A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164\ua0Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models

Modification of gibberellin biosynthesis in apple (<i>Malus</i>) for an improved dwarfing habit

It would be desirable to have dwarfed bittersweet apple cultivars that could be grafted onto semi-vigorous rootstocks, without needing supplementary applications of chemical growth retardants that inhibit gibberellin (GA) biosynthesis. Therefore studies were undertaken in order to manipulate gibberellin biosynthesis in Greensleeves apple and a protocol was developed for genetic transformation of Michelin, a bittersweet variety. Two homologous GA 20-oxidase sequences (94% nucleotide identity) were isolated by RACE from Greensleeves shoot tip mRNA using primers based on a 20-oxidase gene fragment (named 20oxl) previously obtained by degenerate PCR. These sequences named MdGA20oxlA and MdGA20oxlB are highly expressed in developing embryo, at lower levels in shoot tips and young leaves, and at very low levels in ovaries 5 days after pollination. MdGA20oxlA is almost identical to a cDNA previously isolated from Fuji apple (AB07114). Both MdGA20oxlA and MdGA20oxlB were demonstrated to encode enzymes with GA 20-oxidase activity by heterologous expression in Escherichia coli and incubation with [14C]GA12. Using primers based on another fragment isolated by degenerate PCR, a separate GA 20-oxidase cDNA named MdGA20ox2 was obtained by a combination of genome walking and RACE from unpollinated ovary cDNA. It shares 67% and 68% amino acid identity to MdGA20oxlA/B and is expressed in stamens and less so in sepals and unpollinated ovary. Transcripts for a homologous gene were found in unpollinated and pollinated parthenocarpic Wellington Bloomless ovaries 5 days after anthesis. Anti-sense and cosuppression using the original 20oxl fragment driven by the CaMV35S promoter produced dwarf Greensleeves plants. The main effects were reduced intemode length and number. In shoot tissues of one dwarf line the concentration of GA1 was lower, and that of the precursor GA19 higher compared to the control. These results show that it should be feasible to engineer a dwarf habit in bittersweet apple cultivars in the near future

Increasing ascorbate levels in crops to enhance human nutrition and plant abiotic stress tolerance

Ascorbate (or vitamin C) is an essential human micronutrient predominantly obtained from plants. In addition to preventing scurvy, it is now known to have broader roles in human health, for example as a cofactor for enzymes involved in epigenetic programming and as regulator of cellular iron uptake. Furthermore, ascorbate is the major antioxidant in plants and underpins many environmentally induced abiotic stress responses. Biotechnological approaches to enhance the ascorbate content of crops therefore have potential to improve both human health and abiotic stress tolerance of crops. Identifying the genetic basis of ascorbate variation between plant varieties and discovering how some ‘super fruits’ accumulate extremely high levels of ascorbate should reveal new ways to more effectively manipulate the production of ascorbate in crops

An upstream open reading frame Is essential for feedback regulation of ascorbate biosynthesis in arabidopsis

Ascorbate (vitamin C) is an essential antioxidant and enzyme cofactor in both plants and animals. Ascorbate concentration is tightly regulated in plants, partly to respond to stress. Here, we demonstrate that ascorbate concentrations are determined via the posttranscriptional repression of GDP-l-galactose phosphorylase (GGP), a major control enzyme in the ascorbate biosynthesis pathway. This regulation requires a cis-acting upstream open reading frame (uORF) that represses the translation of the downstream GGP open reading frame under high ascorbate concentration. Disruption of this uORF stops the ascorbate feedback regulation of translation and results in increased ascorbate concentrations in leaves. The uORF is predicted to initiate at a noncanonical codon (ACG rather than AUG) and encode a 60- to 65-residue peptide. Analysis of ribosome protection data from Arabidopsis thaliana showed colocation of high levels of ribosomes with both the uORF and the main coding sequence of GGP. Together, our data indicate that the noncanonical uORF is translated and encodes a peptide that functions in the ascorbate inhibition of translation. This posttranslational regulation of ascorbate is likely an ancient mechanism of control as the uORF is conserved in GGP genes from mosses to angiosperms

Diversity and Relative Levels of Actinidin, Kiwellin, and Thaumatin-Like Allergens in 15 Varieties of Kiwifruit (<i>Actinidia</i>)

In the last 30 years the incidence of kiwifruit allergy has increased with the three major allergenic proteins being identified as actinidin, kiwellin, and thaumatin-like protein (TLP). We report wide variation in the levels of actinidin and TLP in 15 kiwifruit varieties from the four most widely cultivated <i>Actinidia</i> species. Acidic and basic isoforms of actinidin were identified in <i>Actinidia deliciosa</i> ‘Hayward’ and <i>Actinidia arguta</i> ‘Hortgem Tahi’, while only a basic isoform of actinidin was identified in <i>Actinidia chinensis</i> ‘Hort16A’. One isoform each of kiwellin and TLP were identified in ripe fruit. The cysteine protease activity of actinidin correlated with protein levels in all species except <i>A. arguta</i>. Protein modeling suggested that modifications to the S2 binding pocket influenced substrate specificity of the <i>A. arguta</i> enzyme. Our results indicate that care is necessary when extrapolating allergenicity results from single varieties to others within the same and between different <i>Actinidia</i> species