The CaMYB340 transcription factor induces chilling injury in post-harvest bell pepper by inhibiting fatty acid desaturation.

Bell pepper (Capsicum annuum L.) is a tradable and desirable crop; however, its perishable nature requires low-temperature handling. Paradoxically, cold causes chilling injury (CI) and post-harvest waste. Current knowledge about CI in pepper is limited. The mechanism of CI is multi-faceted; therefore, we focused on fatty acid (FA) desaturation. We identified an upstream nuclear transcription factor (TF), CaMYB340, belonging to the R2R3 MYB subfamily, that negatively regulates FA desaturation and CaCBF3 expression and whose gene and protein expression is induced by low temperature (4°C). Specifically, McrBC treatment and bisulfite sequencing PCR indicate that exposure to cold triggers DNA methylation on one of the CHH sites in the CaMYB340 promoter. This epigenetic event at least partly contributes to the upregulation of CaMYB340 transcript levels. Increased expression of CaMYB340 results in the formation of protein complexes with CabHLH93 and CaMYB1R1, which in turn downregulate the expression of downstream genes. For peppers held at low temperature, transient overexpression of CaMYB340 reduced unsaturated FA content and membrane fluidity, resulting in cold-induced poor peel texture. Transient CaMYB340 silencing increased FA desaturation and lowered electrolyte leakage, enhancing cold tolerance in CaMYB340 knockdown fruits. Overall, these results underscore the intricacy of transcriptional networks in plants and highlight the role of CaMYB340 in CI occurrence in pepper fruits.

Phenylpropanoid metabolism in relation to peel browning development of cold-stored 'Nanguo' pears.

Cold-stored 'Nanguo' pears are susceptible to peel browning during subsequent shelf life. In this study, 'Nanguo' pears were cold-stored for different periods to elucidate the metabolism of phenylpropanoid accompanying browning. Changes in phenolics and flavonoids and the crucial enzyme activity and related gene expression involved in the phenylpropanoid pathway were monitored. It was found that the fruit that underwent long-term storage showed peel browning symptoms prior to softening, and the symptom got worse with increasing shelf life. Meanwhile, the accumulation of reactive oxygen species (ROS) and the decrease of ROS scavenging ability were noted. The content of phenolics and flavonoids and the activity and expression of shikimate dehydrogenase (SKDH), phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) involved in the phenylpropanoid pathway decreased with prolonged storage. Correlation analysis revealed that browning was positively correlated with ROS accumulation, and the content of phenolics and flavonoids directly affected ROS scavenging ability. In addition, the decrease in phenolics and flavonoids might be owing to the reduced activity of SKDH, PAL, and 4CL and the down-regulated expression of PuPAL and Pu4CL. Collectively, this study indicated that the metabolism of phenylpropanoid is associated with the browning response induced by low-temperature stress.

24-epibrassinolide alleviates postharvest yellowing of broccoli via improving its antioxidant capacity.

Postharvest crop yellowing is a major concern in the broccoli industry. The effect and underlying mechanisms of 24-epibrassinolide (EBR) treatment on yellowing in postharvest broccoli were investigated. Treatment with 2 µM EBR markedly inhibited the increase of the yellowing index and L* values, causing higher retention of the metric hue angle and chlorophyll content compared to the control. Treatment also alleviated oxidative damage by preventing the accumulation of malondialdehyde and superoxide anion (O2•-). The ascorbic acid content of broccoli reached its lowest value at the end of its shelf life, whereas that of the treated sample was obviously higher than the control. Moreover, treated broccoli exhibited higher superoxide dismutase, ascorbate peroxidase, and phenylalanine ammonia-lyase activities. Multivariate statistical analysis further demonstrated the effective enhancement of EBR treatment on antioxidant enzymes. These results indicate that exogenous application of EBR ameliorates postharvest yellowing by improving the antioxidant capacity of broccoli.

Exogenous glutathione alleviates chilling injury in postharvest bell pepper by modulating the ascorbate-glutathione (AsA-GSH) cycle.

We investigated the effect of exogenous glutathione (GSH) on chilling injury (CI) in postharvest bell pepper fruits stored at low temperature and explored the mechanism of this treatment from the perspective of the ascorbate-glutathione (AsA-GSH) cycle. Compared with the control, fruits treated with exogenous GSH before refrigeration displayed only slight CI symptoms and mitigated CI-induced cell damage after 10 d. Moreover, the treated peppers had lower lipid peroxidation product, H2O2, and O2- content than those did the control. Glutathione treatment enhanced the ascorbate-glutathione cycle by upregulating CaAPX1, CaGR2, CaMDHAR1, and CaDHAR1 and the antioxidant enzymes APX, GR, and MDHAR associated with the ascorbate-glutathione cycle. Glutathione treatment also increased ascorbate and glutathione concentrations. Taken together, our results showed that exogenous GSH treatment could alleviate CI in pepper fruits during cold storage by triggering the AsA-GSH cycle and improving antioxidant capacity.

