Transgenic tomato overexpressing ath-miR399d has enhanced phosphorus accumulation through increased acid phosphatase and proton secretion as well as phosphate transporters

By generating and examining transgenic tomato overexpressing ath-miR399d grown in hydroponic conditions, in quartz sand, or in a polytunnel greenhouse vegetable soil culture, this study aimed to investigate the effects of miR399d from Arabidopsis on phosphorus (P) accumulation, P concentrations in transgenic tomato overexpressing ath-miR399d shoots, phosphate transporter expression, and proton secretion and acid phosphatase (APase) activity in roots. In the transgenic tomato, leaf P concentration increased significantly in an agricultural soil, and roots had higher uptake of P, as evidenced by leaf P concentrations and relative expression of the genes LePT1, LePT2, LePT4, and LePT5 in normal-P solution. Enhanced APase activity in transgenic roots and the outside medium led to superior hydrolysis of organic P, and increased proton extrusion by roots led to superior dissolution of AlPO₄. Thus, besides phosphate transporters, higher APase activity and strengthened acidification in the vicinity of the roots may be important mechanisms for transgenic tomato to scavenge or acquire P in soil. These results provide new understanding of miR399-overexpressing plants that accumulate excess P in shoots.     

Increased phosphate transport of Arabidopsis thaliana Pht1;1 by site‐directed mutagenesis of tyrosine 312 may be attributed to the disruption of homomeric interactions

Members of the Pht1 family of plant phosphate (Pi) transporters play vital roles in Pi acquisition from soil and in planta Pi translocation to maintain optimal growth and development. The study of the specificities and biochemical properties of Pht1 transporters will contribute to improving the current understanding of plant phosphorus homeostasis and use‐efficiency. In this study, we show through split in vivo interaction methods and in vitro analysis of microsomal root tissues that Arabidopsis thaliana Pht1;1 and Pht1;4 form homomeric and heteromeric complexes. Transient and heterologous expression of the Pht1;1 variants, Pht1;1^Y312D, Pht1;1^Y312A and Pht1;1^Y312F, was used to analyse the role of a putative Pi binding residue (Tyr 312) in Pht1;1 transporter oligomerization and function. The homomeric interaction among Pht1;1 proteins was disrupted by mutation of Tyr 312 to Asp, but not to Ala or Phe. In addition, the Pht1;1^Y312D variant conferred enhanced Pi transport when expressed in yeast cells. In contrast, mutation of Tyr 312 to Ala or Phe did not affect Pht1;1 transport kinetics. Our study demonstrates that modifications to the Pht1;1 higher‐order structure affects Pi transport, suggesting that oligomerization may serve as a regulatory mechanism for modulating Pi uptake.     

Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter

A cDNA encoding a high-affinity sulphate transporter has been isolated from barley by complementation of a yeast mutant. The cDNA, designated HVST1, encodes a polypeptide of 660 amino acids (M_r = 72 550), which is predicted to have 12 membrane-spanning domains and has extensive sequence homology with other identified eukaryotic sulphate transporters. The K_m for sulphate was 6.9 µM when the HVST1 cDNA was expressed in a yeast mutant deficient in the gene encoding for the yeast SUL1 sulphate transporter. The strong pH-dependency of sulphate uptake when HVST1 was expressed heterologously in yeast suggests that the HVST1 polypeptide is a proton/sulphate co-transporter. The gene encoding HVST1 is expressed specifically in root tissues and the abundance of the mRNA is strongly influenced by sulphur nutrition. During sulphur-starvation of barley, the abundance of mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, both increase. Upon re-supply of sulphate, the abundance of the mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, decrease rapidly, concomitant with rises in tissue sulphate, cysteine and glutathione contents. Addition of the cysteine precursor, O-acetylserine, to plants grown with adequate sulphur supply, leads to increases in sulphate transporter mRNA, sulphate uptake rates and tissue contents of glutathione and cysteine. It is suggested, that whilst sulphate, cysteine and glutathione may be candidates for negative metabolic regulators of sulphate transporter gene expression, this regulation may be overridden by O-acetylserine acting as a positive regulator.     

