Phenotypic changes associated with RNA interference silencing of chalcone synthase in apple (Malus × domestica)

We have identified in apple (Malus × domestica) three chalcone synthase (CHS) genes. In order to understand the functional redundancy of this gene family RNA interference knockout lines were generated where all three of these genes were down-regulated. These lines had no detectable anthocyanins and radically reduced concentrations of dihydrochalcones and flavonoids. Surprisingly, down-regulation of CHS also led to major changes in plant development, resulting in plants with shortened internode lengths, smaller leaves and a greatly reduced growth rate. Microscopic analysis revealed that these phenotypic changes extended down to the cellular level, with CHS-silenced lines showing aberrant cellular organisation in the leaves. Fruit collected from one CHS-silenced line was smaller than the 'Royal Gala' controls, lacked flavonoids in the skin and flesh and also had changes in cell morphology. Auxin transport experiments showed increased rates of auxin transport in a CHS-silenced line compared with the 'Royal Gala' control. As flavonoids are well known to be key modulators of auxin transport, we hypothesise that the removal of almost all flavonoids from the plant by CHS silencing creates a vastly altered environment for auxin transport to occur and results in the observed changes in growth and development.

The genome of the domesticated apple (Malus × domestica Borkh.)

We report a high-quality draft genome sequence of the domesticated apple (Malus × domestica). We show that a relatively recent (>50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.

Endophytic yeasts and filamentous fungi associated with southern Brazilian apple (Malus domestica) orchards subjected to conventional, integrated or organic cultivation

Endophytic fungi were isolated from leaves, flowers and fruit of healthy apple trees (Malus domestica, BORKH.) growing in southern Brazilian orchards under three different cultivation systems (conventional, integrated and organic), during two vegetative cycles. The greatest total number of endophytic isolates was obtained from the orchards under organic cultivation when compared to integrated and conventional cultivation systems. Filamentous fungi from the genera Colletotrichum, Xylaria and Botryosphaeria were the most frequent ones and the most representative yeast genera were Sporobolomyces, Rhodotorula, Debaryomyces and Cryptococcus. It is suggested that some isolates may be used as indicators of the different management systems. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA.

Uso de reguladores de crescimento como alternativa tecnológica na cultura da macieira ‘Eva’ (Malus domestica Borkh.)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise isotópica da variabilidade natural do carbono-13 e análise energética em suco, néctar e refrigerante de maçã (malus domestica, borkh)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diversidade de maçãs (Malus Domestica Borkh.) nas feiras de produtores de Nova Iorque

Visitando feiras de produtores locais na cidade de Nova Iorque por um ano, entre junho de 2008 e maio de 2009, verificou-se significativa amplitude varietal das maçãs, demonstrando-se um aspecto particular da diversidade vegetal. Evidenciadas no verão pela presença volumosa, é no outono e inverno que se nota a diversidade intraespecífica, atravessando todos os períodos sazonais. Verificou-se a existência de mais de quatro centenas de espécies vegetais disponíveis no mercado, escolhendo-se a maçã como um dos tipos mais evidentes para a análise da diversidade, uma vez que se oferecem em 65 cultivares nas feiras do estado ao qual emprestam o apelido. Representam um tipo de diversidade que extrapola ao conceito de especialização, usualmente atribuído à agricultura dos países desenvolvidos, uma vez que convivem com outras espécies nos espaços em que são cultivadas. O objetivo deste artigo é demonstrar a diversidade genética e de usos das maçãs que se expressa na atividade comercial exercida nas ruas e praças nova iorquinas.

Architectural development and dry matter production in a multisite trial on single and multiaxis apple trees (Malus domestica Borkh) grafted on different rootstocks

In two Italian sites, multiaxis trees slightly reduced primary axis length and secondary axis length of newly grafted trees, and increased the number of secondary shoots. The total length, node production, and total dry matter gain were proportional to the number of axis. Growth of both primary and secondary shoots, and dry matter accumulation, have been found to be also well related to rootstock vigour. A great variability in axillary shoot production was recorded among different environments. Grafted trees had higher primary growth, secondary axis growth, and dry matter gain than chip budded trees. Stem water potential measured in the second year after grafting was not affected by rootstocks or number of leaders. Measurements performed in New Zealand (Hawke’s Bay) during the second year after grafting revealed that both final length and growth rate of primary and secondary axis were related to the rootstock rather than to the training system. Dwarfing rootstocks reduced the number of long vegetative shoots and increased the proportion of less vigorous shoots.

