Optimizing the Extraction of the Polyphenolic Fraction from Defatted Strawberry Seeds for Tiliroside Isolation Using Accelerated Solvent Extraction Combined with a Box-Behnken Design.

Tiliroside is a natural polyphenolic compound with a wide range of biological activity, and defatted strawberry seeds are its rich source. The goal of this study was to optimize accelerated solvent extraction (ASE) conditions, including temperature, solvent composition, and the number of extraction cycles, using Box-Behnken design to maximize the yield of tiliroside. UPLC-DAD-MS was applied to investigate the polyphenolic composition of the extracts, and preparative liquid chromatography (pLC) was used for isolation. All obtained mathematical models generally showed an increase in the efficiency of isolating polyphenolic compounds with an increase in temperature, ethanol content, and the number of extraction cycles. The optimal established ASE conditions for tiliroside were as follows: a temperature of 65 °C, 63% ethanol in water, and four extraction cycles. This allowed for the obtainment of a tiliroside-rich fraction, and the recovery of isolated tiliroside from plant material reached 243.2 mg from 100 g. Our study showed that ASE ensures the isolation of a tiliroside-rich fraction with high effectiveness. Furthermore, defatted strawberry seeds proved to be a convenient source of tiliroside because the matrix of accompanying components is relatively poor, which facilitates separation.

Genome-Wide Identification and Abiotic Stress Response Analysis of PP2C Gene Family in Woodland and Pineapple Strawberries.

Protein phosphatase 2C (PP2C) is a negative regulator of serine/threonine residue protein phosphatase and plays an important role in abscisic acid (ABA) and abiotic-stress-mediated signaling pathways in plants. The genome complexity of woodland strawberry and pineapple strawberry is different due to the difference in chromosome ploidy. This study conducted a genome-wide investigation of the FvPP2C (Fragaria vesca) and FaPP2C (Fragaria ananassa) gene family. Fifty-six FvPP2C genes and 228 FaPP2C genes were identified from the woodland strawberry and pineapple strawberry genomes, respectively. FvPP2Cs were distributed on seven chromosomes, and FaPP2Cs were distributed on 28 chromosomes. The size of the FaPP2C gene family was significantly different from that of the FvPP2C gene family, but both FaPP2Cs and FvPP2Cs were localized in the nucleus, cytoplasm, and chloroplast. Phylogenetic analysis revealed that 56 FvPP2Cs and 228 FaPP2Cs could be divided into 11 subfamilies. Collinearity analysis showed that both FvPP2Cs and FaPP2Cs had fragment duplication, and the whole genome duplication was the main cause of PP2C gene abundance in pineapple strawberry. FvPP2Cs mainly underwent purification selection, and there were both purification selection and positive selection effects in the evolution of FaPP2Cs. Cis-acting element analysis found that the PP2C family genes of woodland and pineapple strawberries mainly contained light responsive elements, hormone responsive elements, defense and stress responsive elements, and growth and development-related elements. The results of quantitative real-time PCR (qRT-PCR) showed that the FvPP2C genes showed different expression patterns under ABA, salt, and drought treatment. The expression level of FvPP2C18 was upregulated after stress treatment, which may play a positive regulatory role in ABA signaling and abiotic stress response mechanisms. This study lays a foundation for further investigation on the function of the PP2C gene family.

First report of a new bacterial stem rot disease of strawberry (Fragaria×ananassa) caused by Pantoea ananatis in Jiangsu, China.

