María A. Bañuelos

Inventory and Functional Characterization of the HAK Potassium Transporters of Rice

Plants take up large amounts of K+ from the soil solution and distribute it to the cells of all organs, where it fulfills important physiological functions. Transport of K+from the soil solution to its final destination is mediated by channels and transporters. To better understand K+ movements in plants, we intended to characterize the function of the large KT-HAK-KUP family of transporters in rice (Oryza sativacv Nipponbare). By searching in databases and cDNA cloning, we have identified 17 genes (OsHAK1–17) encoding transporters of this family and obtained evidence of the existence of other two genes. Phylogenetic analysis of the encoded transporters reveals a great diversity among them, and three distant transporters, OsHAK1, OsHAK7, and OsHAK10, were expressed in yeast (Saccharomyces cerevisiae) and bacterial mutants to determine their functions. The three transporters mediate K+ influxes or effluxes, depending on the conditions of the experiment. A comparative kinetic analysis of HAK-mediated K+ influx in yeast and in roots of K+-starved rice seedlings demonstrated the involvement of HAK transporters in root K+ uptake. We discuss that all HAK transporters may mediate K+ transport, but probably not only in the plasma membrane. Transient expression of the OsHAK10-green fluorescent protein fusion protein in living onion epidermal cells targeted this protein to the tonoplast.

Differential expression of two genes encoding isoforms of the ATPase involved in sodium efflux in Saccharomyces cerevisiae

SummaryThe ENA2 gene encoding a P-type ATPase involved in Na+ and Li+ effluxes in Saccharomyces cerevisiae has been isolated. The putative protein encoded by ENA2 differs only in thirteen amino acids from the protein encoded by ENA1/PMR2. However, ENA2 has a very low level of expression and for this reason did not confer significant Li+ tolerance on a Li+ sensitive strain. ENA1 and ENA2 are the first two units of a tandem array of four highly homologous genes with probably homologous functions.

HKT1 mediates sodium uniport in roots. Pitfalls in the expression of HKT1 in yeast.

The function of HKT1 in roots is controversial. We tackled this controversy by studying Na+ uptake in barley (Hordeum vulgare) roots, cloning the HvHKT1 gene, and expressing the HvHKT1 cDNA in yeast (Saccharomyces cerevisiae) cells. High-affinity Na+ uptake was not detected in plants growing at high K+ but appeared soon after exposing the plants to a K+-free medium. It was a uniport, insensitive to external K+ at the beginning of K+ starvation and inhibitable by K+ several hours later. The expression of HvHKT1 in yeast was Na+ (or K+) uniport, Na+-K+ symport, or a mix of both, depending on the construct from which the transporter was expressed. The Na+ uniport function was insensitive to external K+ and mimicked the Na+ uptake carried out by the roots at the beginning of K+ starvation. The K+ uniport function only took place in yeast cells that were completely K+ starved and disappeared when internal K+ increased, which makes it unlikely that HvHKT1 mediates K+ uptake in roots. Mutation of the first in-frame AUG codon of HvHKT1 to CUC changed the uniport function into symport. The expression of the symport from either mutants or constructs keeping the first in-frame AUG took place only in K+-starved cells, while the uniport was expressed in all conditions. We discuss here that the symport occurs only in heterologous expression. It is most likely related to the K+ inhibitable Na+ uptake process of roots that heterologous systems fail to reproduce.

P-TYPE ATPASES MEDIATE SODIUM AND POTASSIUM EFFLUXES IN SCHWANNIOMYCES OCCIDENTALIS

Two genes isolated from Schwanniomyces occidentalis, ENA1 and ENA2, encode P-type ATPases highly homologous to the Na-ATPases ofSaccharomyces cerevisiae and complement the Na+sensitivity of an S. cerevisiae mutant strain lacking its own Na-ATPases. The expression of both ENA1 andENA2 was highly dependent on a high external pH, but whereas a high pH was sufficient for the expression ofENA2, the expression of ENA1 required a high pH and the presence of Na+. Disruption of ENA1rendered the cells less tolerant to Na+ than the wild-type strain and decreased their capacity for Na+ extrusion. Disruption of ENA2 did not affect Na+tolerance, but decreased both the growth at high pH and K+efflux. We discuss these results and propose that fungal Na-ATPases should be considered alkali cation ATPases. By sequence comparison, we found that fungal Na-ATPases form a homogeneous group that can be distinguished from other cation-pumping P-type ATPases, except from the cta3 Ca-ATPase of Schizosaccharomyces pombe.

High-affinity sodium uptake in land plants

High-affinity Na(+) uptake in plants and its mediation by HKT transporters have been studied in very few species. This study expands the knowledge of high-affinity Na(+) uptake in land plants for both uptake characteristics and involvement of HKT transporters. In non-flowering plants, we analyzed the Na(+) content of wild mosses, carried out experiments on K(+) and Na(+) uptake in the micromolar range of concentrations with the moss Physcomitrella patens and the liverwort Riccia fluitans, studied a Deltahkt1 mutant of P. patens and identified the HKT genes of the lycopodiophyta (clubmoss) Selaginella moellendorffii. In flowering plants we studied Na(+) uptake in the micromolar range of concentrations in 16 crop plant species, identified the HKT transporters that could mediate high-affinity Na(+) uptake in several species of the Triticeae tribe, and described some characteristics of high-affinity Na(+) uptake in other species. Our results suggest that high-affinity Na(+) uptake occurs in most land plants. In very few of them, rice and species in the Triticeae and Aveneae tribes of the Poaceae family, it is probably mediated by HKT transporters. In other plants, high-affinity Na(+) uptake is mediated by one or several transporters whose responses to the presence of K(+) or Ba(2+) are fundamentally different from those of HKT transporters.

