Ectopic overexpression of ShCBF1 and SlCBF1 in tomato suggests an alternative view of fruit responses to chilling stress postharvest.

Postharvest chilling injury (PCI) is a physiological disorder that often impairs tomato fruit ripening; this reduces fruit quality and shelf-life, and even accelerates spoilage at low temperatures. The CBF gene family confers cold tolerance in Arabidopsis thaliana, and constitutive overexpression of CBF in tomato increases vegetative chilling tolerance, in part by retarding growth, but, whether CBF increases PCI tolerance in fruit is unknown. We hypothesized that CBF1 overexpression (OE) would be induced in the cold and increase resistance to PCI. We induced high levels of CBF1 in fruit undergoing postharvest chilling by cloning it from S. lycopersicum and S. habrochaites, using the stress-inducible RD29A promoter. Harvested fruit were cold-stored (2.5°C) for up to three weeks, then rewarmed at 20°C for three days. Transgene upregulation was triggered during cold storage from 8.6- to 28.6-fold in SlCBF1-OE, and between 3.1- to 8.3-fold in ShCBF1-OE fruit, but developmental abnormalities in the absence of cold induction were visible. Remarkably, transgenic fruit displayed worsening of PCI symptoms, i.e., failure to ripen after rewarming, comparatively higher susceptibility to decay relative to wild-type (WT) fruit, lower total soluble solids, and the accumulation of volatile compounds responsible for off-odors. These symptoms correlated with CBF1 overexpression levels. Transcriptomic analysis revealed that the ripening and biotic and abiotic stress responses were altered in the cold-stored transgenic fruit. Seedlings grown from 'chilled' and 'non-chilled' WT fruit, in addition to 'non-chilled' transgenic fruit were also exposed to 0°C to test their photosynthetic response to chilling injury. Chilled WT seedlings adjusted their photosynthetic rates to reduce oxidative damage; 'non-chilled' WT seedlings did not. Photosynthetic parameters between transgenic seedlings were similar at 0°C, but SlCBF1-OE showed more severe photoinhibition than ShCBF1-OE, mirroring phenotypic observations. These results suggest that 1) CBF1 overexpression accelerated fruit deterioration in response to cold storage, and 2) Chilling acclimation in fructus can increase chilling tolerance in seedling progeny of WT tomato.

Energy homeostasis mediated by the LcSnRK1α-LcbZIP1/3 signaling pathway modulates litchi fruit senescence.

Cellular energy status is a key factor deciding the switch-on of the senescence of horticultural crops. Despite the established significance of the conserved energy master regulator sucrose non-fermenting 1 (SNF1)-related protein kinase 1 (SnRK1) in plant development, its working mechanism and related signaling pathway in the regulation of fruit senescence remain enigmatic. Here, we demonstrate that energy deficit accelerates fruit senescence, whereas exogenous ATP treatment delays it. The transient suppression of LcSnRK1α in litchi (Litchi chinensis Sonn.) fruit inhibited the expression of energy metabolism-related genes, while its ectopic expression in tomato (Solanum lycopersicum) promoted ripening and a high energy level. Biochemical analyses revealed that LcSnRK1α interacted with and phosphorylated the transcription factors LcbZIP1 and LcbZIP3, which directly bound to the promoters to activate the expression of DARK-INDUCIBLE 10 (LcDIN10), ASPARAGINE SYNTHASE 1 (LcASN1), and ANTHOCYANIN SYNTHASE (LcANS), thereby fine-tuning the metabolic reprogramming to ensure energy and redox homeostasis. Altogether, these observations reveal a post-translational modification mechanism by which LcSnRK1α-mediated phosphorylation of LcbZIP1 and LcbZIP3 regulates the expression of metabolic reprogramming-related genes, consequently modulating litchi fruit senescence.

Salicylic acid delays pear fruit senescence by playing an antagonistic role toward ethylene, auxin, and glucose in regulating the expression of PpEIN3a.

