The m6A reader SlYTH2 negatively regulates tomato fruit aroma by impeding the translation process.

N6-methyladenosine (m6A) is a fundamentally important RNA modification for gene regulation, whose function is achieved through m6A readers. However, whether and how m6A readers play regulatory roles during fruit ripening and quality formation remains unclear. Here, we characterized SlYTH2 as a tomato m6A reader protein and profiled the binding sites of SlYTH2 at the transcriptome-wide level. SlYTH2 undergoes liquid-liquid phase separation and promotes RNA-protein condensate formation. The target mRNAs of SlYTH2, namely m6A-modified SlHPL and SlCCD1B associated with volatile synthesis, are enriched in SlYTH2-induced condensates. Through polysome profiling assays and proteomic analysis, we demonstrate that knockout of SlYTH2 expedites the translation process of SlHPL and SlCCD1B, resulting in augmented production of aroma-associated volatiles. This aroma enrichment significantly increased consumer preferences for CRISPR-edited fruit over wild type. These findings shed light on the underlying mechanisms of m6A in plant RNA metabolism and provided a promising strategy to generate fruits that are more attractive to consumers.

The R2R3 MYB Ruby1 is activated by two cold responsive ethylene response factors, via the retrotransposon in its promoter, to positively regulate anthocyanin biosynthesis in citrus.

Anthocyanins are natural pigments and dietary antioxidants that play multiple biological roles in plants and are important in animal and human nutrition. Low temperature (LT) promotes anthocyanin biosynthesis in many species including blood orange. A retrotransposon in the promoter of Ruby1, which encodes an R2R3 MYB transcription factor, controls cold-induced anthocyanin accumulation in blood orange flesh. However, the specific mechanism remains unclear. In this study, we characterized two LT-induced ETHYLENE RESPONSE FACTORS (CsERF054 and CsERF061). Both CsERF054 and CsERF061 can activate the expression of CsRuby1 by directly binding to a DRE/CRT cis-element within the retrotransposon in the promoter of CsRuby1, thereby positively regulating anthocyanin biosynthesis. Further investigation indicated that CsERF061 also forms a protein complex with CsRuby1 to co-activate the expression of anthocyanin biosynthetic genes, providing a dual mechanism for the upregulation of the anthocyanin pathway. These results provide insights into how LT mediates anthocyanin biosynthesis and increase the understanding of the regulatory network of anthocyanin biosynthesis in blood orange.

Synergistic actions of three MYB transcription factors underpins the high accumulation of myricetin in Morella rubra.

Flavonols are health-promoting bioactive compounds important for human nutrition, health, and plant defense. The transcriptional regulation of kaempferol and quercetin biosynthesis has been studied extensively, while little is known about the regulatory mechanisms underlying myricetin biosynthesis, which has strong antioxidant, anticancer, antidiabetic, and anti-inflammatory activities. In this study, the flavonol-specific MrMYB12 in Morella rubra preferred activating the promoter of flavonol synthase 2 (MrFLS2) (6.4-fold) rather than MrFLS1 (1.4-fold) and upregulated quercetin biosynthesis. Furthermore, two SG44 R2R3-MYB members, MrMYB5 and MrMYB5L, were identified by yeast one-hybrid library screening using the promoter of flavonoid 3',5'-hydroxylase (MrF3'5'H), and transcript levels of these R2R3-MYBs were correlated with accumulation of myricetin derivatives during leaf development. Dual-luciferase and electrophoretic mobility shift assays demonstrated that both MrMYB5 and MrMYB5L could bind directly to MYB recognition sequence elements in promoters of MrF3'5'H or MrFLS1 and activate their expression. Protein-protein interactions of MrMYB5 or MrMYB5L with MrbHLH2 were confirmed by yeast two-hybrid and bimolecular fluorescence complementation assays. MrMYB5L-MrbHLH2 showed much higher synergistic activation of MrF3'5'H or MrFLS1 promoters than MrMYB5-MrbHLH2. Studies with Arabidopsis thaliana homologs AtMYB5 and AtTT8 indicated that similar synergistic regulatory effects occur with promoters of MrF3'5'H or MrFLS1. Transient overexpression of MrMYB5L-MrbHLH2 in Nicotiana benthamiana induced a higher accumulation of myricetin derivatives (57.70 µg g-1 FW) than MrMYB5-MrbHLH2 (7.43 µg g-1 FW) when MrMYB12 was coexpressed with them. This study reveals a novel transcriptional mechanism regulating myricetin biosynthesis with the potential use for future metabolic engineering of health-promoting flavonols.

The microRNA ppe-miR393 mediates auxin-induced peach fruit softening by promoting ethylene production.

