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.

Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10

Anthocyanin concentration is an important determinant of the colour of many fruits. In apple (Malus x domestica), centuries of breeding have produced numerous varieties in which levels of anthocyanin pigment vary widely and change in response to environmental and developmental stimuli. The apple fruit cortex is usually colourless, although germplasm does exist where the cortex is highly pigmented due to the accumulation of either anthocyanins or carotenoids. From studies in a diverse array of plant species, it is apparent that anthocyanin biosynthesis is controlled at the level of transcription. Here we report the transcript levels of the anthocyanin biosynthetic genes in a red-fleshed apple compared with a white-fleshed cultivar. We also describe an apple MYB transcription factor, MdMYB10, that is similar in sequence to known anthocyanin regulators in other species. We further show that this transcription factor can induce anthocyanin accumulation in both heterologous and homologous systems, generating pigmented patches in transient assays in tobacco leaves and highly pigmented apple plants following stable transformation with constitutively expressed MdMYB10. Efficient induction of anthocyanin biosynthesis in transient assays by MdMYB10 was dependent on the co-expression of two distinct bHLH proteins from apple, MdbHLH3 and MdbHLH33. The strong correlation between the expression of MdMYB10 and apple anthocyanin levels during fruit development suggests that this transcription factor is responsible for controlling anthocyanin biosynthesis in apple fruit; in the red-fleshed cultivar and in the skin of other varieties, there is an induction of MdMYB10 expression concurrent with colour formation during development. Characterization of MdMYB10 has implications for the development of new varieties through classical breeding or a biotechnological approach.

MYB transcription factors that colour our fruit

Anthocyanin concentration is a primary determinant of plant colour. Fruit anthocyanin biosynthesis is controlled by a distinct clade of R2R3 MYB transcription factors. In apple, three recent papers describe the discovery of MYB genes activating skin, flesh and foliage anthocyanic colour. These findings lead the way to new approaches in the breeding and biotechnological development of fruit with new colour patterns.

The role of ethylene and cold temperature in the regulation of the apple POLYGALACTURONASE1 gene and fruit softening

Fruit softening in apple (Malus 3 domestica) is associated with an increase in the ripening hormone ethylene. Here, we show that in cv Royal Gala apples that have the ethylene biosynthetic gene ACC OXIDASE1 suppressed, a cold treatment preconditions the apples to soften independently of added ethylene. When a cold treatment is followed by an ethylene treatment, a more rapid softening occurs than in apples that have not had a cold treatment. Apple fruit softening has been associated with the increase in the expression of cell wall hydrolase genes. One such gene, POLYGALACTURONASE1 (PG1), increases in expression both with ethylene and following a cold treatment. Transcriptional regulation of PG1 through the ethylene pathway is likely to be through an ETHYLENE-INSENSITIVE3-like transcription factor, which increases in expression during apple fruit development and transactivates the PG1 promoter in transient assays in the presence of ethylene. A coldrelated gene that resembles a COLD BINDING FACTOR (CBF) class of gene also transactivates the PG1 promoter. The transactivation by the CBF-like gene is greatly enhanced by the addition of exogenous ethylene. These observations give a possible molecular mechanism for the coldand ethylene-regulated control of fruit softening and suggest that either these two pathways act independently and synergistically with each other or cold enhances the ethylene response such that background levels of ethylene in the ethylene-suppressed apples is sufficient to induce fruit softening in apples.

High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex

The biosynthesis of anthocyanin in many plants is affected by environmental conditions. In apple (Malus×domestica Borkh.), concentrations of fruit anthocyanins are lower under hot climatic conditions. We examined the anthocyanin accumulation in the peel of maturing 'Mondial Gala' and 'Royal Gala' apples, grown in both temperate and hot climates, and using artificial heating of on-tree fruit. Heat caused a dramatic reduction of both peel anthocyanin concentration and transcripts of the genes of the anthocyanin biosynthetic pathway. Heating fruit rapidly reduced expression of the R2R3 MYB transcription factor (MYB10) responsible for coordinative regulation for red skin colour, as well as expression of other genes in the transcriptional activation complex. A single night of low temperatures is sufficient to elicit a large increase in transcription of MYB10 and consequently the biosynthetic pathway. Candidate genes that can repress anthocyanin biosynthesis did not appear to be responsible for reductions in anthocyanin content. We propose that temperature-induced regulation of anthocyanin biosynthesis is primarily caused by altered transcript levels of the activating anthocyanin regulatory complex.

