Kiwifruit bZIP transcription factor AcePosF21 elicits ascorbic acid biosynthesis during cold stress.

Cold stress seriously affects plant development, resulting in heavy agricultural losses. L-ascorbic acid (AsA, vitamin C) is an antioxidant implicated in abiotic stress tolerance and metabolism of reactive oxygen species (ROS). Understanding whether and how cold stress elicits AsA biosynthesis to reduce oxidative damage is important for developing cold-resistant plants. Here, we show that the accumulation of AsA in response to cold stress is a common mechanism conserved across the plant kingdom, from single-cell algae to angiosperms. We identified a basic leucine zipper domain (bZIP) transcription factor (TF) of kiwifruit (Actinidia eriantha Benth.), AcePosF21, which was triggered by cold and is involved in the regulation of kiwifruit AsA biosynthesis and defense responses against cold stress. AcePosF21 interacted with the R2R3-MYB TF AceMYB102 and directly bound to the promoter of the gene encoding GDP-L-galactose phosphorylase 3 (AceGGP3), a key conduit for regulating AsA biosynthesis, to up-regulate AceGGP3 expression and produce more AsA, which neutralized the excess ROS induced by cold stress. On the contrary, VIGS or CRISPR-Cas9-mediated editing of AcePosF21 decreased AsA content and increased the generation of ROS in kiwifruit under cold stress. Taken together, we illustrated a model for the regulatory mechanism of AcePosF21-mediated regulation of AceGGP3 expression and AsA biosynthesis to reduce oxidative damage by cold stress, which provides valuable clues for manipulating the cold resistance of kiwifruit.

Kiwifruit MYBS1-like and GBF3 transcription factors influence l-ascorbic acid biosynthesis by activating transcription of GDP-L-galactose phosphorylase 3.

Plant-derived Vitamin C (l-ascorbic acid (AsA)) is crucial for human health and wellbeing and thus increasing AsA content is of interest to plant breeders. In plants GDP-l-galactose phosphorylase (GGP) is a key biosynthetic control step and here evidence is presented for two new transcriptional activators of GGP. AsA measurement, transcriptomics, transient expression, hormone application, gene editing, yeast 1/2-hybrid, and electromobility shift assay (EMSA) methods were used to identify two positively regulating transcription factors. AceGGP3 was identified as the most highly expressed GGP in Actinidia eriantha fruit, which has high fruit AsA. A gene encoding a 1R-subtype myeloblastosis (MYB) protein, AceMYBS1, was found to bind the AceGGP3 promoter and activate its expression. Overexpression and gene-editing show AceMYBS1 effectively increases AsA accumulation. The bZIP transcription factor AceGBF3 (a G-box binding factor), also was shown to increase AsA content, and was confirmed to interact with AceMYBS1. Co-expression experiments showed that AceMYBS1 and AceGBF3 additively promoted AceGGP3 expression. Furthermore, AceMYBS1, but not GBF3, was repressed by abscisic acid, resulting in reduced AceGGP3 expression and accumulation of AsA. This study sheds new light on the roles of MYBS1 homologues and ABA in modulating AsA synthesis, and adds to the understanding of mechanisms underlying AsA accumulation.

Elevating Ascorbate in Arabidopsis Stimulates the Production of Abscisic Acid, Phaseic Acid, and to a Lesser Extent Auxin (IAA) and Jasmonates, Resulting in Increased Expression of DHAR1 and Multiple Transcription Factors Associated with Abiotic Stress Tolerance.

Gene expression and phytohormone contents were measured in response to elevating ascorbate in the absence of other confounding stimuli such as high light and abiotic stresses. Young Arabidopsis plants were treated with 25 mM solutions of l-galactose pathway intermediates l-galactose (l-gal) or l-galactono-1,4-lactone (l-galL), as well as L-ascorbic acid (AsA), with 25 mM glucose used as control. Feeding increased rosette AsA 2- to 4-fold but there was little change in AsA biosynthetic gene transcripts. Of the ascorbate recycling genes, only Dehydroascorbate reductase 1 expression was increased. Some known regulatory genes displayed increased expression and included ANAC019, ANAC072, ATHB12, ZAT10 and ZAT12. Investigation of the ANAC019/ANAC072/ATHB12 gene regulatory network revealed a high proportion of ABA regulated genes. Measurement of a subset of jasmonate, ABA, auxin (IAA) and salicylic acid compounds revealed consistent increases in ABA (up to 4.2-fold) and phaseic acid (PA; up to 5-fold), and less consistently certain jasmonates, IAA, but no change in salicylic acid levels. Increased ABA is likely due to increased transcripts for the ABA biosynthetic gene NCED3. There were also smaller increases in transcripts for transcription factors ATHB7, ERD1, and ABF3. These results provide insights into how increasing AsA content can mediate increased abiotic stress tolerance.

