Eric F. Walton

Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs.

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.

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.

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.

BackgroundKiwifruit (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).ResultsThe 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.ConclusionThis 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.

Postharvest fruit density as an indicator of dry matter and ripened soluble solids of kiwifruit

Abstract The density of unripe kiwifruit ( Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson cv. Hayward) early in storage was investigated as a means of determining the current fruit dry matter (DM) and total sugar-plus-starch concentrations, and of predicting DM and soluble solid concentrations later when the fruit had ripened. To investigate the robustness of the density relationships with DM and ripe fruit soluble solids, measurements were made on fruit collected in a survey involving 208 New Zealand orchards (∼10% of the total) across the normal harvest season (early May–early June). Density measured on unripe fruit early in storage correlated with both DM measured at the same time ( r 2 =82.6%; S.E.=0.68% fresh weight (FW)), and with SSC measured after ripening ( r 2 =85.1%; S.E.=0.57% FW). As fruit taste is related to sugar concentration, and sugars make up the bulk of the soluble solids in fruit, this suggests potential for automated bulk taste sorting of kiwifruit at harvest by floating off low DM fruit in prepared salt solutions. To investigate the reasons for the relationship between density and other fruit measurements, fruit composition was analysed. Fruit taken from storage 2 weeks after harvest were graded non-destructively by density into four distinct dry matter groups, each containing eight fruit (four pairs matched by density) Paired fruit were separated and analysed for DM, soluble solids, starch and sugars (sucrose, glucose and fructose), one fruit immediately and the other after ripening. Equations relating density to both DM and ripe fruit soluble solids in the composition trial had similar parameter values to those of the survey trial and gave S.E. of prediction of about 0.3% FW. DM levels were about 3.2% FW above the sum of soluble solids and starch concentrations in both ripe and unripe fruit, a difference largely independent of DM concentration. Starch lost during ripening was accounted for by the increase in the glucose and fructose sugar pools, and these two sugars had near equal concentrations at each DM level. Sucrose and minor sugar levels were independent of DM and ripeness.

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.

Actinidia DRM1 - An Intrinsically Disordered Protein Whose mRNA Expression Is Inversely Correlated with Spring Budbreak in Kiwifruit

Intrinsically disordered proteins (IDPs) are a relatively recently defined class of proteins which, under native conditions, lack a unique tertiary structure whilst maintaining essential biological functions. Functional classification of IDPs have implicated such proteins as being involved in various physiological processes including transcription and translation regulation, signal transduction and protein modification. Actinidia DRM1 (Ade DORMANCY ASSOCIATED GENE 1), represents a robust dormancy marker whose mRNA transcript expression exhibits a strong inverse correlation with the onset of growth following periods of physiological dormancy. Bioinformatic analyses suggest that DRM1 is plant specific and highly conserved at both the nucleotide and protein levels. It is predicted to be an intrinsically disordered protein with two distinct highly conserved domains. Several Actinidia DRM1 homologues, which align into two distinct Actinidia-specific families, Type I and Type II, have been identified. No candidates for the Arabidopsis DRM1-Homologue (AtDRM2) an additional family member, has been identified in Actinidia.

Relative developmental, environmental, and tree-to-tree variability in buds from field-grown apple trees

High-throughput genomic technologies are becoming more accessible to nonmodel plant species, and therefore, tissue collected outside controlled environments is being increasingly used for microarray analyses. In this study, we present a 15,720-feature apple microarray analysis of the variability of gene expression in buds from field-grown apple trees. Tree-to-tree and day-to-day variances were assessed during two different seasons: summer, when the meristems in the buds were undergoing the first stages of floral development, and autumn, when the buds were undergoing transition to winter dormancy. We found that apple trees with the same scion and rootstock cultivars, grown in a standard orchard environment, had small tree-to-tree variation. Gene expression differences caused by season was the dominant cause of variance while using false discovery rate control with a threshold of α* = 0.01 to select significantly different expression between genes. At this threshold, the environmental and location effects accounted for less than 10% of the genes selected. Consequently, we have shown that field microarray experiments are a viable approach for measuring seasonal changes in gene expression during apple bud development.

An FTIR study of the induction and release of kiwifruit buds from dormancy.

BACKGROUND Many deciduous, perennial fruit crops require winter chilling for adequate budbreak and flowering. Recent research has shown that changes in sugar and amino acid profiles are associated with the release of buds from dormancy. This paper uses FTIR spectrometry to provide an alternative mechanism for tracking metabolic changes in the meristems of kiwifruit buds during winter dormancy. The results suggest that the application of multivariate analysis to FTIR spectra has the potential to be a reliable and fast method for detecting structural and compositional changes in fruit crops. RESULTS Ten wave numbers of the FTIR spectra are used to calculate a bud development function. This function has been validated using data from two seasons and four orchards, and by monitoring the effects of hydrogen cyanamide application, sugar concentrations and soil temperatures on this function. These wave numbers appear to be associated with carbohydrate, pectin and cellulose levels in the meristems. CONCLUSION It is expected that this FTIR signature can be used to advance our understanding of the influence of the various environmental and physiological factors on the breaking of bud dormancy and shoot outgrowth, including the optimum timing and concentrations of applications of budbreak regulators, such as hydrogen cyanamide.

Carbohydrate and Ginsenoside Changes in Ginseng Roots Grown in the Bay of Plenty, New Zealand

Ginseng is traditionally cultivated worldwide in cold continental climates. It is now also being cultivated in maritime environments such as New Zealandis. This paper reports a number of growth and quality parameters for plants grown under those conditions over two growing seasons and the intervening winter dormant period. While shoot biomass peaked mid-summer, in contrast, root biomass peaked late autumn/early winter. Starch, sucrose, fructose, glucose and inositol were detected in the roots. Starch concentrations were highest in early autumn (mean 470 mg/g-¹dry weight) and lowest in mid spring (218 mg/g-¹dry weight). Sucrose concentrations were low during early summer until late autumn but increased rapidly with the onset of winter and peaked during mid spring (168 mg/g-¹dry weight). Fructose and glucose concentrations were similar and peaked in late spring (5.3 and 6.2 mg/g-¹dry weight). Inositol concentrations peaked in mid summer (1.7 mg/g- dry weight). Starch/sugar ratios were high during summer and autumn and low during winter and spring. Ginsenoside concentrations and profiles showed that the six major ginsenosides, Rg1, Re, Rb1, Rc, Rb2 and Rd, were present, but Rf was absent. Concentrations did not vary with sampling date. The most abundant ginsenosides were Re (15.9 to 17.5 mg/g-¹dry weight) and Rb1 (10.7 to 18.1 mg/g-¹dry weight). Combined, they accounted for > 75% of total ginsenoside concentrations. Limited taste tests indicated that highest root quality occurred during late autumn, after the shoots had senesced. However, quality could not be related to plant chemistry.