The NAC transcription factor FaRIF controls fruit ripening in strawberry.

In contrast to climacteric fruits such as tomato, the knowledge on key regulatory genes controlling the ripening of strawberry, a nonclimacteric fruit, is still limited. NAC transcription factors (TFs) mediate different developmental processes in plants. Here, we identified and characterized Ripening Inducing Factor (FaRIF), a NAC TF that is highly expressed and induced in strawberry receptacles during ripening. Functional analyses based on stable transgenic lines aimed at silencing FaRIF by RNA interference, either from a constitutive promoter or the ripe receptacle-specific EXP2 promoter, as well as overexpression lines showed that FaRIF controls critical ripening-related processes such as fruit softening and pigment and sugar accumulation. Physiological, metabolome, and transcriptome analyses of receptacles of FaRIF-silenced and overexpression lines point to FaRIF as a key regulator of strawberry fruit ripening from early developmental stages, controlling abscisic acid biosynthesis and signaling, cell-wall degradation, and modification, the phenylpropanoid pathway, volatiles production, and the balance of the aerobic/anaerobic metabolism. FaRIF is therefore a target to be modified/edited to control the quality of strawberry fruits.

TTL Proteins Scaffold Brassinosteroid Signaling Components at the Plasma Membrane to Optimize Signal Transduction in Arabidopsis.

Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development, and stress responses. BRs are perceived extracellularly by plasma membrane receptor-like kinases that activate an interconnected signal transduction cascade, leading to the transcriptional regulation of BR-responsive genes. TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) genes are specific for land plants, and their encoded proteins are defined by the presence of protein-protein interaction motives, that is, an intrinsic disordered region at the N terminus, six tetratricopeptide repeat domains, and a C terminus with homology to thioredoxins. TTL proteins thus likely mediate the assembly of multiprotein complexes. Phenotypic, molecular, and genetic analyses show that TTL proteins are positive regulators of BR signaling in Arabidopsis (Arabidopsis thaliana). TTL3 directly interacts with a constitutively active BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor kinase, BRI1-SUPPRESSOR1 phosphatase, and the BRASSINAZOLE RESISTANT1 transcription factor and associates with BR-SIGNALING KINASE1, BRASSINOSTEROID INSENSITIVE2 kinases, but not with BRI1-ASSOCIATED KINASE1. A functional TTL3-green fluorescent protein (GFP) shows dual cytoplasmic plasma membrane localization. Depleting the endogenous BR content reduces plasma membrane localization of TTL3-GFP, while increasing BR content causes its plasma membrane relocalization, where it strengthens the association of BR signaling components. Our results reveal that TTL proteins promote BR responses and suggest that TTL proteins may function as scaffold proteins by bringing together cytoplasmic and plasma membrane BR signaling components.

Characterizing the involvement of FaMADS9 in the regulation of strawberry fruit receptacle development.

FaMADS9 is the strawberry (Fragaria x ananassa) gene that exhibits the highest homology to the tomato (Solanum lycopersicum) RIN gene. Transgenic lines were obtained in which FaMADS9 was silenced. The fruits of these lines did not show differences in basic parameters, such as fruit firmness or colour, but exhibited lower Brix values in three of the four independent lines. The gene ontology MapMan category that was most enriched among the differentially expressed genes in the receptacles at the white stage corresponded to the regulation of transcription, including a high percentage of transcription factors and regulatory proteins associated with auxin action. In contrast, the most enriched categories at the red stage were transport, lipid metabolism and cell wall. Metabolomic analysis of the receptacles of the transformed fruits identified significant changes in the content of maltose, galactonic acid-1,4-lactone, proanthocyanidins and flavonols at the green/white stage, while isomaltose, anthocyanins and cuticular wax metabolism were the most affected at the red stage. Among the regulatory genes that were differentially expressed in the transgenic receptacles were several genes previously linked to flavonoid metabolism, such as MYB10, DIV, ZFN1, ZFN2, GT2, and GT5, or associated with the action of hormones, such as abscisic acid, SHP, ASR, GTE7 and SnRK2.7. The inference of a gene regulatory network, based on a dynamic Bayesian approach, among the genes differentially expressed in the transgenic receptacles at the white and red stages, identified the genes KAN1, DIV, ZFN2 and GTE7 as putative targets of FaMADS9. A MADS9-specific CArG box was identified in the promoters of these genes.

