A mutation in amino acid permease AAP6 reduces the amino acid content of the Arabidopsis sieve elements but leaves aphid herbivores unaffected.

The aim of this study was to investigate the role of the amino acid permease gene AAP6 in regulating phloem amino acid composition and then to determine the effects of this altered diet on aphid performance. A genotype of Arabidopsis thaliana (L.) was produced in which the function of the amino acid permease gene AAP6 (At5g49630) was abolished. Plants homozygous for the insertionally inactivated AAP6 gene had a significantly larger mean rosette width than the wild type and a greater number of cauline leaves. Seeds from the aap6 mutant were also significantly larger than those from the wild-type plants. Sieve element (SE) sap was collected by aphid stylectomy and the amino acids derivatized, separated, and quantified using Capillary Electrophoresis with Laser Induced Fluorescence (CE-LIF). In spite of the large variation across samples, the total amino acid concentration of SE sap of the aap6 mutant plants was significantly lower than that of the wild-type plants. The concentrations of lysine, phenylalanine, leucine, and aspartic acid were all significantly lower in concentration in the aap6 mutant plants compared with wild-type plants. This is the first direct demonstration of a physiological role for an amino acid transporter in regulating SE composition in vivo. The amino acid availability in sieve element sap is thought to be the major limiting factor for aphid growth and reproduction. Despite the changes in their diet, the aphid Myzus persicae (Sulzer) displayed only small changes in feeding behaviour on mutant plants when measured using the Electronic Penetration Graph (EPG) technique. Salivation by the aphid into the SE (E1 phase) was increased on mutant plants but there was no significant effect on other feeding EPG behaviours, or in the rate of honeydew production. Consistent with the small effect on aphid feeding behaviour, there was only a small effect of reduced sieve element amino acid concentration on aphid reproduction. The data are discussed in relation to the regulation of phloem composition and the role of phloem amino acids in regulating aphid performance.

Analysis of mono-, di- and oligosaccharides by CE using a two-stage derivatization method and LIF detection.

A sensitive CE with LIF method has been developed for quantitative analysis of small carbohydrates. In this work, 17 carbohydrates including mono-, di- and oligosaccharides were simultaneously derivatized with 4-fluoro-7-nitrobenzofurazane (NBD-F) via a two-step reaction involving reductive amination with ammonia followed by condensation with NBD-F. Under the optimized derivatization conditions all carbohydrates were successfully derivatized within 2.5 h and separated within 15 min using borate buffer (90 mmol/L, pH 9.2). For sugar standards LODs were in the range of 49.7 to 243.6 nmol/L. Migration time and peak area reproducibility were better than RSD 0.1 and 3%, respectively. The method was applied to measure sugars in nanoliter volume samples of phloem sap obtained by stylectomy from wheat and to honeydew samples obtained from aphids feeding from wheat and willow.

Accumulation of calcium in the centre of leaves of coriander (Coriandrum sativum L.) is due to an uncoupling of water and ion transport.

The aim of this study is to understand the parameters regulating calcium ion distribution in leaves. Accumulation of ions in leaf tissue is in part dependent on import from the xylem. This import via the transpiration stream is more important for ions such as calcium that are xylem but not phloem mobile and cannot therefore be retranslocated. Accumulation of calcium was measured on bulk coriander leaf tissue (Coriandrum sativum L. cv. Lemon) using ion chromatography and calcium uptake was visualized using phosphor-images of (45)Ca(2+). Leaves of plants grown in hydroponics had elevated calcium in the centre of the leaf compared with the leaf margin, while K(+) was distributed homogeneously over the leaf. This calcium was shown to be localised to the mesophyll vacuoles using EDAX. Stomatal density and evapotranspiration (water loss per unit area of leaf) were equal at inner and outer sections of the leaf. Unequal ion distribution but uniformity of water loss suggested that there was a difference in the extent of uncoupling of calcium and water transport between the inner and outer leaf. Since isolated tissue from the inner and outer leaf were able to accumulate similar amounts of calcium, it is proposed that the spatial variation of leaf calcium concentration is due to differential ion delivery to the two regions rather than tissue/cell-specific differences in ion uptake capacity. There was a positive correlation between whole leaf calcium concentration and the difference in calcium concentration between inner and outer leaf tissue. Exposing the plants to increased humidity reduced transpiration and calcium delivery to the leaf and abolished this spatial variation of calcium concentration. Mechanisms of calcium delivery to leaves are discussed. An understanding of calcium delivery and distribution within coriander will inform strategies to reduce the incidence of calcium-related syndromes such as tip-burn and provides a robust model for the transport of ions and other substances in the leaf xylem.