Peridermal fruit skin formation in Actinidia sp. (kiwifruit) is associated with genetic loci controlling russeting and cuticle formation.

BACKGROUND: The skin (exocarp) of fleshy fruit is hugely diverse across species. Most fruit types have a live epidermal skin covered by a layer of cuticle made up of cutin while a few create an outermost layer of dead cells (peridermal layer). RESULTS: In this study we undertook crosses between epidermal and peridermal skinned kiwifruit, and showed that epidermal skin is a semi-dominant trait. Furthermore, backcrossing these epidermal skinned hybrids to a peridermal skinned fruit created a diverse range of phenotypes ranging from epidermal skinned fruit, through fruit with varying degrees of patches of periderm (russeting), to fruit with a complete periderm. Quantitative trait locus (QTL) analysis of this population suggested that periderm formation was associated with four loci. These QTLs were aligned either to ones associated with russet formation on chromosome 19 and 24, or cuticle integrity and coverage located on chromosomes 3, 11 and 24. CONCLUSION: From the segregation of skin type and QTL analysis, it appears that skin development in kiwifruit is controlled by two competing factors, cuticle strength and propensity to russet. A strong cuticle will inhibit russeting while a strong propensity to russet can create a continuous dead skinned periderm.

A review of current knowledge about the formation of native peridermal exocarp in fruit.

The outer skin layer in any plant is essential in offering a protective barrier against water loss and pathogen attack. Within fleshy fruit, the skin supports internal cell layers and can provide the initial cues in attracting seed-dispersing animals. The skin of a fruit, termed the exocarp, is a key element of consumer preference and a target for many breeding programs. Across fruiting species there is a huge diversity of exocarp types and these range from a simple single living cell layer (epidermis) often covered with a waxy layer, to complex multicellular suberised and dead cell layers (periderm), with various intermediate russet forms in between. Each exocarp can be interspersed with other structures such as hairs or spines. The epidermis has been well characterised and remains pluripotent with the help of the cells immediately under the epidermis. The periderm, in contrast, is the result of secondary meristematic activity, which replaces the epidermal layers, and is not well characterised in fruits. In this review we explore the structure, composition and mechanisms that control the development of a periderm type fruit exocarp. We draw upon literature from non-fleshy fruit species that form periderm tissue, from which a considerable amount of research has been undertaken.

Molecular Characterisation of a Supergene Conditioning Super-High Vitamin C in Kiwifruit Hybrids.

During analysis of kiwifruit derived from hybrids between the high vitamin C (ascorbic acid; AsA) species Actinidia eriantha and A. chinensis, we observed bimodal segregation of fruit AsA concentration suggesting major gene segregation. To test this hypothesis, we performed whole-genome sequencing on pools of hybrid genotypes with either high or low AsA fruit. Pool-GWAS (genome-wide association study) revealed a single Quantitative Trait Locus (QTL) spanning more than 5 Mbp on chromosome 26, which we denote as qAsA26.1. A co-dominant PCR marker was used to validate this association in four diploid (A. chinensis × A. eriantha) × A. chinensis backcross families, showing that the A. eriantha allele at this locus increases fruit AsA levels by 250 mg/100 g fresh weight. Inspection of genome composition and recombination in other A. chinensis genetic maps confirmed that the qAsA26.1 region bears hallmarks of suppressed recombination. The molecular fingerprint of this locus was examined in leaves of backcross validation families by RNA sequencing (RNASEQ). This confirmed strong allelic expression bias across this region as well as differential expression of transcripts on other chromosomes. This evidence suggests that the region harbouring qAsA26.1 constitutes a supergene, which may condition multiple pleiotropic effects on metabolism.

Enhanced ascorbate level improves multi-stress tolerance in a widely grown indica rice variety without compromising its agronomic characteristics.

