Characterization of Two Divergent Endo-β-1,4-Glucanase cDNA Clones Highly Expressed in the Nonclimacteric Strawberry Fruit

Two cDNAs clones (Cel1 and Cel2) encoding divergent endo-β-1,4-glucanases (EGases) have been isolated from a cDNA library obtained from ripe strawberry (Fragaria x ananassa Duch) fruit. The analysis of the amino acid sequence suggests that Cel1 and Cel2 EGases have different secondary and tertiary structures and that they differ in the presence of potential N-glycosylation sites. By in vitro translation we show that Cel1 and Cel2 bear a functional signal peptide, the cleavage of which yields mature proteins of 52 and 60 kD, respectively. Phylogenetic analysis revealed that the Cel2 EGase diverged early in evolution from other plant EGases. Northern analysis showed that both EGases are highly expressed in fruit and that they have different temporal patterns of accumulation. The Cel2 EGase was expressed in green fruit, accumulating as the fruit turned from green to white and remaining at an elevated, constant level throughout fruit ripening. In contrast, the Cel1 transcript was not detected in green fruit and only a low level of expression was observed in white fruit. The level of Cel1 mRNA increased gradually during ripening, reaching a maximum in fully ripe fruit. The high levels of Cel1 and Cel2 mRNA in ripe fruit and their overlapping patterns of expression suggest that these EGases play an important role in softening during ripening. In addition, the early expression of Cel2 in green fruit, well before significant softening begins, suggests that the product of this gene may also be involved in processes other than fruit softening, e.g. cell wall expansion.

Fruit cell culture as a model system to study cell wall changes during strawberry fruit ripening

Strawberry (Fragaria x ananassa, Duch.) fruit is characterized by its fast ripening and soft texture at the ripen stage, resulting in a short postharvest shelf life and high economic losses. It is generally believed that the disassembly of cell walls, the dissolution of the middle lamella and the reduction of cell turgor are the main factors determining the softening of fleshy fruits. In strawberry, several studies indicate that the solubilisation and depolymerisation of pectins, as well as the depolymerisation of xyloglucans, are the main processes occurring during ripening. Functional analyses of genes encoding pectinases such as polygalacturonase and pectate lyase also point out to the pectin fraction as a key factor involved in textural changes. All these studies have been performed with whole fruits, a complex organ containing different tissues that differ in their cell wall composition and undergo ripening at different rates. Cell cultures derived from fruits have been proposed as model systems for the study of several processes occurring during fruit ripening, such as the production of anthocyanin and its regulation by plant hormones. The main objective of this research was to obtain and characterize strawberry cell cultures to evaluate their potential use as a model for the study of the cell wall disassembly process associate with fruit ripening. Cell cultures were obtained from cortical tissue of strawberry fruits, cv. Chandler, at the stages of unripe-green, white and mature-red. Additionally, a cell culture line derived from strawberry leaves was obtained. All cultures were maintained in solid medium supplemented with 2.5 mg.l-1 2,4-D and incubated in the dark. Cell walls from the different callus lines were extracted and fractionated to obtain CDTA and sodium carbonate soluble pectin fractions, which represent polyuronides located in the middle lamella or the primary cell wall, respectively. The amounts of homogalacturonan in both fractions were estimated by ELISA using LM19 and LM20 antibodies, specific against demethylated and methyl-esterified homogalacturonan, respectively. In the CDTA fraction, the cell line from ripe fruit showed a significant lower amount of demethylated pectins than the rest of lines. By contrast, the content of methylated pectins was similar in green- and red-fruit lines, and lower than in white-fruit and leaf lines. In the sodium carbonate pectin fraction, the line from red fruit also showed the lowest amount of pectins. These preliminary results indicate that cell cultures obtained from fruits at different developmental stages differ in their cell wall composition and these differences resemble to some extent the changes that occur during strawberry softening. Experiments are in progress to further characterize cell wall extracts with monoclonal antibodies against other cell wall epitopes.

