David J. Fairbairn

Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruption of the Na+ transporter AtHKT1

Sodium (Na+) is toxic to most plants, but the molecular mechanisms of plant Na+ uptake and distribution remain largely unknown. Here we analyze Arabidopsis lines disrupted in the Na+ transporter AtHKT1. AtHKT1 is expressed in the root stele and leaf vasculature. athkt1 null plants exhibit lower root Na+ levels and are more salt resistant than wild‐type in short‐term root growth assays. In shoot tissues, however, athkt1 disruption produces higher Na+ levels, and athkt1 and athkt1/sos3 shoots are Na+‐hypersensitive in long‐term growth assays. Thus wild‐type AtHKT1 controls root/shoot Na+ distribution and counteracts salt stress in leaves by reducing leaf Na+ accumulation.

Host-delivered RNAi: an effective strategy to silence genes in plant parasitic nematodes

Root-knot nematodes (Meloidogyne spp.) are obligate, sedentary endoparasites that infect many plant species causing large economic losses worldwide. Available nematicides are being banned due to their toxicity or ozone-depleting properties and alternative control strategies are urgently required. We have produced transgenic tobacco (Nicotiana tabacum) plants expressing different dsRNA hairpin structures targeting a root-knot nematode (Meloidogyne javanica) putative transcription factor, MjTis11. We provide evidence that MjTis11 was consistently silenced in nematodes feeding on the roots of transgenic plants. The observed silencing was specific for MjTis11, with other sequence-unrelated genes being unaffected in the nematodes. Those transgenic plants able to induce silencing of MjTis11, also showed the presence of small interfering RNAs. Even though down-regulation of MjTis11 did not result in a lethal phenotype, this study demonstrates the feasibility of silencing root-knot nematode genes by expressing dsRNA in the host plant. Host-delivered RNA interference-triggered (HD-RNAi) silencing of parasite genes provides a novel disease resistance strategy with wide biotechnological applications. The potential of HD-RNAi is not restricted to parasitic nematodes but could be adapted to control other plant-feeding pests.

Temporal and tissue-specific regulation of a Brassica napus stearoyl-acyl carrier protein desaturase gene.

The nucleotide sequence of a Brassica napus stearoyl-acyl carrier protein desaturase gene (Bn10) is presented. This gene is one member of a family of four closely related genes expressed in oilseed rape. The expression of the promoter of this gene in transgenic tobacco was found to be temporally regulated in the developing seed tissues. However, the promoter was also particularly active in other oleogenic tissues such as the tapetum and pollen grains. This raises the interesting question of whether seed-expressed lipid synthesis genes are regulated by separate tissue-specific determinants or by a single factor common to all oleogenic tissues. Parts of the plants undergoing rapid development such as the components of immature flowers and seedlings also exhibited high levels of promoter activity. These tissues are likely to have an elevated requirement for membrane lipid synthesis. Stearoyl-acyl carrier protein desaturase transcript levels have previously been shown to be temporally regulated in the B. napus embryo (S.P. Slocombe, I. Cummins, R.P. Jarvis, D.J. Murphy [1992] Plant Mol Biol 20: 151–155). Evidence is presented demonstrating the induction of desaturase mRNA by abscisic acid in the embryo.

PineappleDB: An online pineapple bioinformatics resource

BackgroundA world first pineapple EST sequencing program has been undertaken to investigate genes expressed during non-climacteric fruit ripening and the nematode-plant interaction during root infection. Very little is known of how non-climacteric fruit ripening is controlled or of the molecular basis of the nematode-plant interaction. PineappleDB was developed to provide the research community with access to a curated bioinformatics resource housing the fruit, root and nematode infected gall expressed sequences.DescriptionPineappleDB is an online, curated database providing integrated access to annotated expressed sequence tag (EST) data for cDNA clones isolated from pineapple fruit, root, and nematode infected root gall vascular cylinder tissues. The database currently houses over 5600 EST sequences, 3383 contig consensus sequences, and associated bioinformatic data including splice variants, Arabidopsis homologues, both MIPS based and Gene Ontology functional classifications, and clone distributions. The online resource can be searched by text or by BLAST sequence homology. The data outputs provide comprehensive sequence, bioinformatic and functional classification information.ConclusionThe online pineapple bioinformatic resource provides the research community with access to pineapple fruit and root/gall sequence and bioinformatic data in a user-friendly format. The search tools enable efficient data mining and present a wide spectrum of bioinformatic and functional classification information. PineappleDB will be of broad appeal to researchers investigating pineapple genetics, non-climacteric fruit ripening, root-knot nematode infection, crassulacean acid metabolism and alternative RNA splicing in plants.

Manipulation of seed oil content to produce industrial crops

Abstract Three examples are presented of the modification of seed oil quality by molecular genetics, in order to create potentially valuable industrial feedstocks. (a) Cloning of the desaturase gene responsible for the formation of petroselinic acid from the spice plant Coriander has allowed for the insertion of this gene into rapeseed, in an attempt to obtain transgenic rapeseed plants with a high seed petroselinic acid phenotype. The possibility that additional genes other than this desaturase may be required for petroselinic acid formation is discussed. (b) Attempts to achieve very high erucic acid phenotypes in rapeseed are concentrated around cloning an acyltransferase gene from meadowfoam. The transfer of this gene into rapeseed may result in the accumulation of a very high erucic acid seed oil, in contrast to current high erucic varieties which only contain between 45% and 55% of this useful industrial feedstock. (c) Attempts to clone the hydroxylase gene responsible for the accumulation of ricinoleic acid in castorbean are described. A biochemical strategy based upon solubilisation and purification of the hydroxylase protein was unsuccessful due to the loss of hydroxylase activity in the presence of detergents. A differential screening approach was therefore adopted. Antibodies were raised against microsomal membrane fractions from castorbean seeds at a late stage of development corresponding to their maximum oleate hydroxylase activity. The purified IgG fraction was then incubated in the presence of microsomal membrane fractions from either very young or mature castorbean seeds which had no oleate hydroxylase activity. This resulted in the isolation of a population of castorbean IgGs specific for microsomal membrane proteins which were only present at the stage of maximum oleate hydroxylase activity. The IgG fraction was then used to screen a λ-ZAP cDNA expression library prepared from poly A + -enriched RNA, extracted from seeds at the late developmental stage. From these antibody screens, 43 positive cDNA clones were isolated and sequenced from the 5′ end to an average of 250 bases each. All but two of the cDNA clones were eliminated due to (a) sequence similarity with previously known proteins or (b) inappropriate expression patterns of the corresponding mRNAs following Northern blot analysis. Potential sequence motifs in the oleate hydroxylase, which may be useful for the isolation or identification of candidate hydroxylase cDNA clones, such as the two clones isolated in this study, are discussed.