microRNA172 targets APETALA2 to regulate flavonoid biosynthesis in apple (Malus domestica).

MicroRNA172 (miR172) plays a role in regulating a diverse range of plant developmental processes, including flowering, fruit development and nodulation. However, its role in regulating flavonoid biosynthesis is unclear. In this study, we show that transgenic apple plants over-expressing miR172 show a reduction in red coloration and anthocyanin accumulation in various tissue types. This reduction was consistent with decreased expression of APETALA2 homolog MdAP2_1a (a miR172 target gene), MdMYB10, and targets of MdMYB10, as demonstrated by both RNA-seq and qRT-PCR analyses. The positive role of MdAP2_1a in regulating anthocyanin biosynthesis was supported by the enhanced petal anthocyanin accumulation in transgenic tobacco plants overexpressing MdAP2_1a, and by the reduction in anthocyanin accumulation in apple and cherry fruits transfected with an MdAP2_1a virus-induced-gene-silencing construct. We demonstrated that MdAP2_1a could bind directly to the promoter and protein sequences of MdMYB10 in yeast and tobacco, and enhance MdMYB10 promotor activity. In Arabidopsis, over-expression of miR172 reduced flavonoid (including anthocyanins and flavonols) concentration and RNA transcript abundance of flavonoid genes in plantlets cultured on medium containing 7% sucrose. The anthocyanin content and RNA abundance of anthocyanin genes could be partially restored by using a synonymous mutant of MdAP2_1a, which had lost the miR172 target sequences at mRNA level, but not restored by using a WT MdAP2_1a. These results indicate that miR172 inhibits flavonoid biosynthesis through suppressing the expression of an AP2 transcription factor that positively regulates MdMYB10.

Significant improvement of apple (Malus domestica Borkh.) transgenic plant production by pre-transformation with a Baby boom transcription factor.

BABY BOOM (BBM) is a member of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family and its expression has been shown to improve herbaceous plant transformation and regeneration. However, this improvement has not been shown clearly for tree species. This study demonstrated that the efficiency of transgenic apple (Malus domestica Borkh.) plant production was dramatically increased by ectopic expression of the MdBBM1 gene. "Royal Gala" apple plants were first transformed with a CaMV35S-MdBBM1 construct (MBM) under kanamycin selection. These MBM transgenic plants exhibited enhanced shoot regeneration from leaf explants on tissue culture media, with most plants displaying a close-to-normal phenotype compared with CaMV35S-GUS transgenic plants when grown under greenhouse conditions, the exception being that some plants had slightly curly leaves. Thin leaf sections revealed the MBM plants produced more cells than the GUS plants, indicating that ectopic-expression of MdBBM1 enhanced cell division. Transcriptome analysis showed that mRNA levels for cell division activators and repressors linked to hormone (auxin, cytokinin and brassinosteroid) signalling pathways were enhanced and reduced, respectively, in the MBM plants compared with the GUS plants. Plants of eight independent MBM lines were compared with the GUS plants by re-transforming them with an herbicide-resistant gene construct. The number of transgenic plants produced per 100 leaf explants was 0-3% for the GUS plants, 3-8% for five MBM lines, and 20-30% for three MBM lines. Our results provided a solution for overcoming the barriers to transgenic plant production in apple, and possibly in other trees.

Mutagenesis of kiwifruit CENTRORADIALIS-like genes transforms a climbing woody perennial with long juvenility and axillary flowering into a compact plant with rapid terminal flowering.

Annualization of woody perennials has the potential to revolutionize the breeding and production of fruit crops and rapidly improve horticultural species. Kiwifruit (Actinidia chinensis) is a recently domesticated fruit crop with a short history of breeding and tremendous potential for improvement. Previously, multiple kiwifruit CENTRORADIALIS (CEN)-like genes have been identified as potential repressors of flowering. In this study, CRISPR/Cas9- mediated manipulation enabled functional analysis of kiwifruit CEN-like genes AcCEN4 and AcCEN. Mutation of these genes transformed a climbing woody perennial, which develops axillary inflorescences after many years of juvenility, into a compact plant with rapid terminal flower and fruit development. The number of affected genes and alleles and severity of detected mutations correlated with the precocity and change in plant stature, suggesting that a bi-allelic mutation of either AcCEN4 or AcCEN may be sufficient for early flowering, whereas mutations affecting both genes further contributed to precocity and enhanced the compact growth habit. CRISPR/Cas9-mediated mutagenesis of AcCEN4 and AcCEN may be a valuable means to engineer Actinidia amenable for accelerated breeding, indoor farming and cultivation as an annual crop.