Unravelling the nanostructure of strawberry fruit pectins by atomic force microscopy

Atomic force microscopy (AFM) allows the analysis of individual polymers at nanostructural level with a minimal sample preparation. This technique has been used to analyse the pectin disassembly process during the ripening and postharvest storage of several fleshy fruits. In general, pectins analysed by AFM are usually visualized as isolated chains, unbranched or with a low number of branchs and, occasionally, as large aggregates. However, the exact nature of these structures is unknown. It has been suggested that pectin aggregates represent a mixture of rhamnonogalacturonan I and homogalacturonan, while isolated chains and their branches are mainly composed by polygalacturonic acid. In order to gain insight into the nature of these structures, sodium carbonate soluble pectins from ripe strawberry (Fragaria x ananassa, Duch.) fruits were subjected to enzymatic digestion with endo-Polygalacturonase M2 from Aspergillus aculeatus, and the samples visualized by AFM at different time intervals. Pectins isolated from control, non-transformed plants, and two transgenic genotypes with low level of expression of ripening-induced pectinase genes encoding a polygalacturonase (APG) or a pectate lyase (APEL) were also included in this study. Before digestion, isolated pectin chains from control were shorter than those from transgenic fruits, showing number-average (LN) contour length values of 73.2 nm vs. 95.9 nm and 91.4 nm in APG and APEL, respectively. The percentage of branched polymers was significantly higher in APG polyuronides than in the remaining genotypes, 33% in APG vs. 6% in control and APEL. As a result of the endo-PG treatment, a gradual decrease in the main backbone length of isolated chains was observed in the three samples. The minimum LN value was reached after 8 h of digestion, being similar in the three genotypes, 22 nm. By contrast, the branches were not visible after 1.5-2 h of digestion. LN values were plotted against digestion time and the data fitted to a first-order exponential decay curve, obtaining R2 values higher than 0.9. The half digestion time calculated with these equations were similar for control and APG pectins, 1.7 h, but significantly higher in APEL, 2.5 h, indicating that these polymer chains were more resistant to endo-PG digestion. Regarding the pectin aggregates, their volumes were estimated and used to calculate LN molecular weights. Before digestion, control and APEL samples showed complexes of similar molecular weights, 1722 kDa, and slightly higher than those observed in APG samples. After endo-PG digestion, size of complexes diminished significantly, reaching similar values in the three pectin samples, around 650 kDa. These results suggest that isolated polymer chains visualized by AFM are formed by a HG domain linked to a shorter polymer resistant to endo-PG digestion, maybe xylogalacturonan or RG-I. The silencing of the pectate lyase gene slightly modified the structure and/or chemical composition of polymer chains making these polyuronides more resistant to enzymatic degradation. Similarly, polygalacturonic acid is one of the main component of the aggregates.

Studying the role of the strawberry Fra protein family in the flavonoid metabolism during fruit ripening

Strawberry fruits are highly appreciated worldwide due to their pleasant flavor and aroma and to the health benefits associated to their consumption. An important part of these properties is due to their content in secondary metabolites, especially phenolic compounds, of which flavonoids are the most abundant in the strawberry fruit. Although the flavonoid biosynthesis pathway is uncovered, little is known about its regulation. The strawberry Fra a (Fra) genes constitute a large family of homologs of the major birch pollen allergen Bet v 1 and for which no equivalents exist in Arabidopsis. Our group has shown that Fra proteins are involved in the formation of colored compounds in strawberries (Muñoz et al., 2010), which mainly depends on the production of certain flavonoids; that they are structurally homologs to the PYR/PYL/RCAR Arabidopsis ABA receptor, and that they are able to bind flavonoids (Casañal et al., 2013). With these previous results, our working hypothesis is that the Fra proteins are involved in the regulation of the flavonoids pathway. They would mechanistically act as the ABA receptor, binding a protein interactor and a ligand to regulate a signaling cascade and/or act as molecular carriers. The main objective of this research is to characterize the Fra family in strawberry and gain insight into their role in the flavonoid metabolism. By RNAseq expression analysis in ripening fruits we have identified transcripts for 10 members of the Fra family. Although expressed in all tissues analyzed, each family member presents a unique pattern of expression, which suggests functional specialization for each Fra protein. Then, our next approach was to identify the proteins that interact with Fras and their ligands to gain knowledge on the role that these proteins play in the flavonoids pathway. To identify the interacting partners of Fras we have performed a yeast two hybrid (Y2H) screening against cDNA libraries of strawberry fruits at the green and red stages. A protein that shares a 95% homology to the Heat stress transcription factor A-4-C like of Fragaria vesca (HSA4C) interacts specifically with Fra1 and not with other family members, which suggests functional diversification of Fra proteins in specific signaling pathways. The Y2H screening is not yet saturated, so characterization of other interacting proteins with other members of the Fra family will shed light on the functional diversity within this gene family. This research will contribute to gain knowledge on how the flavonoid pathway, and hence, the fruit ripening, is regulated in strawberry; an economically important crop but for which basic research is still very limited. References: Muñoz, C, et al. (2010). The Strawberry Fruit Fra a Allergen Functions in Flavonoid Biosynthesis. Molecular Plant, 3(1): 113–124. Casañal, A, et al (2013). The Strawberry Pathogenesis-related 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding Metabolic Intermediates. Journal of Biological Chemistry, 288(49): 35322–35332.

