Anne L. Rae

Phosphate transport in plants

Transport of inorganic phosphate (Pi) through plant membranes is mediated by a number of families of transporter proteins. Studies on the topology, function, regulation and sites of expression of the genes that encode the members of these transporter families are enabling roles to be ascribed to each of them. The Pht1 family, of which there are nine members in the Arabidopsis genome, includes proteins involved in the uptake of Pi from the soil solution and the redistribution of Pi within the plant. Members of this family are H2PO4−/H+ symporters. Most of the genes of the Pht1 family that are expressed in roots are up-regulated in P-stressed plants. Two members of the Pht1 family have been isolated from the cluster roots of white lupin. These same genes are expressed in non-cluster roots. The evidence available to date suggests that there are no major differences between the types of transport systems that cluster roots and non-cluster roots use to acquire Pi. Differences in uptake rates between cluster and non-cluster roots can be ascribed to more high-affinity Pi transporters in the plasma membranes of cluster roots, rather than any difference in the characteristics of the transporters. The efficient acquisition of Pi by cluster roots arises primarily from their capacity to increase the availability of soil Pi immediately adjacent to the rootlets by excretion of carboxylates, protons and phosphatases within the cluster. This paper reviews Pi transport processes, concentrating on those mediated by the Pht1 family of transporters, and attempts to relate those processes involved in Pi acquisition to likely Pi transport processes in cluster roots.

Identification of a novel sugar transporter homologue strongly expressed in maturing stem vascular tissues of sugarcane by expressed sequence tag and microarray analysis

The ability of sugarcane to accumulate sucrose provides an experimental system for the study of gene expression determining carbohydrate partitioning and metabolism. A sequence survey of 7242 ESTs derived from the sucrose-accumulating, maturing stem revealed that transcripts for carbohydrate metabolism gene sequences (CMGs) are relatively rare in this tissue. However, within the CMG group, putative sugar transporter ESTs form one of the most abundant classes observed. A combination of EST analysis and microarray and northern hybridization revealed that one of the putative sugar transporter types, designated PST type 2a, was the most abundant and most strongly differentially expressed CMG in maturing stem tissue. PST type 2a is homologous to members of the major facilitator super-family of transporters, possessing 12 predicted transmembrane domains and a sugar transport conserved domain, interrupted by a large cytoplasmic loop. Its transcript was localized to phloem companion cells and associated parenchyma in maturing stem, suggesting a role in sugar translocation rather than storage. In addition, other categories of CMGs show evidence of coordinated expression, such as enzymes involved in sucrose synthesis and cleavage, and a majority of enzymes involved in glycolysis and the pentose phosphate pathway. This study demonstrates the utility of genomic approaches using large-scale EST acquisition and microarray hybridization techniques for studies of the developmental regulation of metabolic enzymes and potential transporters in sugarcane.

Molecular mechanisms of phosphate and sulphate transport in plants

The application of molecular techniques in recent years has advanced our understanding of phosphate and sulphate transport processes in plants. Genes encoding phosphate and sulphate transporters have been isolated from a number of plant species. The transporters encoded by these genes are related to the major facilitator superfamily of proteins. They are predicted to contain 12 membrane-spanning domains and function as H(+)/H(2)PO(-4) or H(+)/SO(2/-4) cotransporters. Both high-affinity and low-affinity types have been identified. Most research has concentrated on genes that encode transporters expressed in roots. The expression of many of these genes is transcriptionally regulated by signals that respond to the nutrient status of the plant. Nutrient demand and the availability of precursors needed in the assimilatory pathways also regulate transcription of some of these genes. Information on the cell types in which phosphate and sulphate transporters are expressed is becoming available. These data, together with functional characterisation of the transporters, are enabling the roles of various transporters in the overall phosphate and sulphate nutrition of plants to be defined.

Over-expression of a high-affinity phosphate transporter in transgenic barley plants does not enhance phosphate uptake rates

Transgenic barley plants that over-express the gene encoding a phosphate transporter were generated and used to test the hypothesis that manipulation of transporters may lead to improved phosphate uptake by plant roots. Replicate T2 seedlings from a homozygous line with a single locus insertion were grown in dilute flow culture. The phosphate contents and uptake rates of these plants were compared with control transgenic and wild-type plants. When external phosphate concentration was maintained at 10 μM, all plants including the transgenic over-expressing line displayed low rates of phosphate uptake and contained high levels of phosphate in the shoot tissue. When external phosphate concentration was maintained at 2 μM, the uptake rates increased to a similar level in all plant lines. Three transgenic over-expressing lines were then grown in soil at a range of phosphate concentrations and the dry weights and total phosphorus contents of the shoots were measured and compared to a transgenic control line. The results showed that over-expression of the gene encoding a phosphate transporter did not improve the uptake of phosphate under any of the conditions tested. Transporter activity is likely to be influenced by post-transcriptional mechanisms and will require further investigation before this strategy can be applied to improving plant nutrition.

Regulation of Expression of Genes Encoding Phosphate Transporters in Barley Roots

A family of genes that encode phosphate transporters has been isolated from the barley genome. Three of these genes, HVPT1, HVPT2 and HVPT3 are expressed in the roots. The polypeptides encoded by HVPT1, IIVPT2 and HVPT3 are similar in structure and show significant homology to phosphate transporters that have been isolated from dicotyledonous species. There is a high level of homology between HVPT1 and HVPT2 and these two genes are closely linked, lying on the same 20kB fragment of the genome. Although homologous in conserved regions and having a similar topology to HVPT1 and HVPT2, the polypeptide encoded by HVPT3 differs significantly from HVPT1 and HVPT2.