A diurnal component to the variation in sieve tube amino acid content in wheat.

We have used high-sensitivity capillary electrophoresis coupled to a laser-induced fluorescence detection method to quantify 16 amino acids in wheat (Triticum aestivum) sieve tube (ST) samples as small as 2 nL collected by severing the stylets of feeding aphids. The sensitivity of the method was sufficient to determine a quantitative amino acid profile of individual STs without the need to bulk samples to produce larger volumes for analysis. This allowed the observation of the full range of variation that exists in individual STs. Some of the total concentrations of amino acids recorded are higher than those reported previously. The results obtained show variation in the concentrations of phenylalanine (Phe), histidine/valine (His/Val), leucine/isoleucine (Leu/Ile), arginine, asparagine, glutamine, tyrosine (Tyr), and lysine (Lys) across the ST samples. These could not be explained by plant-to-plant variation. Statistical analyses revealed five analytes (Tyr, Lys, Phe, His/Val, and Leu/Ile) that showed striking covariation in their concentrations across ST samples. A regression analysis revealed a significant relationship between the concentrations of Tyr, Lys, Phe, Leu/Ile, His/Val, asparagine, arginine, and proline and the time of collection of ST samples, with these amino acids increasing in concentration during the afternoon. This increase was confirmed to occur in individual STs by analyzing samples obtained from stylet bundles exuding for many hours. Finally, an apparent relationship between the exudation rate of ST sap and its total amino acid concentration was observed: samples containing higher total amino acid concentrations were observed to exude from the severed stylet bundles more slowly.

A quantitative trait loci analysis of zinc hyperaccumulation in Arabidopsis halleri.

The mechanisms of metal hyperaccumulation are still not understood, so we conducted a quantitative trait locus (QTL) analysis of zinc (Zn) hyperaccumulation in Arabidopsis halleri, in a cross between this and its sister species, A. petraea, in order to determine the number and approximate location of the genomic regions significantly contributing to this adaptation. An F2 cross between the two species was made, and the leaf Zn concentration of 92 individuals was measured at both low (10 microm) and high (100 microm) Zn concentrations. Twenty-five markers were established that were distributed on all of the eight chromosomes. Mapping of the markers established that they were essentially collinear with previous studies. QTLs exceeding a logarithm to the base 10 of the odds (LOD) value of 3 were found on chromosomes 4 (low Zn), 6 (high Zn) and 7 (both high and low Zn). Evidence for a QTL on chromosome 3 (low Zn) was also found. This analysis validates a previously used method of QTL analysis, based on microarray analysis of segregating families. Genes that have altered during the evolution of this character should also be QTL: this analysis calls into question a number of candidate genes from consideration as such primary genes because they do not appear to be associated with QTLs.

Comparison of gene expression in segregating families identifies genes and genomic regions involved in a novel adaptation, zinc hyperaccumulation.