The Arabidopsis synaptotagmin1 is enriched in endoplasmic reticulum-plasma membrane contact sites and confers cellular resistance to mechanical stresses.

Eukaryotic endoplasmic reticulum (ER)-plasma membrane (PM) contact sites are evolutionarily conserved microdomains that have important roles in specialized metabolic functions such as ER-PM communication, lipid homeostasis, and Ca(2+) influx. Despite recent advances in knowledge about ER-PM contact site components and functions in yeast (Saccharomyces cerevisiae) and mammals, relatively little is known about the functional significance of these structures in plants. In this report, we characterize the Arabidopsis (Arabidopsis thaliana) phospholipid binding Synaptotagmin1 (SYT1) as a plant ortholog of the mammal extended synaptotagmins and yeast tricalbins families of ER-PM anchors. We propose that SYT1 functions at ER-PM contact sites because it displays a dual ER-PM localization, it is enriched in microtubule-depleted regions at the cell cortex, and it colocalizes with Vesicle-Associated Protein27-1, a known ER-PM marker. Furthermore, biochemical and physiological analyses indicate that SYT1 might function as an electrostatic phospholipid anchor conferring mechanical stability in plant cells. Together, the subcellular localization and functional characterization of SYT1 highlights a putative role of plant ER-PM contact site components in the cellular adaptation to environmental stresses.

The SUD1 gene encodes a putative E3 ubiquitin ligase and is a positive regulator of 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in Arabidopsis.

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum-associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.

Central role of FaGAMYB in the transition of the strawberry receptacle from development to ripening.

The receptacle of the strawberry (Fragaria × ananassa) fruit accounts for the main properties of the ripe fruit for human consumption. As it ripens, it undergoes changes similar to other fruits in sugar : acid ratio, volatile production and cell wall softening. However, the main regulators of this process have not yet been reported. The white stage marks the initiation of the ripening process, and we had previously reported a peak of expression for a FaGAMYB gene. Transient silencing of FaGAMYB using RNAi and further determination of changes in global gene expression by RNAseq, and composition of primary and secondary metabolites have been used to investigate the role played by this gene during the development of the receptacle. Down-regulation of FaGAMYB caused an arrest in the ripening of the receptacle and inhibited colour formation. Consistent with this, several transcription factors associated with the regulation of flavonoid biosynthetic pathway showed altered expression. FaGAMYB silencing also caused a reduction of ABA biosynthesis and sucrose content. Interestingly, exogenous ABA application to the RNAI-transformed receptacle reversed most defects caused by FaGAMYB down-regulation. The study assigns a key regulatory role to FaGAMYB in the initiation of strawberry receptacle ripening and acting upstream of the known regulator ABA.

Vitamin deficiencies in humans: can plant science help?

The term vitamin describes a small group of organic compounds that are absolutely required in the human diet. Although for the most part, dependency criteria are met in developed countries through balanced diets, this is not the case for the five billion people in developing countries who depend predominantly on a single staple crop for survival. Thus, providing a more balanced vitamin intake from high-quality food remains one of the grandest challenges for global human nutrition in the coming decade(s). Here, we describe the known importance of vitamins in human health and current knowledge on their metabolism in plants. Deficits in developing countries are a combined consequence of a paucity of specific vitamins in major food staple crops, losses during crop processing, and/or overreliance on a single species as a primary food source. We discuss the role that plant science can play in addressing this problem and review successful engineering of vitamin pathways. We conclude that while considerable advances have been made in understanding vitamin metabolic pathways in plants, more cross-disciplinary approaches must be adopted to provide adequate levels of all vitamins in the major staple crops to eradicate vitamin deficiencies from the global population.

The strawberry pathogenesis-related 10 (PR-10) Fra a proteins control flavonoid biosynthesis by binding to metabolic intermediates.

Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low µm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates.

Deciphering gamma-decalactone biosynthesis in strawberry fruit using a combination of genetic mapping, RNA-Seq and eQTL analyses.

