Identification and characterisation of F3GT1 and F3GGT1, two glycosyltransferases responsible for anthocyanin biosynthesis in red-fleshed kiwifruit (Actinidia chinensis)

Much of the diversity of anthocyanins is due to the action of glycosyltransferases, which add sugar moieties to anthocyanidins. We identified two glycosyltransferases, F3GT1 and F3GGT1, from red-fleshed kiwifruit (Actinidia chinensis) that perform sequential glycosylation steps. Red-fleshed genotypes of kiwifruit accumulate anthocyanins mainly in the form of cyanidin 3-O-xylo-galactoside. Genes in the anthocyanin and flavonoid biosynthetic pathway were identified and shown to be expressed in fruit tissue. However, only the expression of the glycosyltransferase F3GT1 was correlated with anthocyanin accumulation in red tissues. Recombinant enzyme assays in vitro and in vivo RNA interference (RNAi) demonstrated the role of F3GT1 in the production of cyanidin 3-O-galactoside. F3GGT1 was shown to further glycosylate the sugar moiety of the anthocyanins. This second glycosylation can affect the solubility and stability of the pigments and modify their colour. We show that recombinant F3GGT1 can catalyse the addition of UDP-xylose to cyanidin 3-galactoside. While F3GGT1 is responsible for the end-product of the pathway, F3GT1 is likely to be the key enzyme regulating the accumulation of anthocyanin in red-fleshed kiwifruit varieties.

Studies on stem cuttings of kiwi (Actinidia chinensis PL. CV Bruno)

O presente trabalho teve como objetivo, estudar o efeito de auxinas sintéticas e do boro, sobre o enraizamento de estacas caulinares de kiwi (Actinidia chinensisPlanch. cv Bruno). As estacas continham dois nós com aproximadamente 10 cm de comprimento, contendo 2 folhas cortadas ao meio. As bases das estacas receberam os seguintes tratamentos: control (H2O); NAA 300 mg.L-1; IBA 300 mg.L-1; NAA 300 mg.L-1 + B; IBA 300 mg.L-1 + B; NAA 0,5%-pó e IBA 0,5%-pó. Após os tratamentos as estacas foram plantadas em bandejas de enraizamento contendo vermiculita pura e colocadas em câmara de nebulização por 120 dias até a coleta das mesmas. Para a avaliação do efeito das auxinas e boro, foram realizadas as seguintes observações: 1. porcentagem de estacas enraizadas; 2. análise de açúcares redutores e açúcares totais (em g/100 g de matéria seca); 3. análise de triptofano (em µg/100 mg de matéria seca). Além disso, foram verificados o efeito dos tratamentos em quatro épocas, que corresponderam às estações do ano (primavera, verão, outono e inverno). Através dos resultados obtidos no processo de enraizamento de estacas caulinares de kiwi (Actinidia chinensis Planch. cv Bruno), conclui-se ser o verão a melhor época de coleta dos ramos para a produção das estacas sem a necessidade do tratamento com auxinas.

Enraizamento de estacas caulinares de kiwi (Actinidia chinensis Planch cv Abbott) tratadas com auxinas e boro

O trabalho teve como finalidade, estudar o efeito de várias auxinas sintéticas em formulações comerciais e do boro, sobre o enraizamento de estacas caulinares de kiwi (Actinidia chinensis Planch, cv Abbott.). As estacas utilizadas continham dois nós e duas folhas cortadas ao meio, com aproximadamente 10 cm de comprimento, onde o corte basal em bisel foi realizado logo abaixo de um nó e o apical acima do outro nó. O efeito das auxinas, sobre o enraizamento de estacas caulinares de kiwi foi verificado mediante os seguintes tratamentos, aplicados sobre as bases das estacas: T1 H(2)0); T2 (NAA 300 ppm); T3 (IBA 300 ppm); T4 (NAA 300 ppm + B); T5 (IBA 300 ppm + B); T6 (NAA 0,5%-pó) e T7 (IBA 0,5%-pó). Após o tratamento das estacas, estas foram plantadas em bandejas de enraizamento, contendo vermiculita pura e colocadas em câmara de nebulização, onde permaneceram por 120 dias, até a sua coleta. Para a avaliação do efeito de auxinas e do ácido bórico, sobre o enraizamento de estacas caulinares de kiwi, foram realizadas as seguintes observações: 1. porcentagem de estacas enraizadas; 2. análise de açúcares redutores e açúcares totais (em g/100 g de matéria seca); 3. análise de triptofano (em µg/100 mg de matéria seca). Os resultados obtidos no processo de enraizamento de estacas caulinares de kiwi (Actinidia chinensis Planch.) variedade Abbott, levou a concluir que o inverno e outono foram as melhores épocas de coleta dos ramos de auxinas para a confecção das estacas. O processo de enraizamento foi ainda incrementado com a aplicação exógena na base das estacas, sendo que o alto teor de açúcares redutores e totais beneficiou a maior porcentagem de enraizamento.