Calcium Treatment Alleviates Pericarp Browning of 'Nanguo' Pears by Regulating the GABA Shunt After Cold Storage.

Long-term storage of pear fruit at low temperature can retard senescence but may result in pericarp browning. We previously reported that increasing endogenous γ-aminobutyrate (GABA) content by exogenous GABA treatment can maintain mitochondrial structure integrity, thereby alleviating pericarp browning of 'Nanguo' pears after cold storage. Here, we tested the effectiveness of Ca2+ treatment on pericarp browning in relation to GABA biosynthesis. Fruit browning was reduced by treatment with Ca2+ after 180 days of storage. Pericarp Ca2+ and calmodulin content in treated fruit increased, and concomitantly, endogenous GABA content, key GABA synthesis-related enzyme activity, and gene expression were upregulated. Moreover, the mitochondrial structure in the pericarp tissue was found to be well preserved. Thus, Ca2+ treatment effectively reduced pericarp browning of refrigerated 'Nanguo' pears owing to improvement in the GABA biosynthesis capacity in the fruit.

Combining salicylic acid and trisodium phosphate alleviates chilling injury in bell pepper (Capsicum annuum L.) through enhancing fatty-acid desaturation efficiency and water retention.

Chilling injury (CI) restricts the quality and shelf life of bell pepper fruits; reducing these CI-induced detrimental effects is therefore of high economic and agricultural relevance. Here, we investigated the effects of trisodium phosphate (TSP), salicylic acid (SA), and TSP + SA treatments on pepper fruits under cold stress at 4 °C for 25 d. Combined TSP + SA treatment performed an optimal effect. Specifically, TSP + SA treatment enhanced fatty-acid desaturation efficiency, as indicated by the increased expression of key fatty acid desaturase genes, and higher content of unsaturated fatty acids. Meanwhile, TSP + SA treatment inhibited the CI-induced membrane damage, manifested as lower electrolyte leakage and malondialdehyde content. Furthermore, low field-nuclear magnetic resonance and proline content also revealed that TSP + SA treatment mitigated CI through enhancing water retention in pepper fruits. Collectively, our results may shed new light on optimizing the low-temperature storage conditions of post-harvest peppers.

Transcriptome analysis of postharvest blueberries (Vaccinium corymbosum 'Duke') in response to cold stress.

BACKGROUND: Blueberry (Vaccinium spp.) is a small berry with high economic value. Although cold storage can extend the storage time of blueberry to more than 60 days, it leads to chilling injury (CI) displaying as pedicle pits; and the samples of 0 °C-30 days was the critical point of CI. However, little is known about the mechanism and the molecular basis response to cold stress in blueberry have not been explained definitely. To comprehensively reveal the CI mechanisms in response to cold stress, we performed high-throughput RNA Seq analysis to investigate the gene regulation network in 0d (control) and 30d chilled blueberry. At the same time, the pitting and decay rate, electrolyte leakage (EL), malondialdehyde (MDA) proline content and GSH content were measured. RESULTS: Two cDNA libraries from 0d (control) and 30d chilled samples were constructed and sequenced, generating a total of 35,060 unigenes with an N50 length of 1348 bp. Of these, 1852 were differentially expressed, with 1167 upregulated and 685 downregulated. Forty-five cold-induced transcription factor (TF) families containing 1023 TFs were identified. The DEGs indicated biological processes such as stress responses; cell wall metabolism; abscisic acid, gibberellin, membrane lipid, energy metabolism, cellular components, and molecular functions were significantly responsed to cold storage. The transcriptional level of 40 DEGs were verified by qRT-PCR. CONCLUSIONS: The postharvest cold storage leads serious CI in blueberry, which substantially decreases the quality, storability and consumer acceptance. The MDA content, proline content, EL increased and the GSH content decreased in this chilled process. The biological processes such as stress responses, hormone metabolic processes were significantly affected by CI. Overall, the results obtained here are valuable for preventing CI under cold storage and could help to perfect the lack of the genetic information of non-model plant species.

Glycine betaine treatment alleviates loss of aroma-related esters in cold-stored 'Nanguo' pears by regulating the lipoxygenase pathway.

