Julie Hayes

Boron-Toxicity Tolerance in Barley Arising from Efflux Transporter Amplification

Both limiting and toxic soil concentrations of the essential micronutrient boron represent major limitations to crop production worldwide. We identified Bot1, a BOR1 ortholog, as the gene responsible for the superior boron-toxicity tolerance of the Algerian barley landrace Sahara 3771 (Sahara). Bot1 was located at the tolerance locus by high-resolution mapping. Compared to intolerant genotypes, Sahara contains about four times as many Bot1 gene copies, produces substantially more Bot1 transcript, and encodes a Bot1 protein with a higher capacity to provide tolerance in yeast. Bot1 transcript levels identified in barley tissues are consistent with a role in limiting the net entry of boron into the root and in the disposal of boron from leaves via hydathode guttation.

Components of organic phosphorus in soil extracts that are hydrolysed by phytase and acid phosphatase

Abstract Extracts were prepared from soil using water, 50 mM citric acid (pH ∼2.3) or 0.5 M NaHCO3 (pH 8.5), and were incubated with excess phytase from Aspergillus niger to determine the amounts of labile P. Two A. niger phytase preparations were used: (1) a purified form which exhibited a narrow substrate specificity and high specific activity against phytate; and (2) a commercial preparation (Sigma) with activity against a broad range of P compounds. A comparatively large proportion (up to 79%, or 5.7 μg g–1 soil) of the organic P (Po) extracted with citric acid was hydrolysed by the commercial phytase, while between 28% and 40% (up to 3.1 μg g–1 soil) was hydrolysed using purified phytase. By comparison, only small quantities of the Po in water and NaHCO3 soil extracts were enzyme labile. While extractable Po was increased both with increasing concentrations of citric acid (up to 50 mM) and increasing pH (pH 2.3–6.0), enzyme-labile P increased only with citric acid concentration. The labile component of Po in citric acid extracts from soils with contrasting fertiliser histories indicated that enzyme-labile Po is a relatively large soil P pool and is potentially an important source of P for plants.

Utilization of phosphorus by pasture plants supplied with myo-inositol hexaphosphate is enhanced by the presence of soil micro-organisms

A range of pasture grass (Danthonia richardsonii and Phalaris aquatica) and legume (Medicago polymorpha, M. sativa, Trifolium repens and T. subterraneum) species showed limited capacity to obtain phosphorus (P) from inositol hexaphosphate (IHP), when grown in either sterile agar (pH 5.0 or 5.5) or sand-vermiculite media (pH 5.0). The total P content of shoots from IHP-supplied plants grown in agar was between 20% and 34% of that for seedlings supplied with an equivalent amount of P as inorganic phosphate (Pi), while in sand-vermiculite, the total P content of IHP-grown plants was between 5 and 10% of control plants. The poor ability of plants to utilize P from IHP resulted in significantly lower tissue P concentrations and, in general, reduced plant dry weight accumulation. In contrast, the P nutrition of plants supplied with IHP was significantly improved by inoculating media with either a cultured population of total soil micro-organisms or with a specific isolate of Pseudomonas sp., selected for its ability to release phosphate from IHP (strain CCAR59; Richardson and Hadobas, 1997 Can. J. Micro. 43, 509-516). In agar and sand-vermiculite media, respectively, the P content of IHP-grown plants increased with inoculation by up to 3.9- and 6.8-fold, such that the dry weight and P content of the plant material were equivalent to those observed for control plants supplied with Pi. However, the response to inoculation was dependent on the growth medium and the source of micro-organisms used. In sand-vermiculite, the cultured population of soil micro-organisms was effective when IHP was supplied at an equivalent level of Pi required for maximum plant growth. By comparison, inoculation of plants with the Pseudomonas strain was only effective at very high levels of IHP supply (×36), whereas in agar a response to inoculation occurred at all levels of IHP. The ability of pasture plants to acquire P from phytate was, therefore, influenced by the availability of IHP substrate, which was further affected by the presence of soil micro-organisms. Our results show that in addition to having an effect on the sorption characteristics of the growth media, soil micro-organisms also provided a source of phytase for the dephosphorylation of phytate for subsequent utilization of Pi by plants.