Salicylic acid (SA) and ethylene (ET) are crucial fruit senescence hormones. SA inhibited ET biosynthesis. However, the mechanism of SA delaying fruit senescence is less known. ETHYLENE INSENSITIVE 3 (EIN3), a key positive switch in ET perception, functions as a transcriptional activator and binds to the primary ET response element that is present in the promoter of the ETHYLENE RESPONSE FACTOR1 gene. In this study, a gene encoding putative EIN3 protein was cloned from sand pear and designated as PpEIN3a. The deduced PpEIN3a contains a conserved EIN3 domain. The evolutionary analysis results indicated that PpEIN3a belonged to the EIN3 superfamily. Real-time quantitative PCR analysis revealed that the accumulation of PpEIN3a transcripts were detected in all tissues of this pear. Moreover, PpEIN3a expression was regulated during fruit development. Interestingly, the expression of PpEIN3a was downregulated by SA but upregulated by ET, auxin, and glucose. Additionally, the contents of free and conjugated SA were higher than those of the control after SA treatment. While the content of ET and auxin (indole-3-acetic acid, IAA) dramatically decreased after SA treatment compared with control during fruit senescence. The content of glucose increased when fruit were treated by SA for 12 h and then there were no differences between SA treatment and control fruit during the shelf life. SA also delayed the decrease in sand pear (Pyrus pyrifolia Nakai. 'Whangkeumbae') fruit firmness. The soluble solid content remained relatively stable between the SA treated and control fruits. This study showed that SA plays an antagonistic role toward ET, auxin, and glucose in regulating the expression of PpEIN3a to delay fruit senescence.

Single and Double Mutations in Tomato Ripening Transcription Factors Have Distinct Effects on Fruit Development and Quality Traits.

Spontaneous mutations associated with the tomato transcription factors COLORLESS NON-RIPENING (SPL-CNR), NON-RIPENING (NAC-NOR), and RIPENING-INHIBITOR (MADS-RIN) result in fruit that do not undergo the normal hallmarks of ripening but are phenotypically distinguishable. Here, we expanded knowledge of the physiological, molecular, and genetic impacts of the ripening mutations on fruit development beyond ripening. We demonstrated through phenotypic and transcriptome analyses that Cnr fruit exhibit a broad range of developmental defects before the onset of fruit ripening, but fruit still undergo some ripening changes similar to wild type. Thus, Cnr should be considered as a fruit developmental mutant and not just a ripening mutant. Additionally, we showed that some ripening processes occur during senescence in the nor and rin mutant fruit, indicating that while some ripening processes are inhibited in these mutants, others are merely delayed. Through gene expression analysis and direct measurement of hormones, we found that Cnr, nor, and rin have alterations in the metabolism and signaling of plant hormones. Cnr mutants produce more than basal levels of ethylene, while nor and rin accumulate high concentrations of abscisic acid. To determine genetic interactions between the mutations, we created for the first time homozygous double mutants. Phenotypic analyses of the double ripening mutants revealed that Cnr has a strong influence on fruit traits and that combining nor and rin leads to an intermediate ripening mutant phenotype. However, we found that the genetic interactions between the mutations are more complex than anticipated, as the Cnr/nor double mutant fruit has a Cnr phenotype but displayed inhibition of ripening-related gene expression just like nor fruit. Our reevaluation of the Cnr, nor, and rin mutants provides new insights into the utilization of the mutants for studying fruit development and their implications in breeding for tomato fruit quality.

Transcriptomic analysis of a near-isogenic line of melon with high fruit flesh firmness during ripening.

