Accelerated fruit deterioration due to CBF1 over expression may be advantageous for seed dispersal Ripening is an early stage in the continuous process leading to senescence , and it may include changes in fruit color, firmness, and flavor. PCI compromises the normal transition between chloroplast and chromoplast during ripening , even after transfer to warmer conditions . However, in the current study, CBF1 over expression not only accentuated the inability of fruit to resume ripening after rewarming that was already observed in WT fruit, but it also increased susceptibility to decay, thus triggering accelerated deterioration and tissue disassembly. Fruits evolved as vehicles for seed dispersal . During ripening, a complex and dynamic set of changes that modify fruit appearance , firmness , aroma and flavor take place . This reconfiguration makes the fruit more attractive for seed dispersing animals, including humans, and is known as the dispersal syndrome . As ripening progresses, fruit becomes more susceptible to pathogen infestation . Since color development is terminated early in CBF1-overexpression lines, we propose that the aggravation of surface pitting, decay, and other symptoms of global fruit disassembly, stackable flower pots might constitute a strategy to facilitate seed dispersal in absence of other traits normally elicited by ripening.

Understanding the basis of fruit deterioration caused by PCI might help to develop solutions for maintaining fruit quality during refrigeration .To explore a potentially advantageous role of PCI in the next plant generation, we collected seeds from WT fruit that were allowed to ripen at room temperature , and from cold-stored fruit followed by RW . Seed color and weight, seedling germination percentages and fresh weight were recorded at room temperature. Cold storage caused seed browning and discoloration, triggered by cell decompartmentalization . The higher weight in chilled seeds could be associated with greater sugar translocation into the seeds during rewarming due to enhanced fruit respiration . We observed similar enhanced respiration in fruit rewarmed after 2.5°C-storage compared to fruit at 12.5°C . Thus, chilled seeds could also have experienced higher sugar translocation from the fruit after rewarming. Treatments like thermal hardening , can support our observations of enhanced germination percentage and seedling weight in samples from chilled fruit. These treatments alter hormonal signaling and also interact with mechanisms crucial to the perception of environmental cues .In the presence of light, chilling stress in cold-sensitive species causes the desynchronization of antenna complexes and PSII , causing photoinhibition and damage .

To better understand how cold acclimation of the seeds in fructa affects the cold tolerance of the resultant seedlings, we exposed WT ‘chilled’ and ‘non-chilled’ seedlings to 0°C for three days and compared their photosynthetic responses. We observed that seed chilling tolerance was enhanced when contained in a maternal organ , thus suggesting that an adaptive mechanism to cope with cold stress was transmitted across generations from the maternal tissue to the embryo. Evidence of chilling acclimation exists, but with the primary stress occurring on seedlings, not fruit contained seeds . Photosynthetic results suggest 1) a reduction of the flow of energy and electrons directed towards PSII, as well as the proportion of photosynthetic inhibition due to excess energy, and relative chlorophyll content, and 2) an increase in mechanisms associated with non-photochemical quenching to minimize over excitation. Chilling-acclimated seedlings may have reduced the amount of light used in photosynthesis to minimize damage, and reducing total chlorophyll is an adaptive mechanism to lower absorbed light .To gain insight into the response of CBF1-OE lines to cold stress during early development, seedlings from lines Sh-13 and Sl-2 were exposed to 0°C for three days. A trend towards higher light absorption and lower non-photochemical quenching was observed in Sl-2 compared to Sh-13. This revealed that Sl-2 experienced more photoinhibition and excitation pressure than Sh-13, suggesting that high levels of SlCBF1 transcript might have weakened the mechanisms against chilling injury in some vegetative tissues.

