Fruit cracking is an important problem in horticultural crop production. Polygalacturonase (SlPG) and expansin (SlEXP1) proteins cooperatively disassemble the polysaccharide network of tomato fruit cell walls during ripening and thereby, enable softening. A Golden 2-like (GLK2) transcription factor, SlGLK2 regulates unripe fruit chloroplast development and results in elevated soluble solids and carotenoids in ripe fruit. To determine whether SlPG, SlEXP1, or SlGLK2 influence the rate of tomato fruit cracking, the incidence of fruit epidermal cracking was compared between wild-type, Ailsa Craig (WT) and fruit with suppressed SlPG and SlEXP1 expression (pg/exp) or expressing a truncated nonfunctional Slglk2 (glk2). Treating plants with exogenous ABA increases xylemic flow into fruit. Our results showed that ABA treatment of tomato plants greatly increased cracking of fruit from WT and glk2 mutant, but not from pg/exp genotypes. The pg/exp fruit were firmer, had higher total soluble solids, denser cell walls and thicker cuticles than fruit of the other genotypes. Fruit from the ABA treated pg/exp fruit had cell walls with less water-soluble and more ionically and covalently-bound pectins than fruit from the other lines, demonstrating that ripening-related disassembly of the fruit cell wall, but not elimination of SlGLK2, influences cracking. Cracking incidence was significantly correlated with cell wall and wax thickness, and the content of cell wall protopectin and cellulose, but not with Ca2+ content.
As fruit ripen, there is a dramatic increase in their tendency to crack13,18. The production of large, uncracked, ripe fruit in cultivars with thin skins and high soluble solids has proven to be an unmet challenge. The complexity and structural plasticity of the ripening process are challenges for approaches designed to understand the relationship between ripening-associated softening, sugar accumulation and cracking.
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Cracking in tomato (Solanum lycopersicum) fruit most commonly begins as they ripen. During ripening, cell wall modifying proteins, including polygalacturonases (PGs) and expansins (EXPs), cooperatively disassemble wall polysaccharide networks and, thereby, contribute to the softening of fruit. Differences in cell wall structure between varieties and between unripe and ripe fruit could be an important factor in fruit tendency to crack.
Quantitative and qualitative changes in the sugars in ripe fruit could influence water potential and also contribute to the tendency of the fruit to crack. Over-expression of a Golden 2-like (GLK2) transcription factor, SlGLK2, in tomato enhances chloroplast elaboration and photosynthesis gene expression in developing fruit, and results in ripe fruit with elevated soluble solids content25,26.
It is desirable to breed or select for varieties whose fruit resist cracking under diverse environmental conditions without hormone treatments. Therefore, we investigated whether reducing the simultaneous expression of SlPG and SlEXP1 genes affects the tendency of fruit to crack. We were also interested to observe cracking of tomato lines with functional or non-functional forms of SlGLK2 to explore the contributions of solutes and sugars to the fruit's predisposition to form cracks. ABA was used as a tool to enhance cracking incidence of the tomato fruit.
In 2012, the incidence of cracking was determined visually on 26 April in all full-sized fruit which were then categorized into different ripeness stages from the same or different clusters based on external fruit color and tagged date. In 2013, the incidence of cracking of fruit at the MG, turning and RR stages were recorded on 30 April.
Analysis of variance (ANOVA) to determine differences in fruit characteristics between the genetic materials and treatments, and correlations between fruit parameters and cracking incidence was determined using statistical product and service solutions (SPSS) 16.0 (SPSS Inc. Chicago, IL, USA).
The incidence of cracking, as evidenced by visible fissures in the epidermis, increased as fruit ripened from MG to turning to RR stages in 2012 and 2013. In both years, no MG fruit were cracked and less than 5% of turning fruit were cracked, regardless of the treatment (data not shown). In 2012, ABA treatment increased the incidence of cracking of the RR glk2 mutant tomatoes (30.2% of ABA treated plants compared to 20% of water treated plants), but not the pg/exp tomatoes (13.1% in ABA treated plants and 12.1% in water treated plants). Similarly, in 2013, treatment of the plant with ABA increased the incidence of cracking of RR fruit of the WT and glk2 mutant genotypes, but not of the pg/exp genotype (Fig. 3).