One of the challenges of comparative genomics is to identify specific genetic changes associated with the evolution of a novel adaptation or trait. We need to be able to disassociate the genes involved with a particular character from all the other genetic changes that take place as lineages diverge. Here we show that by comparing the transcriptional profile of segregating families with that of parent species differing in a novel trait, it is possible to narrow down substantially the list of potential target genes. In addition, by assuming synteny with a related model organism for which the complete genome sequence is available, it is possible to use the cosegregation of markers differing in transcription level to identify regions of the genome which probably contain quantitative trait loci (QTLs) for the character. This novel combination of genomics and classical genetics provides a very powerful tool to identify candidate genes. We use this methodology to investigate zinc hyperaccumulation in Arabidopsis halleri, the sister species to the model plant, Arabidopsis thaliana. We compare the transcriptional profile of A. halleri with that of its sister nonaccumulator species, Arabidopsis petraea, and between accumulator and nonaccumulator F(3)s derived from the cross between the two species. We identify eight genes which consistently show greater expression in accumulator phenotypes in both roots and shoots, including two metal transporter genes (NRAMP3 and ZIP6), and cytoplasmic aconitase, a gene involved in iron homeostasis in mammals. We also show that there appear to be two QTLs for zinc accumulation, on chromosomes 3 and 7.

Identification and characterization of QTL controlling Agrobacterium-mediated transient and stable transformation of Brassica oleracea.

A commonly encountered difficulty with the genetic engineering of crop plants is that different varieties of a particular species can show great variability in the efficiency with which they can be transformed. This increases the effort required to introduce transgenes into particular genetic backgrounds. The use of Substitution Lines has allowed the finer mapping of three Quantitative Trait Loci (tf1, tf2 and tf3) that explain 26% of the variation in the efficiency of Agrobacterium-mediated transformation in Brassica oleracea. Use of an 'orthogonal set' of genotypes (containing all eight possible combinations of 'positive' and 'negative' alleles at the three QTL), along with time course studies of transgene expression, has allowed the determination of the stages at which these genes have their effects during transformation. With regard to control of the level of transient transgene expression, tf1 (on LGO1) alone has no detectable effect, whilst tf2 (on LGO3) and tf3 (on LGO7) have highly significant effects (P < 0.001). All three loci have highly significant (P < 0.001) effects on the levels of expression of stably integrated transgene. The use of RFLP markers has shown that tf1 and tf2 are in duplicated regions of the B. oleracea genome and appear to be paralogous in origin. Colinearity of these regions with the A. thaliana genome has been identified. The results allow the selection of progeny Brassica oleracea genotypes that are more efficiently transformed than either parent used in the original cross.

STAIRS: a new genetic resource for functional genomic studies of Arabidopsis.

Many biologically and economically important traits in plants and animals are quantitative/multifactorial, being controlled by several quantitative trait loci (QTL). QTL are difficult to locate accurately by conventional methods using molecular markers in segregating populations, particularly for traits of low heritability or for QTL with small effects. In order to resolve this, large (often unrealistically large) populations are required. In this paper we present an alternative approach using a specially developed resource of lines that facilitate QTL location first to a particular chromosome, then to successively smaller regions within a chromosome (< or = 0.5 cM) by means of simple comparisons among a few lines. This resource consists of "Stepped Aligned Inbred Recombinant Strains" (STAIRS) plus single whole Chromosome Substitution Strains (CSSs). We explain the analytical power of STAIRS and illustrate their construction and use with Arabidopsis thaliana, although the principles could be applied to many organisms. We were able to locate flowering QTL at the top of chromosome 3 known to contain several potential candidate genes.

Use of aphid stylectomy and RT-PCR for the detection of transporter mRNAs in sieve elements.

Unmodified samples of barley (Hordeum vulgare) sieve tube sap have been obtained by severing the stylets (stylectomy) of feeding aphids and collecting the exuding liquid. Primers were designed to direct the amplification of a series of specific cDNAs encoding barley proteins selected because of their significance in sieve tube function. mRNA encoding the H(+)/sucrose co-transporter SUT1, a putative aquaporin and the H(+)/ATPase PPA1 were detected in sieve tube sap. These mRNA species appear to be present at very low concentrations. mRNA encoding the potassium transporter HAK1 could not be detected. The results strongly suggest that some mRNA species are imported into sieve elements, which are enucleate, from neighbouring companion cells.