Ectopic expression of the PISTILLATA homologous MdPI inhibits fruit tissue growth and changes fruit shape in apple.

Fruit shape represents a key trait that consumers use to identify and select preferred cultivars, and although the manipulation of this trait is an opportunity to create novel, differentiated products, the molecular mechanisms regulating fruit shape are poorly understood in tree fruits. In this study, we have shown that ectopic expression of Malus domestica PISTILLATA (MdPI), the apple ortholog of the floral organ identity gene PISTILLATA (PI), regulates apple fruit tissue growth and shape. MdPI is a single-copy gene, and its expression is high during flower development but barely detectable soon after pollination. Transgenic apple plants with ectopic expression of MdPI produced flowers with white sepals and a conversion of sepals to petals. Interestingly, these plants produced distinctly flattened fruit as a consequence of reduced cell growth at the basipetal position of the fruit. These altered sepal and fruit phenotypes have not been observed in studies using Arabidopsis. This study using apple has advanced our understanding of PI functions outside the control of petal and stamen identity and provided molecular genetic information useful for manipulating fruit tissue growth and fruit shape.

Exogenous cytokinin application to Actinidia chinensis var. deliciosa 'Hayward' fruit promotes fruit expansion through water uptake.

Exogenous application of a cytokinin-like compound forchlorfenuron (CPPU) can promote fruit growth, although often at the expense of dry matter (DM), an important indicator of fruit quality. Actinidia chinensis var. deliciosa 'Hayward' fruit are very responsive to CPPU treatments, but the mechanism underlying the significant fruit weight increase and associated decrease in DM is unclear. In this study, we hypothesised that CPPU-enhanced growth increases fruit carbohydrate demand, but limited carbohydrate supply resulted in decreased fruit DM. During fruit development, CPPU effects on physical parameters, metabolites, osmotic pressure and transcriptional changes were assessed under conditions of both standard and a high carbohydrate supply. We showed that CPPU increased fruit fresh weight but the dramatic DM decrease was not carbohydrate limited. Enhanced glucose and fructose concentrations contributed to an increase in soluble carbohydrate osmotic pressure, which was correlated with increased water accumulation in CPPU-treated fruit and up-regulation of water channel aquaporin gene PIP2.4 at 49 days after anthesis. Transcipt analysis suggested that the molecular mechanism contributing to increased glucose and fructose concentrations was altered by carbohydrate supply. At standard carbohydrate supply, the early glucose increase in CPPU fruit was associated with reduced starch synthesis and increased starch degradation. When carbohydrate supply was high, the early glucose increase in CPPU fruit was associated with a general decrease in starch synthesis but up-regulation of vacuolar invertase and fructokinase genes. We conclude that CPPU affected fruit expansion by increasing the osmotically-driven water uptake and its effect was not carbohydrate supply-limited.

How microRNA172 affects fruit growth in different species is dependent on fruit type.

microRNA172 (miR172) expression has been shown to have a positive effect on Arabidopsis fruit (siliques) growth. In contrast, over-expression of miR172 has a negative influence on fruit growth in apple, resulting in a dramatic reduction in fruit size. This negative influence is supported by the results of analyzing a transposable element (TE) insertional allele of a MIR172 gene that has reduced expression of the miRNA and is associated with an increase in fruit size. Arabidopsis siliques are a dry fruit derived from ovary tissues, whereas apple is a fleshy pome fruit derived mostly from hypanthium tissues. A model has been developed to explain the contrasting impact of miR172 expression in these two plant species based on the differences in their fruit structure. Transgenic apple plants with extremely high levels of miR172 overexpression produced flowers consisting of carpel tissues only, which failed to produce fruit. By comparison, in tomato, a fleshy berry fruit derived from the ovary, high level over-expression of the same miR172 resulted in carpel-only flowers which developed into parthenocarpic fruit. These results further indicate that the influence of miR172 on fruit growth in different plant species depends on its fruit type.

A microRNA allele that emerged prior to apple domestication may underlie fruit size evolution.

