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.

Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples

Mutations in the genes encoding for either the biosynthetic or transcriptional regulation of the anthocyanin pathway have been linked to color phenotypes. Generally, this is a loss of function resulting in a reduction or a change in the distribution of anthocyanin. Here, we describe a rearrangement in the upstream regulatory region of the gene encoding an apple (Malus x domestica) anthocyanin-regulating transcription factor, MYB10. We show that this modification is responsible for increasing the level of anthocyanin throughout the plant to produce a striking phenotype that includes red foliage and red fruit flesh. This rearrangement is a series of multiple repeats, forming a minisatellite-like structure that comprises five direct tandem repeats of a 23-bp sequence. This MYB10 rearrangement is present in all the red foliage apple varieties and species tested but in none of the white fleshed varieties. Transient assays demonstrated that the 23-bp sequence motif is a target of the MYB10 protein itself, and the number of repeat units correlates with an increase in transactivation by MYB10 protein. We show that the repeat motif is capable of binding MYB10 protein in electrophoretic mobility shift assays. Taken together, these results indicate that an allelic rearrangement in the promoter of MYB10 has generated an autoregulatory locus, and this autoregulation is sufficient to account for the increase in MYB10 transcript levels and subsequent ectopic accumulation of anthocyanins throughout the plant.

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.

A meshfree model for plant tissue deformations during drying

Plant tissue has a complex cellular structure which is an aggregate of individual cells bonded by middle lamella. During drying processes, plant tissue undergoes extreme deformations which are mainly driven by moisture removal and turgor loss. Numerical modelling of this problem becomes challenging when conventional grid-based modelling techniques such as Finite Element Methods (FEM) and Finite Difference Methods (FDM) have grid-based limitations. This work presents a meshfree approach to model and simulate the deformations of plant tissues during drying. This method demonstrates the fundamental capabilities of meshfree methods in handling extreme deformations of multiphase systems. A simplified 2D tissue model is developed by aggregating individual cells while accounting for the stiffness of the middle lamella. Each individual cell is simply treated as consisting of two main components: cell fluid and cell wall. The cell fluid is modelled using Smoothed Particle Hydrodynamics (SPH) and the cell wall is modelled using a Discrete Element Method (DEM). During drying, moisture removal is accounted for by reduction of cell fluid and wall mass, which causes local shrinkage of cells eventually leading to tissue scale shrinkage. The cellular deformations are quantified using several cellular geometrical parameters and a favourably good agreement is observed when compared to experiments on apple tissue. The model is also capable of visually replicating dry tissue structures. The proposed model can be used as a step in developing complex tissue models to simulate extreme deformations during drying.

Herbivores and nutrients control grassland plant diversity via light limitation

Human alterations to nutrient cycles1, 2 and herbivore communities3, 4, 5, 6, 7 are affecting global biodiversity dramatically2. Ecological theory predicts these changes should be strongly counteractive: nutrient addition drives plant species loss through intensified competition for light, whereas herbivores prevent competitive exclusion by increasing ground-level light, particularly in productive systems8, 9. Here we use experimental data spanning a globally relevant range of conditions to test the hypothesis that herbaceous plant species losses caused by eutrophication may be offset by increased light availability due to herbivory. This experiment, replicated in 40 grasslands on 6 continents, demonstrates that nutrients and herbivores can serve as counteracting forces to control local plant diversity through light limitation, independent of site productivity, soil nitrogen, herbivore type and climate. Nutrient addition consistently reduced local diversity through light limitation, and herbivory rescued diversity at sites where it alleviated light limitation. Thus, species loss from anthropogenic eutrophication can be ameliorated in grasslands where herbivory increases ground-level light.