A biotechnological approach was adopted for increasing foliar ascorbate levels as a strategy to adapt a widely grown high yielding rice variety to multiple abiotic stresses. The variety IR64 (Oryza sativa L. ssp. indica) was engineered to express the ascorbate biosynthesis gene GDP-L-galactose phosphorylase (AcGGP) from kiwifruit (Actinidia chinensis Planch.) under the control of a leaf-specific promoter of the Leaf Panicle 2 (LP2) gene. Transgene expression increased foliar ascorbate levels up to >2.5 fold but did not lead to any changes in morphological traits (seed yield, sterility rate, grain weight, and biomass) in non-stress conditions. We then hypothesized that enhanced foliar ascorbate would confer multi-stress tolerance. Indeed transgenic lines were more tolerant to salinity in terms of lipid peroxidation and foliar symptoms, and to drought in terms of lipid peroxidation and post-drought recovery (number of dead leaves). A significantly better performance in ozone stress was seen only when ozone coincided with salinity. However, no differences between transgenic lines and wild types occurred when plants were subjected to toxicities in redox-active transition metals, i.e. iron and manganese, although plants showed clear symptoms of oxidative stress. Moreover, no differential response to zinc deficiency was observed, because the background genotype IR64 was not sensitive to this stress. Taken together, our study helps to identify stress conditions that can be mitigated by enhancing foliar ascorbate levels, and therefore facilitates an adaptive breeding approach for multiple stresses that would not imply any yield penalty.

Increasing ascorbate levels in crops to enhance human nutrition and plant abiotic stress tolerance.

Ascorbate (or vitamin C) is an essential human micronutrient predominantly obtained from plants. In addition to preventing scurvy, it is now known to have broader roles in human health, for example as a cofactor for enzymes involved in epigenetic programming and as regulator of cellular iron uptake. Furthermore, ascorbate is the major antioxidant in plants and underpins many environmentally induced abiotic stress responses. Biotechnological approaches to enhance the ascorbate content of crops therefore have potential to improve both human health and abiotic stress tolerance of crops. Identifying the genetic basis of ascorbate variation between plant varieties and discovering how some 'super fruits' accumulate extremely high levels of ascorbate should reveal new ways to more effectively manipulate the production of ascorbate in crops.

The regulation of ascorbate biosynthesis.

We review the regulation of ascorbate (vitamin C) biosynthesis, focusing on the l-galactose pathway. We discuss the regulation of ascorbate biosynthesis at the level of gene transcription (both repression and enhancement) and translation (feedback inhibition of translation by ascorbate concentration) and discuss the eight proteins that have been demonstrated to date to affect ascorbate concentration in plant tissues. GDP-galactose phosphorylase (GGP) and GDP-mannose epimerase are critical steps that regulate ascorbate biosynthesis. These and other biosynthetic genes are controlled at the transcriptional level, while GGP is also controlled at the translational level. Ascorbate feedback on enzyme activity has not been observed unequivocally.

An upstream open reading frame is essential for feedback regulation of ascorbate biosynthesis in Arabidopsis.

Ascorbate (vitamin C) is an essential antioxidant and enzyme cofactor in both plants and animals. Ascorbate concentration is tightly regulated in plants, partly to respond to stress. Here, we demonstrate that ascorbate concentrations are determined via the posttranscriptional repression of GDP-l-galactose phosphorylase (GGP), a major control enzyme in the ascorbate biosynthesis pathway. This regulation requires a cis-acting upstream open reading frame (uORF) that represses the translation of the downstream GGP open reading frame under high ascorbate concentration. Disruption of this uORF stops the ascorbate feedback regulation of translation and results in increased ascorbate concentrations in leaves. The uORF is predicted to initiate at a noncanonical codon (ACG rather than AUG) and encode a 60- to 65-residue peptide. Analysis of ribosome protection data from Arabidopsis thaliana showed colocation of high levels of ribosomes with both the uORF and the main coding sequence of GGP. Together, our data indicate that the noncanonical uORF is translated and encodes a peptide that functions in the ascorbate inhibition of translation. This posttranslational regulation of ascorbate is likely an ancient mechanism of control as the uORF is conserved in GGP genes from mosses to angiosperms.

Diversity and relative levels of actinidin, kiwellin, and thaumatin-like allergens in 15 varieties of kiwifruit (Actinidia).

In the last 30 years the incidence of kiwifruit allergy has increased with the three major allergenic proteins being identified as actinidin, kiwellin, and thaumatin-like protein (TLP). We report wide variation in the levels of actinidin and TLP in 15 kiwifruit varieties from the four most widely cultivated Actinidia species. Acidic and basic isoforms of actinidin were identified in Actinidia deliciosa 'Hayward' and Actinidia arguta 'Hortgem Tahi', while only a basic isoform of actinidin was identified in Actinidia chinensis 'Hort16A'. One isoform each of kiwellin and TLP were identified in ripe fruit. The cysteine protease activity of actinidin correlated with protein levels in all species except A. arguta. Protein modeling suggested that modifications to the S2 binding pocket influenced substrate specificity of the A. arguta enzyme. Our results indicate that care is necessary when extrapolating allergenicity results from single varieties to others within the same and between different Actinidia species.