Species of thrips (Insecta, Thysanoptera) in two strawberry production systems in Rio Grande do Sul State, Brazil

Species of thrips (Insecta, Thysanoptera) in two strawberry production systems in Rio Grande do Sul, Brazil. Thrips are tiny insects responsible for the reduction of strawberry fruit quality. The work aimed to record and quantify the thysanopterofauna present in two strawberry production systems, low tunnel and semi-hydroponic. Leaves, flowers and fruits were collected weekly, from July 2005 to December 2006 in Caxias do Sul and Bom Princípio municipalities, RS. A total of 664 individuals were collected, representing two families, four genus and 10 species: Frankliniella occidentalis (Pergande, 1895), F. schultzei (Trybom, 1910), F. rodeos Moulton, 1933, F. simplex (Priesner, 1924), F. williamsi (Hood, 1915), F. gemina (Bagnall, 1919), Frankliniella sp., Thrips tabaci (Lindeman, 1888), Thrips tabaci (Lindeman, 1888), Caliothrips fasciatus (Pergande 1895) from Thripidae and Heterothrips sp. from Heterothripidae. Frankliniella occidentalis represented 89.7% of the samples with 95.8% of the species collected in flowers, 3.9% in fruits and 0.8% in leaves. The results show that flowers are the most important food resource for these insects on strawberry plants. Frankliniella rodeos, F. simplex, F. williamsi, C. fasciatus, and Heterothrips sp. are new records on strawberry for Brazil.

Cell size in trays for the production of strawberry plug transplants

The objective of this work was to evaluate the influence of cell sizes used for strawberry plug production in trays compared to bare root transplants, regarding initial plant size, harvest timing, and total strawberry fruit yield. Plug transplants were produced from runner tips rooted in trays with cell sizes of 26.5, 50, 100 and 150 cm³ filled with Plantmax HA organic substrate. Bare root transplants (control) were produced in a closed soilless system using sand as substrate. A randomized block design was used, with four replicates with 16 plants per plot. Bare root transplants and plug transplants from 100-cm³ cells had larger crown and higher leaf and root dry mass. Early fruit yield was higher in plants propagated from plugs than in those propagated from bare root transplants. Spring and total fruit yield did not differ among treatments, with an average yield of 435 and 874 g per plant, respectively. Earlier strawberry fruit yield was obtained by using plug transplants, even from trays with small cells of 26.5 or 50 cm³.

QTLs mapping for primary metabolites responsible of the organoleptic and nutritional characteristics of strawberry (Fragaria x ananassa)