When supply does not meet demand-ER stress and plant programmed cell death

The endoplasmic reticulum (ER) is the central organelle in the eukaryotic secretory pathway. The ER functions in protein synthesis and maturation and is crucial for proper maintenance of cellular homeostasis and adaptation to adverse environments. Acting as a cellular sentinel, the ER is exquisitely sensitive to changing environments principally via the ER quality control machinery. When perturbed, ER-stress triggers a tightly regulated and highly conserved, signal transduction pathway known as the unfolded protein response (UPR) that prevents the dangerous accumulation of unfolded/misfolded proteins. In situations where excessive UPR activity surpasses threshold levels, cells deteriorate and eventually trigger programmed cell death (PCD) as a way for the organism to cope with dysfunctional or toxic signals. The programmed cell death that results from excessive ER stress in mammalian systems contributes to several important diseases including hypoxia, neurodegeneration, and diabetes. Importantly, hallmark features and markers of cell death that are associated with ER stress in mammals are also found in plants. In particular, there is a common, conserved set of chaperones that modulate ER cell death signaling. Here we review the elements of plant cell death responses to ER stress and note that an increasing number of plant-pathogen interactions are being identified in which the host ER is targeted by plant pathogens to establish compatibility.

Designing mixed species tree plantations for the tropics : balancing ecological attributes of species with landholder preferences in the Philippines

A mixed species reforestation program known as the Rainforestation Farming system was undertaken in the Philippines to develop forms of farm forestry more suitable for smallholders than the simple monocultural plantations commonly used then. In this study, we describe the subsequent changes in stand structure and floristic composition of these plantations in order to learn from the experience and develop improved prescriptions for reforestation systems likely to be attractive to smallholders. We investigated stands aged from 6 to 11 years old on three successive occasions over a 6 year period. We found the number of species originally present in the plots as trees >5 cm dbh decreased from an initial total of 76 species to 65 species at the end of study period. But, at the same time, some new species reached the size class threshold and were recruited into the canopy layer. There was a substantial decline in tree density from an estimated stocking of about 5000 trees per ha at the time of planting to 1380 trees per ha at the time of the first measurement; the density declined by a further 4.9% per year. Changes in composition and stand structure were indicated by a marked shift in the Importance Value Index of species. Over six years, shade-intolerant species became less important and the native shade-tolerant species (often Dipterocarps) increased in importance. Based on how the Rainforestation Farming plantations developed in these early years, we suggest that mixed-species plantations elsewhere in the humid tropics should be around 1000 trees per ha or less, that the proportion of fast growing (and hence early maturing) trees should be about 30–40% of this initial density and that any fruit tree component should only be planted on the plantation margin where more light and space are available for crowns to develop.

Pattern analysis of the diversity of morphological plant attributes and herbage yield in a world collection of white clover (Trifolium repens L.) germplasm characterised in a summer moisture stress environment of Australia

Information on the variation available for different plant attributes has enabled germplasm collections to be effectively utilised in plant breeding. A world sourced collection of white clover germplasm has been developed at the White Clover Resource Centre at Glen Innes, New South Wales. This collection of 439 accessions was characterised under field conditions as a preliminary study of the genotypic variation for morphological attributes; stolon density, stolon branching, number of nodes. number of rooted nodes, stolon thickness, internode length, leaf length, plant height and plant spread, together with seasonal herbage yield. Characterisation was conducted on different batches of germplasm (subsets of accessions taken from the complete collection) over a period of five years. Inclusion of two check cultivars, Haifa and Huia, in each batch enabled adjustment of the characterisation data for year effects and attribute-by-year interaction effects. The component of variance for seasonal herbage yield among batches was large relative to that for accessions. Accession-by-experiment and accession-by-season interactions for herbage yield were not detected. Accession mean repeatability for herbage yield across seasons was intermediate (0.453). The components of genotypic variance among accessions for all attributes, except plant height, were larger than their respective standard errors. The estimates of accession mean repeatability for the attributes ranged from low (0.277 for plant height) to intermediate (0.544 for internode length). Multivariate techniques of clustering and ordination were used to investigate the diversity present among the accessions in the collection. Both cluster analysis and principal component analysis suggested that seven groups of accessions existed. It was also proposed from the pattern analysis results that accessions from a group characterised by large leaves, tall plants and thick stolons could be crossed with accessions from a group that had above average stolon density and stolon branching. This material could produce breeding populations to be used in recurrent selection for the development of white clover cultivars for dryland summer moisture stress environments in Australia. The germplasm collection was also found to be deficient in genotypes with high stolon density, high number of branches high number of rooted nodes and large leaves. This warrants addition of new germplasm accessions possessing these characteristics to the present germplasm collection.