Compared to Sh-13, line Sl-2 exhibited higher ectopic CBF1 expression and PCI incidence in fruit, along with severe dwarfism and reduced photosynthetic performance in seedlings. Elevated CBF1 expression can be beneficial in vegetative tissues, but in excess, it may suppress the plant’s capacity to cope with cold stress. Further, CBF role in the cold response might be tissue specific and controlled by different mechanisms. While it minimizes photoinhibition and oxidative damage in vegetative tissues, it enhances fruit senescence to facilitate seed dispersal.Post harvest chilling injury is a complex physiological disorder that leads to quantitative and qualitative losses. Knowledge of the CBF1 pathway in the cold-sensitive tomato and molecular mechanisms involved in the development of cold tolerance is needed to develop effective solutions; however, it is still limited compared with that in the cold-tolerant Arabidopsis. In the current study, transgenic tomato lines over expressing the CBF1 gene from Solanum habrochaites and cultivated tomato driven by the RD29A promoter, were generated. Ectopic CBF1 expression data by RT-qPCR confirmed its induction in fruit during cold storage and upregulation for up to one week. Transgenic fruit showed aggravation of PCI symptoms, failure to ripen normally, reduced soluble solids content, abnormal volatile profile and accelerated decay when compared with wild-type fruit, and this correlated with transgenic CBF1 expression. Transcriptomic data suggested that CBF1 over expression in the transgenic lines led to alterations in the ripening process and biotic and abiotic stress responses compared to WT cold stored fruit. Wild-type seedlings originating from chilled fruit displayed signs of acclimation relative to non-chilled samples. Transgenic seedlings photosynthetic performance differed between genotypes, and additional studies are needed to determine their response to cold stress in relation to wild-type seedlings.Fungi are important plant pathogens that cause large economic losses due to their ability to inflict diseases such as rot, rust, and wilt in various plant organs both preharvest and post harvest . Biotrophic fungi feed on living cells and suppress the host immune system by secreting effector proteins . In contrast, necrotrophic fungi feed on dead host cells and cause necrosis by secreting toxins and cell wall degrading enzymes , among other virulence factors . Due to their ability to feed on dead host tissue, necrotrophic fungi are also sometimes grouped into the less defined group of saprotrophic fungi, which includes many fungi that do not actively kill host cells . Additionally, hemibiotrophs are pathogens that start their infection cycle as biotrophs and end as necrotrophs . Biotrophic infection mechanisms are well-studied, whereas those of necrotrophic fungi are less understood. The lower scientific interest in necrotrophic infection mechanisms may be due to their perceived lack of specificity. The brute force strategy of secreting toxins and CWDEs as well as the broad host range of many necrotrophic fungi is often interpreted as indiscriminate killing of host cells without the requirement for host-pathogen compatibility . However, tower garden the reality of necrotrophic infections is multifaceted, as they involve several features initially believed to be unique to biotrophs, e.g., the suppression of the host immune system or symptomless endophytic growth . The relevance of host-pathogen compatibility in necrotrophic infections is also highlighted by the fact that necrotrophic fungi can readily infect ripe fruit but fail to infect unripe fruit or remain quiescent until host and environmental conditions stimulate a successful infection . To develop a better understanding of how fungi attempt to establish infections in fruit, we studied three impactful pathogens with broad host range: Botrytis cinerea, Fusarium acuminatum, and Rhizopus stolonifer. B. cinerea is the causal agent of gray mold, an economically devastating disease, and serves as a model species for plant-necrotroph interactions . In compatible hosts, such as ripe fruit, B. cinerea produces toxins, CWDEs, reactive oxygen species , and other virulence factors to induce rapid death and decay of the plant tissues . In incompatible hosts, such as unripe fruit, B. cinerea establishes quiescent infections while suppressing the host immune system and promoting susceptibility in the host . B. cinerea has been shown to activate fruit ripening processes, including changes in plant hormone biosynthesis and signaling and induction of host CWDEs involved in fruit softening, all of which seem to favor fungal growth and colonization .