The cultivated strawberry (Fragaria x ananassa) is the berry fruit most consumed worldwide and is well-known for its delicate flavour and nutritional properties. However, fruit quality attributes have been lost or reduced after years of traditional breeding focusing mainly on agronomical traits. To face the obstacles encountered in the improvement of cultivated crops, new technological tools, such as genomics and high throughput metabolomics, are becoming essential for the identification of genetic factors responsible of organoleptic and nutritive traits. Integration of “omics” data will allow a better understanding of the molecular and genetic mechanisms underlying the accumulation of metabolites involved in the flavour and nutritional value of the fruit. To identify genetic components affecting/controlling? fruit metabolic composition, here we present a quantitative trait loci (QTL) analysis using a 95 F1 segregating population derived from genotypes ‘1392’, selected for its superior flavour, and ‘232’ selected based in high yield (Zorrilla-Fontanesi et al., 2011; Zorrilla-Fontanesi et al., 2012). Metabolite profiling was performed on red stage strawberry fruits using gas chromatography hyphenated to time-of-flight mass spectrometry, which is a rapid and highly sensitive approach, allowing a good coverage of the central pathways of primary metabolism. Around 50 primary metabolites, including sugars, sugars derivatives, amino and organic acids, were detected and quantified after analysis in each individual of the population. QTL mapping was performed on the ‘232’ x ‘1392’ population separately over two successive years, based on the integrated linkage map (Sánchez-Sevilla et al., 2015). First, significant associations between metabolite content and molecular markers were identified by the non-parametric test of Kruskal-Wallis. Then, interval mapping (IM), as well as the multiple QTL method (MQM) allowed the identification of QTLs in octoploid strawberry. A permutation test established LOD thresholds for each metabolite and year. A total of 132 QTLs were detected in all the linkage groups over the two years for 42 metabolites out of 50. Among them, 4 (9.8%) QTLs for sugars, 9 (25%) for acids and 7 (12.7%) for amino acids were stable and detected in the two successive years. We are now studying the QTLs regions in order to find candidate genes to explain differences in metabolite content in the different individuals of the population, and we expect to identify associations between genes and metabolites which will help us to understand their role in quality traits of strawberry fruit.

Susceptibility and Interactions of Drosophila suzukii and Zaprionus indianus (Diptera: Drosophilidae) in Damaging Strawberry.

Resumo: Drosophila suzukii (Matsumura) foi recentemente detectado causando danos idade para morangos no Brasil. Infestação na cultura de morango tem frequentemente foi observado conjuntamente com a presença de Zaprionus indianus Gupta. este estudo investigou a suscetibilidade de morangos em três amadurecimento estágios para infestação de D. suzukii e Z. indianus e sua interação. Abstracts: Drosophila suzukii (Matsumura) has been recently detected causing damage to strawberries in Brazil. Infestation in strawberry culture has often been observed jointly with the presence of Zaprionus indianus Gupta. This study investigated the susceptibility of strawberries at three ripening stages to infestation of D. suzukii and Z. indianus and their interaction. In the laboratory, strawberries cv. Albion at different ripening stages (green, semi-ripe and ripe) were exposed to D. suzukii and Z. indianus for 24 h in choice and no-choice bioassays. Additionally, we evaluated the effects of mechanical damage incurred artificially or by D. suzukii ovi-position on Z. indianus infestation. In no-choice bioassay, there were no significant differences in fruit susceptibility to D. suzukii infestation at different ripening stages. However, in choice bioassay, D. suzukii adults preferred to oviposit on R fruit. The presence of mechanical damage did not increase susceptibility of fruit to D. suzukii oviposition. For Z. indianus , there was greater susceptibility of R fruit in relation to SR and G fruit in both the choice and no-choice bioassays. There was a significant and positive interaction of mechanical damage and damage caused by D. suzukii to R fruit and infestation by Z. indianus , which was not observed in SR and G fruit. Although infestation of Z. indianus is related to attack damaged or decaying fruit, this work shows that this species has the ability to oviposit and develop in healthy strawberry fruit with and increased infestation level when the fruit has damage to its epidermis.

Susceptibility and Interactions of Drosophila suzukii and Zaprionus indianus (Diptera: Drosophilidae) in Damaging Strawberry.