BACKGROUND: Understanding the basis for volatile organic compound (VOC) biosynthesis and regulation is of great importance for the genetic improvement of fruit flavor. Lactones constitute an essential group of fatty acid-derived VOCs conferring peach-like aroma to a number of fruits including peach, plum, pineapple and strawberry. Early studies on lactone biosynthesis suggest that several enzymatic pathways could be responsible for the diversity of lactones, but detailed information on them remained elusive. In this study, we have integrated genetic mapping and genome-wide transcriptome analysis to investigate the molecular basis of natural variation in γ-decalactone content in strawberry fruit. RESULTS: As a result, the fatty acid desaturase FaFAD1 was identified as the gene underlying the locus at LGIII-2 that controls γ-decalactone production in ripening fruit. The FaFAD1 gene is specifically expressed in ripe fruits and its expression fully correlates with the presence of γ-decalactone in all 95 individuals of the mapping population. In addition, we show that the level of expression of FaFAH1, with similarity to cytochrome p450 hydroxylases, significantly correlates with the content of γ-decalactone in the mapping population. The analysis of expression quantitative trait loci (eQTL) suggests that the product of this gene also has a regulatory role in the biosynthetic pathway of lactones. CONCLUSIONS: Altogether, this study provides mechanistic information of how the production of γ-decalactone is naturally controlled in strawberry, and proposes enzymatic activities necessary for the formation of this VOC in plants.

Eugenol production in achenes and receptacles of strawberry fruits is catalyzed by synthases exhibiting distinct kinetics.

Eugenol is a volatile that serves as an attractant for pollinators of flowers, acts as a defense compound in various plant tissues, and contributes to the aroma of fruits. Its production in a cultivated species such as strawberry (Fragaria × ananassa), therefore, is important for the viability and quality of the fruit. We have identified and functionally characterized three strawberry complementary DNAs (cDNAs) that encode proteins with high identity to eugenol synthases from several plant species. Based on a sequence comparison with the wild relative Fragaria vesca, two of these cDNAs, FaEGS1a and FaEGS1b, most likely correspond to transcripts derived from allelic gene variants, whereas the third cDNA, FaEGS2, corresponds to a different gene. Using coniferyl acetate as a substrate, FaEGS1a and FaEGS1b catalyze the in vitro formation of eugenol, while FaEGS2 catalyzes the formation of eugenol and also of isoeugenol with a lower catalytic efficiency. The expression of these genes is markedly higher in the fruit than in other tissues of the plant, with FaEGS1a and FaEGS1b mostly expressed in the green achenes, whereas FaEGS2 expression is almost restricted to the red receptacles. These expression patterns correlate with the eugenol content, which is highest in the achene at the green stage and in the receptacle at the red stage. The transient expression of the corresponding cDNAs in strawberry fruit and the subsequent volatile analyses confirm FaEGSs as genuine eugenol synthases in planta. These results provide new insights into the diversity of phenylpropene synthases in plants.

Auxin biosynthesis by the YUCCA6 flavin monooxygenase gene in woodland strawberry.

Auxin has been regarded as the main signal molecule coordinating the growth and ripening of fruits in strawberry, the reference genomic system for Rosaceae. The mechanisms regulating auxin biosynthesis in strawberry are largely elusive. Recently, we demonstrated that two YUCCA genes are involved in flower and fruit development in cultivated strawberry. Here, we show that the woodland strawberry (Fragaria vesca L.) genome harbors nine loci for YUCCA genes and eight of them encode functional proteins. Transcription pattern in different plant organs was different for all eight FvYUCs. Functionality of the FvYUC6 gene was studied in transgenic strawberry overexpressing FvYUC6, which showed typical high-auxin phenotypes. Overexpression of FvYUC6 also delayed flowering and led to complete male sterility in F. vesca. Additionally, specific repression of FvYUC6 expression by RNA interference significantly inhibited vegetative growth and reduced plant fertility. The development of leaves, roots, flowers, and fruits was greatly affected in FvYUC6-repressed plants. Expression of a subset of auxin-responsive genes was well correlated with the changes of FvYUC6 transcript levels and free indole-3-acetic acid levels in transgenic strawberry. These observations are consistent with an important role of FvYUC6 in auxin synthesis, and support a main role of the gene product in vegetative and reproductive development in woodland strawberry.

Genetic analysis of strawberry fruit aroma and identification of O-methyltransferase FaOMT as the locus controlling natural variation in mesifurane content.