Functional Specialization of Vacuoles in Sugarcane Leaf and Stem

Plant vacuoles are frequently targeted as a storage site for novel products. We have used environment-sensitive fluorescent dyes and the expression of vacuolar marker proteins to characterize the vacuoles in different organs and cell types of sugarcane. The results demonstrated that the lumen of the vacuole in the parenchyma cells of the stem is acidic (<pH 5) and contains active proteases, characteristic of lytic vacuoles. Western blots and tissue labelling with antibodies to vacuolar H+-ATPase suggest that this proton pump is involved in acidification of the vacuolar lumen. Quantitative real-time PCR was used to show that the expression of vacuolar proteases and a vacuolar sorting receptor is also coordinately regulated. In contrast to the stem parenchyma cells, the cells of sugarcane leaves contain diverse types of vacuoles. The pH of these vacuoles and their capacity to hydrolyze protease substrates varies according to cell type and developmental stage. Sugarcane suspension-cultures contain cells with vacuoles that resemble those of stem parenchyma cells and are thus a useful model system for investigating the properties of the vacuole. Understanding the growth and development of storage capacity will be useful in designing strategies to maximize the production of sucrose or alternative bioproducts.

Antisense suppression of the lignin biosynthetic enzyme, caffeate O-methyltransferase, improves in vitro digestibility of the tropical pasture legume, Stylosanthes humilis

The high lignin content of tropical forage plants reduces digestibility and voluntary feed intake by ruminants. We have used antisense technology to suppress caffeate O-methyltransferase (COMT EC, 2.1.1.68), a lignin biosynthetic enzyme in the tropical forage legume, Stylosanthes humilis Kunth. Plants were transformed using a Ti binary vector containing an antisense COMT construct under the control of the CaMV 35S promoter. From 50 transgenic plants, five were selected on the basis of normal morphology, high levels of antisense gene expression and altered lignin histochemistry. No plants with altered lignin were observed in a population of 20 transgenic plants derived using a binary vector that lacked the COMT cDNA insert. The progeny of lignin-altered plants were analysed for COMT enzyme activity and lignin histochemistry. A variety of COMT and lignin phenotypes was observed. In several T1 plants, COMT activity was specifically suppressed by more than 95% compared to controls. In these plants, expression of antisense mRNA was high while sense mRNA could not be detected on northern blots. The overall lignin content of these plants was unchanged but histochemical tests showed abnormally low levels of the syringyl component, mimicking the pattern of young tissue. Digestibility of these transgenic plants was assessed by incubation of stem material with rumen fluid and acid pepsin in vitro. The digestibility of the antisense material was increased dramatically compared to that of equivalent samples from control transformed plants (72 vs 62%).

A bioinformatic approach to the identification of a conserved domain in a sugarcane legumain that directs GFP to the lytic vacuole

Sugarcane is an ideal candidate as a biofactory for the production of alternate higher value products. One way of achieving this is to direct useful proteins into the vacuoles within the sugarcane storage parenchyma tissue. By bioinformatic analysis of gene sequences from putative sugarcane vacuolar proteins a motif has been identified that displays high conservation across plant legumain homologues that are known to function within vacuolar compartments. This five amino acid motif, represented by the sequence IRLPS in sugarcane is shown to direct an otherwise secreted GFP fusion protein into a large acidic and proteolytic vacuole in sugarcane callus cells as well as in diverse plant species. In mature sugarcane transgenic plants, the stability of GFP appeared to be dependent on cell type, suggesting that the vacuolar environment can be hostile to introduced proteins. This targeting motif will be a valuable tool for engineering plants such as sugarcane for production of novel products.

Localisation of expression of a high-affinity sulfate transporter in barley roots

Abstract. The tissue-specific expression pattern of the gene encoding the high-affinity sulfate transporter, HvST1, was examined in sections of barley (Hordeum vulgare L.) roots by in-situ hybridisation. The results showed expression in all cell layers close to the root tip, with increased expression under conditions of sulfate stress. In mature roots, HvST1 was not expressed in sulfate-sufficient conditions, but under sulfate stress, transcripts were detected in the xylem parenchyma, pericycle and endodermis. This pattern of expression is consistent with a role in sulfate uptake and may indicate that the majority of sulfate uptake occurs close to the root tip. In low-sulfate conditions, HvST1 may have an additional role in scavenging sulfate lost during transport.

A soluble acid invertase is directed to the vacuole by a signal anchor mechanism

Enzyme activities in the vacuole have an important impact on the net concentration of sucrose. In sugarcane (Saccharum hybrid), immunolabelling demonstrated that a soluble acid invertase (β-fructofuranosidase; EC 3.2.1.26) is present in the vacuole of storage parenchyma cells during sucrose accumulation. Examination of sequences from sugarcane, barley and rice showed that the N-terminus of the invertase sequence contains a signal anchor and a tyrosine motif, characteristic of single-pass membrane proteins destined for lysosomal compartments. The N-terminal peptide from the barley invertase was shown to be capable of directing the green fluorescent protein to the vacuole in sugarcane cells. The results suggest that soluble acid invertase is sorted to the vacuole in a membrane-bound form.