EFEITO do GA3 NA GERMINAÇÃO DE SEMENTES DE KIWI (Actinidia chinensis Planch.)

Com o objetivo de analisar a aplicação de ácido giberélico (GA3) na germinação de sementes de Actinidia chinensis Pl, sementes foram extraídas de frutos maduros, lavadas e secas à sombra, recebendo os seguintes tratamentos: T1 - estratificação (5oC por 2 semanas); T2 - testemunha; T3 - GA3 à 50 mg.L-1 ; T4 - GA3 à 100 mg.L-1 e T5 - GA3 à 150 mg.L-1. Durante a primeira semana o substrato dos tratamentos T3, T4 e T5 foi umedecido com GA3. O tratamento mais efetivo para aumentar a porcentagem de germinação e diminuir o tempo médio de germinação foi com 150 mg.L-1 de GA3.

Effects of auxins and boron on rooting of kiwi stem cuttings (Actinidia chinensis Pl cv Matua)

This work studies the effects of some synthetical auxins and boron in the rooting of stem cuttings of kiwi (Actinidia chinensis Planch cv Matua). The stems used had two nodes and two leaves cut in half. The auxin effect was observed through seven different treatments: T1 (H2O); T2 (NAA 300 ppm); T3 (IBA 300 ppm); T4 (NAA 300 ppm + B); T5 (IBA 300 ppm + B); T6 (NAA 0,5%-talc) and T7 (IBA 0,5%-talc), applied to the bases of stem cuttings. After these treatments, the cuttings were placed in suitable rooting dishes, with pure vermiculite in misty nebulization chamber for 120 days until collection day. The evaluation of auxin and boric acid effects were made based on the following observations: 1. The percentage of rooted stem cuttings; 2. reducing sugar and total sugar analyses; and 3. tryptophan analyses. The effects of such treatments were observed in the four seasons. The results showed that winter is best for rooting. Application of IBA talc 0,5% to the cuttings bases increased rooting.

Action of auxins, boron and harvest season on the rooting of kiwi fruit stem cuttings (Actinidia chinensis Pl cv Monty)

Action of auxins on the rooting of stem cuttings of kiwi (Actinidia chinensis P. cv Monty). This work studies the effects of some synthetic auxins and B in the rooting of kiwi (Actinidia chinensis Planch cv Monty) stem cuttings. The treatments used were as follows: T1 (H2O); T2 (NAA 300 ppm); T3 (IBA 300 ppm); T4 (NAA 300 ppm + B); T5 (IBA 300 ppm + B); T6 (NAA 0,5%-talc) and T7 (IBA 0,5%-talc), applied to the bases of the cuttings. These were then placed in rooting dishes with pure vermiculite in a misty nebulization chamber until collection day (120 days). The evaluation of auxin and boric acid effects on kiwi stem cuttings were made based on the following observations: 1. The percentage of rooted stem cuttings; 2. reducing sugars and total sugar analyses (in g/100 g of dry matter); and 3. tryptophan analyses (in mu g/100 mg of dry matter). The results show that summer is the best season for rooting Actinidia chinensis Planch cv Monty stem cuttings. The use of IBA talc 0,5% on the bases of the cuttings shamed positive results too.

