Carbon starvation reduces carbohydrate and anthocyanin accumulation in red-fleshed fruit via trehalose 6-phosphate and MYB27.

Kiwifruit (Actinidia spp.) is a recently domesticated fruit crop with several novel-coloured cultivars being developed. Achieving uniform fruit flesh pigmentation in red genotypes is challenging. To investigate the cause of colour variation between fruits, we focused on a red-fleshed Actinidia chinensis var. chinensis genotype. It was hypothesized that carbohydrate supply could be responsible for this variation. Early in fruit development, we imposed high or low (carbon starvation) carbohydrate supplies treatments; carbohydrate import or redistribution was controlled by applying a girdle at the shoot base. Carbon starvation affected fruit development as well as anthocyanin and carbohydrate metabolite concentrations, including the signalling molecule trehalose 6-phosphate. RNA-Seq analysis showed down-regulation of both gene-encoding enzymes in the anthocyanin and carbohydrate biosynthetic pathways. The catalytic trehalose 6-phosphate synthase gene TPS1.1a was down-regulated, whereas putative regulatory TPS7 and TPS11 were strongly up-regulated. Unexpectedly, under carbon starvation MYB10, the anthocyanin pathway regulatory activator was slightly up-regulated, whereas MYB27 was also up-regulated and acts as a repressor. To link these two metabolic pathways, we propose a model where trehalose 6-phosphate and the active repressor MYB27 are involved in sensing the carbon starvation status. This signals the plant to save resources and reduce the production of anthocyanin in fruits.

Metabolic analysis of kiwifruit (Actinidia deliciosa) berries from extreme genotypes reveals hallmarks for fruit starch metabolism.

Tomato, melon, grape, peach, and strawberry primarily accumulate soluble sugars during fruit development. In contrast, kiwifruit (Actinidia Lindl. spp.) and banana store a large amount of starch that is released as soluble sugars only after the fruit has reached maturity. By integrating metabolites measured by gas chromatography-mass spectrometry, enzyme activities measured by a robot-based platform, and transcript data sets during fruit development of Actinidia deliciosa genotypes contrasting in starch concentration and size, this study identified the metabolic changes occurring during kiwifruit development, including the metabolic hallmarks of starch accumulation and turnover. At cell division, a rise in glucose (Glc) concentration was associated with neutral invertase (NI) activity, and the decline of both Glc and NI activity defined the transition to the cell expansion and starch accumulation phase. The high transcript levels of ß-amylase 9 (BAM9) during cell division, prior to net starch accumulation, and the correlation between sucrose phosphate synthase (SPS) activity and sucrose suggest the occurrence of sucrose cycling and starch turnover. ADP-Glc pyrophosphorylase (AGPase) is identified as a key enzyme for starch accumulation in kiwifruit berries, as high-starch genotypes had 2- to 5-fold higher AGPase activity, which was maintained over a longer period of time and was also associated with enhanced and extended transcription of the AGPase large subunit 4 (APL4). The data also revealed that SPS and galactinol might affect kiwifruit starch accumulation, and suggest that phloem unloading into kiwifruit is symplastic. These results are relevant to the genetic improvement of quality traits such as sweetness and sugar/acid balance in a range of fruit species.

Fruit development of the diploid kiwifruit, Actinidia chinensis 'Hort16A'.

BACKGROUND: With the advent of high throughput genomic tools, it is now possible to undertake detailed molecular studies of individual species outside traditional model organisms. Combined with a good understanding of physiological processes, these tools allow researchers to explore natural diversity, giving a better understanding of biological mechanisms. Here a detailed study of fruit development from anthesis through to fruit senescence is presented for a non-model organism, kiwifruit, Actinidia chinensis ('Hort16A'). RESULTS: Consistent with previous studies, it was found that many aspects of fruit morphology, growth and development are similar to those of the model fruit tomato, except for a striking difference in fruit ripening progression. The early stages of fruit ripening occur as the fruit is still growing, and many ripening events are not associated with autocatalytic ethylene production (historically associated with respiratory climacteric). Autocatalytic ethylene is produced late in the ripening process as the fruit begins to senesce. CONCLUSION: By aligning A. chinensis fruit development to a phenological scale, this study provides a reference framework for subsequent physiological and genomic studies, and will allow cross comparison across fruit species, leading to a greater understanding of the diversity of fruits found across the plant kingdom.