BACKGROUND: A near-isogenic line (NIL) of melon (SC10-2) with introgression in linkage group X was studied from harvest (at firm-ripe stage of maturity) until day 18 of postharvest storage at 20.5 °C together with its parental control ('Piel de Sapo', PS). RESULTS: SC10-2 showed higher flesh firmness and whole fruit hardness but lower juiciness than its parental. SC10-2 showed a decrease in respiration rate accompanied by a decrease in ethylene production during ripening, both of which fell to a greater extent than in PS. The introgression affected 11 volatile organic compounds (VOCs), the levels of which during ripening were generally higher in SC10-2 than in PS. Transcriptomic analysis from RNA-Seq revealed differentially expressed genes (DEGs) associated with the effects studied. For example, 909 DEGs were exclusive to the introgression, and only 23 DEGs were exclusive to postharvest ripening time. Major functions of the DEGs associated with introgression or ripening time were identified by cluster analysis. About 37 genes directly and/or indirectly affected the delay in ripening of SC10-2 compared with PS in general and, more particularly, the physiological and quality traits measured and, probably, the differential non-climacteric response. Of the former genes, we studied in more detail at least five that mapped in the introgression in linkage group (LG) X, and 32 outside it. CONCLUSION: There is an apparent control of textural changes, VOCs and fruit ripening by an expression quantitative trait locus located in LG X together with a direct control on them due to genes presented in the introgression (CmTrpD, CmNADH1, CmTCP15, CmGDSL esterase/lipase, and CmHK4-like) and CmNAC18. © 2020 Society of Chemical Industry.

An integrated metabolomic and gene expression analysis identifies heat and calcium metabolic networks underlying postharvest sweet cherry fruit senescence.

MAIN CONCLUSION: Ηeat and calcium treatments reprogram sweet cherry fruit metabolism during postharvest senescence as evidenced by changes in respiration, amino acid metabolism, sugars, and secondary metabolites shift. Heat and calcium treatments are used to improve postharvest fruit longevity; however, the exact mechanism remains poorly understood. To characterize the impact of these treatments on sweet cherries metabolism, 'Lapins' fruits were treated with heat or CaCl2 solutions and their combination and subsequently were exposed at room temperature, for up to 4 days, defined as senescence period. Single and combined heat and calcium treatments partially delayed fruit senescence, as evidenced by changes in fruit colour darkening, skin penetration force, and respiration activity. Calcium content was noticeably increased by heat in Ca-treated fruit. Several primary metabolites, including amino acids, organic acids, and alcohols, were decreased in response to both treatments, while many soluble sugars and secondary metabolites were increased within 1 day post-treatment. Changes of several metabolites in heat-treated fruits, especially esculetin, peonidin 3-O-glucoside and peonidin 3-O-galactoside, ribose, pyroglutamate, and isorhamnetin-3-O-rutinoside, were detected. The metabolome of fruit exposed to calcium also displayed substantial modulations, particularly in the levels of galactose, glycerate, aspartate, tryptophan, phospharate rutin, and peonidin 3-O-glucoside. The expression of several genes involved in TCA cycle (MDH1, IDH1, OGDH, SUCLA2, and SDH1-1), pectin degradation (ADPG1) as well as secondary (SK1, 4CL1, HCT, and BAN), amino acids (ALDH18A1, ALDH4A1, GS, GAD, GOT2, OPLAH, HSDH, and SDS), and sugar (PDHA1 and DLAT) metabolism were affected by both treatments. Pathway-specific analysis further revealed the regulation of fruit metabolic programming by heat and calcium. This work provides a comprehensive understanding of metabolic regulation in response to heat and calcium during fruit senescence.

Litchi Fruit LcNAC1 is a Target of LcMYC2 and Regulator of Fruit Senescence Through its Interaction with LcWRKY1.

Senescence is a key factor resulting in deterioration of non-climacteric fruit. NAC transcription factors are important regulators in plant development and abiotic stress responses, yet little information regarding the role of NACs in regulating non-climacteric fruit senescence is available. In this study, we cloned 13 NAC genes from litchi (Litchi chinensis) fruit, and analyzed subcellular localization and expression profiles of these genes during post-harvest natural and low-temperature-delayed senescence. Of the 13 NAC genes, expression of LcNAC1 was up-regulated in the pericarp and pulp as senescence progressed, and was significantly higher in senescence-delayed fruit than that in naturally senescent fruit. LcNAC1 was induced by exogenous ABA and hydrogen peroxide. Yeast one-hybrid analysis and transient dual-luciferase reporter assay showed that LcNAC1 was positively regulated by the LcMYC2 transcription factor. LcNAC1 activated the expression of LcAOX1a, a gene associated with reactive oxygen species regulation and energy metabolism, whereas LcWRKY1 repressed LcAOX1a expression. In addition, LcNAC1 interacted with LcWRKY1 in vitro and in vivo. These results indicated that LcNAC1 and LcWRKY1 form a complex to regulate the expression of LcAOX1a antagonistically. Taken together, the results reveal a hierarchical and co-ordinated regulatory network in senescence of harvested litchi fruit.