Functional expression of the ENA1(PMR2) - ATPase of Saccharomyces cerevisiae in Schizosaccharomyces pombe

Na+ efflux and Na+ tolerance depend on a putative P-type ATPase encoded by the gene ENA1(PMR2) in Saccharomyces cerevisiae and on a putative Na+/H+ antiporter encoded by the gene sod2 in Schizosaccharomyces pombe. This report shows that a sod2::ura4 mutant of S. pombe transformed with the ENA1 gene of S. cerevisiae expressed the ENA1 protein, and recovered Na+ efflux and Na+ tolerance. The efflux of Na+ in the wild strain of S. pombe was sensitive to the transmembrane Na+ and H+ gradients, whereas in the sod2::ura4 mutant transformed with ENA1 it was independent of these gradients. The data give further support to the notion that ENA1 and sod2 encode Na+ transporters and not regulators of the process of Na+ export; they show also the physiological consequences of exporting Na+ through an Na(+)-ATPase or an Na+/H+ antiporter.

Individual functions of the HAK and TRK potassium transporters of Schwanniomyces occidentalis

We have cloned the gene encoding the TRK transporter of the soil yeast Schwanniomyces occidentalis and obtained the HAK1 trk1Δ and the hak1Δ TRK1 mutant strains. Analyses of the transport capacities of these mutants have shown that (i) the HAK1 and the TRK1 potassium transporters are the only transporters operating at low and medium K+ concentrations (< 1 mM); (ii) the HAK1 transporter is functional at low pH but fails at high pH; and (iii) the TRK1 transporter functions at neutral and high pH and fails at low pH. At neutral pH, both transporters are functional, but HAK1 is not expressed, except at very low K+ concentrations (< 50 µM) where HAK1 is very effective. TRK1 is also involved in the control of the membrane potential.

Effects of polylinker uATGs on the function of grass HKT1 transporters expressed in yeast cells.

HvHKT1 mediates K(+) or Na(+) uniport in yeast cells if the expression promoter is joined directly to the HvHKT1 cDNA, and Na(+)-K(+) symport if a 59 nucleotide polylinker is inserted. Our results show that three ATG triplets in the polylinker decreased the synthesis of the transporter and that the lower amount of transporter caused the functional change. With the rice HKT1 cDNA, the 59 nt polylinker changed the mode of Na(+) uptake from K(+)-insensitive to K(+)-inhibitable. These two modes of Na(+) uptake also occurred in rice plants.

Use of Schizosaccharomyces strains for wine fermentation—Effect on the wine composition and food safety

Schizosaccharomyces was initially considered as a spoilage yeast because of the production of undesirable metabolites such as acetic acid, hydrogen sulfide, or acetaldehyde, but it currently seems to be of great value in enology.o ced Nevertheless, Schizosaccharomyces can reduce all of the malic acid in must, leading to malolactic fermentation. Malolactic fermentation is a highly complicated process in enology and leads to a higher concentration of biogenic amines, so the use of Schizosaccharomyces pombe can be an excellent tool for assuring wine safety. Schizosaccharomyces also has much more potential than only reducing the malic acid content, such as increasing the level of pyruvic acid and thus the vinylphenolic pyranoanthocyanin content. Until now, few commercial strains have been available and little research on the selection of appropriate yeast strains with such potential has been conducted. In this study, selected and wild Sc. pombe strains were used along with a Saccharomyces cerevisiae strain to ferment red grape must. The results showed significant differences in several parameters including non-volatile and volatile compounds, anthocyanins, biogenic amines and sensory parameters.

Use of non-Saccharomyces in single-culture, mixed and sequential fermentation to improve red wine quality

The use of non-Saccharomyces yeasts can provide advantages in winemaking such as: a reduction of ethanol content, the synthesis of certain metabolites that are important for color stability or a good yield in the production of certain volatile compounds (ethyl lactate, 2,3-butanediol, 2-phenylethyl acetate). The main objective was general evaluation of wines made by non-Saccharomyces yeast under different fermentation trials compared to wines made by Saccharomyces cerevisiae. Single pure fermentations were performed by two strains of S. cerevisiae (used as controls) and two strains of Schizosaccharomyces pombe. The yeasts Lachancea thermotolerans and Torulaspora delbrueckii were tested in sequential fermentations with S. cerevisiae strains. The effect on acidity due to the interaction between L. thermotolerans and S. pombe in a mixed fermentation was studied. Sequential fermentations with L. thermotolerans and S. cerevisiae can reduce the ethanol content and increase the content of glycerol and pyruvic acid; in addition, the use of L. thermotolerans can increase the lactic acid content, while the mixed fermentations of S. pombe and L. thermotolerans may increase the acetaldehyde content and may also reduce the ethanol content. However, these samples require more time to complete the fermentation. Single pure fermentations by S. pombe can increase the amounts of vitisins, acetaldehyde and glycerol and the amounts of acetic acid. The use of L. thermotolerans in a sequential fermentation allowed an increase in fruitiness and body character of red wine.