Resumo: Drosophila suzukii (Matsumura) foi recentemente detectado causando danos idade para morangos no Brasil. Infestação na cultura de morango tem frequentemente foi observado conjuntamente com a presença de Zaprionus indianus Gupta. este estudo investigou a suscetibilidade de morangos em três amadurecimento estágios para infestação de D. suzukii e Z. indianus e sua interação. Abstracts: Drosophila suzukii (Matsumura) has been recently detected causing damage to strawberries in Brazil. Infestation in strawberry culture has often been observed jointly with the presence of Zaprionus indianus Gupta. This study investigated the susceptibility of strawberries at three ripening stages to infestation of D. suzukii and Z. indianus and their interaction. In the laboratory, strawberries cv. Albion at different ripening stages (green, semi-ripe and ripe) were exposed to D. suzukii and Z. indianus for 24 h in choice and no-choice bioassays. Additionally, we evaluated the effects of mechanical damage incurred artificially or by D. suzukii ovi-position on Z. indianus infestation. In no-choice bioassay, there were no significant differences in fruit susceptibility to D. suzukii infestation at different ripening stages. However, in choice bioassay, D. suzukii adults preferred to oviposit on R fruit. The presence of mechanical damage did not increase susceptibility of fruit to D. suzukii oviposition. For Z. indianus , there was greater susceptibility of R fruit in relation to SR and G fruit in both the choice and no-choice bioassays. There was a significant and positive interaction of mechanical damage and damage caused by D. suzukii to R fruit and infestation by Z. indianus , which was not observed in SR and G fruit. Although infestation of Z. indianus is related to attack damaged or decaying fruit, this work shows that this species has the ability to oviposit and develop in healthy strawberry fruit with and increased infestation level when the fruit has damage to its epidermis.

Study of Rapid Alkalinization Factor (RALF)genes as plant susceptibility agents during plant-pathogen interaction in strawberry

Rapid Alkalinization Factor (RALF) are cysteins-rich peptides ubiquitous in plant kingdom. They play multiple roles as hormone signals and recently their involvement in host-pathogen crosstalk as negative regulator of immunity in Arabidopsis has also been recognized. In addition, RALF homologue peptides are secreted by different fungal pathogens as effectors during early stages of infections. The aim of this work was to characterize RALF genes as susceptibility factors during plant pathogen interaction in strawberry. For this, the genomic organization of the RALF gene families in the octoploid strawberry (Fragaria × ananassa) and the re-annotated genome of Fragaria vesca were described , identifying 13 member in F. vesca (FvRALF) and 50 members in F. x ananassa (FaRALF). The changes in expression of fruit FaRALF genes was investigated upon infection with C.acutatum and B. cinerea showing that, among RALF genes expressed in fruit, FaRALF3 was the only one upregulated by fungal infection in the ripe stage. A role of FaRALF3 as susceptibility gene was then assessed trough Agrobacterium-mediated transient FaRALF3 overexpression and silencing in fruits, revealing that FaRALF3 expression promotes fungal growth and hyphae penetration in host tissues. In silico analysis was used to identify distinct pathogen inducible elements upstream of the FaRALF3 gene. Agroinfiltration of strawberry fruit with deletion constructs of the FaRALF3 promoter identified a 5’ region required for FaRALF3 expression in fruit, but failed to identify a region responsible for fungal induced expression. Furthermore, FaRALF3 and strawberry receptor FERONIA (FaMRLK47) were heterologously expressed in E. coli in order to purify active proteins forms and study RALF-FERONIA interaction in strawberry. However, it was not possible to obtain pure and active proteins. Finally RNAi transgenic plants silenced for the FvRALF13 gene were genotypically and phenotypically characterized suggesting a role of FvRALF13 in flowering time regulation and reproductive organs development.

Otimização, validação e aplicação de método para determinação da concentração residual de difenoconazol em morangos após múltiplas aplicações

Difenoconazole residues in strawberry fruit cultivated in pots were estimated using the solid-liquid extraction with low temperature partition (SLE/LTP) method for sample preparation and gas chromatography with electron capture detection (GC/ECD) for analysis. The optimized method presented excellent recovery values from fortified samples and reproducibility (average recovery values ≥ 98%; CV values < 15%). Linearity of response was demonstrated (r = 0.995) with a detection limit of 9 µg kg-1. The method was successfully applied for the determination of difenoconazole residues in strawberries. Based on these results, the fungicide dissipates quickly, but the residual concentration increases after multiple applications.