The molecular genetic mechanisms underlying fruit size remain poorly understood in perennial crops, despite size being an important agronomic trait. Here we show that the expression level of a microRNA gene (miRNA172) influences fruit size in apple. A transposon insertional allele of miRNA172 showing reduced expression associates with large fruit in an apple breeding population, whereas over-expression of miRNA172 in transgenic apple significantly reduces fruit size. The transposon insertional allele was found to be co-located with a major fruit size quantitative trait locus, fixed in cultivated apples and their wild progenitor species with relatively large fruit. This finding supports the view that the selection for large size in apple fruit was initiated prior to apple domestication, likely by large mammals, before being subsequently strengthened by humans, and also helps to explain why signatures of genetic bottlenecks and selective sweeps are normally weaker in perennial crops than in annual crops.

Metabolic analysis of kiwifruit (Actinidia deliciosa) berries from extreme genotypes reveals hallmarks for fruit starch metabolism.

Tomato, melon, grape, peach, and strawberry primarily accumulate soluble sugars during fruit development. In contrast, kiwifruit (Actinidia Lindl. spp.) and banana store a large amount of starch that is released as soluble sugars only after the fruit has reached maturity. By integrating metabolites measured by gas chromatography-mass spectrometry, enzyme activities measured by a robot-based platform, and transcript data sets during fruit development of Actinidia deliciosa genotypes contrasting in starch concentration and size, this study identified the metabolic changes occurring during kiwifruit development, including the metabolic hallmarks of starch accumulation and turnover. At cell division, a rise in glucose (Glc) concentration was associated with neutral invertase (NI) activity, and the decline of both Glc and NI activity defined the transition to the cell expansion and starch accumulation phase. The high transcript levels of ß-amylase 9 (BAM9) during cell division, prior to net starch accumulation, and the correlation between sucrose phosphate synthase (SPS) activity and sucrose suggest the occurrence of sucrose cycling and starch turnover. ADP-Glc pyrophosphorylase (AGPase) is identified as a key enzyme for starch accumulation in kiwifruit berries, as high-starch genotypes had 2- to 5-fold higher AGPase activity, which was maintained over a longer period of time and was also associated with enhanced and extended transcription of the AGPase large subunit 4 (APL4). The data also revealed that SPS and galactinol might affect kiwifruit starch accumulation, and suggest that phloem unloading into kiwifruit is symplastic. These results are relevant to the genetic improvement of quality traits such as sweetness and sugar/acid balance in a range of fruit species.

Genomic analysis of the Kiwifruit pathogen Pseudomonas syringae pv. actinidiae provides insight into the origins of an emergent plant disease.

The origins of crop diseases are linked to domestication of plants. Most crops were domesticated centuries--even millennia--ago, thus limiting opportunity to understand the concomitant emergence of disease. Kiwifruit (Actinidia spp.) is an exception: domestication began in the 1930s with outbreaks of canker disease caused by P. syringae pv. actinidiae (Psa) first recorded in the 1980s. Based on SNP analyses of two circularized and 34 draft genomes, we show that Psa is comprised of distinct clades exhibiting negligible within-clade diversity, consistent with disease arising by independent samplings from a source population. Three clades correspond to their geographical source of isolation; a fourth, encompassing the Psa-V lineage responsible for the 2008 outbreak, is now globally distributed. Psa has an overall clonal population structure, however, genomes carry a marked signature of within-pathovar recombination. SNP analysis of Psa-V reveals hundreds of polymorphisms; however, most reside within PPHGI-1-like conjugative elements whose evolution is unlinked to the core genome. Removal of SNPs due to recombination yields an uninformative (star-like) phylogeny consistent with diversification of Psa-V from a single clone within the last ten years. Growth assays provide evidence of cultivar specificity, with rapid systemic movement of Psa-V in Actinidia chinensis. Genomic comparisons show a dynamic genome with evidence of positive selection on type III effectors and other candidate virulence genes. Each clade has highly varied complements of accessory genes encoding effectors and toxins with evidence of gain and loss via multiple genetic routes. Genes with orthologs in vascular pathogens were found exclusively within Psa-V. Our analyses capture a pathogen in the early stages of emergence from a predicted source population associated with wild Actinidia species. In addition to candidate genes as targets for resistance breeding programs, our findings highlight the importance of the source population as a reservoir of new disease.