Strawberry (Fragaria×ananassa Duch.) is an important economic fruit crop in the world. With the continuous expansion of strawberry planting area, strawberry disease is one of the most important limiting factors, which seriously affects the agronomic performance and leads to significant economic losses. In November 2020, an infected stem rot disease of strawberries was detected in the strawberry growing area of Donghai County, Jiangsu Province, China. The disease incidence ranged from 30 % to 45 %. Initially, infected plants included stunted growth of new leaves, leaflet asymmetry, and holes in the vertical section of the stem, resulting in leaf blight and death in severe cases. To isolate the pathogen, two symptomatic plants were randomly collected. And then infected plants were surface sterilized with 75 % ethanol for 1 min, followed by 2 % sodium hypochlorite for 6 minutes. After that, the infected plants were washed 4-5 times with double sterilized distilled water, cut into 3-5 mm small pieces, and soaked in 2 ml of sterile water for 15 min, after which 100 µl of liquid suspension were spread onto Luria-Bertani medium (LB) and incubated at 28 °C for 12-16 h. All isolates showed yellow, viscous, round, and smooth (Figure S1, C) and the isolates were designated as JX1 and JX2. To identify the pathogen, the genomic DNA were extracted from isolates using the Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, China) and the fragments of gyrB, rpoB and leuS gene were amplified using the primer pairs UP-1S/UP-2Sr (Yamamoto and Harayama 1995), rpoB-F/rpoB-R and leuS-F/leuS-R (Yu et al. 2022), respectively. Sequence analyses showed that the nucleotide sequences of gyrB, rpoB, and leuS fragments of the isolates shared 99.72 %, 99.67 % and 98.37 % identity with the Pantoea ananatis type strain LMG 2665 (KF482590.1, EF988972.1 and KF482626.1, respectively ), which suggests that the isolate could be Pantoea ananatis. To further verify that P. ananatis was identity of these isolates, the whole genome was sequenced using PacBio sequel II technology. The Average Nucleotide Identity (ANI) calculation showed that the whole-genome sequence was 99.0% similar to that of the Pantoea ananatis type strain LMG 2665 (Jain et al. 2018). The isolates were therefore recognized as P. ananatis. To confirm pathogenicity, roots of strawberry plants were inoculated by wounding as described (Wang et al. 2017) with bacterial suspensions (108 CFU/ml) for 30 min, and transplanted into 10 cm ×8.5 cm pots filled with substrate (peat: perlite: vermiculite =3:1:1). The negative control plants were inoculated with sterile distilled water (20 individual plants per group). All infected plants were placed in a greenhouse under the following environmental conditions: 30 ℃/25 ℃ day/night, >70 % relative humidity, 16-h/8-h photoperiod. The experiment was carried out three times. After 3 to 4 weeks of inoculation, the new leaves of the plants were smaller and asymmetrical, while the negative plants remained healthy. After 8 weeks, a significant stem rot pocket developed on all inoculated plants, similar to the symptoms observed in the field. In contrast, no symptoms were observed in negative plants (Figure S2). To fulfill Koch's postulates bacteria were further isolated, purified and identified from the greenhouse inoculated plants. The results proved that the causative agent of strawberry stem rot was P. ananatis. In recent decades, P. ananatis has been found to cause bacterial leaf blight in strawberries (Bajpai et al. 2020). It has also caused other crop diseases, such as maize white spot, peach soft rot and others (Cui et al.2022; Liao et al. 2015). Although other crop diseases caused by P. ananatis, a bacterial pathogen, there has been no report of P. ananatis causing the symptoms of stem rot disease in strawberry. To our knowledge, this is the first report of P. ananatis causing stem rot in strawberry. This study provides solid evidence that strawberry stem rot disease in China can also be caused by the novel pathogen Pantoea ananatis. In conclusion, this report will provide a theoretical reference for the prevention and control measures of P. ananatis causing strawberry stem rot disease in the future.

Non-destructive prediction of total soluble solids in strawberry using near infrared spectroscopy.