Previous research indicates that heritable resistance to cracking can be identified in some tomato breeding lines. However, no single genetic locus seems to be responsible for inheritance of the fruit cracking trait and many genes may contribute to the phenotype2. Many studies point to the involvement of cell wall structure and possibly the cuticle layer in fruit cracking1,34,35,36,37,38,39,40. As cell wall networks weaken with fruit ripening41,42, even as cell turgor falls, resistance to stresses at the fruit surface may require a greater contribution from wax and cuticle layer structures than they can provide; and cell turgor pushing the plasmalemma against the cell wall also creates some stress on the cell wall polysaccharide networks that may be accommodated by the elasticity of the wall "fabric"; this then leading to cracking.
To investigate how PG and EXP may work collaboratively to affect the susceptibility of tomato fruit to cracking, we investigated differences in cell wall composition as influenced by the pg/exp genotype in fruit stressed by increased water uptake following treatment with ABA.
Ripening in tomato is accompanied by a shift in pectins from the CSP and SSP to the WSP45. The clearest impact of simultaneous suppression of PG and EXP was that the pg/exp fruit displayed a substantially reduced breakdown of the cell wall pectin network as they proceed through ripening and the fruit soften less than WT fruit46. The pectin polymers in ripe fruit of the pg/exp genotype are bigger than those in ripe WT fruit, and there was more SSP in pg/exp fruit compared with WT27,46. In our study, the cracking-resistant pg/exp genotype, had more CSP in the mesocarp portion, and more SSP in the exocarp portion of the fruit. In contrast, there was more WSP in fruit of the WT genotype. This observation suggests that both the exocarp and mesocarp cell walls of pg/exp fruit were more intact and thus better able to resist internal stresses that are presumed to promote ripe fruit cracking.
In our study, we used whole-plant sprays of ABA to increase the tendency of tomato fruit to crack. ABA application can decrease stomatal conductance and leaf transpiration, and increases plant water potential49, which results in significantly increased xylemic flow into tomato fruit23. This xylemic flow also carries more Ca2+ into the fruit as has been reported previously and is evident in the higher Ca2+ levels in both whole fruit and cell walls of ABA treated fruit. The higher incidence of cracking in ABA treated RR tomato fruit was likely due to accumulation of water in the fruit when leaf transpiration was reduced by ABA, likely resulting in increases in turgor pressure in the fruit. However, the fruit genotypes showed significant differences in their tendency to crack when cracking was promoted by ABA application. The increase in cracking in response to ABA treatment was not observed in RR pg/exp fruit but was observed in WT and glk2 fruit. And there was no difference in cracking among the three genotypes when plants were treated with water.
ABA treatment also had an influence on tomato cell wall composition, resulting in lower amounts of WSP and SSP in mesocarp and blossom end tissues of all three genotypes. This does not appear to be related to influences on fruit ripening as no visible differences in ripening were observed and ABA is generally reported to enhance ripening, not slow ripening. The higher proportion of chelator soluble cell wall material (CDTA soluble, CSP) may be a response to the higher Ca2+ levels in the fruit due to the higher xylemic flow, but it is unclear what role if any this played in the increased cracking of ABA treated fruit.
Cell walls from pg/exp fruit also appeared thicker and denser than cell walls from WT fruit under electron microscopy, perhaps because of reduced disassembly of the cell wall polysaccharide polymer network. This difference could be another reason for resistance to cracking in this genotype. The thicker and denser cell walls from pg/exp fruit are reflected in the higher levels of CSP, SSP and cellulose in cell wall extracts prepared from the mesocarp of pg/exp fruit. Cantu46 previously demonstrated that the reduction of both PG and EXP activities resulted in isolated and in situ cell walls that swelled much less than walls from WT fruit that soften significantly at the fully ripe stage; which supports the conclusion that pg/exp fruit has a more intact cell wall than WT fruit. In our experiments, pg/exp had a thicker and denser cell wall that may resist swelling.
The cuticular wax layer was thicker in pg/exp fruit, which could also contribute to resistance to cracking. While waxes are part of the overall extracellular matrix, they are not targets of either PG or EXP, which suggests that suppression of the ripening-associated SlPG and SlEXP1 genes may also have impacts on other structures at the fruit surface. It is interesting to note that in addition to changing cell wall network integrity, tomato fruit cuticle chemistry, and structure have been identified as fruit factors that influence ripening-associated fruit softening50. 2ff7e9595c
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