Transformation of apple (Malus × domestica) using mutants of apple acetolactate synthase as a selectable marker and analysis of the T-DNA integration sites.

KEY MESSAGE: Apple acetolactate synthase mutants were generated by site-specific mutagenesis and successfully used as selection marker in tobacco and apple transformation. T-DNA/Apple genome junctions were analysed using genome-walking PCR and sequencing. An Agrobacterium-mediated genetic transformation system was developed for apple (Malus × domestica), using mutants of apple acetolactate synthase (ALS) as a selectable marker. Four apple ALS mutants were generated by site-specific mutagenesis and subsequently cloned under the transcriptional control of the CaMV 35S promoter and ocs 3' terminator, in a pART27-derived plant transformation vector. Three of the four mutations were found to confer resistance to the herbicide Glean(®), containing the active agent chlorsulfuron, in tobacco (Nicotiana tabacum) transformation. In apple transformation, leaf explants infected with Agrobacterium tumefaciens EHA105 containing one of the three ALS mutants resulted in the production of shoots on medium containing 2-8 µg L(-1) Glean(®), whilst uninfected wild-type explants failed to regenerate shoots or survive on medium containing 1 and 3 µg L(-1) Glean(®), respectively. Glean(®)-resistant, regenerated shoots were further multiplied and rooted on medium containing 10 µg L(-1) Glean(®). The T-DNA and apple genome-DNA junctions from eight rooted transgenic apple plants were analysed using genome-walking PCR amplification and sequencing. This analysis confirmed T-DNA integration into the apple genome, identified the genome integration sites and revealed the extent of any vector backbone integration, T-DNA rearrangements and deletions of apple genome DNA at the sites of integration.

A rapid transcriptional activation is induced by the dormancy-breaking chemical hydrogen cyanamide in kiwifruit (Actinidia deliciosa) buds.

Budbreak in kiwifruit (Actinidia deliciosa) can be poor in locations that have warm winters with insufficient winter chilling. Kiwifruit vines are often treated with the dormancy-breaking chemical hydrogen cyanamide (HC) to increase and synchronize budbreak. This treatment also offers a tool to understand the processes involved in budbreak. A genomics approach is presented here to increase our understanding of budbreak in kiwifruit. Most genes identified following HC application appear to be associated with responses to stress, but a number of genes appear to be associated with the reactivation of growth. Three patterns of gene expression were identified: Profile 1, an HC-induced transient activation; Profile 2, an HC-induced transient activation followed by a growth-related activation; and Profile 3, HC- and growth-repressed. One group of genes that was rapidly up-regulated in response to HC was the glutathione S-transferase (GST) class of genes, which have been associated with stress and signalling. Previous budbreak studies, in three other species, also report up-regulated GST expression. Phylogenetic analysis of these GSTs showed that they clustered into two sub-clades, suggesting a strong correlation between their expression and budbreak across species.

Analysis of expressed sequence tags from Actinidia: applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening.

BACKGROUND: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). RESULTS: The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. CONCLUSION: This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Global gene expression analysis of apple fruit development from the floral bud to ripe fruit.

BACKGROUND: Apple fruit develop over a period of 150 days from anthesis to fully ripe. An array representing approximately 13000 genes (15726 oligonucleotides of 45-55 bases) designed from apple ESTs has been used to study gene expression over eight time points during fruit development. This analysis of gene expression lays the groundwork for a molecular understanding of fruit growth and development in apple. RESULTS: Using ANOVA analysis of the microarray data, 1955 genes showed significant changes in expression over this time course. Expression of genes is coordinated with four major patterns of expression observed: high in floral buds; high during cell division; high when starch levels and cell expansion rates peak; and high during ripening. Functional analysis associated cell cycle genes with early fruit development and three core cell cycle genes are significantly up-regulated in the early stages of fruit development. Starch metabolic genes were associated with changes in starch levels during fruit development. Comparison with microarrays of ethylene-treated apple fruit identified a group of ethylene induced genes also induced in normal fruit ripening. Comparison with fruit development microarrays in tomato has been used to identify 16 genes for which expression patterns are similar in apple and tomato and these genes may play fundamental roles in fruit development. The early phase of cell division and tissue specification that occurs in the first 35 days after pollination has been associated with up-regulation of a cluster of genes that includes core cell cycle genes. CONCLUSION: Gene expression in apple fruit is coordinated with specific developmental stages. The array results are reproducible and comparisons with experiments in other species has been used to identify genes that may play a fundamental role in fruit development.