An R2R3 MYB transcription factor determines red petal colour in an Actinidia (kiwifruit) hybrid population

Background Red colour in kiwifruit results from the presence of anthocyanin pigments. Their expression, however, is complex, and varies among genotypes, species, tissues and environments. An understanding of the biosynthesis, physiology and genetics of the anthocyanins involved, and the control of their expression in different tissues, is required. A complex, the MBW complex, consisting of R2R3-MYB and bHLH transcription factors together with a WD-repeat protein, activates anthocyanin 3-O-galactosyltransferase (F3GT1) to produce anthocyanins. We examined the expression and genetic control of anthocyanins in flowers of Actinidia hybrid families segregating for red and white petal colour. Results Four inter-related backcross families between Actinidia chinensis Planch. var. chinensis and Actinidia eriantha Benth. were identified that segregated 1:1 for red or white petal colour. Flower pigments consisted of five known anthocyanins (two delphinidin-based and three cyanidin-based) and three unknowns. Intensity and hue differed in red petals from pale pink to deep magenta, and while intensity of colour increased with total concentration of anthocyanin, no association was found between any particular anthocyanin data and hue. Real time qPCR demonstrated that an R2R3 MYB, MYB110a, was expressed at significant levels in red-petalled progeny, but not in individuals with white petals. A microsatellite marker was developed that identified alleles that segregated with red petal colour, but not with ovary, stamen filament, or fruit flesh colour in these families. The marker mapped to chromosome 10 in Actinidia. The white petal phenotype was complemented by syringing Agrobacterium tumefaciens carrying Actinidia 35S::MYB110a into the petal tissue. Red pigments developed in white petals both with, and without, co-transformation with Actinidia bHLH partners. MYB110a was shown to directly activate Actinidia F3GT1 in transient assays. Conclusions The transcription factor, MYB110a, regulates anthocyanin production in petals in this hybrid population, but not in other flower tissues or mature fruit. The identification of delphinidin-based anthocyanins in these flowers provides candidates for colour enhancement in novel fruits.

MICROPROPAGAÇÃO do KIWI CV. HAYWARD

O presente trabalho teve como objetivo desenvolver um protocolo para a obtenção de mudas de kiwi (Actinidia chinensis Planch), cv. Hayward, por meio do cultivo in vitro de cotilédones. Utilizou-se o meio de MURASHIGE & SKOOG (1962) -- MS, suplementado com dois tipos de auxina (AIA e AIB) e uma citocinina (BAP). Foram verificados os efeitos de três doses de auxinas (0,125; 0,250 e 0,375 mg.L-1), combinadas com três doses de citocinina (0,5; 1,0 e 1,5 mg.L-1) na capacidade morfogênica dos explantes. Procedeu-se o estudo histológico dos órgãos das plântulas obtidas in vitro, e verificou-se, também, a capacidade de aclimatação das mudas ex vitro. A menor dose de AIB (0,125 mg.L-1), independentemente das doses de BAP, foi a mais eficaz na morfogênese dos explantes. Não foram verificadas alterações histológicas e anatômicas das plântulas obtidas in vitro. Aos três meses após o cultivo ex vitro dos explantes, verificou-se a sobrevivência de 88% das plantas transplantadas em condições de campo.

Efeito de auxinas e boro no enraizamento de estacas caulinares de kiwi retiradas em diferentes épocas

This work was carried out to study the effects of some synthetical auxins and boron on the rooting of stem cuttings of kiwi (Actinidia deliciosa Pl. cv. Tomuri). Cuttings of semi-woody stems with two knots and leaves divided in two, with approximately 10 cm of length were utilized. The base of the cuttings received the following treatments: 1) water only; 2) NAA 300 ppm; 3) IBA 300 ppm; 4) NAA 300 ppm + B; 5) IBA 300 ppm + B; 6) NAA 0,5%-talc and 7) IBA 0,5%-talc. After these treatments, the stems were placed in suitable rooting dishes, with pure vermiculite in misty nebulization chamber for 120 days. Evaluations were made based on the following observations: percentage of rooted stem cuttings; reductor sugar and total sugar analyses and tryptophan analyses. The results showed that the autumm season is the best for rooting for kiwi stem cuttings. The exogenous application of 0.5% of IBA talc on the bases of the cuttings showed positive results.

