A.C. Allan

Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

BackgroundWe describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes.ResultsWe give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs.ConclusionIn developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

MYB transcription factors that colour our fruit

Anthocyanin concentration is a primary determinant of plant colour. Fruit anthocyanin biosynthesis is controlled by a distinct clade of R2R3 MYB transcription factors. In apple, three recent papers describe the discovery of MYB genes activating skin, flesh and foliage anthocyanic colour. These findings lead the way to new approaches in the breeding and biotechnological development of fruit with new colour patterns.

Mapping a candidate gene (MdMYB10) for red flesh and foliage colour in apple

BackgroundIntegrating plant genomics and classical breeding is a challenge for both plant breeders and molecular biologists. Marker-assisted selection (MAS) is a tool that can be used to accelerate the development of novel apple varieties such as cultivars that have fruit with anthocyanin through to the core. In addition, determining the inheritance of novel alleles, such as the one responsible for red flesh, adds to our understanding of allelic variation. Our goal was to map candidate anthocyanin biosynthetic and regulatory genes in a population segregating for the red flesh phenotypes.ResultsWe have identified the Rni locus, a major genetic determinant of the red foliage and red colour in the core of apple fruit. In a population segregating for the red flesh and foliage phenotype we have determined the inheritance of the Rni locus and DNA polymorphisms of candidate anthocyanin biosynthetic and regulatory genes. Simple Sequence Repeats (SSRs) and Single Nucleotide Polymorphisms (SNPs) in the candidate genes were also located on an apple genetic map. We have shown that the MdMYB10 gene co-segregates with the Rni locus and is on Linkage Group (LG) 09 of the apple genome.ConclusionWe have performed candidate gene mapping in a fruit tree crop and have provided genetic evidence that red colouration in the fruit core as well as red foliage are both controlled by a single locus named Rni. We have shown that the transcription factor MdMYB10 may be the gene underlying Rni as there were no recombinants between the marker for this gene and the red phenotype in a population of 516 individuals. Associating markers derived from candidate genes with a desirable phenotypic trait has demonstrated the application of genomic tools in a breeding programme of a horticultural crop species.

Heat-induced oxidative activity protects suspension-cultured plant cells from low temperature damage

A short heat pre-treatment (1u2009h at 38°C) was found to protect both suspension-cultured apple fruit cells and tobacco cells from cold-induced cell death. Tobacco cells were more sensitive to low temperatures than apple cells, with significant cell death after 48u2009h at 0 or -2°C. Real-time measurements of H2O2 levels during the heat pre-treatment revealed a substantial burst of this reactive oxygen species in both cell types. Real-time and longer-term measurements also showed a large burst of H2O2 production from tobacco cells, but not apple cells, when exposed to low temperatures. Lower temperatures reduced levels of peroxidase activity (both total and intracellular), with the heat pre-treatment preventing some of the cold-induced reduction of this activity in both apple and tobacco cells. The greater sensitivity to low temperature of the tobacco cells may be related to higher H2O2 production, with the heat treatment maintaining higher peroxidase activity. The lesser sensitivity of the apple cells may be due to the lack of a H2O2 burst and maintenance of peroxidase activity by the heat treatment. These results support a role for oxidative metabolism in the beneficial effects of heat in inducing low temperature tolerance.

THE CONTROL OF KIWIFRUIT RED FLESH COLOUR

Red-fleshed fruit occur in a few taxa of the genus Actinidia. In such taxa, anthocyanins can accumulate in different parts of the fruit including the skin, the whole pericarp or only the inner pericarp. Differences in the relative amounts of cyanidin- and delphinidin-based anthocyanins account for the different shades of red observed. Red-fleshed fruit of A. chinensis accumulate mainly cyanidin 3-Oxylo-galactoside and cyanidin 3-O-galactoside, which usually are restricted to the inner pericarp of the fruit, creating a characteristic and colourful pattern in mature fruit. In order to understand the process of anthocyanin synthesis in Actinidia fruit, we have studied the expression of the anthocyanin biosynthetic genes identified in our Actinidia EST database. Of the different genes of the pathway, only the expression of one glycosyltransferase (F3GT1), confirmed functionally to be cyanidin 3-O-galactosyltransferase, was strongly correlated with red pigmentation of the fruit. However, inheritance of the gene could not be associated with the segregation of the red phenotype of the fruit, suggesting that other factors are able to regulate the biosynthetic pathway. We show that the F3GT1 gene is regulated by a MYB-bHLH mechanism; allelic variation on these genes is likely to account for the expression of the different phenotype.