Improvement of strawberry (Fragaria × ananassa) fruit flavor is an important goal in breeding programs. To investigate genetic factors controlling this complex trait, a strawberry mapping population derived from genotype '1392', selected for its superior flavor, and '232' was profiled for volatile compounds over 4 years by headspace solid phase microextraction coupled to gas chromatography and mass spectrometry. More than 300 volatile compounds were detected, of which 87 were identified by comparison of mass spectrum and retention time to those of pure standards. Parental line '1392' displayed higher volatile levels than '232', and these and many other compounds with similar levels in both parents segregated in the progeny. Cluster analysis grouped the volatiles into distinct chemically related families and revealed a complex metabolic network underlying volatile production in strawberry fruit. Quantitative trait loci (QTL) detection was carried out over 3 years based on a double pseudo-testcross strategy. Seventy QTLs covering 48 different volatiles were detected, with several of them being stable over time and mapped as major QTLs. Loci controlling γ-decalactone and mesifurane content were mapped as qualitative traits. Using a candidate gene approach we have assigned genes that are likely responsible for several of the QTLs. As a proof of concept we show that one homoeolog of the O-methyltransferase gene (FaOMT) is the locus responsible for the natural variation of mesifurane content. Sequence analysis identified 30 bp in the promoter of this FaOMT homoeolog containing putative binding sites for basic/helix-loop-helix, MYB, and BZIP transcription factors. This polymorphism fully cosegregates with both the presence of mesifurane and the high expression of FaOMT during ripening.

The Arabidopsis tetratricopeptide thioredoxin-like gene family is required for osmotic stress tolerance and male sporogenesis.

TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins are characterized by the presence of six tetratricopeptide repeats in conserved positions and a carboxyl-terminal region known as the thioredoxin-like domain with homology to thioredoxins. In Arabidopsis (Arabidopsis thaliana), the TTL gene family is composed by four members, and the founder member, TTL1, is required for osmotic stress tolerance. Analysis of sequenced genomes indicates that TTL genes are specific to land plants. In this study, we report the expression profiles of Arabidopsis TTL genes using data mining and promoter-reporter ß-glucuronidase fusions. Our results show that TTL1, TTL3, and TTL4 display ubiquitous expression in normal growing conditions but differential expression patterns in response to osmotic and NaCl stresses. TTL2 shows a very different expression pattern, being specific to pollen grains. Consistent with the expression data, ttl1, ttl3, and ttl4 mutants show reduced root growth under osmotic stress, and the analysis of double and triple mutants indicates that TTL1, TTL3, and TTL4 have partially overlapping yet specific functions in abiotic stress tolerance while TTL2 is involved in male gametophytic transmission.

Ethylene is involved in strawberry fruit ripening in an organ-specific manner.

The fruit of the strawberry Fragaria×ananassa has traditionally been classified as non-climacteric because its ripening process is not governed by ethylene. However, previous studies have reported the timely endogenous production of minor amounts of ethylene by the fruit as well as the differential expression of genes of the ethylene synthesis, reception, and signalling pathways during fruit development. Mining of the Fragaria vesca genome allowed for the identification of the two main ethylene biosynthetic genes, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Their expression pattern during fruit ripening was found to be stage and organ (achene or receptacle) specific. Strawberry plants with altered sensitivity to ethylene could be employed to unravel the role of ethylene in the ripening process of the strawberry fruit. To this end, independent lines of transgenic strawberry plants were generated that overexpress the Arabidopsis etr1-1 mutant ethylene receptor, which is a dominant negative allele, causing diminished sensitivity to ethylene. Genes involved in ethylene perception as well as in its related downstream processes, such as flavonoid biosynthesis, pectin metabolism, and volatile biosynthesis, were differently expressed in two transgenic tissues, the achene and the receptacle. The different transcriptional responsiveness of the achene and the receptacle to ethylene was also revealed by the metabolic profiling of the primary metabolites in these two organs. The free amino acid content was higher in the transgenic lines compared with the control in the mature achene, while glucose and fructose, and citric and malic acids were at lower levels. In the receptacle, the most conspicuous change in the transgenic lines was the depletion of the tricarboxylic acid cycle intermediates at the white stage of development, most probably as a consequence of diminished respiration. The results are discussed in the context of the importance of ethylene during strawberry fruit ripening.

Purification, crystallization and preliminary X-ray analysis of the strawberry allergens Fra a 1E and Fra a 3 in the presence of catechin.