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.

The kiwifruit lycopene beta-cyclase plays a significant role in carotenoid accumulation in fruit

The composition of carotenoids, along with anthocyanins and chlorophyll, accounts for the distinctive range of colour found in the Actinidia (kiwifruit) species. Lutein and beta-carotene are the most abundant carotenoids found during fruit development, with beta-carotene concentration increasing rapidly during fruit maturation and ripening. In addition, the accumulation of beta-carotene and lutein is influenced by the temperature at which harvested fruit are stored. Expression analysis of carotenoid biosynthetic genes among different genotypes and fruit developmental stages identified Actinidia lycopene beta-cyclase (LCY-β) as the gene whose expression pattern appeared to be associated with both total carotenoid and beta-carotene accumulation. Phytoene desaturase (PDS) expression was the least variable among the different genotypes, while zeta carotene desaturase (ZDS), beta-carotene hydroxylase (CRH-β), and epsilon carotene hydroxylase (CRH-ε) showed some variation in gene expression. The LCY-β gene was functionally tested in bacteria and shown to convert lycopene and delta-carotene to beta-carotene and alpha-carotene respectively. This indicates that the accumulation of beta-carotene, the major carotenoid in these kiwifruit species, appears to be controlled by the level of expression of LCY-β gene.

Maternal feeding self-efficacy and fruit and vegetable intakes in infants. Results from the SAIDI study

Adequate consumption of fruits and vegetables (FV) is a characteristic of a healthy diet but remains a challenge in nutrition interventions. This cross-sectional study explored the multi-directional relationships between maternal feeding self-efficacy, parenting confidence, child feeding behaviour, exposure to new food and FV intake in a cohort of 277 infants. Mothers with healthy infants weighing ≥2500 g and ≥37 weeks gestation were recruited post-natally from 11 South Australian hospitals. Socio-demographic datawere collected at recruitment. At 6 months postnatal, infantswereweighed and measured, andmothers completed a questionnaire exploring their perceptions of child feeding behaviour and child exposure to newfoods. The questionnaire also included the Short Temperament Scale for Infants, Kessler 10 to measure maternal psychological distress and 5 items measuring maternal feeding self-efficacy. The number of occasions and variety of FV (number of subgroups within food groups) consumed by infants were estimated from a 24-hour dietary recall and 2 days food record. Structural equation modellingwas performed using Mplus version 6.11. Median (IQR) variety scores were 2 (1–3) for fruit and 3 (2–5) for vegetable intake. The most popular FV consumed were apple (n = 108, 45.0%) and pumpkin (n = 143, 56.3%). None of the variables studied predicted the variety of child fruit intake. Parenting confidence, exposure to new foods and child feeding behaviourwere indirectly related to child vegetable intake through maternal feeding self-efficacy while total number of children negatively predicted child vegetable variety (p < 0.05). This highlights the need for addressing antecedents of maternal feeding self-efficacy and the family eating environment as key strategies towards development of healthy eating in children.

The functional significance of fruit exocarp on host selection and oviposition by Queensland fruit fly, Bactrocera tryoni (Froggatt) (Tephritidae: Diptera)

Queensland fruit fly is Australia's most serious insect pest of horticulture. The fly lays its eggs into fruit, where they hatch into maggots which destroy the fruit. Understanding egg laying behaviour, known as oviposition, is a critical but under-researched aspect of fruit fly biology. This thesis focused on three aspects of oviposition: the role of fruit peel as a physical barrier to oviposition; the quality of fruit for maggot development; and the structure and wear of the egg laying organ – the ovipositor. Results showed that flies selected fruit based on their suitability for offspring survival, not because of the softness or hardness of fruit peel. Previously reported use of holes or wounds in fruit peel by ovipositing females was determined to be a mechanism which saved the female time, not a mechanism to reduce ovipositor wear. The results offer insights into the evolution of host use by fruit flies and their sustainable management.

