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Acid soils restrict plant production around the world. One of the major limitations to plant growth on acid soils is the prevalence of soluble aluminium (Al(3+)) ions which can inhibit root growth at micromolar concentrations. Species that show a natural resistance to Al(3+) toxicity perform better on acid soils. Our understanding of the physiology of Al(3+) resistance in important crop plants has increased greatly over the past 20 years, largely due to the application of genetics and molecular biology. Fourteen genes from seven different species are known to contribute to Al(3+) tolerance and resistance and several additional candidates have been identified. Some of these genes account for genotypic variation within species and others do not. One mechanism of resistance which has now been identified in a range of species relies on the efflux of organic anions such as malate and citrate from roots. The genes controlling this trait are members of the ALMT and MATE families which encode membrane proteins that facilitate organic anion efflux across the plasma membrane. Identification of these and other resistance genes provides opportunities for enhancing the Al(3+) resistance of plants by marker-assisted breeding and through biotechnology. Most attempts to enhance Al(3+) resistance in plants with genetic engineering have targeted genes that are induced by Al(3+) stress or that are likely to increase organic anion efflux. In the latter case, studies have either enhanced organic anion synthesis or increased organic anion transport across the plasma membrane. Recent developments in this area are summarized and the structure-function of the TaALMT1 protein from wheat is discussed.

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By comparison with dicot plant species, relatively little work has been reported on the phosphate transporter (Pht1) gene family from monocot species. Initial studies have shown that barley contains at least eight homologous genes. The promoters of six of these genes were analysed for the presence of regulatory elements potentially associated with expression specificity. In particular, the P1BS-like elements (implicated in phosphorus-regulated expression of genes in plants) was identified in all HvPht1 promoters examined. For two members of the family (HvPht1;1 and HvPht1;2), promoter fusions to beta-glucuronidase and green fluorescent protein reporter genes were constructed, transformed into rice, and the expression profiles observed. The inclusion of an intron derived from Adh1 enhanced gene expression approximately 20-fold, but did not appear to affect the specificity of expression. The HvPht1;1 and HvPht1;2 promoters showed minor differences in expression patterns but, in general, expression was observed at high levels in trichoblast cells (root hairs) and stele of the nodal root, throughout secondary roots, and at a relatively low level in leaf tissues. Under phosphorus deficiency, expression was induced by up to 5-fold. These observations are consistent with a primary role for the encoded genes in the uptake of phosphate by root hairs from soil solution and further current understanding of the mechanisms involved. The promoters also have application for providing a new resource for cereal transformation, ideally suited for driving the expression of foreign genes associated with nutrient uptake.

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The aluminium cation Al(3+) is toxic to many plants at micromolar concentrations. A range of plant species has evolved mechanisms that enable them to grow on acid soils where toxic concentrations of Al(3+) can limit plant growth. Organic acids play a central role in these aluminium tolerance mechanisms. Some plants detoxify aluminium in the rhizosphere by releasing organic acids that chelate aluminium. In at least two species, wheat and maize, the transport of organic acid anions out of the root cells is mediated by aluminium-activated anion channels in the plasma membrane. Other plants, including species that accumulate aluminium in their leaves, detoxify aluminium internally by forming complexes with organic acids.

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The rhizosphere is the zone of soil immediately surrounding plant roots that is modified by root activity. In this critical zone, plants perceive and respond to their environment. As a consequence of normal growth and development, a large range of organic and inorganic substances are exchanged between the root and soil, which inevitably leads to changes in the biochemical and physical properties of the rhizosphere. Plants also modify their rhizosphere in response to certain environmental signals and stresses. Organic anions are commonly detected in this region, and their exudation from plant roots has now been associated with nutrient deficiencies and inorganic ion stresses. This review summarizes recent developments in the understanding of the function, mechanism, and regulation of organic anion exudation from roots. The benefits that plants derive from the presence of organic anions in the rhizosphere are described and the potential for biotechnology to increase organic anion exudation is highlighted.