The strawberry Fra a proteins belong to the pathogenesis-related PR-10 protein family and share a common fold with the Bet v 1 major pollen allergen and the START/PYR/PYL proteins, which are characterized by the presence of a central cavity and are often involved in the binding of a variety of natural compounds. The Fra a proteins play a key role in the control of flavonoid biosynthesis in strawberries and are essential for pigment formation in fruits. In order to understand Fra a protein function, full-length Fra a 1E and Fra a 3 cDNAs were cloned and expressed in Escherichia coli, and the proteins were purified to homogeneity using metal-affinity chromatography. Diffraction-quality crystals of Fra a 1E and of Fra a 3 in the presence of (+)-catechin were obtained by the sitting-drop vapour-diffusion method. X-ray diffraction data from single crystals of Fra a 1E and Fra a 3 were processed to 2.2 and 3.0 Šresolution in space groups P212121 and P2221, with unit-cell parameters a = 70.02, b = 74.42, c = 84.04 Šand a = 137.91, b = 206.61, c = 174.7 Šfor Fra a 1E and Fra a 3, respectively.

Genetic and genome-wide transcriptomic analyses identify co-regulation of oxidative response and hormone transcript abundance with vitamin C content in tomato fruit.

BACKGROUND: L-ascorbic acid (AsA; vitamin C) is essential for all living plants where it functions as the main hydrosoluble antioxidant. It has diverse roles in the regulation of plant cell growth and expansion, photosynthesis, and hormone-regulated processes. AsA is also an essential component of the human diet, being tomato fruit one of the main sources of this vitamin. To identify genes responsible for AsA content in tomato fruit, transcriptomic studies followed by clustering analysis were applied to two groups of fruits with contrasting AsA content. These fruits were identified after AsA profiling of an F8 Recombinant Inbred Line (RIL) population generated from a cross between the domesticated species Solanum lycopersicum and the wild relative Solanum pimpinellifollium. RESULTS: We found large variability in AsA content within the RIL population with individual RILs with up to 4-fold difference in AsA content. Transcriptomic analysis identified genes whose expression correlated either positively (PVC genes) or negatively (NVC genes) with the AsA content of the fruits. Cluster analysis using SOTA allowed the identification of subsets of co-regulated genes mainly involved in hormones signaling, such as ethylene, ABA, gibberellin and auxin, rather than any of the known AsA biosynthetic genes. Data mining of the corresponding PVC and NVC orthologs in Arabidopis databases identified flagellin and other ROS-producing processes as cues resulting in differential regulation of a high percentage of the genes from both groups of co-regulated genes; more specifically, 26.6% of the orthologous PVC genes, and 15.5% of the orthologous NVC genes were induced and repressed, respectively, under flagellin22 treatment in Arabidopsis thaliana. CONCLUSION: Results here reported indicate that the content of AsA in red tomato fruit from our selected RILs are not correlated with the expression of genes involved in its biosynthesis. On the contrary, the data presented here supports that AsA content in tomato fruit co-regulates with genes involved in hormone signaling and they are dependent on the oxidative status of the fruit.

Two strawberry miR159 family members display developmental-specific expression patterns in the fruit receptacle and cooperatively regulate Fa-GAMYB.

• We have reported previously that the gibberellin (GA) content in strawberry receptacle is high, peaking at specific stages, pointing to a role of this hormone in fruit development. In Arabidopsis, miR159 levels are dependent on GA concentration. This prompted us to investigate the role of two members of the miR159 family and their putative strawberry target gene, GAMYB, in relation to changes in GA content during the course of fruit development. • The highest expression level of the two Fa-MIR159 genes was in the fruit's receptacle tissue, with dramatic changes observed throughout development. The lowest levels of total mature miR159 (a and b) were observed during the white stage of receptacle development, which was concurrent with the highest expression of Fa-GAMYB. A functional interaction between miR159 and Fa-GAMYB has been demonstrated in receptacle tissue. • The application of bioactive GA (i.e. GA(3) ) to strawberry plants caused the down-regulated expression of Fa-MIR159a, but the expression of Fa-MIR159b was not affected significantly. Clear discrepancies between Fa-MIR159b and mature Fa-miR159b levels were indicative of post-transcriptional regulation of Fa-MIR159b gene expression. • We propose that Fa-miR159a and Fa-miR159b interact with Fa-GAMYB during the course of strawberry receptacle development, and that they act in a cooperative fashion to respond, in part, to changes in GA endogenous levels.