A Jasmonate-Induced Defense Elicitation in Mature Leaves Reduces Carbon Export and Alters Sink Priority in Grape (Vitis vinifera Chardonnay).

This work aims to understand how Vitis vinifera (Chardonnay) vines prioritise the export and distribution of recently fixed photoassimilate between root tissue, fruit, and defence, following the elicitation of a defence response. Jasmonic acid (JA) and its methyl ester, MeJA, are endogenous plant hormones, known collectively as jasmonates, that have signalling roles in plant defence and consequently are often used to prime plant defence systems. Here, we use exogenous jasmonate application to mature source leaves of Chardonnay grapevines to elucidate the prioritisation strategy of carbon allocation between plant defence and growth. Our results demonstrate that jasmonate application to Chardonnay leaves can elicit a defence response to Botrytis cinerea, but the effect was localised to the jasmonate-treated area. We found no evidence of a systemic defence response in non-treated mature leaves or young growing tissue. JA application reduced the photosynthetic rate of the treated leaf and reduced the export rate of recently fixed carbon-11 from the leaf. Following JA application, a greater proportion of available recently fixed carbon was allocated to the roots, suggesting an increase in sink strength of the roots. Relative sink strength of the berries did not change; however, an increase in berry sugar was observed seven days after JA treatment. We conclude that the data provide evidence for a "high sugar resistance" model in the mature treated leaves of the vine, since the export of carbon was reduced to ensure an elevated defence response in the treated leaf. The increase in berry sugar concentration seven days after treatment can be explained by the initial prioritisation of a greater portion of the exported carbon to storage in the roots, making it available for remobilisation to the berries once the challenge to defence had passed.

Phytohormone and Transcriptomic Analysis Reveals Endogenous Cytokinins Affect Kiwifruit Growth under Restricted Carbon Supply.

Following cell division, fruit growth is characterized by both expansion through increases in cell volume and biomass accumulation in cells. Fruit growth is limited by carbon starvation; however, the mechanism controlling fruit growth under restricted carbohydrate supply is poorly understood. In a previous study using red-fleshed kiwifruit, we showed that long-term carbon starvation had detrimental effects on carbohydrate, anthocyanin metabolism, and fruit growth. To elucidate the mechanisms underlying the reduction in fruit growth during kiwifruit development, we integrated phytohormone profiling with transcriptomic and developmental datasets for fruit under high or low carbohydrate supplies. Phytohormone profiling of the outer pericarp tissue of kiwifruit showed a 6-fold reduction in total cytokinin concentrations in carbon-starved fruit, whilst other hormones were less affected. Principal component analysis visualised that cytokinin composition was distinct between fruit at 16 weeks after mid bloom, based on their carbohydrate supply status. Cytokinin biosynthetic genes (IPT, CYP735A) were significantly downregulated under carbon starvation, in agreement with the metabolite data. Several genes that code for expansins, proteins involved in cell wall loosening, were also downregulated under carbon starvation. In contrast to other fleshy fruits, our results suggest that cytokinins not only promote cell division, but also drive fruit cell expansion and growth in kiwifruit.

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.

Apple dwarfing rootstocks exhibit an imbalance in carbohydrate allocation and reduced cell growth and metabolism.