Elicitors of induced disease resistance in postharvest horticultural crops: a brief review

Increasing loss of conventional fungicides due to pathogen resistance and general unacceptability in terms of public and environmental risk have favoured the introduction of integrated pest management (IPM) programmes. Induction of natural disease resistance (NDR) in harvested horticultural crops using physical, biological and/or chemical elicitors has received increasing attention over recent years, it being considered a preferred strategy for disease management. This article reviews the enhancement of constitutive and inducible antifungal compounds and suppression of postharvest diseases through using elicitors. The effect of timing of pre- and/or postharvest elicitor treatment and environment on the degree of elicitation and the potential for inducing local acquired resistance, systemic acquired resistance and/or induced systemic resistance to reduce postharvest disease is discussed. The review highlights that more applied and basic research is required to understand the role that induced NDR can play in achieving practical suppression of postharvest diseases as part of an IPM approach. (C) 2003 Elsevier B.V. All rights reserved.

Methyl jasmonate vapour treatment suppresses specking caused by Botrytis cinerea on cut Freesia hybrida L. flowers

Treatment of cut freesia var. Cote d'Azur flowers with methyl jasmonate (MeJA, 0.1 mu l MeJA l(-1)) vapour suppressed petal specking caused by Botrytis cinerea infection. MeJA efficacy was concentration and incubation temperature dependent. Disease severity, lesion numbers and lesion diameters decreased with increasing MeJA concentration from 0.025 to 0.1 mu l MeJA l(-1). However, there were no significant (P > 0.05) differences among MeJA concentrations examined. MeJA was more effective in reducing B. cinerea flower specking at 20 degrees C than at 12 degrees C. MeJA treatment was ineffective at 5 degrees C. At 20 degrees C, MeJA treatment at 0.1 mu l MeJA l(-1) reduced disease severity, lesion numbers and lesion diameters by 58, 50 and 48%, respectively, as compared to untreated controls. In a repeat experiment, disease severity, lesion numbers and lesion diameters on MeJA vapour treated flowers after 12 h of incubation were reduced by 68, 56 and 50%, respectively. MeJA did not exert direct antifungal activity in-vitro, suggesting that treatment in-vivo reduced B. cinerea-induced flower specking by induction of host defence responses. MeJA at 0.1 mu l MeJA l(-1) significantly (P < 0.05) increased vase life of cut freesia flowers and delayed senescence judged by lower wilt scores and higher fresh weights as compared to untreated controls. (c) 2005 Elsevier B.V. All rights reserved.

Development and fruit yield of strawberry plants as affected by crown diameter and plantlet growing period

The objective of this work was to evaluate the influence of crown diameter and growing period of runner tips in 128 cell-trays on growth and yield of strawberry in the field. Treatments consisted of three classes of runner tip crown diameters, between 2.0 to 3.9 mm; 4.0 to 5.5 mm and 5.6 to 7.0 mm, respectively, and four growing periods in trays, 24; 39; 54 and 69 days, respectively. Higher shoot dry mass of transplants at planting and earlier yield of plants in the field were obtained in transplants grown for 69 days in trays. Larger runner tips lead to more vigorous transplants at planting and plants with higher vegetative growth in the field, with minor impact on yield. Increasing the growing time of runner tips in trays improved early fruit yield and decreased plant vegetative growth in the field.

Analysis of shade and temperature effects on assimilate partitioning in the everbearing strawberry 'Everest' as the basis for optimised long-season fruit production

Growth patterns and cropping were evaluated over the season for the everbearing strawberry 'Everest' at a range of temperatures (15-27degreesC) in two light environments (ambient and 50% shade). The highest yield was recorded for unshaded plants grown at 23degreesC, but the optimum temperature for vegetative growth was 15degreesC. With increasing temperature fruit number increased, but fruit weight decreased. Fruit weight was also significantly reduced by shade, and although 'Everest' showed a degree of shade tolerance in vegetative growth, yield was consistently reduced by shade. Shade also reduced the number of crowns developed by the plants over the course of the season, emphasising that crown number was ultimately the limiting factor for yield potential. We conclude that, in contrast to Junebearers which partition more assimilates to fruit at temperatures around 15degreesC (Le Miere et al., 1998), optimised cropping in the everbearer 'Everest' is achieved at the significantly higher temperature of 23degreesC. These findings have significance for commercial production, in which protection tends to reduce light levels but increase average temperature throughout the season.