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Actinidia arguta and related species produce small edible kiwifruit (kiwiberry) with attractive consumer features and high nutritional value. As a recently developed crop, kiwiberry can be improved further by classical breeding or genetic manipulation. The currently available Agrobacterium-mediated transformation protocol is only applicable to a limited number of kiwiberry genotypes such as the female cultivar ‘Hortgem Tahi’. We developed protocols for regeneration and Agrobacterium-mediated transformation of two A. arguta genotypes, female AA06-01 and male AA05-06, also applicable to ‘Hortgem Tahi’. Altered composition of basal salts in the callus-induction media was sufficient to prevent callus browning in AA06-01 and ‘Hortgem Tahi’, but not in AA05-06. Altering the hormone composition to avoid a prolonged callus stage was successfully used to prevent browning and induce shoot development in all three genotypes. Using our improved protocols, Agrobacterium-mediated transformation of a binary vector carrying the neomycin phosphotransferase II (nptII) expression cassette gave rise to kanamycin-resistant plants of both AA06-01 and AA05-06 and the presence of the nptII transgene was confirmed by genomic PCR. The improved protocols were also used to ectopically overexpress the anthocyanin-related transcription factor AcMYB110 in ‘Hortgem Tahi’ giving rise to purple callus with elevated anthocyanin accumulation. AcMYB110 expression and increased levels of anthocyanins were detected in mature leaves of established transgenic plants compared to controls, clearly demonstrating the important role for MYB110 in regulation of anthocyanin accumulation in kiwiberry. The protocols developed during this study provide tools for further functional analyses and genetic manipulation of kiwiberry genotypes. Using a newly developed transformation method we were able to generate transgenic lines of three Actinidia arguta genotypes and demonstrate an important role for MYB110 in anthocyanin accumulation in kiwiberry.

Lolium perenne (ryegrass) is a perennial forage grass of worldwide importance. It is a temperate climate grass and flowering is induced by long day photoperiods. The agronomic productivity of ryegrass is strongly influenced by flowering time, but less is known about ryegrass flowering time regulators in other temperate Poaceae like wheat, barley and the model grass Brachypodium. The GIGANTEA (GI) gene was first identified in Arabidopsis and is an important regulator of photoperiodic flowering in angiosperms. GI usually exists as a conserved, single-copy gene. However, recently, genome sequencing has revealed that some plants, including the tropical grass maize, carry more than one full-length copy of GI. Here we describe the isolation and characterisation of the ryegrass L. perenne GIGANTEA gene (LpGI) gene. Comparative genomic analysis indicates that LpGI gene structure is very well conserved overall with GI genes from monocots and eudicots. LpGI protein clusters with GI proteins from other temperate grasses and is most closely related to the GI protein from meadow fescue. Genetic mapping locates LpGI to a chromosomal region that is syntenic with rice and Brachypodium GI. Our functional characterisation shows that LpGI shows a diurnal pattern of expression, continues to oscillate under constant light and adjusts its phase in response to changes in day length as seen in other plants. Constitutive expression of LpGI completely rescues the Arabidopsis gigantea-3 allele (gi-3) mutation, confirming that the isolated ryegrass gene is fully functional. Taken together, it is highly likely that LpGI is orthologous to Arabidopsis GI and involved in photoperiodic flowering time control in ryegrass.

Carboxylesterases hydrolyze esters of short-chain fatty acids and have roles in animals ranging from signal transduction to xenobiotic detoxification. In plants, however, little is known of their roles. We have systematically mined the genome from the model plant Arabidopsis thaliana for carboxylesterase genes and studied their distribution in the genome and expression profile across a range of tissues. Twenty carboxylesterase genes (AtCXE) were identified. The AtCXE family shares conserved sequence motifs and secondary structure characteristics with carboxylesterases and other members of the larger alpha/beta hydrolase fold superfamily of enzymes. Phylogenetic analysis of the AtCXE genes together with other plant carboxylesterases distinguishes seven distinct clades, with an Arabidopsis thaliana gene represented in six of the seven clades. The AtCXE genes are widely distributed across the genome (present in four of five chromosomes), with the exception of three clusters of tandemly duplicated genes. Of the interchromosomal duplication events, two have been mediated through newly identified partial chromosomal duplication events that also include other genes surrounding the AtCXE loci. Eighteen of the 20 AtCXE genes are expressed over a broad range of tissues, while the remaining 2 (unrelated) genes are expressed only in the flowers and siliques. Finally, hypotheses for the functional roles of the AtCXE family members are presented based on the phylogenetic relationships with other plant carboxylesterases of known function, their expression profile, and knowledge of likely esterase substrates found in plants.