Gibberellin biosynthesis and signalling during development of the strawberry receptacle.

The enlargement of receptacle cells during strawberry (Fragaria × ananassa) fruit development is a critical factor determining fruit size, with the increase in cell expansion being one of the most important physiological processes regulated by the phytohormone gibberellin (GA). Here, we studied the role of GA during strawberry fruit development by analyzing the endogenous content of bioactive GAs and the expression of key components of GA signalling and metabolism. Bioactive GA(1) , GA(3) and GA(4) were monitored during fruit development, with the content of GA(4) being extremely high in the receptacle, peaking at the white stage of development. •Genes with high homology to genes encoding GA pathway components, including receptors (FaGID1(GIBBERELLIN-INSENSITIVE DWARF1)b and FaGID1c), DELLA (FaRGA(REPRESSOR OF GA) and FaGAI(GA-INSENSITIVE)), and enzymes involved in GA biosynthesis (FaGA3ox) and catabolism (FaGA2ox), were identified, and their expression in different tissues and developmental stages of strawberry fruit was studied in detail. The expression of all of these genes showed a stage-specific pattern during fruit development and was highest in the receptacle. FaGID1c bound GA in vitro, interacted with FaRGA in vitro and in vivo, and increased GA responses when ectopically expressed in Arabidopsis. This study thus reveals key elements of GA responses in strawberry and points to a critical role for GA in the development of the receptacle.

Regulation of L-ascorbic acid content in strawberry fruits.

Plants have several L-ascorbic acid (AsA) biosynthetic pathways, but the contribution of each one to the synthesis of AsA varyies between different species, organs, and developmental stages. Strawberry (Fragaria×ananassa) fruits are rich in AsA. The pathway that uses D-galacturonate as the initial substrate is functional in ripe fruits, but the contribution of other pathways to AsA biosynthesis has not been studied. The transcription of genes encoding biosynthetic enzymes such as D-galacturonate reductase (FaGalUR) and myo-inositol oxygenase (FaMIOX), and the AsA recycling enzyme monodehydroascorbate reductase (FaMDHAR) were positively correlated with the increase in AsA during fruit ripening. Fruit storage for 72 h in a cold room reduced the AsA content by 30%. Under an ozone atmosphere, this reduction was 15%. Ozone treatment increased the expression of the FaGalUR, FaMIOX, and L-galactose-1-phosphate phosphatase (FaGIPP) genes, and transcription of the L-galactono-1,4-lactone dehydrogenase (FaGLDH) and FAMDHAR genes was higher in the ozone-stored than in the air-stored fruits. Analysis of AsA content in a segregating population from two strawberry cultivars showed high variability, which did not correlate with the transcription of any of the genes studied. Study of GalUR protein in diverse cultivars of strawberry and different Fragaria species showed that a correlation between GalUR and AsA content was apparent in most cases, but it was not general. Three alleles were identified in strawberry, but any sequence effect on the AsA variability was eliminated by analysis of the allele-specific expression. Taken together, these results indicate that FaGalUR shares the control of AsA levels with other enzymes and regulatory elements in strawberry fruit.

Demethylation of oligogalacturonides by FaPE1 in the fruits of the wild strawberry Fragaria vesca triggers metabolic and transcriptional changes associated with defence and development of the fruit.

Ectopic expression of the strawberry (Fragaria×ananassa) gene FaPE1 encoding pectin methyl esterase produced in the wild species Fragaria vesca partially demethylated oligogalacturonides (OGAs), which conferred partial resistance of ripe fruits to the fungus Botrytis cinerea. Analyses of metabolic and transcriptional changes in the receptacle of the transgenic fruits revealed channelling of metabolites to aspartate and aromatic amino acids as well as phenolics, flavanones, and sesquiterpenoids, which was in parallel with the increased expression of some genes related to plant defence. The results illustrate the changes associated with resistance to B. cinerea in the transgenic F. vesca. These changes were accompanied by a significant decrease in the auxin content of the receptacle of the ripe fruits of transgenic F. vesca, and enhanced expression of some auxin-repressed genes. The role of these OGAs in fruit development was revealed by the larger size of the ripe fruits in transgenic F. vesca. When taken together these results show that in cultivated F. ananassa FaPE1 participates in the de-esterification of pectins and the generation of partially demethylated OGAs, which might reinforce the plant defence system and play an active role in fruit development.