Cloning of a Picea abies monosaccharide transporter gene and expression – analysis in plant tissues and ectomycorrhizas

Ectomycorrhizas are formed between certain soil fungi and fine roots of woody plants. An important feature of this symbiosis is the supply of photoassimilates to the fungus. Hexoses, formed from sucrose in the common apoplast at the root/fungus interface, can be taken up by both plant and fungal monosaccharide transporters. Recently we characterised a monosaccharide transporter from the ectomycorrhizal fungus Amanita muscaria. This transporter was up-regulated in mycorrhizas, thus increasing the hexose uptake capacity of the fungal partner in symbiosis. In order to characterise host (Picea abies) root monosaccharide transporters, degenerate oligonucleotide primers, designed to match conserved regions from known plant hexose transporters, were used to isolate a cDNA fragment of a transporter by PCR. This fragment was used to identify a presumably full length clone (PaMST1) in a P. abies/A. muscaria mycorrhizal cDNA library. The entire cDNA code for an open reading frame of 513 amino acids, revealing best homology to H⁺/monosaccharide transporters from Ara- bidopsis, Saccharum and Ricinus. PaMST1 was highly expressed in the hypocotyl and in roots of P. abies seedlings, but not in needles. Mycorrhiza formation led to a slight reduction of PaMST1 expression. The results are discussed with special reference to carbon allocation in ectomycorrhizas. Received: 9 October 1999 / Accepted: 22 December 1999     

Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max)

A full-length cDNA, GmNRT2, encoding a putative high-affinity nitrate transporter was isolated from a Glycine max (L.) root cDNA library and sequenced. The deduced GmNRT2 protein is 530 amino acids in length and contains 12 putative membrane-spanning domains and a long, hydrophilic C-terminal domain. GmNRT2 is related to high-affinity nitrate transporters in the eukaryotes Chlamydomonas reinhardtii and Aspergillus nidulans, and to putative high-affinity nitrate transporters in barley and tobacco. Southern blot analysis indicated that GmNRT2 is part of a small, multigene family in soybean. Expression of GmNRT2 in roots was regulated by the type of nitrogen source provided to plants: GmNRT2 mRNA levels were barely detectable in ammonium-grown plants, higher in nitrogen-deprived plants, and highest in nitrate-grown plants. Induction of GmNRT2 mRNA levels in roots occurred within 1 h after exposure of plants to nitrate. Nitrate induction of GmNRT2 mRNA levels was accompanied by a fourfold increase in net nitrate uptake by soybean roots at 100 μM external nitrate. The molecular and physiological evidence indicates that GmNRT2 is probably a high-affinity nitrate transporter involved in nitrate uptake by soybean roots. Received: 22 November 1997 / Accepted: 26 January 1998     

Hexose transporters of tomato: molecular cloning,expression analysis and functional characterization

A full-length (LeHT2) and two partial (LeHT1 and LeHT3) cDNA clones, encoding hexose transporters, were isolated from tomato (Lycopersicon esculentum) fruit and flower cDNA libraries. Southern blot analysis confirmed the presence of a gene family of hexose transporters in tomato consisting of at least three members. The full-length cDNA (LeHT2) encodes a protein of 523 amino acids, with a calculated molecular mass of 57.6 kDa. The predicted protein has 12 putative membrane-spanning domains and belongs to the Major Facilitator Superfamily of membrane carriers. The three clones encode polypeptides that are homologous to other plant monosaccharide transporters and contain conserved amino acid motifs characteristic of this superfamily. Expression of the three genes in different organs of tomato was investigated by quantitative PCR. LeHT1 and LeHT3 are expressed predominantly in sink tissues, with both genes showing highest expression in young fruit and root tips. LeHT2 is expressed at relatively high levels in source leaves and certain sink tissues such as flowers. LeHT2 was functionally expressed in a hexose transport-deficient mutant (RE700A) of Saccharomyces cerevisiae. LeHT2-dependent transport of glucose in RE700A exhibited properties consistent with the operation of an energy-coupled transporter and probably a H⁺/hexose symporter. The K _m of the symporter for glucose is 45 M.     

Identification and characterization of a rhizosphere β-galactosidase from Pisum sativum L.