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Aluminum (Al) toxicity and poor phosphorus (P) availability are factors that limit plant growth on many agricultural soils. Previous work reported that expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco (Nicotiana tabacum; CSb lines) resulted in improved Al tolerance (J.M. de la Fuente, V. Ramírez-Rodríguez, J.L. Cabrera-Ponce, L. Herrera-Estrella [1997] Science 276: 1566-1568) and an enhanced ability to acquire P from alkaline soils (J. López-Bucio, O. Martínez de la Vega, A. Guevara-García, L. Herrera-Estrella [2000] Nat Biotechnol 18: 450-453). These effects were attributed to the P. aeruginosa citrate synthase increasing the biosynthesis and efflux of citrate from roots. To verify these findings we: (a) characterized citrate efflux from roots of wild-type tobacco; (b) generated tobacco lines expressing the citrate synthase gene from P. aeruginosa; and (c) analyzed selected CSb lines described above. Al stimulated citrate efflux from intact roots of wild-type tobacco and root apices were found to be responsible for most of the efflux. Despite generating transgenic tobacco lines that expressed the citrate synthase protein at up to a 100-fold greater level than the previously described CSb lines, these lines did not show increased accumulation of citrate in roots or increased Al-activated efflux of citrate from roots. Selected CSb lines, similarly, failed to show differences compared with controls in either citrate accumulation or efflux. We conclude that expression of the P. aeruginosa citrate synthase gene in plants is unlikely to be a robust and easily reproducible strategy for enhancing the Al tolerance and P-nutrition of crop and pasture species.

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We describe the cloning of a wheat cDNA (TaPSS1) that encodes a phosphatidylserine synthase (PSS) and provides the first strong evidence for the existence of this enzyme in a higher eukaryotic cell. The cDNA was isolated on its ability to confer increased resistance to aluminum toxicity when expressed in yeast. The sequence of the predicted protein encoded by TaPSS1 shows homology to PSS from both yeast and bacteria but is distinct from the animal PSS enzymes that catalyze base-exchange reactions. In wheat, Southern blot analysis identified the presence of a small family of genes that cross-hybridized to TaPSS1, and Northern blots showed that aluminum induced TaPSS1 expression in root apices. Expression of TaPSS1 complemented the yeast cho1 mutant that lacks PSS activity and altered the phospholipid composition of wild type yeast, with the most marked effect being increased abundance of phosphatidylserine (PS). Arabidopsis thaliana leaves overexpressing TaPSS1 showed a marked enhancement in PSS activity, which was associated with increased biosynthesis of PS at the expense of both phosphatidylinositol and phosphatidylglycerol. Unlike mammalian cells where PS accumulation is tightly regulated even when the capacity for PS biosynthesis is increased, plant cells accumulated large amounts of PS when TaPSS1 was overexpressed. High levels of TaPSS1 expression in Arabidopsis and tobacco (Nicotiana tabacum) led to the appearance of necrotic lesions on leaves, which may have resulted from the excessive accumulation of PS. The cloning of TaPSS1 now provides evidence that the yeast pathway for PS synthesis exists in some plant tissues and provides a tool for understanding the pathways of phospholipid biosynthesis and their regulation in plants.

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The pho2 mutant of Arabidopsis thaliana (L.) Heynh. accumulates excessive Pi (inorganic phosphate) concentrations in shoots compared to wild-type plants (E. Delhaize and P. Randall, 1995, Plant Physiol. 107: 207-213). In this study, a series of experiments was conducted to compare the uptake and translocation of Pi by pho2 with that of wild-type plants. The pho2 mutants had about a twofold greater Pi uptake rate than wild-type plants under P-sufficient conditions and a greater proportion of the Pi taken up accumulated in shoots of pho2. When shoots were removed, the uptake rate by roots was found to be similar for both genotypes, suggesting that the greater Pi uptake by the intact pho2 mutant is due to a greater shoot sink for Pi. Although pho2 mutants could recycle 32Pi from shoots to roots through phloem the proportion of 32Pi translocated to roots was less than half of that found in wild-type plants. When transferred from P-sufficient to P-deficient solutions, Pi concentrations in pho2 roots had a similar depletion rate to wild-type roots despite pho2 shoots having a fourfold greater Pi concentration than wild-type shoots throughout the experiment. We suggest that the pho2 phenotype could result from a partial defect in Pi transport in the phloem between shoots and roots or from an inability of shoot cells to regulate internal Pi concentrations.

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We describe an anion channel in the plasmalemma of protoplasts isolated from wheat (Triticum aestivum L.) roots that is activated by aluminum (Al3+). In the whole-cell configuration, addition of 20-50 microM AlCl3 to the external solution depolarized the membrane and activated an inward current that could remain active for more than 60 min. The activation by Al3+ was rapid in 20% of protoplasts examined, whereas in another 30% a delay of more than 10 min occurred after Al3+ was added. Once the current was activated, changing the external Cl- concentration shifted the membrane reversal potential with ECl, showing that the channel is more selective for anions than cations (Ca2+, K+, tetraethylammonium+). The channel could be activated by Al3+, but not by La3+, and was observed in protoplasts isolated from the root apex but not in protoplasts isolated from mature root tissue. The anion channel antagonist niflumate inhibited the current in whole cell measurements by 83% at 100 microM. Outside-out patch recordings revealed a multistate channel with single-channel conductances of between 27 and 66 pS.