BACKGROUND: Near-infrared spectroscopy (NIRS) is considered to be a fast and reliable non-destructive technique for fruit analysis. Considering that consumers are looking for strawberries with good sweetness, texture, and appearance, producers need to effectively measure the ripeness stage of strawberries to guarantee their final quality. Therefore, the use of this technique can contribute to decreasing the high level of waste and delivering good ripe strawberries to consumers. The present study aimed to evaluate the predictive capacity of NIRS technology, as a possible alternative to conventional methodology, for the analysis of the main organoleptic parameters of strawberries (Fragaria × ananassa Duch.). RESULTS: Spectroscopic measurements and physicochemical analyses [total soluble solids (TSS), titratable acidity, colour, texture] of 'Victory' strawberries were carried out. The predictive models developed for titratable acidity, colour and texture were not good enough to quantify those parameters. By contrast, in the NIRS quantitative prediction analysis of TSS, it was observed that the spectral pre-treatment with the highest predictive capacity was the first derivative 1-5-5. The coefficients of determination were: 0.9277 for the calibration model; 0.5755 for the validation model; and 0.8207 for the prediction model, using a seven-factor partial least squares multivariate regression analysis. CONCLUSION: Therefore, these results demonstrate that NIR analysis could be used to predict the TSS in strawberry, and further work on sampling is desirable to improve the prediction obtained in the present study. It is shown that NIRS technology is a suitable tool for determining quality attributes of strawberry in a fast, economic, and environmentally friendly way. © 2022 Society of Chemical Industry.

Phytophthora Crown Rot of Florida Strawberry: Inoculum Sources and Thermotherapy of Transplants for Disease Management.

Phytophthora crown rot, caused mainly by Phytophthora cactorum but also by P. nicotianae, reported in 2018, is an important disease in the Florida strawberry annual production system. Mefenoxam is the most effective and widely used fungicide to manage this disease. However, because of pathogen resistance, alternatives to chemical control are needed. Phytophthora spp. were rarely recovered during the summer from soil of commercial farms where the disease was observed during the season. In a more detailed survey on research plots, neither of the two species was recovered 1 month after the crop was terminated and water was shut off. Therefore, Phytophthora spp. does not seem to survive in the soil over summer in Florida. In a field trial, asymptomatic nursery transplants harboring quiescent infections were confirmed as the major source of inoculum for these pathogens in Florida. Heat treatment of P. cactorum zoospores at 44°C for as little as 5 min was effective in inhibiting germination and colony formation; however, oospore germination was not inhibited by any of the tested temperatures in vitro. In the field, thermotherapy treatment of inoculated plants was shown to have great potential to serve as a nonchemical approach for managing Phytophthora crown rot in production fields and reducing mefenoxam-resistant populations in nursery transplants.

Optimising anthocyanin extraction from strawberry fruits using response surface methodology and application in yoghurt as natural colorants and antioxidants.

The aim of the present study was to optimise the extraction conditions of anthocyanins from strawberry fruits and incorporate them in yoghurt to achieve a natural coloration as well as enrich the product with antioxidants. The response surface methodology (RSM) based on Box-Behnken design was studied to assess the influence of the three factors being agitation speed (400-800 rpm), sample to solvent ratio (0.5-2 g/40 mL), and extraction time (1-15 min) on total anthocyanin content and antioxidant activity of strawberries. According to the results, the linear, quadratic and interaction effects of the studied factors on total anthocyanin content and antioxidant activity were determined by the response surface methodology, and the optimal conditions for anthocyanin extraction were 586 rpm for agitation speed, 1.26 g/40 mL for sample to solvent ratio, and 9.36 min for extraction time. Under these extraction conditions, the total anthocyanin content and antioxidant activity recorded by the two validated models were 38.04 mg C3GE/100 g FW and 21.38 mg AAE/100 g FW, respectively. The enriched natural yoghurt contains anthocyanins with a content of 36.50 µg C3GE/100 g and an antioxidant activity of 21.22 µg AAE/100 g. The anthocyanin enriched yoghurt developed in this study may be considered as a functional food with an interesting source of natural antioxidants, and these anthocyanins can substitute synthetic (industrial) colorants.

Higher expression of the strawberry xyloglucan endotransglucosylase/hydrolase genes FvXTH9 and FvXTH6 accelerates fruit ripening.