Apple dwarfing rootstocks cause earlier shoot termination and reduced root and shoot mass. To identify physiological factors responsible for rootstock-induced growth restriction, we compared vascular-enriched gene expression between two dwarfing rootstocks ('M27' and 'M9') and the vigorous rootstock 'M793' using RNA sequencing and quantitative reverse transcriptase PCR. Differentially expressed genes common to both dwarfing rootstocks belonged to five main biological processes: (1) primary metabolism, (2) cell wall synthesis and modification, (3) secondary metabolism, (4) hormone signalling and response and (5) redox homeostasis. Genes promoting the biosynthesis of amino acids, lipids and cell walls were downregulated in dwarfing rootstocks, whereas genes promoting the breakdown of these compounds were upregulated. The only exception to this trend was the upregulation of starch synthesis genes in dwarfing rootstocks. Non-structural carbohydrate analysis demonstrated that starch concentrations in 'M9' roots, stems and grafted 'Royal Gala' ('RG') scions were double that of equivalent tissues from 'RG' homo-grafted trees ('RG'/'RG'). Fructose and glucose concentrations were much lower in all three tissues of the 'RG'/'M9' trees. Together, these data indicate that dwarfing rootstocks are in a state of sugar depletion and reduced cellular activity despite having large starch reserves. Another significant finding was the over-accumulation of flavonoids and the downregulation of auxin influx transporters MdAUX1 and MdLAX2 in dwarfing rootstocks. We propose that both factors reduce polar auxin transport. The results of this study contribute novel information about the physiological state of dwarfing rootstocks.

Exogenous cytokinin application to Actinidia chinensis var. deliciosa 'Hayward' fruit promotes fruit expansion through water uptake.

Exogenous application of a cytokinin-like compound forchlorfenuron (CPPU) can promote fruit growth, although often at the expense of dry matter (DM), an important indicator of fruit quality. Actinidia chinensis var. deliciosa 'Hayward' fruit are very responsive to CPPU treatments, but the mechanism underlying the significant fruit weight increase and associated decrease in DM is unclear. In this study, we hypothesised that CPPU-enhanced growth increases fruit carbohydrate demand, but limited carbohydrate supply resulted in decreased fruit DM. During fruit development, CPPU effects on physical parameters, metabolites, osmotic pressure and transcriptional changes were assessed under conditions of both standard and a high carbohydrate supply. We showed that CPPU increased fruit fresh weight but the dramatic DM decrease was not carbohydrate limited. Enhanced glucose and fructose concentrations contributed to an increase in soluble carbohydrate osmotic pressure, which was correlated with increased water accumulation in CPPU-treated fruit and up-regulation of water channel aquaporin gene PIP2.4 at 49 days after anthesis. Transcipt analysis suggested that the molecular mechanism contributing to increased glucose and fructose concentrations was altered by carbohydrate supply. At standard carbohydrate supply, the early glucose increase in CPPU fruit was associated with reduced starch synthesis and increased starch degradation. When carbohydrate supply was high, the early glucose increase in CPPU fruit was associated with a general decrease in starch synthesis but up-regulation of vacuolar invertase and fructokinase genes. We conclude that CPPU affected fruit expansion by increasing the osmotically-driven water uptake and its effect was not carbohydrate supply-limited.

Mannans in tomato fruit are not depolymerized during ripening despite the presence of endo-ß-mannanase.

Cell walls of tomato fruit contain hemicellulosic mannans that may fulfill a structural role. Two populations were purified from cell walls of red ripe tomato tissue and named galactoglucomannan-glucuronoxylan I and II (GGM-GX I and II), respectively. Both polysaccharides not only consisted of mannose, glucose and galactose, indicating the presence of GGM, but also contained xylose and glucuronic acid, indicating the presence of GX. Treatment of both polysaccharides with xylanase or endo-ß-mannanase showed that the GX and the GGM were associated in a complex. The composition of GGM-GX II changed slightly during tomato ripening, but both GGM-GX I and II showed no change in molecular weight, indicating that they were not hydrolyzed during ripening. Ripe tomato fruit also possess an endo-ß-mannanase, an enzyme that in vitro was capable of either hydrolyzing GGM-GX I and II (endo-ß-mannanase activity), or transglycosylating them in the presence of mannan oligosaccharides (mannan transglycosylase activity). The lack of evidence for hydrolysis of these potential substrates in vivo suggests either that the enzyme and potential substrates are not accessible to each other for some reason, or that the main activity of endo-ß-mannanase is not hydrolysis but transglycosylation, a reaction in which polysaccharide substrates and end-products are indistinguishable. Transglycosylation would remodel rather than weaken the cell wall and allow the fruit epidermis to possibly retain flexibility and plasticity to resist cracking and infection when the fruit is ripe.