Jasmonates and abscisic acid influence fruit ripening and plant water use: practical, physiological and morphological aspects

The aim of the present thesis was to better understand the physiological role of the phytohormones jasmonates (JAs) and abscisic acid (ABA) during fruit ripening in prospect of a possible field application of JAs and ABA to improve fruit yield and quality. In particular, the effects of exogenous application of these substances at different fruit developmental stages and under different experimental conditions were evaluated. Some aspects of the water relations upon ABA treatment were also analysed. Three fruit species, peach (Prunus persica L. Batsch), golden (Actinidia chinensis) and green kiwifruit (Actinidia deliciosa), and several of their cvs, were used for the trials. Different experimental models were adopted: fruits in planta, detached fruit, detached branches with fruit, girdled branches and micropropagated plants. The work was structured into four sets of experiments as follows: (i) Pre-harvest methyl jasmonate (MJ) application was performed at S3/S4 transition under field conditions in Redhaven peach; ethylene production, ripening index, fruit quality and shelf-life were assessed showing that MJ-treated fruit were firmer and thus less ripe than controls as confirmed by the Index of Absorbance Difference (IAD), but exhibited a shorter shelf-life due to an increase in ethylene production. Moreover, the time course of the expression of ethylene-, auxin- and other ripening-related genes was determined. Ripening-related ACO1 and ACS1 transcript accumulation was inhibited though transiently by MJ, and gene expression of the ethylene receptor ETR2 and of the ethylene-related transcription factor ERF2 was also altered. The time course of the expression of several auxin-related genes was strongly affected by MJ suggesting an increase in auxin biosynthesis, altered auxin conjugation and release as well as perception and transport; the need for a correct ethylene/auxin balance during ripening was confirmed. (ii) Pre- and post-harvest ABA applications were carried out under field conditions in Flaminia and O’Henry peach and Stark Red Gold nectarine fruit; ethylene production, ripening index, fruit quality and shelf-life were assessed. Results show that pre-harvest ABA applications increase fruit size and skin color intensity. Also post-harvest ABA treatments alter ripening-related parameters; in particular, while ethylene production is impaired in ABA-treated fruit soluble solids concentration (SSC) is enhanced. Following field ABA applications stem water potential was modified since ABA-treated peach trees retain more water. (iii) Pre- and post-harvest ABA and PDJ treatments were carried out in both kiwifruit species under field conditions at different fruit developmental stages and in post-harvest. Ripening index, fruit quality, plant transpiration, photosynthesis and stomatal conductance were assessed. Pre-harvest treatments enhance SSC in the two cvs and flesh color development in golden kiwifruit. Post-harvest applications of either ABA or ABA plus PDJ lead to increased SSC. In addition, ABA reduces gas exchanges in A. deliciosa. (iv) Spray, drench and dipping ABA treatments were performed in micropropagated peach plants and in peach and nectarine detached branches; plant water use and transpiration, biomass production and fruit dehydration were determined. In both plants and branches ABA significantly reduces water use and fruit dehydration. No negative effects on biomass production were detected. The present information, mainly arising from plant growth regulator application in a field environment, where plants have to cope with multiple biotic and abiotic stresses, may implement the perspectives for the use of these substances in the control of fruit ripening.

Determinación de la calidad interna del kiwi mediante técnicas no destructivas: impacto e imagen hiperspectral.