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Two genes, APT1 and APT2, with DNA sequences that exhibit significant sequence identity to yeast and fungal H+/orthophosphate co-transporters, have been isolated from Arabidopsis thaliana. These genes are genetically linked and map to chromosome 5 between markers g4028 and m435. The genes encode almost identical 524 amino acid polypeptides and are predicted to contain 12 membrane-spanning domains. Both APT1 and APT2 are predominantly expressed in A. thaliana root tissues. The level of expression of both genes in roots is regulated by the phosphorus status of the plant, being considerably enhanced when the plants were deprived of an external phosphate supply. The APT1 and APT2 polypeptides are likely to be associated with the membrane transport of phosphate within A. thaliana roots.

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A mutation designated man1 (for manganese accumulator) was found to cause Arabidopsis thaliana seedlings to accumulate a range of metals. The man1 mutation segregated as a single recessive locus located on chromosome 3. When grown on soil, mutant seedlings accumulated Mn (7.5 times greater than wild type), Cu (4.6 times greater than wild type), Zn (2.8 times greater than wild type), and Mg (1.8 times greater than wild type) in leaves. In addition to these metals, the man1 mutant accumulated 2.7-fold more S in leaves, primarily in the oxidized form, than wild-type seedlings. Analysis of seedlings grown by hydroponic culture showed a similar accumulation of metals in leaves of man1 mutants. Roots of man1 mutants also accumulated metals, but unlike leaves they accumulated 10-fold more total Fe (symplasmic and apoplasmic combined) than wild-type roots. Roots of man1 mutants possessed greater (from 1.8- to 20-fold) ferric-chelate reductase activity than wild-type seedings, and this activity was not responsive to changes of Mn nutrition in either genotype. Taken together, these results suggest that the man1 mutation disrupts the regulation of metal-ion uptake or homeostasis in Arabidopsis.

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We have characterized a novel mutation of Arabidopsis thaliana at a locus designated pho2. pho2 mutants accumulated up to 3-fold more total P in leaves, mostly as inorganic phosphate (Pi), than wild-type seedlings. In addition, we isolated a mutant (locus designated pho1-2, an allelle of pho1-1 described by Y. Poirier, S. Thoma, C. Somerville, J. Schiefelbein [1991] Plant Physiol 97: 1087-1093) with low Pi concentrations in leaves. When grown under high transpiration conditions, leaves of pho2 seedlings became severely P intoxicated, whereas shoots of pho1-2 mutants were P deficient and wild-type seedlings were normal. A pho1/pho2 double mutant resulting from a cross between the single mutants was identified in the F2 generation and shown to have a pho1 phenotype. Prior to the development of P toxicity symptoms, P was the only mineral nutrient whose concentration was greater in pho2 mutants than wild-type seedlings. Compared to wild-type, pho2 mutants had greater Pi concentrations in stems, siliques, and seeds, but roots of pho2 mutants had similar or lower Pi concentrations than either pho1 mutants or wild-type seedlings. We suggest that the pho2 mutation affects a function normally involved in regulating the concentration of Pi in shoots of Arabidopsis.

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We investigated the uptake and distribution of Al in root apices of near-isogenic wheat (Triticum aestivum L.) lines differing in Al tolerance at a single locus (Alt1: aluminum tolerance). Seedlings were grown in nutrient solution that contained 100 [mu]M Al, and the roots were subsequently stained with hematoxylin, a compound that binds Al in vitro to form a colored complex. Root apices of Al-sensitive genotypes stained after short exposures to Al (10 min and 1 h), whereas apices of Al-tolerant seedlings showed less intense staining after equivalent exposures. Differential staining preceded differences observed in either root elongation or total Al concentrations of root apices (terminal 2-3 mm of root). After 4 h of exposure to 100 [mu]M Al in nutrient solution, Al-sensitive genotypes accumulated more total Al in root apices than Al-tolerant genotypes, and the differences became more marked with time. Analysis of freeze-dried root apices by x-ray microanalysis showed that Al entered root apices of Al-sensitive plants and accumulated in the epidermal layer and in the cortical layer immediately below the epidermis. Long-term exposure of sensitive apices to Al (24 h) resulted in a distribution of Al coinciding with the absence of K. Quantitation of Al in the cortical layer showed that sensitive apices accumulated 5- to 10-fold more Al than tolerant apices exposed to Al solutions for equivalent times. These data are consistent with the hypothesis that Alt1 encodes a mechanism that excludes Al from root apices.

