Search

SUMMARY: During fruit ripening, polygalacturonases (PGs) are key contributors to the softening process in many species. Apple is a crisp fruit that normally exhibits only minor changes to cell walls and limited fruit softening. Here, we explore the effects of PG overexpression during fruit development using transgenic apple lines overexpressing the ripening‐related endo‐POLYGALACTURONASE1 gene. MdPG1‐overexpressing (PGox) fruit displayed early maturation/ripening with black seeds, conversion of starch to sugars and ethylene production occurring by 80 days after pollination (DAP). PGox fruit exhibited a striking, white‐skinned phenotype that was evident from 60 DAP and most likely resulted from increased air spaces and separation of cells in the hypodermis due to degradation of the middle lamellae. Irregularities in the integrity of the epidermis and cuticle were also observed. By 120 DAP, PGox fruit cracked and showed lenticel‐associated russeting. Increased cuticular permeability was associated with microcracks in the cuticle around lenticels and was correlated with reduced cortical firmness at all time points and extensive post‐harvest water loss from the fruit, resulting in premature shrivelling. Transcriptomic analysis suggested that early maturation was associated with upregulation of genes involved in stress responses, and overexpression of MdPG1 also altered the expression of genes involved in cell wall metabolism (e.g. β‐galactosidase, MD15G1221000) and ethylene biosynthesis (e.g. ACC synthase, MD14G1111500). The results show that upregulation of PG not only has dramatic effects on the structure of the fruit outer cell layers, indirectly affecting water status and turgor, but also has unexpected consequences for fruit development. [ABSTRACT FROM AUTHOR]

Knowledge of the transcriptional regulation of the carotenoid metabolic pathway is still emerging and here, we have misexpressed a key biosynthetic gene in apple to highlight potential transcriptional regulators of this pathway. We overexpressed phytoene synthase (PSY1), which controls the key rate-limiting biosynthetic step, in apple and analyzed its effects in transgenic fruit skin and flesh using two approaches. Firstly, the effects of PSY overexpression on carotenoid accumulation and gene expression was assessed in fruit at different development stages. Secondly, the effect of light exclusion on PSY1-induced fruit carotenoid accumulation was examined. PSY1 overexpression increased carotenoid content in transgenic fruit skin and flesh, with beta-carotene being the most prevalent carotenoid compound. Light exclusion by fruit bagging reduced carotenoid content overall, but carotenoid content was still higher in bagged PSY fruit than in bagged controls. In tissues overexpressing PSY1, plastids showed accelerated chloroplast to chromoplast transition as well as high fluorescence intensity, consistent with increased number of chromoplasts and carotenoid accumulation. Surprisingly, the expression of other carotenoid pathway genes was elevated in PSY fruit, suggesting a feed-forward regulation of carotenogenesis when this enzyme step is mis-expressed. Transcriptome profiling of fruit flesh identified differentially expressed transcription factors (TFs) that also were co-expressed with carotenoid pathway genes. A comparison of differentially expressed genes from both the developmental series and light exclusion treatment revealed six candidate TFs exhibiting strong correlation with carotenoid accumulation. This combination of physiological, transcriptomic and metabolite data sheds new light on plant carotenogenesis and TFs that may play a role in regulating apple carotenoid biosynthesis. [ABSTRACT FROM AUTHOR]

Fruit accumulate a diverse set of volatiles including esters and phenylpropenes. Volatile esters are synthesised via fatty acid degradation or from amino acid precursors, with the final step being catalysed by alcohol acyl transferases ( AATs). Phenylpropenes are produced as a side branch of the general phenylpropanoid pathway. Major quantitative trait loci ( QTLs) on apple ( Malus × domestica) linkage group ( LG)2 for production of the phenylpropene estragole and volatile esters (including 2-methylbutyl acetate and hexyl acetate) both co-located with the Md AAT1 gene. Md AAT1 has previously been shown to be required for volatile ester production in apple ( Plant J., 2014, ), and here we show it is also required to produce p-hydroxycinnamyl acetates that serve as substrates for a bifunctional chavicol/eugenol synthase ( MdoPhR5) in ripe apple fruit. Fruit from transgenic 'Royal Gala' Md AAT1 knockdown lines produced significantly reduced phenylpropene levels, whilst manipulation of the phenylpropanoid pathway using Md CHS (chalcone synthase) knockout and Md MYB10 over-expression lines increased phenylpropene production. Transient expression of Md AAT1, MdoPhR5 and Mdo OMT1 ( O-methyltransferase) genes reconstituted the apple pathway to estragole production in tobacco. AATs from ripe strawberry ( SAAT1) and tomato ( Sl AAT1) fruit can also utilise p-coumaryl and coniferyl alcohols, indicating that ripening-related AATs are likely to link volatile ester and phenylpropene production in many different fruit. [ABSTRACT FROM AUTHOR]