El kiwi es un fruto con epidermis pilosa de color pardo-verdoso cuyo aspecto externo no sufre cambios significativos a lo largo del proceso de maduración. El objetivo de este trabajo fue evaluar el potencial de distintas técnicas no destructivas para la determinación de la calidad interna de kiwi. Se trabajó con frutos de pulpa verde (Actinidia deliciosa) ?Hayward? y de pulpa amarilla (Actinidia chinensis) ?Hort16A? y tres estados de madurez. Se realizaron determinaciones mecánicas y acústicas con el impactador lateral LPF y el equipo comercial AWETA, ambos para la estimación no destructiva de la firmeza, y se adquirieron imágenes con cámara hiperespectral (rango 400-1000 nm), como herramienta para la estimación global del estado de madurez del fruto. Se encontró que existe una correlación positiva y significativa (0,94? rpearson ?0,97) entre las variables Fuerzamáx. (impactador) y Frecuenciaresonante máx. (AWETA) con la Fuerzamáx registrada en el ensayo de referencia ?compresión con bola? realizado sobre los mismos frutos con una máquina universal de ensayos. Las imágenes hiperespectrales permitieron evidenciar el distinto nivel de madurez de los frutos en evolución y dentro de cada lote e incluso en distintas zonas de un mismo fruto, apreciándose cierto patrón espacial común en la evolución de la madurez dentro de los frutos.

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.

Detection and molecular characterization of viruses infecting Actinidia spp.

Kiwifruit (genus Actinidia) is an important horticultural crop grown in the temperate regions. The four world’s largest producers are China, Italy, New Zealand and Chile. More than 50 species are recognized in the genus but the principal species in cultivation are A. deliciosa and A. chinensis. In Italy, as well as in many other countries, the kiwifruit crop has been considered to be relatively disease free and then no certification system for this species has been developed to regulate importation of propagation plant material in the European Union. During the last years a number of fungal and bacterial diseases have been recorded such as Botrytis cinerea and Pseudomonas syringae pv. actinidiae. Since 2003, several viruses and virus-like diseases have been identified and more recent studies demonstrated that Actinidia spp can be infected by a wide range of viral agents. In collaboration with the University of Auckland we have been detected thirteen different viral species on kiwifruit plants. During the three years of my PhD I worked on the characterization of Cucumber mosaic virus (CMV) and Pelargonium zonate spot virus (PZSV). The determination of causal agents has been based on host range, symptom expression in the test plant species and morphological properties of the virus particles using transmission electron microscopy (TEM) and using specific oligonucleotide primers in reverse transcription-polymerase chain reaction (RT-PCR). Both viruses induced several symptoms on kiwifruit plants. Moreover with new technologies such as high-throughput sequencing we detected additional viruses, a new member of the family Closteroviridae and a new member of the family Totiviridae. Taking together all results of my studies it is clear that, in order to minimize the risk of serious viral disease in kiwifruit, it is vital to use virus-free propagation material in order to prevent the spread of these viruses.

Influence of nitrogen fertiliser application and timing on greenhouse gas emissions from a lychee (Litchi chinensis) orchard in humid subtropical Australia

Nitrous oxide emissions from intensive, fertilised agricultural systems have been identified as significant contributors to both Australia's and the global greenhouse gas (GHG) budget. This is expected to increase as rates of agriculture intensification and land use change accelerate to support population growth and food production. Limited data exists on N2O trace gas fluxes from subtropical or tropical tree cropping soils critical for the development of effective mitigation strategies.This study aimed to quantify GHG emissions over two consecutive years (March 2007 to March 2009) from a 30 year (lychee) orchard in the humid subtropical region of Australia. GHG fluxes were measured using a combination of high temporal resolution automated sampling and manually sampled chambers. No fertiliser was added to the plots during the 2007 measurement season. A split application of nitrogen fertiliser (urea) was added at the rate of 265kgNha-1 during the autumn and spring of 2008. Emissions of N2O were influenced by rainfall events and seasonal temperatures during 2007 and the fertilisation events in 2008. Annual N2O emissions from the lychee canopy increased from 1.7kgN2O-Nha-1yr-1 for 2007, to 7.6kgN2O-Nha-1yr-1 following fertiliser application in 2008. This represented an emission factor of 1.56%, corrected for background emissions. The timing of the split application was found to be critical to N2O emissions, with over twice as much lost following an application in spring (2.44%) compared to autumn (EF: 1.10%). This research suggests that avoiding fertiliser application during the hot and moist spring/summer period can reduce N2O losses without compromising yields.