The total soluble solids of the transgenic fruit was consistently lower than non-transgenic samples throughout cold storage, as well as after rewarming . Respiration rates were significantly higher in Sh-13 compared to non-transgenic fruit throughout cold storage, except for day 4 . However, after rewarming all three genotypes were the same. Interestingly, CO2 emission in Sh-13 only increased 512.8% which was lower than in Sl-2 and WT fruit. Ethylene emission rates were similar between transgenic lines and the WT, with changes on days 2 and 6. After transfer to 20°C, values increased ~3,500% .The level of volatile organic compounds in Sh-13, Sl-2, and WT fruit chilled at 2.5°C for 2 weeks followed by RW was determined by solid phase microextraction coupled with gas chromatography–mass spectrometry . A total of 34 VOCs were identified from the fruit headspace . Principal component analysis revealed 52.8% and 15.3% of the variance was explained by the first and second principal components, respectively. Samples from the transgenic fruit did not separate from those of the WT . However, transgenic fruit had a lower relative abundance of the monoterpenes a- terpineol, 3,7-dimethyl-1,6- octadien-3-ol, and b- damascenone, hydroponic bucket higher levels of the fatty acid 3- methylbutanoic acid .Differentially expressed genes in the Sh-13, Sl-2 transgenic fruit vs. the WT after storage at 2.5°C for either 6 hours or 1 week were identified.
The data from the WT fruit were compared against the pooled data of the transgenics. After 6 hours and 1 week of cold storage, 5,169 genes were induced collectively in the transgenics, whereas 5,007 genes were repressed . In Sh-13 and Sl-2 fruit, a range of 5,832- 7,141 DEGs were shared by both genotypes in the cold . The similarity of these results strengthened the justification for treating the combined results from the CBF1-OE lines as biological replicates of the same genotype.Seedlings at the six-leaf stage, obtained from WT chilled and non-chilled fruit, were stored at 2.5 or 0°C for up to 72 hours, and their photosynthetic performance was monitored every 24 hours on the fourth leaf. No differences were detected between the 2.5°C and control seedlings , however, photosynthetic responses differed after storage at 0°C, and particularly after 72 hours . Responses to cold were heterogeneous among parameters. Some were lower in chilled, compared to non-chilled seedlings: 1) quantum yield of PSII, or Phi2, which is the percentage of incoming light directed into PSII; 2) Linear Electron Flow or the flow of electrons from antennae complexes into PSII; 3) PhiNO, a ratio of processes that inhibit photosynthesis, when absorbed light is not dissipated by non-photochemical quenching . Two parameters related to Non-Photochemical Quenching were also recorded i.e., NPQt and PhiNPQ . They denote the ratio of incoming light in terms of excess energy that is dissipated as heat .
These two variables progressively increased in both ‘chilled’ and ‘non-chilled’ samples over the course of the experiment, however, after 72 hours, they were higher in ‘chilled’ compared to ‘non-chilled’ seedlings. The relative chlorophyll content of seedlings was consistently higher in ‘non-chilled’ compared to ‘chilled’ seedlings after 24, 48, and 72 hours at 0°C, matching qualitative observations . However, at 2.5°C, values did not differ . Overall, visual attributes of non-chilled seedlings were more affected by cold storage after three days than chilled samples. Wilting and leaf curling was accentuated in non-chilled seedlings.Of the engineered genotypes studied, Sl-2 and Sh-13 fruit had the highest and lowest incidence of PCI symptoms, respectively , and Sl-12 showed more aberrant vegetative growth compared to Sh-13 . Therefore, Sl-2 and Sh-13 were selected to characterize their photosynthetic performance during cold storage at the seedling stage. The aim of this test was to determine if fruit and seedling response to cold stress were correlated, and it was instructive to choose lines with distinct PCI responses. Seedlings from Sh-13 and Sl-2 lines at the 6th-leaf stage, obtained from non-chilled fruit, were exposed to 0°C for up to 72 hours. Photosynthetic parameters were monitored every 24 hours on the fourth leaf. LEF progressively decreased over time in both lines and reached the lowest level on day three, with Sl-2 being higher than Sh-13 . The rest of the parameters were similar between genotypes at all time points, but indicators of non-photochemical quenching were higher in Sh-13 . Phenotypically, Sl-2, which showed the highest severity PCI symptoms in the fruit, had a greater incidence of leaf curling and wilting after cold-storage than Sh-13 .