Glycine betaine (GB) is known to alleviate chilling injury in many fruit species. Therefore, we studied how GB affects the biosynthesis of esters in 'Nanguo' pears. Based on the kinds of esters, total esters, and the quantity of the main esters, it was evident that aroma losses were alleviated by GB treatment. In addition, unsaturated fatty acids contents (linoleic and linolenic acid) and the activities of lipoxygenase (LOX) and alcohol acyltransferase (AAT) enzymes were also increased. Meanwhile, comparing with the control fruit, the genes directly involved in ester synthesis were up-regulated in the GB-treated fruit. In addition, an increase in the activities and gene expression of antioxidant enzymes was observed in the treated samples. Thus, GB treatment promotes the synthesis of esters by regulating the LOX pathway and increasing antioxidant capacity, thereby effectively improving the quality of esters in cold-stored fruit.

Exogenous glycine betaine treatment alleviates low temperature-induced pericarp browning of 'Nanguo' pears by regulating antioxidant enzymes and proline metabolism.

The effect of glycine betaine (GB) on chilling injury (CI)-induced pericarp browning in 'Nanguo' pears was investigated during shelf life at 20 °C after storage at 0 °C for 120 d. GB treatment alleviated the severity of browning in 'Nanguo' pears as represented by lower browning index (BI) and browning incidence. Membrane lipid peroxidation in GB-treated fruit was lower than that in the control, and membrane integrity was maintained in good condition. The activities and expression of ascorbate peroxidase (APX), catalase (CAT), and superoxide dismutase (SOD) were higher in GB-treated fruit than in control fruit. Furthermore, significantly higher proline content, proline synthesis key enzyme activities, and gene expression were observed in the treated fruit, including ornithine d-aminotransferase (OAT) and Δ1-pyrroline-5-carbox-ylate synthetase (P5CS), which were consistent with the browning tendency. In a nutshell, GB treatment can effectively alleviate pericarp browning of cold-stored 'Nanguo' pears by regulating antioxidant enzymes and proline metabolism.

Transcription factor CaNAC1 regulates low-temperature-induced phospholipid degradation in green bell pepper.

Phospholipids constitute the main component of biomembranes. During low-temperature storage and transportation of harvested bell peppers (Capsicum annuum), chilling injury participates in their decay. A primary cause of this chilling injury is phospholipid degradation. In this study, three genes encoding phospholipase D (PLD) were identified from bell peppers and their activities were examined under cold stress. Low temperature (4 °C) induced strong accumulation of the CaPLDα4 transcript, suggesting that it is associated with the phenomenon of phospholipid degradation and destruction of cell membranes. Low temperature also significantly induced increased amounts of NAM-ATAF1/2-CUC2 (NAC) domain transcription factors. CaNAC1 was found to interact with the promoter of CaPLD4 in a yeast one-hybrid screen. Electrophoretic mobility shift and ß-glucuronidase reporter assays demonstrated that CaNAC1 binds to the CTGCAG motif in the CaPLDα4 promoter, thereby activating its transcription and controlling phospholipid degradation. The ubiquitination sites of the CaNAC1 protein were characterized by liquid chromatography-tandem mass spectrometry. We conclude that CaNAC1 is a transcriptional activator of CaPLDα4 and suggested that it participates in the degradation of membrane lipids in bell peppers when they are stored at low temperature.

Potential of jasmonic acid (JA) in accelerating postharvest yellowing of broccoli by promoting its chlorophyll degradation.

Chlorophyll degradation is the main reason for postharvest yellowing of broccoli. To uncover the role of jasmonic acid (JA) on the degradation of chlorophyll, broccoli flowers were treated with exogenous methyl jasmonate (MeJA) and diethyldithiocarbamic acid (DIECA). We found a surge of endogenous JA content with the yellowing process, and a significant correlation between JA and chlorophyll content. MeJA treatments led to increased endogenous JA, increased allene oxide cyclase (AOC) activity, and enhanced expression of JA synthesis genes. MeJA caused a stronger reduction in the maximum quantum yield (Fv/Fm), fluorescence decline ratio (Rfd), and total chlorophyll content, advanced the peak of pheide a oxygenase (PAO) activity, and up-regulated the expression of chlorophyll degradation genes. The DIECA treatment resulted in lower endogenous levels of JA, and AOC and 12-oxo-phytodienoic acid reductase (OPR) activity. This study revealed that the potential role of JA on broccoli yellowing is to promote the chlorophyll degradation.

Changes in Membrane Lipid Metabolism Accompany Pitting in Blueberry During Refrigeration and Subsequent Storage at Room Temperature.