A genomics approach reveals that aroma production in apple is controlled by ethylene predominantly at the final step in each biosynthetic pathway.

Ethylene is the major effector of ripening in many fleshy fruits. In apples (Malus x domestica) the addition of ethylene causes a climacteric burst of respiration, an increase in aroma, and softening of the flesh. We have generated a transgenic line of 'Royal Gala' apple that produces no detectable levels of ethylene using antisense ACC OXIDASE, resulting in apples with no ethylene-induced ripening attributes. In response to external ethylene these antisense fruits undergo a normal climacteric burst and produced increasing concentrations of ester, polypropanoid, and terpene volatile compounds over an 8-d period. A total of 186 candidate genes that might be involved in the production of these compounds were mined from expressed sequence tags databases and full sequence obtained. Expression patterns of 179 of these were assessed using a 15,720 oligonucleotide apple microarray. Based on sequence similarity and gene expression patterns we identified 17 candidate genes that are likely to be ethylene control points for aroma production in apple. While many of the biosynthetic steps in these pathways were represented by gene families containing two or more genes, expression patterns revealed that only a single member is typically regulated by ethylene. Only certain points within the aroma biosynthesis pathways were regulated by ethylene. Often the first step, and in all pathways the last steps, contained enzymes that were ethylene regulated. This analysis suggests that the initial and final enzymatic steps with the biosynthetic pathways are important transcriptional regulation points for aroma production in apple.

Analyses of expressed sequence tags from apple.

The domestic apple (Malus domestica; also known as Malus pumila Mill.) has become a model fruit crop in which to study commercial traits such as disease and pest resistance, grafting, and flavor and health compound biosynthesis. To speed the discovery of genes involved in these traits, develop markers to map genes, and breed new cultivars, we have produced a substantial expressed sequence tag collection from various tissues of apple, focusing on fruit tissues of the cultivar Royal Gala. Over 150,000 expressed sequence tags have been collected from 43 different cDNA libraries representing 34 different tissues and treatments. Clustering of these sequences results in a set of 42,938 nonredundant sequences comprising 17,460 tentative contigs and 25,478 singletons, together representing what we predict are approximately one-half the expressed genes from apple. Many potential molecular markers are abundant in the apple transcripts. Dinucleotide repeats are found in 4,018 nonredundant sequences, mainly in the 5'-untranslated region of the gene, with a bias toward one repeat type (containing AG, 88%) and against another (repeats containing CG, 0.1%). Trinucleotide repeats are most common in the predicted coding regions and do not show a similar degree of sequence bias in their representation. Bi-allelic single-nucleotide polymorphisms are highly abundant with one found, on average, every 706 bp of transcribed DNA. Predictions of the numbers of representatives from protein families indicate the presence of many genes involved in disease resistance and the biosynthesis of flavor and health-associated compounds. Comparisons of some of these gene families with Arabidopsis (Arabidopsis thaliana) suggest instances where there have been duplications in the lineages leading to apple of biosynthetic and regulatory genes that are expressed in fruit. This resource paves the way for a concerted functional genomics effort in this important temperate fruit crop.

Transformation of Actinidia eriantha: a potential species for functional genomics studies in Actinidia.

Protocols were developed for regeneration and Agrobacterium-mediated transformation of Actinidia eriantha Benth. A. eriantha has a number of features that make it a useful tool for functional genomics in Actinidia: the vines are relatively small and non-vigorous in nature, flowers form all over the vine including on lower axillary branches and the species flowers prolifically in greenhouse conditions. Flowering and fruiting of transgenic A. eriantha plants was obtained within 2 years of transformation in a containment greenhouse. GUS (beta-glucuronidase) activity indicating stable expression of the uidA gene was observed in leaf, stem, root, petal and fruit tissues. Molecular evidence for incorporation of transgenes into the A. eriantha genome was obtained by PCR and DNA gel blot analysis. Inheritance of transgenic phenotypes was demonstrated in seedling progeny. Functional genomic studies in kiwifruit have been initiated using transgenic A. eriantha plants.

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

BACKGROUND: We 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. RESULTS: We 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. CONCLUSION: In 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.

