and Amara, Division of Biology California Institute of Technology 1996). For example, the 5-HT transporter is not electrogenic; yet it displays at least four distinct conducting Pasadena, California 91125 states (Mager et al., 1994). Three of these states are permeable to Na ϩ ions, and the other to protons; and recent results show that at least two of the Na ϩ conduc-Our title recalls Listening to Prozac, the best-seller describing clinical case histories of patients who have been tances arise from unitary events that resemble single channel openings, with conductances of a few pS and aided by fluoxetine (Kramer, 1993). Fluoxetine typifies a new class of antidepressants called serotonin-selective open durations of a few ms (Lin et al., 1996). If one assumes that these events arise uniformly from all trans-reuptake inhibitors (SSRIs). The fraction of adults who take SSRIs may be exceeded only by the fraction of porters in the membrane, then the open probability is about 10 Ϫ6 ; and an individual transporter produces an children diagnosed with attention-deficit disorder or attention deficit-hyperactivity disorder who benefit from opening only once per 350-700 transport cycles. One of these channel-based conducting states occurs in the methylphenidate (Ritalin) or other drugs thought to act on monoamine transporters either by blocking uptake presence of substrate, and one "leakage" state in its absence. Evidently channel events are not an obligate or by enhancing release.Modern concepts of therapeutics would imply that part of each transport cycle. However, we do not know whether channel events arise from a subpopulation of such success stories arise because of highly specific actions on one subtype of target molecule. Yet the transporters with abnormal properties. Channel-like unitary events are confined neither to known neurotransmitter transporter families are much smaller than the superfamilies that comprise 7-helix re-5-HT transporters nor to oocyte expression systems and, in the case of glutamate transporters, also occur ceptors and ligand-gated channels. There are, for example, at least fourteen known serotonin receptor genes, in native cells. All cloned mammalian monoamine transporters also display "excess transport-associated cur-but only one known serotonin transporter gene, and another one apiece for dopamine and noradrenaline/ rent," which may be explained by the channel-like events. When measurements of macroscopic current adrenaline (the last is relatively nonspecific). Because the unique serotonin transporter is widely expressed, a during transport are integrated, the charge exceeds the value expected from simultaneously measured uptake fascinating question in molecular neuroscience concerns the mechanism for the exquisite antidepressant of radiolabeled organic substrate. The discrepancy ranges from a factor of 3 to more than 100. Presumably effects of SSRIs.Might this specificity arise because a given neu...

The evolution of glyphosate resistance in weedy species places an environmentally benign herbicide in peril. The first report of a dicot plant with evolved glyphosate resistance was horseweed, which occurred in 2001. Since then, several species have evolved glyphosate resistance and genomic information about nontarget resistance mechanisms in any of them ranges from none to little. Here, we report a study combining iGentifier transcriptome analysis, cDNA sequencing, and a heterologous microarray analysis to explore potential molecular and transcriptomic mechanisms of nontarget glyphosate resistance of horseweed. The results indicate that similar molecular mechanisms might exist for nontarget herbicide resistance across multiple resistant plants from different locations, even though resistance among these resistant plants likely evolved independently and available evidence suggests resistance has evolved at least four separate times. In addition, both the microarray and sequence analyses identified non–target-site resistance candidate genes for follow-on functional genomics analysis.

The rat and human serotonin transporters (rSERT and hSERT, respectively) were expressed in Xenopus oocytes and studied using site-directed mutagenesis, electrophysiological recordings, and [3H]5-HT uptake measurements. rSERT, but not hSERT, displayed increased transport-associated current at low pH. Chimeras and point mutations showed that, of the 52 nonidentical residues, a single residue at position 490 (threonine in rSERT and lysine in hSERT) governs this difference. Furthermore, potentiation required the glutamate residue at position 493. Cysteine substitution and alkylation experiments showed that residue 493 is extracellular. Cysteine at 493 increased, whereas aspartate decreased, the net charge movement per transported 5-HT molecule. The mutations at this region did not significantly affect other aspects of SERT function, including agonist-independent leakage current, voltage-dependent transient current, and H+ current. This region may therefore be part of an external gate required for rSERT function. The data and analyses show that, in the absence of detailed structural information, a gate-lumen-gate scheme is useful for interpreting results from mutations that alter functional properties of neurotransmitter transporters.