The annual growth cycle of trees is the result of seasonal cues. The onset of winter triggers an endodormant state preventing bud growth and, once a chilling requirement is satisfied, these buds enter an ecodormant state and resume growing. MADS-box genes with similarity to Arabidopsis SHORT VEGETATIVE PHASE (SVP) [the SVP-like and DORMANCY ASSOCIATED MADS-BOX (DAM) genes] have been implicated in regulating flowering and growth-dormancy cycles in perennials. Here, we identified and characterized three DAM-like (MdDAMs) and two SHORT VEGETATIVE PHASElike (MdSVPs) genes from apple (Malus x domestica 'Royal Gala'). The expression of MdDAMa and MdDAMc indicated they may play a role in triggering autumn growth cessation. In contrast, the expression of MdDAMb, MdSVPa and MdSVPb suggested a role in maintaining bud dormancy. Consistent with this, ectopic expression of MdDAMb and MdSVPa in 'Royal Gala' apple plants resulted in delayed budbreak and architecture change due to constrained lateral shoot outgrowth, but normal flower and fruit development. The association of MdSVPa and MdSVPb expression with floral bud development in the low fruiting 'Off' trees of a biennial bearing cultivar 'Sciros' suggested the SVP genes might also play a role in floral meristem identity. [ABSTRACT FROM AUTHOR]

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 micro RNA gene ( mi RNA172) influences fruit size in apple. A transposon insertional allele of mi RNA172 showing reduced expression associates with large fruit in an apple breeding population, whereas over-expression of mi RNA172 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. [ABSTRACT FROM AUTHOR]

Phenylpropenes, such as eugenol and trans-anethole, are important aromatic compounds that determine flavour and aroma in many herbs and spices. Some apple varieties produce fruit with a highly desirable spicy/aromatic flavour that has been attributed to the production of estragole, a methylated phenylpropene. To elucidate the molecular basis for estragole production and its contribution to ripe apple flavour and aroma we characterised a segregating population from a Royal Gala ( RG, estragole producer) × Granny Smith ( GS, non-producer) apple cross. Two quantitative trait loci ( QTLs; accounting for 9.2 and 24.8% of the variation) on linkage group ( LG) 1 and LG2 were identified that co-located with seven candidate genes for phenylpropene O-methyltransferases ( Mdo OMT1-7). Of these genes, only expression of Mdo OMT1 on LG1 increased strongly with ethylene and could be correlated with increasing estragole production in ripening RG fruit. Transient over-expression in tobacco showed that Mdo OMT1 utilised a range of phenylpropene substrates and catalysed the conversion of chavicol to estragole. Royal Gala carried two alleles ( Mdo OMT1a, Mdo OMT1b) whilst GS appeared to be homozygous for Mdo OMT1b. Mdo OMT1a showed a higher affinity and catalytic efficiency towards chavicol than Mdo OMT1b, which could account for the phenotypic variation at the LG1 QTL. Multiple transgenic RG lines with reduced Mdo OMT1 expression produced lower levels of methylated phenylpropenes, including estragole and methyleugenol. Differences in fruit aroma could be perceived in these fruit, compared with controls, by sensory analysis. Together these results indicate that Mdo OMT1 is required for the production of methylated phenylpropenes in apple and that phenylpropenes including estragole may contribute to ripe apple fruit aroma. [ABSTRACT FROM AUTHOR]

Consumers of whole foods, such as fruits, demand consistent high quality and seek varieties with enhanced health properties, convenience or novel taste. We have raised the polyphenolic content of apple by genetic engineering of the anthocyanin pathway using the apple transcription factor MYB10. These apples have very high concentrations of foliar, flower and fruit anthocyanins, especially in the fruit peel. Independent lines were examined for impacts on tree growth, photosynthesis and fruit characteristics. Fruit were analysed for changes in metabolite and transcript levels. Fruit were also used in taste trials to study the consumer perception of such a novel apple. No negative taste attributes were associated with the elevated anthocyanins. Modification with this one gene provides near isogenic material and allows us to examine the effects on an established cultivar, with a view to enhancing consumer appeal independently of other fruit qualities. [ABSTRACT FROM AUTHOR]