Spatial control of transgene expression in rice (Oryza sativa L.) using the GAL4 enhancer trapping system.

We 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.

Sodium influx and accumulation in Arabidopsis.

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+ entry and the effects of Ca2+ on plant growth and net Na+ accumulation in saline conditions. Unidirectional Na+ influx was measured carefully using very short influx times in roots of 12-d-old seedlings. Influx showed three components with distinct sensitivities to Ca2+, diethylpyrocarbonate, and osmotic pretreatment. Pharmacological agents and known mutants were used to test the contribution of different transport pathways to Na+ 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+ and Ca2+ 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+ accumulation in plants grown in 50 mM NaCl was strongly reduced by increasing Ca2+ activity (from 0.05-3.0 mM), and plant survival was improved. However, plant biomass was not affected by shoot Na+ concentration, suggesting that in Arabidopsis Na+ toxicity is not dependent on shoot Na+ accumulation. These data suggest that Arabidopsis is a good model for investigation of Na+ transport, but may be of limited utility as a model for the study of Na+ toxicity.

Functional analysis of AtHKT1 in Arabidopsis shows that Na(+) recirculation by the phloem is crucial for salt tolerance.

Two allelic recessive mutations of Arabidopsis, sas2-1 and sas2-2, were identified as inducing sodium overaccumulation in shoots. The sas2 locus was found (by positional cloning) to correspond to the AtHKT1 gene. Expression in Xenopus oocytes revealed that the sas2-1 mutation did not affect the ionic selectivity of the transporter but strongly reduced the macro scopic (whole oocyte current) transport activity. In Arabidopsis, expression of AtHKT1 was shown to be restricted to the phloem tissues in all organs. The sas2-1 mutation strongly decreased Na(+) concentration in the phloem sap. It led to Na(+) overaccumulation in every aerial organ (except the stem), but to Na(+) underaccumulation in roots. The sas2 plants displayed increased sensitivity to NaCl, with reduced growth and even death under moderate salinity. The whole set of data indicates that AtHKT1 is involved in Na(+) recirculation from shoots to roots, probably by mediating Na(+) loading into the phloem sap in shoots and unloading in roots, this recirculation removing large amounts of Na(+) from the shoot and playing a crucial role in plant tolerance to salt.