Fruit softening in Fragaria (strawberry) is proposed to be associated with the modification of cell wall components such as xyloglucan by the action of cell wall-modifying enzymes. This study focuses on the in vitro and in vivo characterization of two recombinant xyloglucan endotransglucosylase/hydrolases (XTHs) from Fragaria vesca, FvXTH9 and FvXTH6. Mining of the publicly available F. vesca genome sequence yielded 28 putative XTH genes. FvXTH9 showed the highest expression level of all FvXTHs in a fruit transcriptome data set and was selected with the closely related FvXTH6 for further analysis. To investigate their role in fruit ripening in more detail, the coding sequences of FvXTH9 and FvXTH6 were cloned into the vector pYES2 and expressed in Saccharomyces cerevisiae. FvXTH9 and FvXTH6 displayed xyloglucan endotransglucosylase (XET) activity towards various acceptor substrates using xyloglucan as the donor substrate. Interestingly, FvXTH9 showed activity of mixed-linkage glucan:xyloglucan endotransglucosylase (MXE) and cellulose:xyloglucan endotransglucosylase (CXE). The optimum pH of both FvXTH9 and FvXTH6 was 6.5. The prediction of subcellular localization suggested localization to the secretory pathway, which was confirmed by localization studies in Nicotiana tabacum. Overexpression showed that Fragaria × ananassa fruits infiltrated with FvXTH9 and FvXTH6 ripened faster and showed decreased firmness compared with the empty vector control pBI121. Thus FvXTH9 and also FvXTH6 might promote strawberry fruit ripening by the modification of cell wall components.

Transcriptome analysis of strawberry fruit in response to exogenous arginine.

MAIN CONCLUSION: Transcriptome and physiological analysis showed that exogenous arginine can delay the ripening process of postharvest strawberry fruit. Arginine (Arg) plays an important role in the growth and development of plants, but its growth and development regulatory mechanisms in strawberry fruit are unknown. In this study, we found that the content of Arg decreased after the onset of fruit coloration and exogenous Arg inhibited fruit coloration. We comprehensively analyzed the transcriptome of 'Sweet Charlie' strawberry fruit with or without Arg treatment and identified a large number of differential genes and metabolites. Based on the transcriptome data, we also found that Arg inhibited ripening, which coincided with changes in several physiological parameters and their corresponding gene transcripts, including firmness, anthocyanin content, sugar content, Arg content, indole-acetic acid (IAA) content, abscisic acid (ABA) content, and ethylene emissions. We also found that Arg induced the expression of heat-shock proteins (HSPs) and antioxidant enzyme genes, which improved strawberry stress resistance. This study elucidated the molecular mechanism by which exogenous Arg delays strawberry fruit ripening, providing some genetic information to help guide the future improvement and cultivation of strawberry.

The control of red colour by a family of MYB transcription factors in octoploid strawberry (Fragaria × ananassa) fruits.

Octoploid strawberry (Fragaria × ananassa Duch.) is a model plant for research and one of the most important non-climacteric fruit crops throughout the world. The associations between regulatory networks and metabolite composition were explored for one of the most critical agricultural properties in octoploid strawberry, fruit colour. Differences in the levels of flavonoids are due to the differences in the expression of structural and regulatory genes involved in flavonoid biosynthesis. The molecular mechanisms underlying differences in fruit colour were compared between red and white octoploid strawberry varieties. FaMYB genes had combinatorial effects in determining the red colour of fruit through the regulation of flavonoid biosynthesis in response to the increase in endogenous ABA at the final stage of fruit development. Analysis of alleles of FaMYB10 and FaMYB1 in red and white strawberry varieties led to the discovery of a white-specific variant allele of FaMYB10, FaMYB10-2. Its coding sequence possessed an ACTTATAC insertion in the genomic region encoding the C-terminus of the protein. This insertion introduced a predicted premature termination codon, which suggested the loss of intact FaMYB10 protein playing a critical role in the loss of red colour in white octoploid strawberry.

Genetic modulation of RAP alters fruit coloration in both wild and cultivated strawberry.