Differential display was used to isolate genes differentially expressed early in fruit development of apple (Malus domestica Borkh.). This approach resulted in the isolation of MDH1, a homeobox gene with a homeodomain similar to that of BELL1 (BEL1), which is involved in regulation of ovule development in Arabidopsis. However, outside the homeodomain MDH1 is quite different from BEL1. In apple, MDH1 mRNA was predominantly found in flowers, expanding leaves and expanding fruit. In pre-anthesis flowers, in situ hybridization showed that MDH1 mRNA accumulated in ovules. To further investigate the function of this new homeobox gene, MDH1 was transformed into Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. The transgenic Arabidopsis plants showed dwarfing, reduced fertility and changes in carpel and fruit (silique) shape. The size and shape of the cells in the transgenic fruit was irregular. Both the transgenic phenotypes in Arabidopsis and the expression pattern of this gene in apple are consistent with the idea that MDH1 is likely to play an important role in control of plant fertility.

The co mutation of Arabidopsis thaliana causes a late-flowering phenotype that is insensitive to day-length. The mutation was mapped previously to the upper arm of chromosome 5, approximately 1.6 cM from the chalcone synthase gene (CHS). We were provided with five yeast artificial chromosome (YAC) libraries and used these to perform a chromosome walk from CHS to the CO gene. In this paper we report the isolation of 1700 kb of contiguous Arabidopsis DNA, which represents approximately 1%-2% of the genome, inserted in YACs. This required the detailed analysis of 67 YACs, from which 87 end probes were isolated and examined in hybridisation experiments. This analysis showed that approximately 40% of the YACs presented problems in chromosome walking experiments because they contained repetitive sequence at one of their termini, were chimaeric or because part of the plant DNA was deleted. DNA fragments isolated from YACs were used as restriction fragment length polymorphism (RFLP) markers to localize CO to a 300 kb region within the cloned DNA. We compare the physical distance between CHS and CO with the genetic distance and find that in this region 1 cM is equivalent to approximately 200 kb.

Background To investigate the link between the flowering time gene GIGANTEA (GI) and downstream genes, an inducible GI system was developed in Arabidopsis thaliana L. Heynh. Transgenic Arabidopsis plant lines were generated with a steroid-inducible post-translational control system for GI. The gene expression construct consisted of the coding region of the GI protein fused to that of the ligand binding domain of the rat glucocorticoid receptor (GR). This fusion gene was expressed from the constitutive cauliflower mosaic virus 35S promoter and was introduced into plants carrying the gi-2 mutation. Application of the steroid dexamethasone (DEX) was expected to result in activation of the GI-GR protein and its relocation from the cytoplasm to the nucleus. Results Application of DEX to the transgenic plant lines rescued the late flowering phenotype conferred by the gi-2 mutation. However, despite their delayed flowering in the absence of steroid, the transgenic lines expressed predicted GI downstream genes such as CONSTANS (CO) to relatively high levels. Nevertheless, increased CO and FLOWERING LOCUS T (FT) transcript accumulation was observed in transgenic plants within 8 h of DEX treatment compared to controls which was consistent with promotion of flowering by DEX. Unlike CO and FT, there was no change in the abundance of transcript of two other putative GI downstream genes HEME ACTIVATOR PROTEIN 3A (HAP3A) or TIMING OF CHLOROPHYLL A/B BINDING PROTEIN 1 (TOC1) after DEX application. Conclusion The post-translational activation of GI and promotion of flowering by steroid application supports a nuclear role for GI in the floral transition. Known downstream flowering time genes CO and FT were elevated by DEX treatment, but not other proposed targets HAP3A and TOC1, indicating that the expression of these genes may be less directly regulated by GI.