Managed livestock grazing is compatible with the maintenance of plant diversity in semidesert grasslands

Even when no baseline data are available, the impacts of 150 years of livestock grazing on natural grasslands can be assessed using a combined approach of grazing manipulation and regional-scale assessment of the flora. Here, we demonstrate the efficacy of this method across 18 sites in the semidesert Mitchell grasslands of northeastern Australia. Fifteen-year-old exclosures (ungrazed and macropod grazed) revealed that the dominant perennial grasses in the genus Astrebla do not respond negatively to grazing disturbance typical of commercial pastoralism. Neutral, positive, intermediate, and negative responses to grazing disturbance were recorded amongst plant species with no single life-form group associated with any response type. Only one exotic species, Cenchrus ciliaris, was recorded at low frequency. The strongest negative response was from a native annual grass, Chionachne hubbardiana, an example of a species that is highly sensitive to grazing disturbance. Herbarium records revealed only scant evidence that species with a negative response to grazing have declined through the period of commercial pastoralism. A regional analysis identified 14 from a total of 433 plant species in the regional flora that may be rare and potentially threatened by grazing disturbance. However, a targeted survey precluded grazing as a cause of decline for seven of these based on low palatability and positive responses to grazing and other disturbance. Our findings suggest that livestock grazing of semidesert grasslands with a short evolutionary history of ungulate grazing has altered plant composition, but has not caused declines in the dominant perennial grasses or in species richness as predicted by the preceding literature. The biggest impact of commercial pastoralism is the spread of woody leguminous trees that can transform grassland to thorny shrubland. The conservation of plant biodiversity is largely compatible with commercial pastoralism provided these woody weeds are controlled, but reserves strategically positioned within water remote areas are necessary to protect grazing-sensitive species. This study demonstrates that a combination of experimental studies and regional surveys can be used to understand anthropogenic impacts on natural ecosystems where reference habitat is not available.

Overexpression of the kiwifruit SVP3 gene affects reproductive development and suppresses anthocyanin biosynthesis in petals, but has no effect on vegetative growth, dormancy, or flowering time

SVP-like MADS domain transcription factors have been shown to regulate flowering time and both inflorescence and flower development in annual plants, while having effects on growth cessation and terminal bud formation in perennial species. Previously, four SVP genes were described in woody perennial vine kiwifruit (Actinidia spp.), with possible distinct roles in bud dormancy and flowering. Kiwifruit SVP3 transcript was confined to vegetative tissues and acted as a repressor of flowering as it was able to rescue the Arabidopsis svp41 mutant. To characterize kiwifruit SVP3 further, ectopic expression in kiwifruit species was performed. Ectopic expression of SVP3 in A. deliciosa did not affect general plant growth or the duration of endodormancy. Ectopic expression of SVP3 in A. eriantha also resulted in plants with normal vegetative growth, bud break, and flowering time. However, significantly prolonged and abnormal flower, fruit, and seed development were observed, arising from SVP3 interactions with kiwifruit floral homeotic MADS-domain proteins. Petal pigmentation was reduced as a result of SVP3-mediated interference with transcription of the kiwifruit flower tissue-specific R2R3 MYB regulator, MYB110a, and the gene encoding the key anthocyanin biosynthetic step, F3GT1. Constitutive expression of SVP3 had a similar impact on reproductive development in transgenic tobacco. The flowering time was not affected in day-neutral and photoperiod-responsive Nicotiana tabacum cultivars, but anthesis and seed germination were significantly delayed. The accumulation of anthocyanin in petals was reduced and the same underlying mechanism of R2R3 MYB NtAN2 transcript reduction was demonstrated.

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.