Absolute parallels between the responses of wild-type and transgenic seedlings cannot be established, due to differences in light intensity between experiments.Constitutive over expression of AtCBF1 , AtCBF3 or SlCBF1 in tomato increased abiotic stress tolerance, but at the cost of growth and development. The use of stress-inducible promoters yielded variable results. In ABRC1::AtCBF1 and RD29A::AtCBF1 plants, developmental abnormalities were absent, but it is unclear if growth chambers or greenhouse were used in these studies . Growth chamber-grown RD29A:: AtCBF3 plants had a phenotype consistent with CBF over expression , whereas dexamethasoneinducible lines over expressing AtCBF1 did not manifest abnormalities during vegetative growth . In this study, we used RD29A as a cold-inducible promoter to preferentially over express ShCBF1 or SlCBF1 in harvested fruit under chilling, to minimize plant pleiotropic effects associated with constitutive over expression. The RD29A promoter has been extensively studied in Arabidopsis and widely used in ectopic expression systems, including tomato , potato and tobacco . In this study, RD29A was selected as a promoter based on its responsiveness to cold stress, and the minimized leakiness reported by other authors. Our plants were phenotypically abnormal, similar to transgenic lines constitutively expressing CBF1 . Therefore, our greenhouse led to variable, ‘stressful’ conditions, that activated RD29A and the transcription of CBF1 in the absence of an imposed, designed stress, i.e., cold. The RD29A promoter contains ciselements that are responsive to various abiotic stresses involving changes in osmotic potential, low temperature, dehydration, salinity, and ABA application , consisted of two dehydrins , and one putative proteinase inhibitor . A higher synthesis of protective molecules may explain increases in cold tolerance due to CBF constitutive over expression . We hypothesized that the induction of additional CBF1 transcripts in tomato fruit during post harvest chilling, would increase cold tolerance and ameliorate PCI symptoms due to increased synthesis of transcripts associated with protective proteins. Furthermore, the regulated over expression of CBF1 would circumvent pleiotropic effects during plant growth and development. We were able to induce CBF1 over expression in fruit post harvest chilling, consistent with previous reports , stackable planters thus validating the use of this regulated-expression approach. Expression of CBF1 peaked after three days of cold exposure relative to wild-type fruit: 8-fold in ShCBF1-OE, and 28- fold higher in SlCBF1-OE lines. Remarkably, CBF1 over expression aggravated PCI symptoms. Fruit were unable to complete the ripening process, and deterioration was accelerated when compared to wild-type fruit under the same conditions. Lines Sl-2, Sl-12 and Sh-36 had the highest incidence of pitting and decay, which correlated with CBF1 over expression data. These observations corroborate the connection between CBF1 upregulation and worsening of PCI symptoms. Expression and phenotypic differences between transformation events of the same transgenic construct may be due to position effects , somaclonal variation due to chromosomal rearrangements during tissue culture , or transgene copy number variations. Insertion of multiple copies of a transgene could lead to silencing and hinder the interpretation of progeny segregation ratios . Previous reports altering the expression of CBFs in tomato fruit have revealed mixed results. The absence of a severe phenotype during fruit post harvest chilling of AtCBF1-overexpression lines may be due to the limited storage length used . On the other hand, fruit from slcbf1 mutants showed aggravated PCI symptoms after rewarming, compared to the wild-type . Fruit decay susceptibility is commonly associated with pathogen accessibility to cell wall components, which is facilitated by ripening . However, CBF1 transgenic fruit were ripening-impaired, yet had a higher incidence of decay.
