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Plant rhamnogalacturonan lyases (RGLyases) cleave the backbone of rhamnogalacturonan I (RGI), the "hairy" pectin and polymer of the disaccharide rhamnose (Rha)-galacturonic acid (GalA) with arabinan, galactan or arabinogalactan side chains. It has been suggested that RGLyases could participate in remodeling cell walls during fruit softening, but clear evidence has not been reported. To investigate the role of RGLyases in strawberry softening, a genome-wide analysis of RGLyase genes in the genus Fragaria was performed. Seventeen genes encoding RGLyases with functional domains were identified in Fragaria × ananassa. FaRGLyase1 was the most expressed in the ripe receptacle of cv. Chandler. Transgenic strawberry plants expressing an RNAi sequence of FaRGLyase1 were obtained. Three transgenic lines yielded ripe fruits firmer than controls without other fruit quality parameters being significantly affected. The highest increase in firmness achieved was close to 32%. Cell walls were isolated from ripe fruits of two selected lines. The amount of water-soluble and chelated pectins was higher in transgenic lines than in the control. A carbohydrate microarray study showed a higher abundance of RGI epitopes in pectin fractions and in the cellulose-enriched fraction obtained from transgenic lines. Sixty-seven genes were differentially expressed in transgenic ripe fruits when compared with controls. These genes were involved in various physiological processes, including cell wall remodeling, ion homeostasis, lipid metabolism, protein degradation, stress response, and defense. The transcriptomic changes observed in FaRGLyase1 plants suggest that senescence was delayed in transgenic fruits., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Firmness is one of the most important fruit quality traits in strawberries. The postharvest shelf life of this soft fruit is highly limited by the loss of firmness, where cell wall disassembly plays an important role. Previous studies demonstrated that the polygalacturonase FaPG1 has a key role in remodelling pectins during strawberry softening. In this study, FaPG1 knockout strawberry plants have been generated using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens . Ten independent lines, cv. "Chandler", were obtained, and all of them were successfully edited as determined by PCR amplification and T7 endonuclease assay. The targeted mutagenesis insertion and deletion rates were analyzed using targeted deep sequencing. The percentage of edited sequences varied from 47% up to almost 100%, being higher than 95% for seven of the selected lines. Phenotypic analyses showed that 7 out of the eight lines analyzed produced fruits significantly firmer than the control, ranging from 33 to 70% increase in firmness. There was a positive relationship between the degree of FaPG1 editing and the rise in fruit firmness. Minor changes were observed in other fruit quality traits, such as colour, soluble solids, titratable acidity or anthocyanin content. Edited fruits showed a reduced softening rate during postharvest, displayed a reduced transpirational water loss, and were less damaged by Botrytis cinerea inoculation. The analysis of four potential off-target sites revealed no mutation events. In conclusion, editing the FaPG1 gene using the CRISPR/Cas9 system is an efficient method for improving strawberry fruit firmness and shelf life., Competing Interests: The authors declare that there are no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Key Message: Agronomical characterization of a RIL population for fruit mineral contents allowed for the identification of QTL controlling these fruit quality traits, flanked by co-dominant markers useful for marker-assisted breeding. Tomato quality is a multi-variant attribute directly depending on fruit chemical composition, which in turn determines the benefits of tomato consumption for human health. Commercially available tomato varieties possess limited variability in fruit quality traits. Wild species, such as Solanum pimpinellifolium, could provide different nutritional advantages and can be used for tomato breeding to improve overall fruit quality. Determining the genetic basis of the inheritance of all the traits that contribute to tomato fruit quality will increase the efficiency of the breeding program necessary to take advantage of the wild species variability. A high-density linkage map has been constructed from a recombinant inbred line (RIL) population derived from a cross between tomato Solanum lycopersicum and the wild-relative species S. pimpinellifolium. The RIL population was evaluated for fruit mineral contents during three consecutive growing seasons. The data obtained allowed for the identification of main QTL and novel epistatic interaction among QTL controlling fruit mineral contents on the basis of a multiple-environment analysis. Most of the QTL were flanked by candidate genes providing valuable information for both tomato breeding for new varieties with novel nutritional properties and the starting point to identify the genes underlying these QTL, which will help to reveal the genetic basis of tomato fruit nutritional properties.