iTRAQ-based quantitative proteomic analysis reveals the role of the tonoplast in fruit senescence.

UNLABELLED: The vacuole is by far the largest multifunctional organelle in fruits and plays a functional role in fruit development and fruit quality. Despite its significance, little information exists pertaining to the role of the vacuolar membrane (tonoplast) in the process of fruit senescence. In the present study, an iTRAQ-based quantitative proteomic approach was used to characterize the dynamic alterations in the tonoplast proteome during fruit senescence. Tonoplasts were purified from apple fruit at various stages of senescence using an iodixanol step gradient protocol. A total of 345 tonoplast-related proteins were identified with diverse functions such as transporters and proton pumps, signal transduction, membrane fusion or vesicle trafficking, cellular metabolic process, defense response, protein folding and degradation, and cytoskeleton. Changes in protein abundance during storage were characterized for the identified proteins. A total of 22 proteins displayed differential levels of abundance during storage. The senescence-related tonoplast proteins mostly function in the transportation of metabolites, signal transduction, membrane trafficking, and stress response. RT-qPCR analysis was used to quantify the level of expression of nine genes encoding some of the differentially abundant proteins. The results of this study provide new information regarding the function of the tonoplast during fruit senescence. BIOLOGICAL SIGNIFICANCE: Studies on the postharvest physiology and biochemistry of apple fruit have been conducted for several decades. Little proteomic information is available, however, pertaining to the role of the vacuole in fruit ripening and senescence. In the present study, an iTRAQ-based quantitative proteomic analysis was conducted on tonoplasts isolated from apple fruit in order to gain a global view of alterations in the tonoplast proteome during fruit senescence. The information obtained in the present study not only provides basic information about the tonoplast proteome in apple fruit but also characterized the quantitative changes that occur in the abundance of tonoplast proteins during the course of fruit senescence. This study provides a deeper insight into the cellular functions of the vacuole during fruit senescence that can serve as a basis for the development of future biotechnological strategies for the improvement of fruit quality.

Proteomic analysis of changes in mitochondrial protein expression during peach fruit ripening and senescence.

UNLABELLED: Ripening and senescence define the last step of fruit development, which directly affects its commercial value, and mitochondria play a crucial role in these processes. To better understand mitochondrial roles in maintaining and regulating metabolism in storage tissues, highly purified mitochondria were isolated from peach tissues (Prunus persica. cv. Xiahui-8) stored at 4°C and 25°C, respectively, and their proteome was conducted using the method of 2-DE and MALDI-TOF/TOF. Twenty-four (24) differentially expressed proteins (2-fold, p≤0.01) were identified out of more than 300 spots and were divided into six categories by PIR and Uniprot, including oxidative stress (34%), carbon metabolism (29%), respiratory chain (17%), amino acid metabolism and protein biosynthesis (8%), heat shock protein (4%), ion channels (4%). Proteins involved in antioxidative systems, gluconeogenesis, glycolysis, ethanol fermentation were changed significantly in response to high temperature. Storage at 4°C dramatically delayed ripening and senescence processes by postponing the climacteric peak, slowing down carbon metabolism and degradation of cell structure. Besides, low temperature induced the expression of formate dehydrogenase and some amino acid metabolism proteins. Proteins classified in respiratory chain, ion channels showed high coherence with climacteric respiratory burst, and the antioxidative enzymes showed relatively important symptoms on ROS scavenging through orderly expressions. SIGNIFICANCE: With the advent of proteomics and mass spectrometry (MS), it becomes possible to identify the specific functions of differentially abundant proteins in peach mitochondria. In the present study, a procedure to isolate mitochondria from peach fruits was established, and the mitochondrial proteome was systematically analyzed by 2-D gel electrophoresis procedures in combination with protein identification by mass spectrometry. Differentially expressed proteins in peach mitochondria during different stages of peach fruit ripening and senescence were characterized. Our data provide a great deal of information likely to enhance the understanding of the mitochondrial function in peach ripening and senescent process during storage.