Future Bloom and Blossom Frost Risk for Malus domestica Considering Climate Model and Impact Model Uncertainties

The future bloom and risk of blossom frosts for Malus domestica were projected using regional climate realizations and phenological (= impact) models. As climate impact projections are susceptible to uncertainties of climate and impact models and model concatenation, the significant horizon of the climate impact signal was analyzed by applying 7 impact models, including two new developments, on 13 climate realizations of the IPCC emission scenario A1B. Advancement of phenophases and a decrease in blossom frost risk for Lower Saxony (Germany) for early and late ripeners was determined by six out of seven phenological models. Single model/single grid point time series of bloom showed significant trends by 2021-2050 compared to 1971-2000, whereas the joint signal of all climate and impact models did not stabilize until 2043. Regarding blossom frost risk, joint projection variability exceeded the projected signal. Thus, blossom frost risk cannot be stated to be lower by the end of the 21st century despite a negative trend. As a consequence it is however unlikely to increase. Uncertainty of temperature, blooming date and blossom frost risk projection reached a minimum at 2078-2087. The projected phenophases advanced by 5.5 d K-1, showing partial compensation of delayed fulfillment of the winter chill requirement and faster completion of the following forcing phase in spring. Finally, phenological model performance was improved by considering the length of day.

Determinação da origem das lenticelas e sua relação com a lenticelose em maçãs (Malus domestica Borkh.).

2011

RNA interference silencing of CHS greatly alters the growth pattern of apple (Malus x domestica)

Plants produce a vast array of phenolic compounds which are essential for their survival on land. One major class of polyphenols are the flavonoids and their formation is dependent on the enzyme chalcone synthase (CHS). In a recent study we silenced the CHS genes of apple (Malus × domestica Borkh.) and observed a loss of pigmentation in the fruit skin, flowers and stems. More surprisingly, highly silenced lines were significantly reduced in size, with small leaves and shortened internode lengths. Chemical analysis also revealed that the transgenic shoots contained greatly reduced concentrations of flavonoids which are known to modulate auxin flow. An auxin transport study verified this, with an increased auxin transport in the CHS-silenced lines. Overall, these findings suggest that auxin transport in apple has adapted to take place in the presence of high endogenous concentrations of flavonoids. Removal of these compounds therefore results in abnormal auxin movement and a highly disrupted growth pattern. © 2013 Landes Bioscience.

Análise funcional e potencial biotecnológico do gene DHN11 de macieira (Malus x domestica Borkh.)

A deficiência hídrica é o principal fator limitante à agricultura, causando significativas perdas de produtividade nas mais diversas culturas vegetais. Novas tecnologias agrícolas, tais como a geração de plantas geneticamente modificadas melhor adaptadas a condições adversas de cultivo, são necessárias. Em um estudo anterior do grupo caracterizando a família gênica das desidrinas (DHNs) em macieira (Malus x domestica Borkh.), foi verificado que o gene MdDHN11 possui níveis de expressão cerca de 6 mil vezes maior em sementes em relação a outros tecidos. Sugere-se que o acúmulo nos últimos estágios de desenvolvimento de sementes possua papel protetor na tolerância à dessecação. Além disso, as DHNs são proteínas caracterizadas por seu envolvimento na resposta a mudanças na disponibilidade de água, principalmente os causados por estresses abióticos.

The role of enoyl reductase genes in phloridzin biosynthesis in apple

Phloridzin is the predominant polyphenol in apple (Malus× domestica Borkh.) where it accumulates to high concentrations in many tissues including the leaves, bark, roots and fruit. Despite its relative abundance in apple the biosynthesis of phloridzin and other related dihydrochalcones remains only partially understood. The key unidentified enzyme in phloridzin biosynthesis is a putative carbon double bond reductase which is thought to act on p-coumaroyl-CoA to produce the dihydro p-coumaroyl-CoA precursor. A functional screen of six apple enoyl reductase-like (ENRL) genes was carried out using transient infiltration into tobacco and gene silencing by RNA interference (RNAi) in order to determine carbon double bond reductase activity and contribution to foliar phloridzin concentrations. The ENRL-3 gene caused a significant increase in phloridzin concentration when infiltrated into tobacco leaves whilst a second protein ENRL-5, with over 98% amino acid sequence similarity to ENRL-3, showed p-coumaroyl-CoA reductase activity in enzyme assays. Finally, an RNAi study showed that reducing the transcript levels of ENRL-3 in transgenic 'Royal Gala' led to a 66% decrease in the concentration of dihydrochalcones in the leaves in the one available silenced line. Overall these results suggest that ENRL-3, and its close homolog ENRL-5, may contribute to the biosynthesis of phloridzin in apple.