Volatile organic compounds (VOCs) are major determinants of fruit flavor, a primary objective in tomato breeding. A recombinant inbred line (RIL) population consisting of 169 lines derived from a cross between Solanum lycopersicum and a red-fruited wild tomato species Solanum pimpinellifolium accession (SP) was characterized for VOCs in three different seasons. Correlation and hierarchical cluster analyses were performed on the 52 VOCs identified, providing a tool for the putative assignation of individual compounds to metabolic pathways. Quantitative trait locus (QTL) analysis, based on a genetic linkage map comprising 297 single nucleotide polymorphisms (SNPs), revealed 102 QTLs (75% not described previously) corresponding to 39 different VOCs. The SP alleles exerted a positive effect on most of the underlying apocarotenoid volatile QTLs-regarded as desirable for liking tomato-indicating that alleles inherited from SP are a valuable resource for flavor breeding. An introgression line (IL) population developed from the same parental genotypes provided 12 ILs carrying a single SP introgression and covering 85 VOC QTLs, which were characterized at three locations. The results showed that almost half of the QTLs previously identified in the RILs maintained their effect in an IL form, reinforcing the value of these QTLs for flavor/aroma breeding in cultivated tomato., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Key Message: QTL and codominant genetic markers for fruit cracking have been identified in a tomato genetic map derived from a RIL population, providing molecular tools for marker-assisted breeding of this trait. In tomato, as well as in other fleshy fruits, one of the main disorders that widely limit quality and production is fruit cracking or splitting of the epidermis that is observed on the fruit skin and flesh at any stage of fruit growth and maturation. To elucidate the genetic basis of fruit cracking, a quantitative trait loci (QTL) analysis was conducted in a recombinant inbred line (RIL) population derived from a cross between tomato (Solanum lycopersicum) and the wild-relative species S. pimpinellifolium. The RIL population was evaluated for fruit cracking during three consecutive growing seasons. Construction of a high-density linkage map based on codominant markers, covering more than 1000 cM of the whole genome, led to the identification of both main and epistatic QTL controlling fruit cracking on the basis of a single-environment as well as multiple-environment analysis. This information will enhance molecular breeding for novel cracking resistant varieties and simultaneously assist the identification of genes underlying these QTL, helping to reveal the genetic basis of fruit cracking in tomato.

Premise of the Study: Interspecific reproductive barriers (IRBs) often prevent hybridization between closely related species in sympatry. In the tomato clade (Solanum section Lycopersicon), interspecific interactions between natural sympatric populations have not been evaluated previously. In this study, we assessed IRBs between members of the tomato clade from nine sympatric sites in Peru., Methods: Coflowering was assessed at sympatric sites in Peru. Using previously collected seeds from sympatric sites in Peru, we evaluated premating prezygotic (floral morphology), postmating prezygotic (pollen-tube growth), and postzygotic barriers (fruit and seed development) between sympatric species in common gardens. Pollen-tube growth and seed development were examined in reciprocal crosses between sympatric species., Key Results: We confirmed coflowering of sympatric species at five sites in Peru. We found three types of postmating prezygotic IRBs during pollen-pistil interactions: (1) unilateral pollen-tube rejection between pistils of self-incompatible species and pollen of self-compatible species; (2) potential conspecific pollen precedence in a cross between two self-incompatible species; and (3) failure of pollen tubes to target ovules. In addition, we found strong postzygotic IRBs that prevented normal seed development in 11 interspecific crosses, resulting in seed-like structures containing globular embryos and aborted endosperm and, in some cases, overgrown endothelium. Viable seed and F 1 hybrid plants were recovered from three of 19 interspecific crosses., Conclusions: We have identified diverse prezygotic and postzygotic IRBs that would prevent hybridization between sympatric wild tomato species, but interspecific hybridization is possible in a few cases., (© 2016 Botanical Society of America.)

We have studied a genomic library of introgression lines from the Solanum pimpinellifolium accession TO-937 into the genetic background of the "Moneymaker" cultivar in order to evaluate the accession's breeding potential. Overall, no deleterious phenotypes were observed, and the plants and fruits were phenotypically very similar to those of "Moneymaker," which confirms the feasibility of translating the current results into elite breeding programs. We identified chromosomal regions associated with traits that were both vegetative (plant vigor, trichome density) and fruit-related (morphology, organoleptic quality, color). A trichome-density locus was mapped on chromosome 10 that had not previously been associated with insect resistance, which indicates that the increment of trichomes by itself does not confer resistance. A large number of quantitative trait loci (QTLs) have been identified for fruit weight. Interestingly, fruit weight QTLs on chromosomes 1 and 10 showed a magnitude effect similar to that of QTLs previously defined as important in domestication and diversification. Low variability was observed for fruit-shape-related traits. We were, however, able to identify a QTL for shoulder height, although the effects were quite low, thus demonstrating the suitability of the current population for QTL detection. Regarding organoleptic traits, consistent QTLs were detected for soluble solid content (SSC). Interestingly, QTLs on chromosomes 2 and 9 increased SSC but did not affect fruit weight, making them quite promising for introduction in modern cultivars. Three ILs with introgressions on chromosomes 1, 2, and 10 increased the internal fruit color, making them candidates for increasing the color of modern cultivars. Comparing the QTL detection between this IL population and a recombinant inbred line population from the same cross, we found that QTL stability across generations depended on the trait, as it was very high for fruit weight but low for organoleptic traits. This difference in QTL stability may be due to a predominant additive gene action for QTLs involved in fruit weight, whereas epistatic and genetic background interactions are most likely important for the other traits.

