Stephen R. Mudge

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.

Root-specific and phosphate-regulated expression of phytase under the control of a phosphate transporter promoter enables Arabidopsis to grow on phytate as a sole P source

The efficient use of transgenic technology for the improvement of phosphorus (P) nutrition in crop plants may require the expression of introduced genes only under conditions of phosphate deficiency and only in specific tissues. We have investigated the use of the promoter from the Arabidopsis Pht1;2 phosphate transporter to drive the expression of a secretable Aspergillus niger phytase gene only in the root epidermis of phosphate-deprived plants. Deletion analysis of this promoter indicated that, while the 933 bp upstream of the start codon is sufficient for root-specific and phosphate-responsive reporter gene expression, the 2000 bp upstream of the start codon are required for maximum expression levels. We show that transgenic Arabidopsis plants in which this 2000 bp promoter drives the Aspergillus phytase gene secrete phytase enzyme only from roots when grown on medium containing low phosphate concentrations, and that this transgene enables the plants to grow on medium containing phytate as a sole P source. The growth rates and shoot P concentrations of these plants were similar when grown on phytate or phosphate as the P source, and were similar to transgenic lines in which the phytase was driven by the constitutive CaMV 35S promoter. Our results demonstrate the potential usefulness of the promoter from the Pht1;2 phosphate transporter for driving genes that may improve crop P uptake and nutrition.

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.

Efficient silencing of reporter transgenes coupled to known functional promoters in sugarcane, a highly polyploid crop species.

Sugarcane is a crop of great interest for engineering of sustainable biomaterials and biofuel production. Isolated sugarcane promoters have generally not maintained the expected patterns of reporter transgene expression. This could arise from defective promoters on redundant alleles in the highly polyploid genome, or from efficient transgene silencing. To resolve this question we undertook detailed analysis of a sugarcane gene that combines a simple pattern in genomic Southern hybridization analysis with potentially useful, sink-specific, expression. Sequence analysis indicates that this gene encodes a member of the SHAQYF subfamily of MYB transcription factors. At least eight alleles were revealed by PCR analysis in sugarcane cultivar Q117 and a similar level of heterozygosity was seen in BAC clones from cultivar Q200. Eight distinct promoter sequences were isolated from Q117, of which at least three are associated with expressed alleles. All of the isolated promoter variants were tested for ability to drive reporter gene expression in sugarcane. Most were functional soon after transfer, but none drove reporter activity in mature stems of regenerated plants. These results show that the ineffectiveness of previously tested sugarcane promoters is not simply due to the isolation of non-functional promoter copies from the polyploid genome. If the unpredictable onset of silencing observed in most other plant species is associated with developmental polyploidy, approaches that avoid efficient transgene silencing in polyploid sugarcane are likely to have much wider utility in molecular improvement.

DNA Is Taken Up by Root Hairs and Pollen, and Stimulates Root and Pollen Tube Growth

Phosphorus (P) enters roots as inorganic phosphate (Pi) derived from organic and inorganic P compounds in the soil. Nucleic acids can support plant growth as the sole source of P in axenic culture but are thought to be converted into Pi by plant-derived nucleases and phosphatases prior to uptake. Here, we show that a nuclease-resistant analog of DNA is taken up by plant cells. Fluorescently labeled S-DNA of 25 bp, which is protected against enzymatic breakdown by its phosphorothioate backbone, was taken up and detected in root cells including root hairs and pollen tubes. These results indicate that current views of plant P acquisition may have to be revised to include uptake of DNA into cells. We further show that addition of DNA to Pi-containing growth medium enhanced the growth of lateral roots and root hairs even though plants were P replete and had similar biomass as plants supplied with Pi only. Exogenously supplied DNA increased length growth of pollen tubes, which were studied because they have similar elongated and polarized growth as root hairs. Our results indicate that DNA is not only taken up and used as a P source by plants, but ironically and independent of Pi supply, DNA also induces morphological changes in roots similar to those observed with P limitation. This study provides, to our knowledge, first evidence that exogenous DNA could act nonspecifically as signaling molecules for root development.