An ancient duplication of apple MYB transcription factors is responsible for novel red fruit-flesh phenotyp

Anthocyanin accumulation is coordinated in plants by a number of conserved transcription factors. In apple (Malus × domestica), an R2R3 MYB transcription factor has been shown to control fruit flesh and foliage anthocyanin pigmentation (MYB10) and fruit skin color (MYB1). However, the pattern of expression and allelic variation at these loci does not explain all anthocyanin-related apple phenotypes. One such example is an open-pollinated seedling of cv Sangrado that has green foliage and develops red flesh in the fruit cortex late in maturity. We used methods that combine plant breeding, molecular biology, and genomics to identify duplicated MYB transcription factors that could control this phenotype. We then demonstrated that the red-flesh cortex phenotype is associated with enhanced expression of MYB110a, a paralog of MYB10. Functional characterization of MYB110a showed that it was able to up-regulate anthocyanin biosynthesis in tobacco (Nicotiana tabacum). The chromosomal location of MYB110a is consistent with a whole-genome duplication event that occurred during the evolution of apple within the Maloideae family. Both MYB10 and MYB110a have conserved function in some cultivars, but they differ in their expression pattern and response to fruit maturity.

Apple skin patterning is associated with differential expression of MYb10

Background Some apple (Malus × domestica Borkh.) varieties have attractive striping patterns, a quality attribute that is important for determining apple fruit market acceptance. Most apple cultivars (e.g. 'Royal Gala') produce fruit with a defined fruit pigment pattern, but in the case of 'Honeycrisp' apple, trees can produce fruits of two different kinds: striped and blushed. The causes of this phenomenon are unknown. Results Here we show that striped areas of 'Honeycrisp' and 'Royal Gala' are due to sectorial increases in anthocyanin concentration. Transcript levels of the major biosynthetic genes and MYB10, a transcription factor that upregulates apple anthocyanin production, correlated with increased anthocyanin concentration in stripes. However, nucleotide changes in the promoter and coding sequence of MYB10 do not correlate with skin pattern in 'Honeycrisp' and other cultivars differing in peel pigmentation patterns. A survey of methylation levels throughout the coding region of MYB10 and a 2.5 Kb region 5' of the ATG translation start site indicated that an area 900 bp long, starting 1400 bp upstream of the translation start site, is highly methylated. Cytosine methylation was present in all three contexts, with higher methylation levels observed for CHH and CHG (where H is A, C or T) than for CG. Comparisons of methylation levels of the MYB10 promoter in 'Honeycrisp' red and green stripes indicated that they correlate with peel phenotypes, with an enrichment of methylation observed in green stripes. Conclusions Differences in anthocyanin levels between red and green stripes can be explained by differential transcript accumulation of MYB10. Different levels of MYB10 transcript in red versus green stripes are inversely associated with methylation levels in the promoter region. Although observed methylation differences are modest, trends are consistent across years and differences are statistically significant. Methylation may be associated with the presence of a TRIM retrotransposon within the promoter region, but the presence of the TRIM element alone cannot explain the phenotypic variability observed in 'Honeycrisp'. We suggest that methylation in the MYB10 promoter is more variable in 'Honeycrisp' than in 'Royal Gala', leading to more variable color patterns in the peel of this cultivar.

Transcriptional analysis of apple fruit proanthocyanidin biosynthesis

Proanthocyanidins (PAs) are products of the flavonoid pathway, which also leads to the production of anthocyanins and flavonols. Many flavonoids have antioxidant properties and may have beneficial effects for human health. PAs are found in the seeds and fruits of many plants. In apple fruit (Malus × domestica Borkh.), the flavonoid biosynthetic pathway is most active in the skin, with the flavan-3-ols, catechin, and epicatechin acting as the initiating units for the synthesis of PA polymers. This study examined the genes involved in the production of PAs in three apple cultivars: two heritage apple cultivars, Hetlina and Devonshire Quarrenden, and a commercial cultivar, Royal Gala. HPLC analysis shows that tree-ripe fruit from Hetlina and Devonshire Quarrenden had a higher phenolic content than Royal Gala. Epicatechin and catechin biosynthesis is under the control of the biosynthetic enzymes anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR1), respectively. Counter-intuitively, real-time quantitative PCR analysis showed that the expression levels of Royal Gala LAR1 and ANR were significantly higher than those of both Devonshire Quarrenden and Hetlina. This suggests that a compensatory feedback mechanism may be active, whereby low concentrations of PAs may induce higher expression of gene transcripts. Further investigation is required into the regulation of these key enzymes in apple.