Flavonoids accumulate in tomato (Solanum lycopersicum L.) fruit cuticles during ripening. Their quantitative contribution to the biomechanical properties of the cuticle is studied in six tomato genotypes which show presence or absence of these compounds at the red ripe stage of fruit development. Tomato cuticles with flavonoids at red ripe showed a dramatic increase of these compounds between mature green and red ripe stages together with a significant increase in the elastic modulus. On the other hand, cuticles without flavonoids displayed a similar biomechanical behaviour at mature green and red ripe stages. The absence of flavonoids could also be related with a predominance of the viscoelastic performance of the cuticle. Thus, the increase of phenolics in tomato fruit cuticles during ripening is correlated with a more rigid cutin network that reinforces the mechanical function of polysaccharides which tend to diminish at this stage due to cell wall disassembly. A role of phenolics as biomechanical modulators of the cuticle behaviour is proposed.

The cuticle of a plant plays an important role in many physiological events of fruit development and ripening. Despite this, little is known about cuticle formation and development. We include a detailed morphological study at the microscopic level of cuticle during fruit growth and ripening using tomato as a fruit model. In addition, a study of the differences in cuticle thickness and composition during development is included. The four genotypes studied in this work showed a similar timing of the main morphological events: initiation of epidermal differentiation, changes in the distribution of the lipid, pectin and cellulose material within the cuticle, appearance of pegs, beginning of cuticle invaginations, maximum thickness and loss of polysaccharidic material. Fruit growth, measured by fruit diameter, showed a positive correlation with the increase of cuticle thickness and the amount of cuticle and their cutin and polysaccharide components per fruit unit during development. By contrast, cuticle waxes showed a different behaviour. Two important characteristics of cuticle growth were observed during tomato fruit development. First, the amount of cuticle per surface area reached its maximum in the first 15 days after anthesis and remained more or less constant until ripening. Second, there was a significant loss of polysaccharidic material from the beginning of ripening (breaker stage) to full red ripe.

The mechanical characteristics of the cuticular membrane (CM), a complex composite biopolymer basically composed of a cutin matrix, waxes, and hydrolysable polysaccharides, have been described previously. The biomechanical behaviour and quantitative contribution of cutin and polysaccharides have been investigated here using as experimental material mature green and red ripe tomato fruits. Treatment of isolated CM with anhydrous hydrogen fluoride in pyridine allowed the selective elimination of polysaccharides attached to or incrusted into the cutin matrix. Cutin samples showed a drastic decrease in elastic modulus and stiffness (up to 92%) compared with CM, which clearly indicates that polysaccharides incorporated into the cutin matrix are responsible for the elastic modulus, stiffness, and the linear elastic behaviour of the whole cuticle. Reciprocally, the viscoelastic behaviour of CM (low elastic modulus and high strain values) can be assigned to the cutin. These results applied both to mature green and red ripe CM. Cutin elastic modulus, independently of the degree of temperature and hydration, was always significantly higher for the ripe than for the green samples while strain was lower; the amount of phenolics in the cutin network are the main candidates to explain the increased rigidity from mature green to red ripe cutin. The polysaccharide families isolated from CM were pectin, hemicellulose, and cellulose, the main polymers associated with the plant cell wall. The three types of polysaccharides were present in similar amounts in CM from mature green and red ripe tomatoes. Physical techniques such as X-ray diffraction and Raman spectroscopy indicated that the polysaccharide fibres were mainly randomly oriented. A tomato fruit CM scenario at the supramolecular level that could explain the observed CM biomechanical properties is presented and discussed.

The mechanical properties of enzymatically isolated cuticular membrane (CM) from ripe tomato fruits were investigated at 10 to 45°C and relative humidity (RH) of 40 to wet. CM samples were stressed by uniaxial tension loads to determine their tensile modulus, E, breaking stress (strength), σ(max), and maximum elongation, ε(max). The CM stress-strain curves revealed a biphasic behavior when tested at RH values below wet conditions. In the first phase, CM responded to the loads by instantaneous extension with no further extension recorded until a further load was added: defined as pure elastic strain (E(e)). In the second phase, CM responded by instantaneous extension and by some additional time-dependent extension, defined as viscoelastic strain (E(v)). When CMs were submerged in aqueous solution (wet), the stress-strain curves were monophasic, with both elastic and viscoelastic strain. E(e) depended on RH and was higher than E(v), which was independent of RH. Temperature decreased E(e) and σ(max) of tomato fruit CM. Temperature response was not linear but consisted of two temperature-independent phases separated by a transition temperature. This transition zone has been related previously to the presence of a secondary phase transition in the cutin matrix of the tomato fruit CM.