Investigation of ascorbate metabolism during inducement of storage disorders in pear.

In pear and apple, depletion of ascorbate has previously been associated with development of stress-related flesh browning. This disorder occurs in intact fruit and differs from browning associated with tissue maceration and processing. We investigated changes in ascorbate content, ascorbate peroxidase (APX) activities and gene expression of l-galactose pathway genes, ascorbate recycling genes and APXs from harvest to 30 days storage for three pear varieties ['Williams Bon Chretien' (WBC), 'Doyenne du Comice' and 'Beurre Bosc']. The pears were stored at 0.5°C in air or controlled atmosphere (CA, 2 kPa O(2) and 5 kPa CO(2)). Storage in CA caused significant amounts of storage disorders in WBC only. Ascorbate content generally declined after harvest, although a transient increase in ascorbate in the form of dehydroascorbate (DHA) between harvest and 3 days was observed in CA stored WBC, possibly due to low at-harvest monodehydroascorbate reductase and CA-decreased dehydroascorbate reductase expression. Quantitative polymerase chain reaction indicated that all cultivars responded to CA storage by increasing transcripts for APXs, and surprisingly the pre-l-galactose pathway gene GDP-mannose pyrophosphorylase, of which the product GDP mannose, is utilized either for cell wall polysaccharides, protein N-glycosylation or ascorbate production. Overall, the small differences in ascorbate we observed suggest how ascorbate is utilized, rather than ascorbate content, determines the potential to develop internal browning. Moreover, a transitory increase in DHA postharvest may indicate that fruits are at risk of developing the disorder.

Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase.

Ascorbate, or vitamin C, is obtained by humans mostly from plant sources. Various approaches have been made to increase ascorbate in plants by transgenic means. Most of these attempts have involved leaf material from model plants, with little success reported using genes from the generally accepted l-galactose pathway of ascorbate biosynthesis. We focused on increasing ascorbate in commercially significant edible plant organs using a gene, GDP-l-galactose phosphorylase (GGP or VTC2), that we had previously shown to increase ascorbate concentration in tobacco and Arabidopsis thaliana. The coding sequence of Actinidia chinensis GGP, under the control of the 35S promoter, was expressed in tomato and strawberry. Potato was transformed with potato or Arabidopsis GGP genes under the control of the 35S promoter or a polyubiquitin promoter (potato only). Five lines of tomato, up to nine lines of potato, and eight lines of strawberry were regenerated for each construct. Three lines of tomato had a threefold to sixfold increase in fruit ascorbate, and all lines of strawberry showed a twofold increase. All but one line of each potato construct also showed an increase in tuber ascorbate of up to threefold. Interestingly, in tomato fruit, increased ascorbate was associated with loss of seed and the jelly of locular tissue surrounding the seed which was not seen in strawberry. In both strawberry and tomato, an increase in polyphenolic content was associated with increased ascorbate. These results show that GGP can be used to raise significantly ascorbate concentration in commercially significant edible crops.

Component-resolved diagnosis of kiwifruit allergy with purified natural and recombinant kiwifruit allergens.

BACKGROUND: Kiwifruit is one of the most common causes of food allergic reactions. Component-resolved diagnostics may enable significantly improved detection of sensitization to kiwifruit. OBJECTIVE: To evaluate the use of individual allergens for component-resolved in vitro diagnosis of kiwifruit allergy. METHODS: Thirty patients with a positive double-blind placebo-controlled food challenge to kiwifruit, 10 atopic subjects with negative open provocation to kiwifruit, and 5 nonatopic subjects were enrolled in the study. Specific IgE to 7 individual allergens (nAct d 1-5 and rAct d 8-9) and allergen extracts was measured by ImmunoCAP. RESULTS: The diagnostic sensitivities of the commercial extract and of the sum of single allergens were 17% and 77%, respectively, whereas diagnostic specificities were 100% and 30%. A combination of the kiwi allergens Act d 1, Act d 2, Act d 4, and Act d 5 gave a diagnostic sensitivity of 40%, whereas diagnostic specificity remained high (90%). Exclusion of the Bet v 1 homolog recombinant (r) Act d 8 and profilin rAct d 9 from this allergen panel reduced sensitivity to 50% but increased specificity to 40%. Kiwifruit-monosensitized patients reacted more frequently (P < .001) with Act d 1 than polysensitized patients, whereas the latter group reacted more frequently with rAct d 8 (P = .004). CONCLUSION: Use of single kiwifruit allergen ImmunoCAP increases the quantitative test performance and diagnostic sensitivity compared with the commercial extract. Bet v 1 homolog and profilin are important allergens in pollen-related kiwifruit allergy, whereas actinidin is important in monoallergy to kiwifruit, in which symptoms are often more severe.