Fruit colour affects consumer preference and is an important trait for breeding in strawberry. Previously, we isolated the Reduced Anthocyanins in Petioles (RAP) gene encoding a glutathione S-transferase (GST) that binds anthocyanins to facilitate their transport from cytosol to vacuole in the diploid strawberry Fragaria vesca. The parent of rap was the F. vesca variety 'Yellow Wonder' that develops white fruit due to a natural mutation in the FveMYB10 gene. Here, we investigated the application potential of RAP in modulating fruit colours by overexpression of RAP in F. vesca and knockout of RAP in the cultivated strawberry Fragaria × ananassa. Unexpectedly, the RAP overexpression in Yellow Wonder background caused formation of red fruit. In addition, the red coloration occurs precociously at floral stage 10 and continues in the receptacle during early fruit development. Transcriptome analysis revealed that the anthocyanin biosynthesis genes were not up-regulated in RAP-ox; rap myb10 flowers at anthesis and largely inhibited at the turning stage in fruit, suggesting a coloration mechanism independent of FveMYB10. Moreover, we used CRISPR/Cas9 to knockout RAP in cultivated strawberry which is octoploid. Six copies of RAP were simultaneously knocked out in the T0 generation leading to the green stem and white-fruited phenotype. Several T1 progeny have segregated away the CRISPR/Cas9 transgene but maintain the green stem trait. Our results indicate that enhancing the anthocyanin transport could redirect the metabolic flux from proanthocyanidin to anthocyanin production at early developmental stages of fruit and that RAP is one promising candidate gene in fruit colour breeding of strawberry.

Elucidating the role of polygalacturonase genes in strawberry fruit softening.

To disentangle the role of polygalacturonase (PG) genes in strawberry softening, the two PG genes most expressed in ripe receptacles, FaPG1 and FaPG2, were down-regulated. Transgenic ripe fruits were firmer than those of the wild type when PG genes were silenced individually. Simultaneous silencing of both PG genes by transgene stacking did not result in an additional increase in firmness. Cell walls from ripe fruits were characterized by a carbohydrate microarray. Higher signals of homogalacturonan and rhamnogalacturonan I pectin epitopes in polysaccharide fractions tightly bound to the cell wall were observed in the transgenic genotypes, suggesting a lower pectin solubilization. At the transcriptomic level, the suppression of FaPG1 or FaPG2 alone induced few transcriptomic changes in the ripe receptacle, but the amount of differentially expressed genes increased notably when both genes were silenced. Many genes encoding cell wall-modifying enzymes were down-regulated. The expression of a putative high affinity potassium transporter was induced in all transgenic genotypes, indicating that cell wall weakening and loss of cell turgor could be linked. These results suggest that, besides the disassembly of pectins tightly linked to the cell wall, PGs could play other roles in strawberry softening, such as the release of oligogalacturonides exerting a positive feedback in softening.

RNAi-mediated endogene silencing in strawberry fruit: detection of primary and secondary siRNAs by deep sequencing.

RNA interference (RNAi) has been exploited as a reverse genetic tool for functional genomics in the nonmodel species strawberry (Fragaria × ananassa) since 2006. Here, we analysed for the first time different but overlapping nucleotide sections (>200 nt) of two endogenous genes, FaCHS (chalcone synthase) and FaOMT (O-methyltransferase), as inducer sequences and a transitive vector system to compare their gene silencing efficiencies. In total, ten vectors were assembled each containing the nucleotide sequence of one fragment in sense and corresponding antisense orientation separated by an intron (inverted hairpin construct, ihp). All sequence fragments along the full lengths of both target genes resulted in a significant down-regulation of the respective gene expression and related metabolite levels. Quantitative PCR data and successful application of a transitive vector system coinciding with a phenotypic change suggested propagation of the silencing signal. The spreading of the signal in strawberry fruit in the 3' direction was shown for the first time by the detection of secondary small interfering RNAs (siRNAs) outside of the primary targets by deep sequencing. Down-regulation of endogenes by the transitive method was less effective than silencing by ihp constructs probably because the numbers of primary siRNAs exceeded the quantity of secondary siRNAs by three orders of magnitude. Besides, we observed consistent hotspots of primary and secondary siRNA formation along the target sequence which fall within a distance of less than 200 nt. Thus, ihp vectors seem to be superior over the transitive vector system for functional genomics in strawberry fruit.

Protective Effect of Fragaria ananassa Crude Extract on Cadmium-Induced Lipid Peroxidation, Antioxidant Enzymes Suppression, and Apoptosis in Rat Testes.