A particle based model to simulate microscale morphological changes of plant tissues during drying

Fundamental understanding on microscopic physical changes of plant materials is vital to optimize product quality and processing techniques, particularly in food engineering. Although grid-based numerical modelling can assist in this regard, it becomes quite challenging to overcome the inherited complexities of these biological materials especially when such materials undergo critical processing conditions such as drying, where the cellular structure undergoes extreme deformations. In this context, a meshfree particle based model was developed which is fundamentally capable of handling extreme deformations of plant tissues during drying. The model is built by coupling a particle based meshfree technique: Smoothed Particle Hydrodynamics (SPH) and a Discrete Element Method (DEM). Plant cells were initiated as hexagons and aggregated to form a tissue which also accounts for the characteristics of the middle lamella. In each cell, SPH was used to model cell protoplasm and DEM was used to model the cell wall. Drying was incorporated by varying the moisture content, the turgor pressure, and cell wall contraction effects. Compared to the state of the art grid-based microscale plant tissue drying models, the proposed model can be used to simulate tissues under excessive moisture content reductions incorporating cell wall wrinkling. Also, compared to the state of the art SPH-DEM tissue models, the proposed model better replicates real tissues and the cell-cell interactions used ensure efficient computations. Model predictions showed good agreement both qualitatively and quantitatively with experimental findings on dried plant tissues. The proposed modelling approach is fundamentally flexible to study different cellular structures for their microscale morphological changes at dehydration.

A novel approach for numerical simulation of plant tissue shrinkage during drying

Drying is a key processing techniques used in food engineering which demands continual developments on advanced analysis techniques in order to optimize the product and the process. In this regard, plant based materials are a frequent subject of interest where microstructural studies can provide a clearer understanding on the fundamental physical mechanisms involved. In this context, considering numerous challenges of using conventional numerical grid-based modelling techniques, a meshfree particle based model was developed to simulate extreme deformations of plant microstructure during drying. The proposed technique is based on a particle based meshfree method: Smoothed Particle Hydrodynamics (SPH) and a Discrete Element Method (DEM). A tissue model was developed by aggrading individual cells modelled with SPH-DEM coupled approach by initializing the cells as hexagons and aggregating them to form a tissue. The model also involves a middle lamella resembling real tissues. Using the model, different dried tissue states were simulated with different moisture content, the turgor pressure, and cell wall contraction effects. Compared to the state of the art grid-based microscale plant tissue drying models, the proposed model is capable of simulating plant tissues at lower moisture contents which results in excessive shrinkage and cell wall wrinkling. Model predictions were compared with experimental findings and a fairly good agreement was observed both qualitatively and quantitatively.

A coupled SPH-DEM model for micro-scale structural deformations of plant cells during drying

A single plant cell was modeled with smoothed particle hydrodynamics (SPH) and a discrete element method (DEM) to study the basic micromechanics that govern the cellular structural deformations during drying. This two-dimensional particle-based model consists of two components: a cell fluid model and a cell wall model. The cell fluid was approximated to a highly viscous Newtonian fluid and modeled with SPH. The cell wall was treated as a stiff semi-permeable solid membrane with visco-elastic properties and modeled as a neo-Hookean solid material using a DEM. Compared to existing meshfree particle-based plant cell models, we have specifically introduced cell wall–fluid attraction forces and cell wall bending stiffness effects to address the critical shrinkage characteristics of the plant cells during drying. Also, a moisture domain-based novel approach was used to simulate drying mechanisms within the particle scheme. The model performance was found to be mainly influenced by the particle resolution, initial gap between the outermost fluid particles and wall particles and number of particles in the SPH influence domain. A higher order smoothing kernel was used with adaptive smoothing length to improve the stability and accuracy of the model. Cell deformations at different states of cell dryness were qualitatively and quantitatively compared with microscopic experimental findings on apple cells and a fairly good agreement was observed with some exceptions. The wall–fluid attraction forces and cell wall bending stiffness were found to be significantly improving the model predictions. A detailed sensitivity analysis was also done to further investigate the influence of wall–fluid attraction forces, cell wall bending stiffness, cell wall stiffness and the particle resolution. This novel meshfree based modeling approach is highly applicable for cellular level deformation studies of plant food materials during drying, which characterize large deformations.