Gene expression studies in kiwifruit and gene over-expression in Arabidopsis indicates that GDP-L-galactose guanyltransferase is a major control point of vitamin C biosynthesis.

Vitamin C (L-ascorbic acid, AsA) is an essential metabolite for plants and animals. Kiwifruit (Actinidia spp.) are a rich dietary source of AsA for humans. To understand AsA biosynthesis in kiwifruit, AsA levels and the relative expression of genes putatively involved in AsA biosynthesis, regeneration, and transport were correlated by quantitative polymerase chain reaction in leaves and during fruit development in four kiwifruit genotypes (three species; A. eriantha, A. chinensis, and A. deliciosa). During fruit development, fruit AsA concentration peaked between 4 and 6 weeks after anthesis with A. eriantha having 3-16-fold higher AsA than other genotypes. The rise in AsA concentration typically occurred close to the peak in expression of the L-galactose pathway biosynthetic genes, particularly the GDP-L-galactose guanyltransferase gene. The high concentration of AsA found in the fruit of A. eriantha is probably due to higher expression of the GDP-mannose-3',5'-epimerase and GDP-L-galactose guanyltransferase genes. Over-expression of the kiwifruit GDP-L-galactose guanyltransferase gene in Arabidopsis resulted in up to a 4-fold increase in AsA, while up to a 7-fold increase in AsA was observed in transient expression studies where both GDP-L-galactose guanyltransferase and GDP-mannose-3',5'-epimerase genes were co-expressed. These studies show the importance of GDP-L-galactose guanyltransferase as a rate-limiting step to AsA, and demonstrate how AsA can be significantly increased in plants.

Characterization of Bet v 1-related allergens from kiwifruit relevant for patients with combined kiwifruit and birch pollen allergy.

Allergy to kiwifruit appears to have become more common in Europe and elsewhere during the past several years. Seven allergens have been identified from kiwifruit so far, with actinidin, kiwellin and the thaumatin-like protein as the most relevant ones. In contrast to other fruits, no Bet v 1 homologues were characterized from kiwifruit so far. We cloned, purified, and characterized recombinant Bet v 1-homologous allergens from green (Actinidia deliciosa, Act d 8) and gold (Actinidia chinensis, Act c 8) kiwifruit, and confirmed the presence of its natural counterpart by inhibition assays. Well-characterized recombinant Act d 8 and Act c 8 were recognized by birch pollen/kiwifruit (confirmed by double-blind placebo-controlled food challenge) allergic patients in IgE immunoblots and ELISA experiments. The present data point out that Bet v 1 homologues are allergens in kiwifruit and of relevance for patients sensitized to tree pollen and kiwifruit, and might have been neglected so far due to low abundance in the conventional extracts used for diagnosis.

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.

Cardiac chamber hypertrophy following hematopoietic stem cell transplantation for primary immunodeficiency.

Children with primary immune deficiency (PID) who receive hematopoietic stem cell transplantation (HSCT) often suffer from graft-versus-host disease (GVHD), which is commonly treated with corticosteroids (CS). CS may cause hypertension, development of cardiac chamber hypertrophy (CCH), and left ventricular outflow tract obstruction (LVOTO). We followed the development of CCH and LVOTO by serial echocardiograms in 10 children with PID before and 6 to 12 weeks after HSCT, and correlated their development with age of transplant, GVHD, use of CS and hypertension. CCH developed in all 4 children transplanted before 1 year of age who received high dose CS treatment for grade III or IV acute GVHD (aGVHD), but not in the 6 children who were transplanted at later ages or who had not received high-dose CS (P = .07). Significant correlation (P < .002) was found between CCH and blood pressure measurements that deviated above the 99th percentile. One child also suffered from severe LVOTO. CCH and LVOTO improved when CS treatment was discontinued and blood pressure normalized. We conclude that following HSCT, young children who suffer from aGVHD, treated with high CS doses, and have excessive hypertension are at risk of developing CCH.