This study of the Arabidopsis thaliana nitrate transporter NRT1.6 indicated that nitrate is important for early embryo development. Functional analysis of cDNA-injected Xenopus laevis oocytes showed that NRT1.6 is a low-affinity nitrate transporter and does not transport dipeptides. RT-PCR, in situ hybridization, and b-glucuronidase reporter gene analysis showed that expression of NRT1.6 is only detectable in reproductive tissue (the vascular tissue of the silique and funiculus) and that expression increases immediately after pollination, suggesting that NRT1.6 is involved in delivering nitrate from maternal tissue to the developing embryo. In nrt1.6 mutants, the amount of nitrate accumulated in mature seeds was reduced and the seed abortion rate increased. In the mutants, abnormalities (i.e., excessive cell division and loss of turgidity), were found mainly in the suspensor cells at the one-or two-cell stages of embryo development. The phenotype of the nrt1.6 mutants revealed a novel role of nitrate in early embryo development. Interestingly, the seed abortion rate of the mutant was reduced when grown under N-deficient conditions, suggesting that nitrate requirements in early embryo development can be modulated in response to external nitrogen changes.

The effects of KCl, NaCl, and LiCl on the growth of Debaryomyces hansenii, usually considered a halotolerant yeast, and Saccharomyces cerevisiae were compared. KCl and NaCl had similar effects on D. hansenii, indicating that NaCl created only osmotic stress, while LiCl had a specific inhibitory effect, although relatively weaker than in S. cerevisiae. In media with low K ؉ , Na ؉ was able to substitute for K ؉ , restoring the specific growth rate and the final biomass of the culture. The intracellular concentration of Na ؉ reached values up to 800 mM, suggesting that metabolism is not affected by rather high concentrations of salt. The ability of D. hansenii to extrude Na ؉ and Li ؉ was similar to that described for S. cerevisiae, suggesting that this mechanism is not responsible for the increased halotolerance. Also, the kinetic parameters of Rb ؉ uptake in D. hansenii (V max , 4.2 nmol mg [dry weight] ؊1 min ؊1 ; K m , 7.4 mM) indicate that the transport system was not more efficient than in S. cerevisiae. Sodium (50 mM) activated the transport of Rb ؉ by increasing the affinity for the substrate in D. hansenii, while the effect was opposite in S. cerevisiae. Lithium inhibited Rb ؉ uptake in D. hansenii. We propose that the metabolism of D. hansenii is less sensitive to intracellular Na ؉ than is that of S. cerevisiae, that Na ؉ substitutes for K ؉ when K ؉ is scarce, and that the transport of K ؉ is favored by the presence of Na ؉. In low K ؉ environments, D. hansenii behaved as a halophilic yeast.

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Two genes encoding Na؉ -ATPases from Debaryomyces hansenii were cloned and sequenced. The genes, designated ENA1 from D. hansenii (DhENA1) and DhENA2, exhibited high homology with the corresponding genes from Schwanniomyces occidentalis. DhENA1 was expressed in the presence of high Na ؉ concentrations, while the expression of DhENA2 also required high pH. A mutant of Saccharomyces cerevisiae lacking the Na ؉ efflux systems and sensitive to Na ؉ , when transformed with DhENA1 or DhENA2, recovered Na ؉ tolerance and also the ability to extrude Na ؉ .

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The yeast Debaryomyces hansenii has been chosen as a model for molecular studies of tolerance to NaCl. A gene library was built and transformants of Saccharomyces cerevisiae W303 containing genes from D. hansenii were selected for their ability to grow in the presence of high concentrations of NaCl and/or low concentrations of KCl. In three of these transformants 500 mM NaCl improved growth at pH 7.6 like in D. hansenii but not in S. cerevisiae. One of the plasmids restored growth at 50 microM KCl and K(+) uptake in a mutant of S. cerevisiae lacking genes that encode K(+) transporters.

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