Boron Tolerance in Barley Is Mediated by Efflux of Boron from the Roots

Many plants are known to reduce the toxic effects of high soil boron (B) by reducing uptake of B, but no mechanism for limiting uptake has previously been identified. The B-tolerant cultivar of barley (Hordeum vulgare L.), Sahara, was shown to be able to maintain root B concentrations up to 50% lower than in the B-sensitive cultivar, Schooner. This translated into xylem concentrations that were approximately 64% lower and leaf concentrations 73% lower in the tolerant cultivar. In both cultivars, B accumulation was rapid and reached a steady-state concentration in roots within 3 h. In Schooner, this concentration was similar to the external medium, whereas in Sahara, the root concentration was maintained at a lower concentration. For this to occur, B must be actively extruded from the root in Sahara, and this is presumed to be the basis for B tolerance in barley. The extrusion mechanism was inhibited by sodium azide but not by treatment at low temperature. Several anion channel inhibitors were also effective in limiting extrusion, but it was not clear whether they acted directly or via metabolic inhibition. The ability of Sahara to maintain lower root B concentrations was constitutive and occurred across a wide range of B concentrations. This ability was lost at high pH, and both Schooner and Sahara then had similar root B concentrations. A predictive model that is consistent with the empirical results and explains the tolerance mechanism based on the presence of a borate anion efflux transporter in Sahara is presented.

Phytase and acid phosphatase activities in extracts from roots of temperate pasture grass and legume seedlings

Phytase and acid phosphatase activities were measured in extracts from roots of 14- to 22- day old seedlings of a range of temperate pasture species that were grown aseptically in sand culture. Phytase activity from roots of phosphorus- (P-)-deficient Trifolium subterraneum L. was characterised. Activity was enhanced by 40% when extracts were passed through Sephadex G-25, and increased by a further 20–30% with the addition of either 1 mМ EDTA or 5 mМ cysteine to assay solutions. The optimum temperature for phytase activity was 50°C and the optimum pH was 5.3. When compared with phosphatase activity measured in the roots of T. subterraneum, phytase activity exhibited narrower pH and temperature optima, and was also more strongly inhibited by Co2+, Zn2+ and AsO42− ions. Significantly, for the five pasture species examined, phytase activity was less than 5% of the total acid phosphatase activity in extracts of plant roots. Measured phytase activity ranged between 0.13 and 1.7 nkat g–1 root fresh wt and was enhanced under P-deficient relative to P-sufficient growth conditions in all of the pasture species with the exception of Trifolium repens L., for which the Km constant for activity was 50% lower in P-deficient plants. When expressed on a root fresh wt basis, increases in phytase activity of ~1.25-fold were observed for extracts from T. subterraneum and Medicago polymorpha L., and of up to 3.3-fold for Danthonia richardsonii A.B. Cashmore and Phalaris aquatica L. Increases in acid phosphatase activity with P deficiency were less evident. Between 3.1% and 4.3% only of the total phytase activity measured in root extracts was eluted from intact roots into 0.1 М NaCl.

Boron Toxicity Tolerance in Barley through Reduced Expression of the Multifunctional Aquaporin HvNIP2;1

Boron (B) toxicity is a significant limitation to cereal crop production in a number of regions worldwide. Here we describe the cloning of a gene from barley (Hordeum vulgare), underlying the chromosome 6H B toxicity tolerance quantitative trait locus. It is the second B toxicity tolerance gene identified in barley. Previously, we identified the gene Bot1 that functions as an efflux transporter in B toxicity-tolerant barley to move B out of the plant. The gene identified in this work encodes HvNIP2;1, an aquaporin from the nodulin-26-like intrinsic protein (NIP) subfamily that was recently described as a silicon influx transporter in barley and rice (Oryza sativa). Here we show that a rice mutant for this gene also shows reduced B accumulation in leaf blades compared to wild type and that the mutant protein alters growth of yeast (Saccharomyces cerevisiae) under high B. HvNIP2;1 facilitates significant transport of B when expressed in Xenopus oocytes compared to controls and to another NIP (NOD26), and also in yeast plasma membranes that appear to have relatively high B permeability. We propose that tolerance to high soil B is mediated by reduced expression of HvNIP2;1 to limit B uptake, as well as by increased expression of Bot1 to remove B from roots and sensitive tissues. Together with Bot1, the multifunctional aquaporin HvNIP2;1 is an important determinant of B toxicity tolerance in barley.