Cadmium is a deleterious environmental pollutant that threats both animals and human health. Oxidative stress and elevated levels of reactive oxygen species (ROS) have recently been reported to be the main cause of cellular damage as a result of cadmium exposure. We investigate, here, the protective effect of strawberry crude extracts on cadmium-induced oxidative damage of testes in rats. Four groups (n = 8) of 32 adult male Wistar rats weighing 160-180 g were used. The control group received 0.9% saline solution all over the experimental period (5 days). Group 2 was intraperitoneally injected with 6.5 mg/kg CdCl2. Group 3 was provided only with an oral administration of strawberry methanolic extract (SME) at a dose of 250 mg/kg. Group 4 was treated with SME before cadmium injection with the same mentioned doses. It was shown that cadmium exposure results in a significant decrease in both relative testicular weight and serum testosterone level. Analyzing the oxidative damaging effect of cadmium on the testicular tissue revealed the induction of oxidative stress markers represented in the elevated level of lipid peroxidation (LPO), nitric oxide (NO), and a decrease in the reduced glutathione (GSH) content. Considering cadmium toxicity, the level of the antioxidant enzyme activities including catalase (CAT), superoxide dismutase (SOD2), glutathione peroxidase (GPx1), and glutathione reductase (GR) were markedly decreased. Moreover, gene expression analysis indicated significant upregulation of the pro-apoptotic proteins, bcl-2-associated-X-protein (BAX), and tumor necrosis factor-α (TNFA) in response to cadmium intoxication, while significant downregulation of the anti-apoptotic, B-cell lymphoma 2 (BCL2) gene was detected. Immunohistochemistry of the testicular tissue possessed positive immunostaining for the increased level of TNF-α, but decreased number of proliferating cell nuclear antigen (PCNA) stained cells. Administration of SME debilitated the deleterious effect of cadmium via reduction of both LPO and NO levels followed by a significant enhancement in the gene expression level of CAT, SOD2, GPX1, GR, nuclear factor-erythroid 2-related factor 2 (NFE2L2), heme oxygenase-1 (HMOX1), Bcl-2, and PCNA. In addition, the SME treated group revealed a significant increase in the level of testosterone and GSH accompanied by a marked decrease in the gene expression level of Bax and TNF-α. In terms of the summarized results, the SME of Fragaria ananassa has a protective effect against cadmium-induced oxidative damage of testes.

Iodide and iodate effects on the growth and fruit quality of strawberry.

BACKGROUND: Iodine deficiency is an environmental health problem affecting one-third of the global population. An iodine biofortification hydroponic experiment was conducted to explore the iodide and iodate uptake characteristics of strawberry plants, to measure the dosage effects of iodine on plant growth and to evaluate the influence of I- or IO3- application on fruit quality. RESULTS: After biofortification, the iodine contents of the fresh strawberry fruits were 600-4000 µg kg-1 , covering the WHO dietary iodine allowance of 150 µg · day-1 for adults. The iodine uptake of the strawberry plants increased with increasing I- or IO3- concentration of the culture solution. At the same iodine concentration, the iodate uptakes of various plant organs under I- treatments were apparently more than those under IO3- treatments. Low-level exogenous iodine (I- ≤ 0.25 mg L-1 or IO3- ≤ 0.50 mg L-1 ) not only promoted plant growth and increased biomass per plant, but also improved fruit quality by enhancing the vitamin C and soluble sugar contents of the strawberry fruits. Nevertheless, excessive exogenous iodine inhibited plant growth and reduced biomass per plant. IO3- uptake apparently increased the total acidity and nitrate content of the fruits, reducing the quality of the strawberry fruits. Conversely, I- uptake obviously decreased the total acidity and nitrate content of the strawberry fruits, improving the fruit quality. CONCLUSION: The strawberry can be used as a target crop for iodine biofortification. Furthermore, applying an appropriate dose of KI can improve the fruit quality of the strawberry plants. © 2016 Society of Chemical Industry.

Protective effect of Fragaria ananassa methanolic extract on cadmium chloride (CdCl2)-induced hepatotoxicity in rats.