The Decreased apical dominance1/Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE8 gene affects branch production and plays a role in leaf senescence, root growth, and flower development.

Carotenoids and carotenoid cleavage products play an important and integral role in plant development. The Decreased apical dominance1 (Dad1)/PhCCD8 gene of petunia (Petunia hybrida) encodes a hypothetical carotenoid cleavage dioxygenase (CCD) and ortholog of the MORE AXILLARY GROWTH4 (MAX4)/AtCCD8 gene. The dad1-1 mutant allele was inactivated by insertion of an unusual transposon (Dad-one transposon), and the dad1-3 allele is a revertant allele of dad1-1. Consistent with its role in producing a graft-transmissible compound that can alter branching, the Dad1/PhCCD8 gene is expressed in root and shoot tissue. This expression is upregulated in the stems of the dad1-1, dad2, and dad3 increased branching mutants, indicating feedback regulation of the gene in this tissue. However, this feedback regulation does not affect the root expression of Dad1/PhCCD8. Overexpression of Dad1/PhCCD8 in the dad1-1 mutant complemented the mutant phenotype, and RNA interference in the wild type resulted in an increased branching phenotype. Other differences in phenotype associated with the loss of Dad1/PhCCD8 function included altered timing of axillary meristem development, delayed leaf senescence, smaller flowers, reduced internode length, and reduced root growth. These data indicate that the substrate(s) and/or product(s) of the Dad1/PhCCD8 enzyme are mobile signal molecules with diverse roles in plant development.

Serpins in fruit and vegetative tissues of apple (Malus domestica): expression of four serpins with distinct reactive centres and characterisation of a major inhibitory seed form, MdZ1b.

Most serpins irreversibly inhibit serine proteinases of the chymotrypsin family using a suicide-substrate-based mechanism. Serpins are present in all domains of life, but physiological functions in the plant kingdom are yet to be elucidated. Inhibitory properties of many abundant cereal grain serpins are well characterised, but serpins have not been identified in eudicot seeds. In apple (Malus domestica Borkh.), the origin of 88 serpin expressed sequence tags (ESTs) identified among 160 000 ESTs from 30 cultivar-, tissue- and time-specific libraries showed that serpin genes are expressed in a wide variety of tissues, including developing and mature fruits, seeds and vegetative buds as well as developing, mature and senescing leaves. Analysis of 46 sequences, most full-length, identified serpins with four distinct reactive centres belonging to two subfamilies (MdZ1 and MdZ2) with ~85% amino acid sequence identity. MdZ1 included three molecular forms with identical reactive centre loop (RCL) sequences except for three different, but related, residues at P2 (Asp, Asn or Glu). A major seed serpin, MdZ1b, with P2-P1' Glu-Arg-Arg was purified from decorticated seeds and characterised kinetically. MdZ1b was a fast inhibitor of bovine and porcine trypsin (second-order association rate constant k a ~4 × 106 m -1 s-1 and stoichiometry of inhibition SI = 1). Human plasmin and urokinase-type plasminogen activator (u-PA), but not thrombin, were inhibited at lower rates (k a ~104 m -1 s-1). Chymotrypsin was inhibited at the same site (k a~4 × 103 m -1 s-1), but a significant part of MdZ1b was cleaved as substrate (SI > 2). Unexpectedly, the MdZ1b-trypsin complex was relatively short-lived with a first-order dissociation rate constant k d in the order of 10-4 s-1. The bulk of mature seed MdZ1b was localised to the cotyledons. The content of MdZ1b in ripe apples was 5-26 µg per seed, whereas MdZ1b could not be detected in the cortex or skin. Localisation and inhibitory specificity of serpins in monocot and eudicot plants are compared and putative functions are discussed.

Agrobacterium and PEG-mediated transformation of the phytopathogen Venturia inaequalis.

We report the development of two new transformation systems, polyethylene glycol (PEG)-mediated transformation of protoplasts and Agrobacterium tumefaciens-mediated transformation of mycelium, for the filamentous ascomycete Venturia inaequalis. New binary vectors have been created for the latter. Although transformation was initially achieved using a PEG-mediated method, this was superseded by the A. tumefaciens-mediated approach. The advantages of the latter include: ease of the protocol, no requirement for protoplasts; higher transformation efficiency; and single-site integration. A comparison between the two transformation methods is presented.