Plant enzyme activities in the rhizosphere potentially are a resource for improved plant nutrition, soil fertility, bioremediation, and disease resistance. Here we report that a border cell specific β-galactosidase is secreted into the acidic extracellular environment surrounding root tips of pea, as well as bean, alfalfa, barrel medic, sorghum, and maize. No enzyme activity was detected in radish and Arabidopsis, species that do not produce viable border cells. The secreted enzyme activity was inhibited by galactose and 2-phenylethyl 1-thio-β-d-galactopyranoside (PETG) at concentrations that altered root growth without causing cell death. A tomato galactanase encoding gene was used as a probe to isolate a full length pea cDNA clone (BRDgal1) from a root cap-border cell cDNA library. Southern blot analysis using full length BRDgal1 as a probe revealed 1–2 related sequences within the pea genome. BRDgal1 mRNA expression was analysed by whole mount in situ hybridization (WISH) and found to occur in the outermost peripheral layer of the cap and in suspensions of detached border cells. No expression was detected within the body of the root cap. Repeated efforts to develop viable hairy root clones expressing BRDgal1 antisense mRNA under the control of the CaMV35S promoter, whose expression in the root cap is limited to cells at the root cap periphery only during root emergence, were unsuccessful. These data suggest that altered expression of this enzyme is deleterious to early root development. The first two authors contributed equally to the completion of this project.     

The Pht1;9 and Pht1;8 transporters mediate inorganic phosphate acquisition by the Arabidopsis thaliana root during phosphorus starvation

? The activation of high-affinity root transport systems is the best-conserved strategy employed by plants to cope with low inorganic phosphate (Pi) availability, a role traditionally assigned to Pi transporters of the Pht1 family, whose respective contributions to Pi acquisition remain unclear. ? To characterize the Arabidopsis thaliana Pht1;9 transporter, we combined heterologous functional expression in yeast with expression/subcellular localization studies and reverse genetics approaches in planta. Double Pht1;9/Pht1;8 silencing lines were also generated to gain insight into the role of the closest Pht1;9 homolog. ? Pht1;9 encodes a functional plasma membrane-localized transporter that mediates high-affinity Pi/H? symport activity in yeast and is highly induced in Pi-starved Arabidopsis roots. Null pht1;9 alleles exhibit exacerbated responses to prolonged Pi limitation and enhanced tolerance to arsenate exposure, whereas Pht1;9 overexpression induces the opposite phenotypes. Strikingly, Pht1;9/Pht1;8 silencing lines display more pronounced defects than the pht1;9 mutants. ? Pi and arsenic plant content analyses confirmed a role of Pht1;9 in Pi acquisition during Pi starvation and arsenate uptake at the root-soil interface. Although not affecting plant internal Pi repartition, Pht1;9 activity influences the overall Arabidopsis Pi status. Finally, our results indicate that both the Pht1;9 and Pht1;8 transporters function in sustaining plant Pi supply on environmental Pi depletion.     

Arabidopsis IRT2 gene encodes a root-periphery iron transporter

Iron uptake from the soil is a tightly controlled process in plant roots, involving specialized transporters. One such transporter, IRT1, was identified in Arabidopsis thaliana and shown to function as a broad-range metal ion transporter in yeast. Here we report the cloning and characterization of the IRT2 cDNA, a member of the ZIP family of metal transporters, highly similar to IRT1 at the amino-acid level. IRT2 expression in yeast suppresses the growth defect of iron and zinc transport yeast mutants and enhances iron uptake and accumulation. However, unlike IRT1, IRT2 does not transport manganese or cadmium in yeast. IRT2 expression is detected only in roots of A. thaliana plants, and is upregulated by iron deficiency. By fusing the IRT2 promoter to the uidA reporter gene, we show that the IRT2 promoter is mainly active in the external cell layers of the root subapical zone, and therefore provide the first tissue localization of a plant metal transporter. Altogether, these data support a role for the IRT2 transporter in iron and zinc uptake from the soil in response to iron-limited conditions.     

Cloning, expression and function of phosphate transporter encoded gene in Oryza sativa L.