This study investigated the protective effect of Fragaria ananassa methanolic extract on cadmium chloride (CdCl2)-induced hepatotoxicity in rats. CdCl2 was intraperitoneally injected at a dose of 6.5 mg/kg of body weight for 5 d with or without methanol extract of Fragaria ananassa (250 mg/kg). The hepatic cadmium concentration, lipid peroxidation, nitric oxide, glutathione (GSH) content, and antioxidant enzyme activities, including superoxide dismutase, catalase (CAT), GSH peroxidase, and GSH reductase, were estimated. CdCl2 injection induced a significant elevation in cadmium concentration, lipid peroxidation, and nitric oxide and caused a significant depletion in GSH content compared to controls, along with a remarkable decrease in antioxidant enzymes. Oxidative stress induction and cadmium accumulation in the liver were successfully ameliorated by F. ananassa (strawberry) pre-administration. In addition, the pre-administration of strawberry decreased the elevated gene expression of the pro-apoptotic Bax gene as well as the protein expression of caspases-3 in the liver of CdCl2-injected rats. In addition, the reduced gene expression of anti-apoptotic Bcl-2 was increased. Our results show an increase in the expression of tumor necrosis factor α in the liver of rats treated with cadmium. In sum, our results suggested that F. ananassa successfully prevented deleterious effects on liver function by reinforcing the antioxidant defense system, inhibiting oxidative stress and reducing apoptosis.

Transcriptome profiling of postharvest strawberry fruit in response to exogenous auxin and abscisic acid.

MAIN CONCLUSION: Auxin and abscisic acid regulate strawberry fruit ripening and senescence through cross-talk of their signal transduction pathways that further modulate the structural genes related to physico-chemical properties of fruit. The physiological and transcriptomic changes in harvested strawberry fruits in responses to IAA, ABA and their combination were analyzed. Exogenous IAA delayed the ripening process of strawberries after harvest while ABA promoted the postharvest ripening. However, treatment with a combination of IAA and ABA did not slow down nor accelerate the postharvest ripening in the strawberry fruits. At the molecular level, exogenous IAA up regulated the expressions of genes related to IAA signaling, including AUX/IAA, ARF, TOPLESS and genes encoding E3 ubiquitin protein ligase and annexin, and down regulated genes related to pectin depolymerization, cell wall degradation, sucrose and anthocyanin biosyntheses. In contrast, exogenous ABA induced genes related to fruit softening, and genes involved in signaling pathways including SKP1, HSPs, CK2, and SRG1. Comparison of transcriptomes in responses to individual treatments with IAA or ABA or the combination revealed that there were cooperative and antagonistic actions between IAA and ABA in fruit. However, 17% of the differentially expressed unigenes in response to the combination of IAA and ABA were unique and were not found in those unigenes responding to either IAA or ABA alone. The analyses also found that receptor-like kinases and ubiquitin ligases responded to both IAA and ABA, which seemed to play a pivotal role in both hormones' signaling pathways and thus might be the cross-talk points of both hormones.

Involvement of three annexin genes in the ripening of strawberry fruit regulated by phytohormone and calcium signal transduction.

KEY MESSAGE: Three annexin genes may be involved in the ripening progress of strawberry fruit. Phytohormones and calcium regulate the expressions of three annexin genes during strawberry fruit ripening. Plant annexins are multi-functional membrane- and Ca(2+)-binding proteins that are involved in various developmental progresses and stress responses. Three annexins FaAnn5a, FaAnn5b and FaAnn8 cDNA obtained from strawberry fruit encode amino acid sequences of approximately 35 kDa containing four annexin repeats, Ca(2+)-binding site, GTP-binding motif, peroxidase residue, and conserved amino acid residues of tryptophan, arginine and cysteine. During fruit development, the transcript levels of FaAnn5a and FaAnn5b increased while FaAnn5b declined after 3/4R stage. The expression patterns of annexins suggested their potential roles in strawberry fruit development and ripening. Expressions of annexin genes were also highly correlated with hormone levels. In addition, exogenous abscisic acid (ABA) enhanced the expressions of FaAnn5a and FaAnn8 while exogenous auxin (IAA) retarded it. However, both ABA and IAA promoted the transcript levels of FaAnn5b, indicating the independent regulation of annexins in fruit likely due to multi-functions of their large family. The responses of annexin genes to exogenous ABA and IAA inhibitors verified the involvement of annexins in plant hormone signaling. Besides, calcium restrained the expressions of FaAnn5s (FaAnn5a and FaAnn5b) but promoted the expression of FaAnn8. Effects of calcium and ethylene glycol tetraacetic acid (EGTA) on the transcript levels of annexins confirmed that calcium likely mediated hormone signal transduction pathways, which helped to elucidate the mechanism of calcium in fruit ripening. Therefore, FaAnn5s and FaAnn8 might be involved in plant hormones' regulation in the development and ripening of strawberry fruit through calcium signaling in the downstream.

