HKT-type transporters appear to play key roles in Na + accumulation and salt sensitivity in plants. In Arabidopsis HKT1;1 has been proposed to influx Na + into roots, recirculate Na + in the phloem and control root : shoot allocation of Na + . We tested these hypotheses using 22 Na + flux measurements and ion accumulation assays in an hkt1;1 mutant and demonstrated that AtHKT1;1 contributes to the control of both root accumulation of Na + and retrieval of Na + from the xylem, but is not involved in root influx or recirculation in the phloem. Mathematical modelling indicated that the effects of the hkt1;1 mutation on root accumulation and xylem retrieval were independent. Although AtHKT1;1 has been implicated in regulation of K + transport and the hkt1;1 mutant showed altered net K + accumulation, 86 Rb + uptake was unaffected by the hkt1;1 mutation. The hkt1;1 mutation has been shown previously to rescue growth of the sos1 mutant on low K + ; however, HKT1;1 knockout did not alter K + or 86 Rb + accumulation in sos1.

Arabidopsis is frequently used as a genetic model in plant salt tolerance studies, however, its physiological responses to salinity remain poorly characterized. This study presents a characterization of initial Na ϩ entry and the effects of Ca 2ϩ on plant growth and net Na ϩ accumulation in saline conditions. Unidirectional Na ϩ influx was measured carefully using very short influx times in roots of 12-d-old seedlings. Influx showed three components with distinct sensitivities to Ca 2ϩ , diethylpyrocarbonate, and osmotic pretreatment. Pharmacological agents and known mutants were used to test the contribution of different transport pathways to Na ϩ uptake. Influx was stimulated by 4-aminobutyric acid and glutamic acid; was inhibited by flufenamate, quinine, and cGMP; and was insensitive to modulators of K ϩ and Ca 2ϩ channels. Influx did not differ from wild type in akt1 and hkt1 insertional mutants. These data suggested that influx was mediated by several different types of nonselective cation channels. Na ϩ accumulation in plants grown in 50 mm NaCl was strongly reduced by increasing Ca 2ϩ activity (from 0.05-3.0 mm), and plant survival was improved. However, plant biomass was not affected by shoot Na ϩ concentration, suggesting that in Arabidopsis Na ϩ toxicity is not dependent on shoot Na ϩ accumulation. These data suggest that Arabidopsis is a good model for investigation of Na ϩ transport, but may be of limited utility as a model for the study of Na ϩ toxicity.

There is increasing evidence of the important roles of glutamate receptors (GLRs) in plant development and in adaptation to stresses. However, the studies of these putative ion channels, both in planta and in Xenopus oocytes, may have been limited by our lack of knowledge of possible GLR heteromer formation in plants. We have developed a modification of the single-cell sampling technique to investigate GLR co-expression, and thus potential heteromer formation, in single cells of Arabidopsis thaliana leaves. MicroEXpression amplification (MEX) has allowed us to amplify gene transcripts from a single cell, enabling expression of up to 100 gene transcripts to be assayed. We measured, on average, the transcripts of five to six different AtGLRs in a single cell. However, no consistent patterns of co-expression or cell-type-specific expression were detected, except that cells sampled from the same plant showed similar expression profiles. The only discernible feature was the detection of AtGLR3.7 in every cell examined, an observation supported by GUS staining patterns in plants stably expressing promoter::uidA fusions. In addition, we found AtGLR3.7 expression in oocytes induces a Ba 2+ -, Ca 2+ -and Na + -permeable plasma membrane conductance.

The effect of glutamate on plant plasma membrane cation transport was studied in roots of Arabidopsis thaliana (L.) Heynh. Patch-clamp experiments using root protoplasts, (22)Na(+) unidirectional fluxes into intact roots and measurements of cytosolic Ca(2+) activity using plants expressing cytosolically-targeted aequorin in specific cell types were carried out. It was demonstrated that low-millimolar concentrations of glutamate activate within seconds both Na(+) and Ca(2+) currents in patch-clamped protoplasts derived from roots. The probability of observing glutamate-activated currents increased with increasing glutamate concentration (up to 29% at 3 mM); half-maximal activation was seen at 0.2-0.5 mM glutamate. Glutamate-activated currents were voltage-insensitive, 'instantaneous' (completely activated within 2-3 ms of a change in voltage) and non-selective for monovalent cations (Na(+), Cs(+) and K(+)). They also allowed the permeation of Ca(2+). Half-maximal Na(+) currents occurred at 20-30 mM Na(+). Glutamate-activated currents were sensitive to non-specific blockers of cation channels (quinine, La(3+), Gd(3+)). Although low-millimolar concentrations of glutamate did not usually stimulate unidirectional influx of (22)Na(+) into intact roots, they reliably caused an increase in cytosolic Ca(2+) activity in protoplasts isolated from the roots of aequorin-transformed Arabidopsis plants. The response of cytosolic Ca(2+) activity revealed a two-phase development, with a rapid large transient increase (lasting minutes) and a prolonged subsequent stage (lasting hours). Use of plants expressing aequorin in specific cell types within the root suggested that the cell types most sensitive to glutamate were in the mature epidermis and cortex. The functional significance of these glutamate-activated currents for both cation uptake into plants and cell signaling remains the subject of speculation, requiring more knowledge about the dynamics of apoplastic glutamate in plants.

SummaryWe used enhancer trapping with the GAL4 transcriptional activator from yeast to obtain spatial control of transgene expression in all organs of the model monocotyledonous species rice (Oryza sativa L. cv. Nipponbare). Our T-DNA enhancer trapping cassette consisted of two principle components: (1) the minimal promoter-equipped gal4 gene placed adjacent to the right border, and (2) the green fluorescent protein gene (gfp) fused to the upstream activation sequence element (UAS) to which GAL4 binds and activates expression, so that gfp expression corresponds to gal4 expression. Agrobacterium-mediated integration of the cassette into the rice genome often brings the gal4 gene under transcriptional control of local genomic enhancers and promoters, resulting in gal4/gfp expression patterns ranging in specificity from single-cell types to constitutive expression. We produced more than 13 000 enhancer trap lines with this cassette and screened T 0 adult plants (1982 lines), T 1 seed (2684 lines) and T 1 seedlings (2667 lines) for gfp expression. Approximately 30% of the lines produced GFP, and we identified lines with gfp expression in specific cell types of all major organs of the rice plant. Subsequently, using the GUS reporter gene (uidA), we demonstrated that UAS:geneX constructs can be transactivated in specific cell types where gal4 and gfp are expressed, thus providing an excellent system for the manipulation of gene expression and physiological function in specific cell types of rice.