Comparison of Vibrio and firefly luciferases as reporter gene systems for use in bacteria and plants

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

RNAi Mediated Down-Regulation of PDS Gene Expression in Sugarcane (Saccharum), a Highly Polyploid Crop

Sugarcane is a crop with great potential for metabolic engineering, but progress has been limited by highly efficient transgene silencing. The potential exists to utilize efficient gene silencing in molecular improvement through down-regulation of sugarcane genes. However, sugarcane is highly polyploid and heterozygous, which might complicate efforts to employ transgene-mediated silencing of endogenous genes. To explore this issue, we tested in sugarcane a construct designed for hairpin-mediated silencing of the phytoene desaturase (PDS) gene in sugarcane. The hairpin construct was designed based on the sugarcane PDS EST collection containing multiple alleles, to down-regulate the suite of PDS alleles. Three out of four plants transformed using the PDS hairpin construct showed detectable hairpin transcripts when analyzed by northern analysis, and displayed the photo-bleaching phenotype characteristic of PDS knockout lines. There was near-complete reduction in the levels of endogenous PDS transcripts in leaf tissue, indicating efficient hairpin-mediated down-regulation despite the highly polyploid and heterozygous sugarcane genome. Hairpin-mediated gene silencing should therefore be a powerful tool for the molecular improvement of this important crop.

Design rules for efficient transgene expression in plants.

Sustained expression of transgenes in specified developmental patterns is commonly needed in plant biotechnology, but obstructed by transgene silencing. Here, we present a set of gene design rules, tested on the silencing-susceptible beetle luc and bacterial ims genes, expressed in sugarcane. Designs tested independently or in combination included removal of rare codons, removal of RNA instability sequences, blocking of likely endogenous sRNA binding sites and randomization of non-rare codons. Stable transgene expression analyses, on multiple independent lines per construct, showed greatest improvement from the removal of RNA instability sequences, accompanied by greatly reduced transcript degradation evident in northern blot analysis. We provide a set of motifs that readily can be eliminated concurrently with rare codons and undesired structural features such as repeat sequences, using Gene Designer 2.0 software. These design rules yielded 935- and 5-fold increased expression in transgenic callus, relative to the native luc and ims sequences; and gave sustained expression under the control of sugarcane and heterologous promoters over several years in greenhouse and field trials. The rules can be applied easily with codon usage tables from any plant species, providing a simple and effective means to achieve sustained expression of otherwise silencing-prone transgenes in plants.

Genomic Approaches for Improving Grain Quality of Sorghum

Sorghum grain provides an important calorific source for millions of people living in developing countries and is a principal animal feed and source of gluten-free flour for the livestock and food processing industries of developed nations. A versatile grain, sorghum is also widely utilized in the production of alcoholic beverages in countries such as China and several countries in sub-Saharan Africa, where the liquor baijiu and beer are a major end-use, respectively. Renowned as a hardy crop, sorghum is relatively drought tolerant and can be grown on marginal lands and is adaptable to a wide range of environmental conditions, giving this species particular advantages over other cereals. Despite its inherent benefits, sorghum has not proven to be a major alternative to the other notable cereals such as wheat and maize, due to significant problems concerning the low amount of specific essential amino acids, for example, lysine, lower protein content, lower starch digestibility, and smaller grain size, which has implications for the traits mentioned above as well as acting as an impediment to efficient grain handling in cereal-processing industries. The challenges in enhancing sorghum grain quality are not insurmountable and great strides have already been achieved in a relatively short time via scientific breeding to enhance grain yield and provide abiotic and biotic stress resistance. As the sorghum market has matured, demand for higher quality grain, whether for alcohol production or animal and human consumption, is increasing. Although yield and disease resistance are still the primary focus of breeders, advances in genomics, online bioinformatic data repositories, high-throughput phenotypic screening such as near-infrared reflectance (NIR), and the increasing affordability of next-generation sequencing, have allowed breeders to incorporate improved grain quality parameters into their programs. This chapter elaborates recent advances in genomics that have provided researchers with the tools to solve several of the issues surrounding grain quality in sorghum as well as future directions for experimentation.