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.

Phosphorus and iron deficiencies induce a metabolic reprogramming and affect the exudation traits of the woody plant Fragaria×ananassa.

Strawberries are a very popular fruit among berries, for both their commercial and economic importance, but especially for their beneficial effects for human health. However, their bioactive compound content is strictly related to the nutritional status of the plant and might be affected if nutritional disorders (e.g. Fe or P shortage) occur. To overcome nutrient shortages, plants evolved different mechanisms, which often involve the release of root exudates. The biochemical and molecular mechanisms underlying root exudation and its regulation are as yet still poorly known, in particular in woody crop species. The aim of this work was therefore to characterize the pattern of root exudation of strawberry plants grown in either P or Fe deficiency, by investigating metabolomic changes of root tissues and the expression of genes putatively involved in exudate extrusion. Although P and Fe deficiencies differentially affected the total metabolism, some metabolites (e.g. raffinose and galactose) accumulated in roots similarly under both conditions. Moreover, P deficiency specifically affected the content of galactaric acid, malic acid, lysine, proline, and sorbitol-6-phosphate, whereas Fe deficiency specifically affected the content of sucrose, dehydroascorbic acid, galactonate, and ferulic acid. At the same time, the citrate content did not change in roots under both nutrient deficiencies with respect to the control. However, a strong release of citrate was observed, and it increased significantly with time, being +250% and +300% higher in Fe- and P-deficient plants, respectively, compared with the control. Moreover, concomitantly, a significant acidification of the growth medium was observed in both treatments. Gene expression analyses highlighted for the first time that at least two members of the multidrug and toxic compound extrusion (MATE) transporter family and one member of the plasma membrane H(+)-ATPase family are involved in the response to both P and Fe starvation in strawberry plants.

Expression of the ß-1,3-glucanase gene bgn13.1 from Trichoderma harzianum in strawberry increases tolerance to crown rot diseases but interferes with plant growth.

The expression of antifungal genes from Trichoderma harzianum, mainly chitinases, has been used to confer plant resistance to fungal diseases. However, the biotechnological potential of glucanase genes from Trichoderma has been scarcely assessed. In this research, transgenic strawberry plants expressing the ß-1,3-glucanase gene bgn13.1 from T. harzianum, under the control of the CaMV35S promoter, have been generated. After acclimatization, five out of 12 independent lines analysed showed a stunted phenotype when growing in the greenhouse. Moreover, most of the lines displayed a reduced yield due to both a reduction in the number of fruit per plant and a lower fruit size. Several transgenic lines showing higher glucanase activity in leaves than control plants were selected for pathogenicity tests. When inoculated with Colletotrichum acutatum, one of the most important strawberry pathogens, transgenic lines showed lower anthracnose symptoms in leaf and crown than control. In the three lines selected, the percentage of plants showing anthracnose symptoms in crown decreased from 61 % to a mean value of 16.5 %, in control and transgenic lines, respectively. Some transgenic lines also showed an enhanced resistance to Rosellinia necatrix, a soil-borne pathogen causing root and crown rot in strawberry. These results indicate that bgn13.1 from T. harzianum can be used to increase strawberry tolerance to crown rot diseases, although its constitutive expression affects plant growth and fruit yield. Alternative strategies such as the use of tissue specific promoters might avoid the negative effects of bgn13.1 expression in plant performance.