Low-temperature storage is the primary postharvest method employed to maintain fruit quality and commercial value. However, pitting can develop during refrigeration, especially during the shelf life. In this study, a membrane lipidomic approach was employed to analyze the potential relationship between pitting and membrane lipid metabolism during post-cold-storage shelf life. We also determined the changes in ultrastructure and water distribution by low-field nuclear magnetic resonance (LF-NMR) and assessed the permeability of membrane, membrane lipid peroxidation, proline and malondialdehyde contents, and the activity and gene expression of phospholipase D and lipoxygenase, which are involved in membrane lipid metabolism. The results indicated that the changes in blueberry phospholipids during storage could be caused by cold stress. Furthermore, dehydration is a manifestation of chilling injury. Finally, the significant increase in electrolyte leakage, content of malondialdehyde and proline, and activity of phospholipase D and lipoxygenase in chilled blueberry also indicated that membrane lipid metabolism plays an important role in cold stress response.

Chlorophyll degradation and carotenoid biosynthetic pathways: Gene expression and pigment content in broccoli during yellowing.

Broccoli undergoes yellowing in unfavorable conditions, thereby diminishing the sensory quality and commodity value. This study aimed to investigate systematically cellular and/or biomolecular changes involved in broccoli yellowing by analyzing changes in microstructural integrity, pigment content, and gene expression. On day-5 of storage at 20 °C, the buds turned yellow without blooming and showed structural damage; ultrastructural analysis revealed plastid transformation and abnormal chloroplast development. Genes regulating pigment content and chloroplast structure directly were identified. More specifically, BoCAO and BoNYC1 regulated chlorophyll turnover, affecting chlorophyll a and b contents. Changes in the ß-cryptoxanthin content were influenced by the combined action of up- (BoHYD) and downstream (BoZEP) genes. BoZEP and BoVDE were activated after cold-temperature induction. High BoHO1 expression delayed yellowing at low temperature, inducing BoZEP expression. Color intensity correlated significantly with the chlorophyll b, ß-cryptoxanthin, and ß-carotene contents, which were associated with increased yellowing of plant tissues.

GABA application improves the mitochondrial antioxidant system and reduces peel browning in 'Nanguo' pears after removal from cold storage.

Although refrigeration is commonly used in the storage of 'Nanguo' pears (Pyrus ussuriensis Maxim.), long-term refrigeration can result in browning of the pericarp. In this study, we aimed to determine how γ-aminobutyrate (GABA) affects the mitochondrial oxidation defense system in 'Nanguo' pears and how it might be used to prevent post-refrigeration peel browning. We found that fruit treated with GABA browned slower, and had lower browning indices and reactive oxygen and malondialdehyde content; increased peroxidase, superoxide dismutase, alternating oxidase, and catalase enzyme activities; and heightened enzyme-related gene expression. The mitochondria of GABA-treated fruit also showed less damage following cold storage, and there were decreases in mitochondrial membrane permeability transition pore concentrations. Furthermore, we detected an increase in the endogenous GABA content of fruits following GABA treatment. These observations indicate that, by regulating the mitochondrial oxidative defense system and maintaining mitochondrial structure, GABA is effective in terms of reducing peel browning.

Insights into the metabolism of membrane lipid fatty acids associated with chilling injury in post-harvest bell peppers.

Bell peppers are susceptible to chilling injury (CI). To uncover the metabolism of membrane lipid fatty acids (FAs) accompanying CI, a gas chromatography-mass spectrometry (GC-MS)-based approach was used to quantitatively profile major membrane lipid FAs in bell peppers. RT-qPCR was performed to investigate the expression of the key genes that regulate the synthesis of unsaturated FAs. Additionally, we used microstructural, organoleptic, and physicochemical investigations to monitor the primary physiological metabolism of bell peppers. The study revealed that CI symptoms mostly resulted from the destabilization of the cytomembrane, which was induced by decreasing FA desaturation. Moreover, three times lower level of the double bond index in chilled fruits, than the control, further proved that membrane FA unsaturation can be considered a key factor during CI. In conclusion, this study revealed that the metabolism of membrane lipid FAs is involved in responses to CI.

Transcriptome analysis of harvested bell peppers (Capsicum annuum L.) in response to cold stress.