Even though B. cinerea infection strategies have been studied in various pathosystems , it is mostly unknown whether F. acuminatum and R. stolonifer, two understudied fungal pathogens, implement similar mechanisms when interacting with compatible and incompatible hosts. F. acuminatum has been reported to infect roots and fruit . Within the Fusarium genus, F. acuminatum is among the most toxic species as it produces strong mycotoxins, such as trichothecene and fumonisins, to kill host cells and induce tissue necrosis . R. stolonifer causes rotting of fruit and other fresh products, mainly by secreting CWDEs, and is considered to be one of the most destructive post harvest pathogens due to its extremely fast growth rate . We leveraged the fact that tomato fruit display an increase in susceptibility to necrotrophic fungal infection as a result of ripening to develop a system for studying compatible and incompatible host-pathogen interactions. The transition from unripe to ripe fruit results in a markedly different physicochemical environment for colonization. In comparison to unripe fruit, ripe fruit have higher levels of total soluble solids, greater titratable acidity , lower firmness, and a different composition of secondary metabolites and volatiles . In light of this, we anticipated that these pathogens would exhibit specific patterns of gene expression based on the fruit ripening stage and that the functions of these genes would reflect important strategies for interaction with the different host environments. First, we evaluated the incidence and progression of fungal infections caused by B. cinerea, F. acuminatum, and R. stolonifer when inoculated in tomato fruit. Then, to determine if the pathogens adapted their infection strategies as a function of the host developmental stage, we analyzed the transcriptomes of each fungus at two points post-inoculation in unripe and ripe tomato fruit and compared these against their transcriptomes when grown under in vitro conditions. This approach allowed us to identify specific pathogenicity and virulence factors, e.g., CWDEs and toxin biosynthetic genes, that are differentially or commonly deployed by the pathogens in each host tissue. As necrotrophic infection strategies may be evolutionarily conserved as well as highly specific, we used the transcriptomic data toexamine virulence functions among the three fungi and identified similarities in the adaptations of the pathogens to the different environments of ripe and unripe fruit. Finally, to further validate necrotrophic strategies dependent on the ripening stage of the fruit host, we evaluated the pathogenicity of the three fungal pathogens in fruit in a non-ripening tomato mutant. Overall, the approach followed in this study provides an initial platform to perform comparative transcriptomics among three fungi that cause economically relevant fruit diseases and sheds light into how pathogens with necrotrophic lifestyles adapt their infection mechanisms during compatible and incompatible interactions.Tomato fruit from the cultivar Ailsa Craig and the isogenic mutant non-ripening were used in this study. Plants were grown under field conditions in Davis, CA, United States, during the 2017 season. Mature green fruit were harvested 31 days post-anthesis and red ripe fruit at 42 dpa. The fungal pathogens studied were B. cinerea strain B05.10, an isolate of R. stolonifer, and an isolate of F. acuminatum. The isolates of R. stolonifer and F. acuminatum were obtained from post harvest infections of fresh produce and identified using morphological and sequencing methods. All fungi were grown on 1% potato dextrose agar plates at room temperature until sporulation. Spore suspensions were prepared in 0.01% TweenR 20 . Fungi from axenic in vitro cultures were grown on 1% PDA plates at RT, and mycelium for RNA extraction was harvested before the fungi reached the sporulation stage.Tomato fruit from AC and nor were collected at MG and RR or RR-like stage. Selected AC MG fruit were green, firm, and had soluble solids content of 5.24 ± 0.44 g sucrose/100 g solution and TA of 5.20 ± 1.24%. AC RR fruit were bright red, pliable when squeezed, and had SSC of 6.27 ± 0.42 g sucrose/100 g solution and a TA of 3.47 ± 0.26%. nor MG fruit were similar to AC MG fruit, and nor RR-like fruit were selected that were green in color slightly soft at the blossom end. Fruit were sterilized in 0.6% sodium hypochlorite, wounded four to six times on the blossom end with a sterile pipette tip and inoculated with 10 µl per wound using a 500 spores/µl suspension for B. cinerea, a 30 spores/µl suspension of R. stolonifer, and a 1,000 spores/µl suspension of F. acuminatum.