OsPT6:1, a phosphate transporter encoding gene from the leaf samples of Oryza sativa, was identified through PCR with specifically designed primers. The phylogenetic analysis and the conserved amino acid residue site detection suggested OsPT6:1 a possible high-affinity phosphate transporter encoding gene. In situ hybridization and RT-PCR demonstrated the expression of OsPT6:1 in both roots and leaves. The peak expression signal was observed in mesophyll cells under low phosphorus (P) induction. A homologous recombination study indicated that OsPT6:1 can enhance the Pi uptake efficiency of Pichia pastoris. At the meantime, the introduction of OsPT6:1 was able to complement the Pi uptake function of yeast cells with high-affinity phosphate transporters deficient. Those results substantiated our contention that OsPT6:1 encoded a high-affinity phosphate transporter of Oryza sativa.     

Phosphate transporters OsPHT1;9 and OsPHT1;10 are involved in phosphate uptake in rice

We characterized the function of two rice phosphate (Pi) transporters: OsPHT1;9 (OsPT9) and OsPHT1;10 (OsPT10). OsPT9 and OsPT10 were expressed in the root epidermis, root hairs and lateral roots, with their expression being specifically induced by Pi starvation. In leaves, expression of the two genes was observed in both mesophyll and vasculature. High‐affinity Km values for Pi transport of OsPT9 and OsPT10 were determined by yeast experiments and two‐electrode voltage clamp analysis of anion transport in Xenopus oocytes expressing OsPT9 and OsPT10. Pi uptake and Pi concentrations in transgenic plants harbouring overexpressed OsPT9 and OsPT10 were determined by Pi concentration analysis and ³³P‐labelled Pi uptake rate analysis. Significantly higher Pi uptake rates in transgenic plants compared with wild‐type plants were observed under both high‐Pi and low‐Pi solution culture conditions. Conversely, although no alterations in Pi concentration were found in OsPT9 or OsPT10 knockdown plants, a significant reduction in Pi concentration in both shoots and roots was observed in double‐knockdown plants grown under both high‐ and low‐Pi conditions. Taken together, our results suggest that OsPT9 and OsPT10 redundantly function in Pi uptake.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> IRT2 cooperates with the high-affinity iron uptake system to maintain iron homeostasis in root epidermal cells

Iron is an essential nutrient for all organisms but toxic when present in excess. Consequently, plants carefully regulate their iron uptake, dependent on the FRO2 ferric reductase and the IRT1 transporter, to control its homeostasis. Arabidopsis IRT2 gene, whose expression is induced in root epidermis upon iron deprivation, was shown to encode a functional iron/zinc transporter in yeast, and proposed to function in iron acquisition from the soil. In this study, we demonstrate that, unlike its close homolog IRT1, IRT2 is not involved in iron absorption from the soil since overexpression of IRT2 does not rescue the iron uptake defect of irt1-1 mutant and since a null irt2 mutant shows no chlorosis in low iron. Consistently, an IRT2-green fluorescent fusion protein, transiently expressed in culture cells, localizes to intracellular vesicles. However, IRT2 appears strictly co-regulated with FRO2 and IRT1, supporting the view that IRT2 is an integral component of the root response to iron deficiency in root epidermal cells. We propose a model where IRT2 likely prevents toxicity from IRT1-dependent iron fluxes in epidermal cells, through compartmentalization.     

Cloning, expression and function of phosphate transporter encoded gene in Oryza sativa L.

OsPT6:1, a phosphate transporter encoding gene from the leaf samples of Oryza sativa, was identified through PCR with specifically designed primers. The phylogenetic analysis and the conserved amino acid residue site detection suggested OsPT6:1 a possible high-affinity phosphate transporter encoding gene. In situ hybridization and RT-PCR demonstrated the expression of OsPT6:1 in both roots and leaves. The peak expression signal was observed in mesophyll cells under low phosphorus (P) induction. A homologous recombination study indicated that OsPT6:1 can enhance the Pi uptake efficiency of Pichia pastoris. At the meantime, the introduction of OsPT6:1 was able to complement the Pi uptake function of yeast cells with high-affinity phosphate transporters deficient. Those results substantiated our contention that OsPT6:1 encoded a high-affinity phosphate transporter of Oryza sativa. These authors contributed equally to this work.