Bell peppers are valued for their plentiful vitamin C and nutritional content. Pepper fruits are susceptible to cold storage, which leads to chilling injury (CI); however, the crucial metabolic product and molecular basis response to cold stress have not been elucidated definitely yet. To comprehensively understand the gene regulation network and CI mechanisms in response to cold stress on a molecular level, we performed high-throughput RNA-Seq analysis to investigate genome-wide expression profiles in bell peppers at different storage temperatures (4 °C and 10 °C). A total of 61.55 Gb of clean data were produced; 3863 differentially expressed genes (DEGs) including 1669 up-regulated and 2194 down-regulated were annotated and classified between the CI group and control. Together, a total of 41 cold-induced transcription factor families comprising 250 transcription factors (TFs) were identified. Notably, numerous DEGs involved in biomembrane stability, dehydration and osmoregulation, and plant hormone signal transduction processes were discovered. The transcriptional level of 20 DEGs was verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Our results present transcriptome profiles of bell peppers in response to cold stress; the data obtained may be useful for the identification of key candidate genes and elucidation of the mechanisms underlying membrane damage during chilling injury.

Low temperature conditioning alleviates loss of aroma-related esters of 'Nanguo' pears by regulation of ethylene signal transduction.

The optimum harvest time for 'Nanguo' pears lasts for only 20 days. Refrigeration is a common technique used to extend the fruit supply. However, with low-temperature storage, the proper aroma of the pear is lost when it matures during shelf-life at room temperature. In this study, we investigated the ability of low-temperature conditioning (LTC) to regulate aroma esters of 'Nanguo' pears, and identified the key genes in the ethylene signal transduction pathway based on RNA-seq analysis. LTC caused higher ethylene production and effectively alleviated the loss of aroma-related esters in 'Nanguo' pears during refrigeration and during the subsequent shelf-life at room temperature. Furthermore, the expression levels of PuERS1, PuEIN4, PuEIN2, and PuERF were increased under LTC treatment. Thus, we speculate that the alleviation effect of LTC on fruit aroma esters is closely related to ethylene signal transduction, including up-regulated expression of PuERS1, PuEIN4, PuEIN2, and PuERF.

The effect of ethylene absorbent treatment on the softening of blueberry fruit.

The potential of ethylene absorbent (EA) to delay softening of 'Lanfeng' blueberry (Vaccinium spp.) fruit in conjunction with cold storage was evaluated. The fruit quality was evaluated after 60 days of storage at 0 °C again kept at 20 °C, with or without EA. Changes in quality attributes and ethylene biosynthesis and fruit softening indicators were assessed. The results indicated that EA treatment inhibited fruit softening, reduced weight loss and decay, and prevented the loss of total phenolic content. It also decreased the fruit ethylene production by inhibiting 1-aminocyclopropane-1-carboxylic acid oxidase and 1-aminocyclopropane-1- carboxylic acid synthase activities, whilst maintaining firmness by hampering cell wall-degrading enzyme activities, especially after more than 30 days of cold storage. In conclusion, EA treatment can inhibit the softening of harvested blueberry fruit during storage at 0 °C and shelf life after cold storage. After being refrigerated for more than 30 days at 0 °C, the EA has a good effect on blueberries storage.

Membrane lipid metabolism changes and aroma ester loss in low-temperature stored Nanguo pears.

Cold storage is an effective method used to retard the senescence of Nanguo pears after harvest. However, this causes aroma loss in the fruit. To elucidate the role of membrane lipid metabolism in aroma reduction, we investigated the contents of total aroma eaters and major fatty acid components, the membrane permeability, and the activity and gene expression of key enzymes in membrane lipid metabolism and aroma formation. The results showed that the contents of total aroma esters, oleic acid and linoleic acid, and alcohol dehydrogenase activity were at a lower level in cold stored fruit than that in control fruit. However, the palmitic acid content, membrane permeability, and the activities and gene expression of alcohol acyltransferase, lipoxygenase, phospholipase D, and lipase were higher. In conclusion, the loss of aroma esters may be caused by membrane lipid metabolism disruption during cold storage.

Changes in Membrane Lipid Composition and Function Accompanying Chilling Injury in Bell Peppers.

Bell peppers are vulnerable to low temperature (<7°C) and subject to chilling injury (CI). To elucidate the relationship between cell membrane lipid composition and CI, a membrane lipidomic approach was taken. In addition, we performed microstructural analysis and low-field nuclear magnetic resonance to better understand CI. We also monitored primary physiological metabolism parameters to explain lipidomics. Our study indicated that cellular structure damage was more serious at 4°C, mostly represented by damage to the plasmalemma and plastid degradation. Membrane lipidomic data analysis reveals monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid as crucial biomarkers during CI. Furthermore, the significant increase in proline, electrolyte leakage and phospholipase D in chilled fruits also proved that membrane lipid metabolism is involved in the response to low temperature stress. To our knowledge, this study is the first attempt to describe the CI mechanisms in bell peppers based on membrane lipidomics.