Molecular basis of adaptation to high soil boron in wheat landraces and elite cultivars

Environmental constraints severely restrict crop yields in most production environments, and expanding the use of variation will underpin future progress in breeding. In semi-arid environments boron toxicity constrains productivity, and genetic improvement is the only effective strategy for addressing the problem. Wheat breeders have sought and used available genetic diversity from landraces to maintain yield in these environments; however, the identity of the genes at the major tolerance loci was unknown. Here we describe the identification of near-identical, root-specific boron transporter genes underlying the two major-effect quantitative trait loci for boron tolerance in wheat, Bo1 and Bo4 (ref. 2). We show that tolerance to a high concentration of boron is associated with multiple genomic changes including tetraploid introgression, dispersed gene duplication, and variation in gene structure and transcript level. An allelic series was identified from a panel of bread and durum wheat cultivars and landraces originating from diverse agronomic zones. Our results demonstrate that, during selection, breeders have matched functionally different boron tolerance alleles to specific environments. The characterization of boron tolerance in wheat illustrates the power of the new wheat genomic resources to define key adaptive processes that have underpinned crop improvement.

Mechanisms and control of nutrient uptake in plants.

This review is a distillation of the vast amount of physiological and molecular data on plant membrane transport, to provide a concise overview of the main processes involved in the uptake of mineral nutrients in plants. Emphasis has been placed on transport across the plasma membrane, and on the primary uptake from soil into roots, or in the case of aquatic plants, from their aqueous environment. Control of uptake has been mainly considered in terms of local effects on the rate of transport and not in terms of long-distance signaling. The general picture emerging is of a large array of membrane transporters, few of which display any strong selectivity for individual nutrients. Instead, many transporters allow low-affinity uptake of several different nutrients. These features, plus the huge number of potential transporter genes that has been revealed by sequencing of plant genomes, raise some interesting questions about their evolution and likely function.

HvZIP7 mediates zinc accumulation in barley (Hordeum vulgare) at moderately high zinc supply

High expression of zinc (Zn)-regulated, iron-regulated transporter-like protein (ZIP) genes increases root Zn uptake in dicots, leading to high accumulation of Zn in shoots. However, none of the ZIP genes tested previously in monocots could enhance shoot Zn accumulation. In this report, barley (Hordeum vulgare) HvZIP7 was investigated for its functions in Zn transport. The functions of HvZIP7 in planta were studied using in situ hybridization and transient analysis of subcellular localization with a green fluorescent protein (GFP) reporter. Transgenic barley lines overexpressing HvZIP7 were also generated to further understand the functions of HvZIP7 in metal transport. HvZIP7 is strongly induced by Zn deficiency, primarily in vascular tissues of roots and leaves, and its protein was localized in the plasma membrane. These properties are similar to its closely related homologs in dicots. Overexpression of HvZIP7 in barley plants increased Zn uptake when moderately high concentrations of Zn were supplied. Significantly, there was a specific enhancement of shoot Zn accumulation, with no measurable increase in iron (Fe), manganese (Mn), copper (Cu) or cadmium (Cd). HvZIP7 displays characteristics of low-affinity Zn transport. The unique function of HvZIP7 provides new insights into the role of ZIP genes in Zn homeostasis in monocots, and offers opportunities to develop Zn biofortification strategies in cereals.

Root Exudates in Phosphorus Acquisition by Plants

This chapter discusses the processes in the rhizosphere that are determined by exudates from roots and that in turn affect the availability of phosphorus to plants. These include control of rhizosphere pH, exudation of organic acids and root phosphatases. Possibilities for manipulating these processes in order to improve phosphorus acquisition in agricultural plants are discussed in the context of the phosphorus cycle in agriculture.