It is tempting to hypothesize that CBF1 regulates cell wall degradation via a ripening-independent pathway. Higher decay and deterioration of transgenic fruit mirrored our volatile data. CBF1-overexpression fruit had a higher relative abundance of 3-methylbutanoic acid, which is associated with an unpleasant ‘cheesy’, ‘sweaty’ aroma in tomato pomace samples fermented by yeast, compared to unfermented samples . Other compounds were less abundant in the transgenics than in WT fruit. a-terpineol is a defense VOC over-emitted in tomato plants that resisted a bacterial disease , and it is perceived as a pine-like, woody aroma, valued in the flavors and fragrances industry . This correlates with the lower decay incidence observed in WT fruit. Synthesis of bdamascenone and 3,7-dimethyl-1,6-octadien-3-ol was greater in ripe fruit and it is associated with the lower color development in our chilled transgenic samples compared to the WT. The CBF family of transcription factors integrate environmental and hormonal signals, to promote plant survival under stresses . There is evidence of an interaction between ethylene biosynthesis and SlCBF1 expression in response to post harvest chilling stress, and, during ripening . Exogenous application of ethylene in addition to cold storage, led to SlCBF1 upregulation . Further, the suppression of ethylene biosynthesis was linked to lower SlCBF1 transcript accumulation and an increase in chilling sensitivity in tomato fruit . Our results are consistent with an increase in fruit susceptibility to chilling due to CBF1 upregulation, but they did not correlate with ethylene biosynthetic rates. CBFs repress the cellular content of the hormone gibberellin , and this may lead to pleiotropic effects seen in CBF1 transgenic lines. Lower GA levels have been shown to underscore the dwarfism observed in transgenic lines over expressing CBF cloned from Arabidopsis . This phenotype has also been observed in potato , rice , apple , and in our transgenic plants. When we treated CBF1-OE seedlings with 5 μL L-1 GA they grew taller than water-treated samples but not to WT levels . GAs may also influence fruit ripening, although the mechanism is not yet clear . In our lines, fruit GA levels were not assessed, however, fruit sensitivity and responsivity to altered GA may differ, causing differential responses in fruit ripening. Based on our results, we propose that CBFs are involved in critical processes in plant growth and development. The higher rate of fruit deterioration and susceptibility to post harvest decay suggest CBFs are important regulators in fruit ripening and senescence under stress conditions. Further research should address its interplay with plant hormones and other key players in the cold stress response pathway.Lines Sh-13 and Sl-2 were selected for RNASeq analysis after short- or long- term cold storage as they developed similar PCI symptoms. Upregulation of genes involved in abiotic and biotic stress response and hormone biosynthesis was detected after cold storage. ARGINASE2 is involved in arginine metabolism and has been linked to methyl jasmonate-induced defense responses to Botrytis cinerea , and PCI tolerance in heat-shock exposed tomato fruit . Modulation of aquaporin expression has been seen in tomato plants exposed to abiotic stresses, including salt, drought, and elevated carbon dioxide . The LURP1 gene is involved in biotic stress responses in Arabidopsis . Interestingly, TLOG1 codes for a cytokinin-activating enzyme associated with tuber formation in tomato plants overexpressing this gene and suggests that hormone homeostasis was compromised in our transgenic lines. Other genes related to biotic and abiotic stress response, and developmental processes, were down regulated in CBF1-OE fruit in response to cold. For instance, AHL genes are positive regulators of cold tolerance in trifoliate orange . Acyl-CoAbinding domain-containing proteins and EXLB1 are involved in stress responses and ripening in tomato , Arabidopsis , and apple . Vestitone reductase-like proteins participate in the biosynthesis of the defense compounds phytoalexins . Repression of these genes is consistent with the higher chilling sensitivity and impaired stress response presented by our transgenic fruit. Down regulation of the transcription factor and master ripening regulator, RIN, can partly explain the inability of CBF1-OE fruit to resume ripening to WT levels after cold storage, and its early repression suggests it may be a candidate for predicting PCI onset. Consistent with this, DDTFR18, with putative roles in ripening and ethylene response was also repressed. In WT Micro-Tom fruit, DDTFR18 was coldinduced , but ectopic CBF1 over expression may have altered this response.