Sexual selection in true fruit flies (Diptera: Tephritidae) : transcriptome and experimental evidences for phytochemicals increasing male competitive ability

In male tephritid fruit flies of the genus Bactrocera, feeding on secondary plant compounds (sensu lato male lures = methyl eugenol, raspberry ketone and zingerone) increases male mating success. Ingested male lures alter the male pheromonal blend, normally making it more attractive to females and this is considered the primary mechanism for the enhanced mating success. However, the male lures raspberry ketone and zingerone are known, across a diverse range of other organisms, to be involved in increasing energy metabolism. If this also occurs in Bactrocera, then this may represent an additional benefit to males as courtship is metabolically expensive and lure feeding may increase a fly's short-term energy. We tested this hypothesis by performing comparative RNA-seq analysis between zingerone-fed and unfed males of Bactrocera tryoni. We also carried out behavioural assays with zingerone- and cuelure-fed males to test whether they became more active. RNA-seq analysis revealed, in zingerone-fed flies, up-regulation of 3183 genes with homologues transcripts to those known to regulate intermale aggression, pheromone synthesis, mating and accessory gland proteins, along with significant enrichment of several energy metabolic pathways and gene ontology terms. Behavioural assays show significant increases in locomotor activity, weight reduction and successful mating after mounting; all direct/indirect measures of increased activity. These results suggest that feeding on lures leads to complex physiological changes, which result in more competitive males. These results do not negate the pheromone effect, but do strongly suggest that the phytochemical-induced sexual selection is governed by both female preference and male competitive mechanisms.

One and the same: Integrative taxonomic evidence that Bactrocera invadens (Diptera: Tephritidae) is the same species as the Oriental fruit fly Bactrocera dorsalis

The invasive fruit fly Bactrocera invadens Drew, Tsuruta & White, and the Oriental fruit fly Bactrocera dorsalis (Hendel) are highly destructive horticultural pests of global significance. Bactrocera invadens originates from the Indian subcontinent and has recently invaded all of sub-Saharan Africa, while B. dorsalis principally occurs from the Indian subcontinent towards southern China and South-east Asia. High morphological and genetic similarity has cast doubt over whether B. invadens is a distinct species from B. dorsalis. Addressing this issue within an integrative taxonomic framework, we sampled from across the geographic distribution of both taxa and: (i) analysed morphological variation, including those characters considered diagnostic (scutum colour, length of aedeagus, width of postsutural lateral vittae, wing size, and wing shape); (ii) sequenced four loci (ITS1, ITS2, cox1 and nad4) for phylogenetic inference, and; (iii) generated a cox1 haplotype network to examine population structure. Molecular analyses included the closely related species, Bactrocera kandiensis Drew & Hancock. Scutum colour varies from red-brown to fully black for individuals from Africa and the Indian subcontinent. All individuals east of the Indian subcontinent are black except for a few red-brown individuals from China. The postsutural lateral vittae width of B. invadens is narrower than B. dorsalis from eastern Asia, but the variation is clinal, with subcontinent B. dorsalis populations intermediate in size. Aedeagus length, wing shape and wing size cannot discriminate between the two taxa. Phylogenetic analyses failed to resolve B. invadens from B. dorsalis, but did resolve B. kandiensis. Bactrocera dorsalis and B. invadens shared cox1 haplotypes, yet the haplotype network pattern does not reflect current taxonomy or patterns in thoracic colour. Some individuals of B. dorsalis/B. invadens possessed haplotypes more closely related to B. kandiensis than to conspecifics, suggestive of mitochondrial introgression between these species. The combined evidence fails to support the delimitation of B. dorsalis and B. invadens as separate biological species. Consequently, existing biological data for B. dorsalis may be applied to the invasive population in Africa. Our recommendation, in line with other recent publications, is that B. invadens be synonymized with B. dorsalis.