Changes in quinic acid metabolism during fruit development in three kiwifruit species.

Kiwifruit are novel in that they contain high levels of quinic acid (1-2% w/w), which contributes to the flavour, sugar/acid balance and health-giving properties of the fruit. In a study of quinic acid storage and metabolism in three kiwifruit species (Actinidia chinensis Planch. var. chinensis, Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson var. deliciosa and Actinidia arguta (Sieb. et Zucc.) Planch. ex Miq. var. arguta) quinic acid accumulation occurred principally in the early stages (<60 days after anthesis; (DAA)) of fruit development. The present study established that there are separate quinate dehydrogenase (QDH) and shikimate dehydrogenase (SDH) activities in kiwifruit, probably representing different proteins. Quinate dehydrogenase activity was at a maximum around the time of greatest quinic acid accumulation and declined markedly in late fruit development, and was also higher in the species that accumulated the largest amounts of quinic acid (A. chinensis and A. deliciosa). In contrast, SDH activity was highest in the early stages of fruit development and only declined to 30-50% at later stages of fruit development in all three species. Dehydroquinate synthase gene expression levels measured by quantitative real-time PCR showed a high level in the early season, which was sustained through the mid-season. The quantitative real-time PCR results for a kiwifruit EST that had homology to chloroplastic isoforms of SDH showed an induction in the middle to late season; therefore, the high level of SDH activity in the early season (<30 DAA) may have resulted from the expression of a cytosolic isoform of the enzyme. The results are also consistent with the relative levels of the bifunctional dehydroquinate dehydratase/SDH enzyme and QDH enzyme controlling the accumulation and utilisation of quinic acid in kiwifruit.

Planteose is a short-term storage carbohydrate in Actinidia leaves.

The polyol myo-inositol constitutes 10-20% of soluble carbohydrates in mature leaves of Actinidia deliciosa (A.Chev.) C.F. Liang et A.R. Ferguson var. deliciosa 'Hayward' and A. arguta (Sieb. et Zucc.) Planch. ex Miq. var. arguta. In contrast with other non-structural carbohydrates, myo-inositol concentrations in A. deliciosa leaves increase only slightly during development from sink to source, and are not affected in source leaves by increased sink demand upon fruit set. In mature fruit-bearing leaves myo-inositol concentrations fluctuate diurnally, increasing during the night and declining towards morning, but in plants with less sink demand a diurnal pattern is not observed. In potted A. arguta seedlings subjected to extended dark periods, leaf concentrations of sugars and starch decline rapidly while myo-inositol concentrations are maintained. Labelling studies with 14CO2 revealed that myo-inositol in leaves is not a primary photosynthetic product and is turned over more slowly than other soluble carbohydrates. A suggested role of myo-inositol as a precursor in mucilage synthesis was not substantiated, as radioactivity was incorporated into mucilage more rapidly than into free myo-inositol. Planteose, a trisaccharide comprising sucrose and galactose, incorporated substantial amounts of radioactivity and accumulated to high levels, indicating a role in short-term storage of sucrose. Planteose was synthesised during the day and degraded during the night in a manner that was opposite to that of sucrose while starch and myo-inositol levels remained relatively constant. Planteose has been reported in Cyclamen persicum, ash and sesame seed. This is the first report of planteose in Actinidia, and the first time it has been identified as a major short-term storage carbohydrate in Actinidia leaves.