Ethylene negatively regulates transcript abundance of ROP-GAP rheostat-encoding genes and affects apoplastic reactive oxygen species homeostasis in epicarps of cold stored apple fruits.

Apple (Malus×domestica Borkh) fruits are stored for long periods of time at low temperatures (1 °C) leading to the occurrence of physiological disorders. 'Superficial scald' of Granny Smith apples, an economically important ethylene-dependent disorder, was used as a model to study relationships among ethylene action, the regulation of the ROP-GAP rheostat, and maintenance of H2O2 homeostasis in fruits during prolonged cold exposure. The ROP-GAP rheostat is a key module for adaptation to low oxygen in Arabidopsis through Respiratory Burst NADPH Oxidase Homologs (RBOH)-mediated and ROP GTPase-dependent regulation of reactive oxygen species (ROS) homeostasis. Here, it was shown that the transcriptional expression of several components of the apple ROP-GAP machinery, including genes encoding RBOHs, ROPs, and their ancillary proteins ROP-GEFs and ROP-GAPs, is coordinately and negatively regulated by ethylene in conjunction with the progressive impairment of apoplastic H2O2 homeostatic levels. RNA sequencing analyses showed that several components of the known ROP- and ROS-associated transcriptional networks are regulated along with the ROP-GAP rheostat in response to ethylene perception. These findings may extend the role of the ROP-GAP rheostat beyond hypoxic responses and suggest that it may be a functional regulatory node involved in the integration of ethylene and ROS signalling pathways in abiotic stress.

Comprehensive insights on how 2,4-dichlorophenoxyacetic acid retards senescence in post-harvest citrus fruits using transcriptomic and proteomic approaches.

Auxin-like 2,4-dichlorophenoxyacetic acid (2,4-D), a high-efficiency anti-stalling agent for the post-harvest fresh fruit industry, has had its use restricted due to environmental concerns. However, no other substitutes for 2,4-D are available to the post-harvest industry. Insights into the molecular mechanism underlying the effects of 2,4-D on fruit quality preservation will provide a theoretical basis for exploring new safe and effective anti-stalling agents. This study comprehensively analysed changes in the peel of Olinda Valencia orange [Citrus sinensis (L.) Osbeck] induced by 500 ppm 2,4-D using 'omic'-driven approaches. Transcriptional profiling revealed that transcriptional factor (mainly AP2/ERF, WRKY, and NAC family members), transport, and hormone metabolism genes were over-represented and up-regulated within 24h post-treatment (HPT). Stress defence genes were up-regulated, while cell wall metabolism genes were down-regulated after 48 HPT. However, secondary metabolism genes, especially phenylpropanoid and lignin biosynthesis-related genes, were over-represented at all the time points. Comparative proteomic analysis indicated that the expression of proteins implicated in stress responses (25%), hormone metabolism, and signal transduction (12%) significantly accumulated at the post-transcriptional level. Hormone levels detected by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) showed that abscisic acid, salicylic acid, and 2,4-D significantly increased, while ethylene production (detected by gas chromatography) decreased after 2,4-D treatment. In addition, lignin and water content in the fruit peel also increased and the epicuticle wax ultrastructure was modified. In conclusion, 2,4-D retarded fruit senescence by altering the levels of many endogenous hormones and by improving stress defence capabilities by up-regulating defence-related genes and proteins.