Indirect effects of phytochemicals on offspring performance of Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae)

Phytochemical lures such as methyl eugenol (ME) and cue-lure are used in the management of Bactrocera fruit flies for monitoring and control. These lures are not just attractants, but also trigger physiological changes in males that lead to enhanced mating success. Additionally, in the cue-lure-responsive Bactrocera tryoni, females mated with lure-fed males exhibit changes in fecundity, remating receptivity and longevity. While the lures show current generation effects, no research has been carried out on possible multigenerational effects, although such effects have been hypothesized within a ‘sexy-son’ sexual selection model. In this study, we test for indirect, cross-generational effects of lure exposure in F1offspring of B. tryoni females mated with cue-lure-fed, zingerone-fed and lure-unfed (=control) males. The F1 attributes we recorded were immature development time, immature survival, adult survival and adult male lure foraging. No significant differences were found between treatments for any of the three life-history measurements, except that the offspring sired by zingerone-fed males had a longer egg development time than cue-lure and control offspring. However, indirect exposure to lures significantly enhanced the lure-foraging ability of F1 adult males. More offspring of cue-lure-fed males arrived at a lure source in both large flight cages and small laboratory cages over a 2-h period than did control males. The offspring of zingerone-fed males were generally intermediate between cue-lure and control offspring. This study provides the first evidence of a next generation effect of fruit fly male lures. While the results of this study support a ‘sexy-son’ sexual selection mechanism for the evolution of lure response in Bactrocera fruit flies, our discussion urges caution in interpreting our results in this way.

Fruit-feeding behaviour and use of olfactory cues by the fruit bat Rousettus leschenaulti: an experimental study

This paper describes the feeding behaviour ofRousettus leschenaulti Desmarest, 1820 on lychees, the preferred cultivated food of this bat in captive conditions. We found that feeding comprised 25–30% of the total activity of these animals in a flight cage and that feeding durations were not significantly different between two sexes. To evaluate the role of odor and vision in foraging behaviour, we provided animals with artificial lychees, real lychees and artificial lychees soaked in the juice of real lychees and we recorded the number of feeding approaches to the different “fruit” types. The results indicated that bats approached real fruit significantly more than artificial fruit, and that the number of approaches to the soaked artificial fruit was also significantly higher than to the unsoaked artificial fruit. There were no significant differences between sexes in approach rates to any “fruit” type. We discuss the role of different sensory cues in the foraging behaviour of these bats and emphasize that the olfactory cue is important in detecting food resources and discriminating between different kinds of food items.

Estimating the burden of disease attributable to low fruit and vegetable intake in South Africa in 2000

Objectives To estimate the burden of disease attributed to low fruit and vegetable intake by sex and age group in South Africa for the year 2000. Design The analysis follows the World Health Organization comparative risk assessment (CRA) methodology. Populationattributable fractions were calculated from South African prevalence data from dietary surveys and applied to the revised South African burden of disease estimates for 2000. A theoretical maximum distribution of 600 g per day for fruit and vegetable intake was chosen. Monte Carlo simulationmodelling techniques were used for uncertainty analysis. Setting South Africa. Subjects Adults ≥ 15 years. Outcome measures Mortality and disability-adjusted life years (DALYs), from ischaemic heart disease, ischaemic stroke, lung cancer, gastric cancer, colorectal cancer and oesophageal cancer. Results Low fruit and vegetable intake accounted for 3.2% of total deaths and 1.1% of the 16.2 million attributable DALYs. For both males and females the largest proportion of total years of healthy life lost attributed to low fruit and vegetable intake was for ischaemic heart disease (60.6% and 52.2%, respectively). Ischaemic stroke accounted for 17.8% of attributable DALYs for males and 32.7% for females. For the related cancers, the leading attributable DALYs for men and women were oesophageal cancer (9.8% and 7.0%, respectively) and lung cancer (7.8% and 4.7%, respectively). Conclusions A high intake of fruit and vegetables can make a significant contribution to decreasing mortality from certain diseases. The challenge lies in creating the environment that facilitates changes in dietary habits such as the increased intake of fruit and vegetables.