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We investigated the role of organic acids in conferring Al tolerance in near-isogenic wheat (Triticum aestivum L.) lines differing in Al tolerance at the Al tolerance locus (Alt1). Addition of Al to nutrient solutions stimulated excretion of malic and succinic acids from roots of wheat seedlings, and Al-tolerant genotypes excreted 5- to 10-fold more malic acid than Al-sensitive genotypes. Malic acid excretion was detectable after 15 min of exposure to 200 [mu]M Al, and the amount excreted increased linearly over 24 h. The amount of malic acid excreted was dependent on the external Al concentration, and excretion was stimulated by as little as 10 [mu]M Al. Malic acid added to nutrient solutions was able to protect Al-sensitive seedlings from normally phytotoxic Al concentrations. Root apices (terminal 3-5 mm of root) were the primary source of the malic acid excreted. Root apices of Al-tolerant and Al-sensitive seedlings contained similar amounts of malic acid before and after a 2-h exposure to 200 [mu]M Al. During this treatment, Al-tolerant seedlings excreted about four times the total amount of malic acid initially present within root apices, indicating that continual synthesis of malic acid was occurring. Malic acid excretion was specifically stimulated by Al, and neither La, Fe, nor the absence of Pi was able to elicit this response. There was a consistent correlation of Al tolerance with high rates of malic acid excretion stimulated by Al in a population of seedlings segregating for Al tolerance. These data are consistent with the hypothesis that the Alt1 locus in wheat encodes an Al tolerance mechanism based on Al-stimulated excretion of malic acid.

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The effects of Cd on poly(gamma-glutamylcysteinyl)glycine [(gammaEC)(n)G] biosynthesis and formation of (gammaEC)(n)G:Cd complexes were measured in two cell lines of Datura innoxia with differing Cd tolerance. In addition, RNA synthesis, protein synthesis, and GSH concentrations were measured during a 48 hour exposure to Cd. Exposure to 250 micromolar CdCl(2) was toxic to the sensitive line, whereas the tolerant line survived and grew in its presence. Cd-sensitive cells synthesized the same amount of (gammaEC)(n)G as tolerant cells during an initial 24 hour exposure to 250 micromolar CdCl(2). However, rates of (gammaEC)(n)G:Cd complex formation differed between the two cell lines with the sensitive cells forming complexes later than tolerant cells. In addition, the complexes formed by sensitive cells were of lower molecular weight than those of tolerant cells and did not bind all of the cellular Cd. Pulse-labeling of cells with l-[(35)S]cysteine resulted in equivalent rates of incorporation into the (gammaEC)(n)G of both cell lines during the initial 24 hours after Cd. Rates of protein and RNA synthesis were similar for both cell lines during the initial 8 hours after Cd but thereafter declined rapidly in sensitive cells. This was reflected by a decline in viability of sensitive cells. The GSH content of both cell lines declined rapidly upon exposure to Cd but was higher in sensitive cells throughout the experiment. These results show that the biosynthetic pathway for (gammaEC)(n)G synthesis in sensitive cells is operational and that relative overproduction of (gammaEC)(n)G is not the mechanism of Cd-tolerance in a Cd-tolerant cell line of D. innoxia. Rapid formation of (gammaEC)(n)G:Cd complexes that bind all of the cellular Cd within 24 hours appears to correlate with tolerance in these cells.

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Suspension cultures of Datura innoxia cells were pulse-labeled with [(35)S]cysteine, then exposed to Cd to determine whether there is a direct precursor-product relationship amongst the different forms of the Cd-induced polypeptides, poly(γ-glutamylcysteinyl)glycines [(γEC)nG, n=2 to 5]. Degradation of the polypeptides and possible regeneration of the [(35)S]-labeled glutathione and cysteine pools were also examined. After 2 h of exposure to [(35)S]cysteine, about 70% of the [(35)S]cysteine in the soluble fraction of the cell was incorporated into [(35)S]glutathione before exposure of the cells to Cd. One h after Cd exposure, most of the cellular [(35)S]glutathione was depleted and label was incorporated into (γEC)nG. Analysis of [(35)S](γEC)nG by reverse phase HPLC showed no direct precursor-product relationship between the synthesis of the shorter and longer chain forms. However, the rate of synthesis of the different polypeptides was linear for 32 h after Cd exposure. There was no evidence of degradation of [(35)S](γEC)nG nor was it excreted into the medium within this period. From these results it is suggested that in the presence of Cd, a large pool of (γEC)nG is unavailable for elongation to (γEC)n+1G.

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The effect of Cd on gene expression in suspension cultures of twoDatura innoxia cell lines with differing Cd tolerance was studied.In vivo labeling experiments using [(3)H] leucine showed that Cd induced the synthesis of a similar range of proteins in both cell lines at a concentration which will kill the sensitive but not the tolerant cells. Corresponding changes in levels of translatable mRNA were also observed. The induction of the synthesis of proteins by Cd was transient since Cd-tolerant cells growing continuously in 250 µM CdCl2 contained a similar set ofin vitro translation products to cells growing in the absence of Cd. Although Cd had a similar effect on gene expression in both cell lines, Cd-tolerant cells possess two abundant mRNAs which are constitutively produced. These mRNAs encode proteins of low molecular weight (about 11 kDa) and are either absent or present at a low level in Cd-sensitive cells. The functions of these proteins are not known but they may be involved in the tolerance mechanism. Two-dimensional gel electrophoresis ofin vitro translation products showed that many of the Cd-induced proteins are also induced by heat shock. A 42°C heat shock resulted in agreater range and more intense induction of translatable mRNAs than 4 h exposure to 250 µM CdCl2. However a subset of mRNAs were induced specifically by Cd while other mRNAs were heat shock-specific. There was no difference in the ability of the two cell lines to tolerate heat shock. This was also reflected by the same pattern of major proteins induced by heat shock in the two cell lines.

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The biosynthesis of diamine oxidase (DAO; EC 1.4.3.6) in leaf blades of subterranean clover (Trifolium subterraneum L. cv Seaton Park) was followed by labeling whole plants with (14)CO(2). A pulse-chase experiment where DAO was immunoprecipitated with anti-DAO antibodies showed that only leaf primordia and the youngest emerged leaves were able to synthesize the enzyme. The amount of DAO in young leaves of clover grown with a range of Cu treatments was determined by its enzymic activity and by single radial immunodiffusion against anti-DAO antibodies; both parameters were highly correlated with the Cu concentration of the leaf. Further, anti-DAO antibodies reacted against apo-DAO prepared in vitro indicating that apo-DAO was absent from Cu-deficient leaves. These results suggest that the biosynthesis of DAO in young clover leaves is controlled by the Cu concentrations of the leaves. Poly(A) mRNA purified from leaf primordia and young emerging leaves of plants with either a high Cu or low Cu supply was translated in wheat germ and rabbit reticulocyte cell-free systems. No differences between the two Cu treatments could be seen in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the translation products after fluorography. However, anti-DAO antibodies did not detect any DAO synthesized in vitro from either treatment.

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Subterranean clover (Trifolium subterraneum L. cv Seaton Park) was grown in solution cultures containing adequate nitrogen both with and without Cu. After Cu deficiency had developed, Cu(2+) was added to some deficient plants and Cu content, protein content, and activities of three Cu metalloenzymes (diamine oxidase [EC1.4.3.6], ascorbate oxidase [EC1.10.3.3] and o-diphenol oxidase [EC1.10.3.1]) were assayed in young and recently matured leaf blades over 11 days during the development of the next three leaves.Copper deficiency had little effect on protein concentrations, but markedly depressed enzyme activities and Cu concentration in all leaf blades assayed. Within 4 d of adding Cu(2+) to Cu-deficient plants, Cu concentrations of all the leaf blades increased to adequate values. Enzyme activities only increased to control levels in leaves which had not yet emerged at the time that Cu(2+) was added.The results suggest that active holoenzymes of diamine oxidase, ascorbate oxidase, and o-diphenol oxidase can only be synthesized in leaf blades during very early stages of their development.

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The superovulated rat model was used to investigate the enzymic focus for the decrease in oestrogen synthesis which occurs in ovary at the time of ovulation. Radioimmunoassays of progesterone, 17 alpha-hydroxyprogesterone, androstenedione, testosterone and 17 beta-oestradiol were used to measure the steroid concentrations in plasma for 6 days after the initiation of follicular development with pregnant mare's gonadotropin, and the long-term and acute effects of choriogonadotropin on these circulatory concentrations. The results showed that the cross-over point following the mid-cycle administration of gonadotropin was between 17 alpha-hydroxyprogesterone and androstenedione, and suggested that choriogonadotropin affected the 17 alpha-hydroxyprogesterone 17:20 lyase. In vitro assay of this microsomal enzyme confirmed that choriogonadotropin given in vivo at intervals before death caused 50% reduction in 17:20 lyase activity in 4 h and 93% reduction in 6 h. It was concluded that the synthesis of oestrogens declined following ovulation because the substrate (testosterone) was not available in sufficient concentration for the aromatase enzymes to use it.

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