Auxin can inhibit or promote fruit ripening, depending on the species. Melting flesh (MF) peach fruit (Prunus persica L. Batsch) cultivars produce high levels of ethylene caused by high concentrations of indole-3-acetic acid (IAA), which leads to rapid fruit softening at the late stage of development. In contrast, due to the low concentrations of IAA, the fruit of stony hard (SH) peach cultivars does not soften and produces little ethylene. Auxin seems necessary to trigger the biosynthesis of ethylene in peach fruit; however, the mechanism is not well understood. In this study, we identified miRNA gene family members ppe-miR393a and ppe-miR393b that are differentially expressed in SH and MF fruits. RNA ligase-mediated 5' rapid amplification of cDNA ends and transient transformation of Nicotiana benthamiana revealed TRANSPORT INHIBITOR RESPONSE 1 (PpTIR1), part of the auxin perception and response system, as a target of ppe-miR393a and b. Yeast 2-hybrid assay and bimolecular fluorescence complementation assay revealed that PpTIR1 physically interacts with an Aux/IAA protein PpIAA13. The results of yeast 1-hybrid assay, electrophoretic mobility shift assay, and dual-luciferase assay indicated that PpIAA13 could directly bind to and trans-activate the promoter of 1-aminocyclopropane-1-carboxylic acid synthase 1 (PpACS1), required for ethylene biosynthesis. Transient overexpression and suppression of ppe-miR393a and PpIAA13 in peach fruit induced and repressed the expression of PpACS1, confirming their regulatory role in ethylene synthesis. Gene expression analysis in developing MF and SH fruits, combined with postharvest α-naphthalene acetic acid (NAA) treatment, supports a role for a ppe-miR393-PpTIR1-PpIAA13-PpACS1 module in regulating auxin-related differences in ethylene production and softening extent in different types of peach.

Genome-wide analysis of UDP-glycosyltransferase gene family and identification of members involved in flavonoid glucosylation in Chinese bayberry (Morella rubra).

Glycosylation was catalyzed by UDP-glycosyltransferase (UGT) and was important for enriching diversity of flavonoids. Chinese bayberry (Morella rubra) has significant nutritional and medical values because of diverse natural flavonoid glycosides. However, information of UGT gene family was quite limited in M. rubra. In the present study, a total of 152 MrUGT genes clustered into 13 groups were identified in M. rubra genome. Among them, 139 MrUGT genes were marked on eight chromosomes and 13 members located on unmapped scaffolds. Gene duplication analysis indicated that expansion of MrUGT gene family was mainly forced by tandem and proximal duplication events. Gene expression patterns in different tissues and under UV-B treatment were analyzed by transcriptome. Cyanidin 3-O-glucoside (C3Glc) and quercetin 3-O-glucoside (Q3Glc) were two main flavonoid glucosides accumulated in M. rubra. UV-B treatment significantly induced C3Glc and Q3Glc accumulation in fruit. Based on comprehensively analysis of transcriptomic data and phylogenetic homology together with flavonoid accumulation patterns, MrUFGT (MrUGT78A26) and MrUGT72B67 were identified as UDP-glucosyltransferases. MrUFGT was mainly involved in C3Glc and Q3Glc accumulation in fruit, while MrUGT72B67 was mainly involved in Q3Glc accumulation in leaves and flowers. Gln375 and Gln391 were identified as important amino acids for glucosyl transfer activity of MrUFGT and MrUGT72B67 by site-directed mutagenesis, respectively. Transient expression in Nicotiana benthamiana tested the function of MrUFGT and MrUGT72B67 as glucosyltransferases. The present study provided valuable source for identification of functional UGTs involved in secondary metabolites biosynthesis in M. rubra.

Identification of UDP-rhamnosyltransferases and UDP-galactosyltransferase involved in flavonol glycosylation in Morella rubra.

Flavonol glycosides are health-promoting phytochemicals important for human nutrition and plant defense against environmental stresses. Glycosylation modification greatly enriches the diversity of flavonols. Morella rubra, a member of the Myricaceae, contains high amounts of myricetin 3-O-rhamnoside (M3Rha), quercetin 3-O-rhamnoside (Q3Rha), and quercetin 3-O-galactoside (Q3Gal). In the present study, MrUGT78R1 and MrUGT78R2 were identified as two functional UDP-rhamnosyltransferases, while MrUGT78W1 was identified as a UDP-galactosyltransferase. Site-directed mutagenesis identified Pro143 and Asn386 as important residues for rhamnosyl transfer activity of MrUGT78R1, while the two corresponding positions in MrUGT78W1 (i.e. Ser147 and Asn370) also play important roles in galactosyl transfer activity. Transient expression data for these three MrUGTs in Nicotiana benthamiana tested the function of MrUGT78R1 and MrUGT78R2 as rhamnosyltransferases and MrUGT78W1 as a galactosyltransferase in glycosylation of flavonols. This work enriches knowledge of the diversity of UDP-rhamnosyltransferase in planta and identifies two amino acid positions important for both rhamnosyltransferase and galactosyltransferase.

Genome-Wide Analysis of Calmodulin Binding Transcription Activator (CAMTA) Gene Family in Peach (Prunus persica L. Batsch) and Ectopic Expression of PpCAMTA1 in Arabidopsis camta2,3 Mutant Restore Plant Development.

Calmodulin-binding transcription activator (CAMTA) is a transcription factor family containing calmodulin (CaM) binding sites and is involved in plant development. Although CAMTAs in Arabidopsis have been extensively investigated, the functions of CAMTAs remain largely unclear in peaches. In this study, we identified five peach CAMTAs which contained conserved CG-1 box, ANK repeats, CaM binding domain (CaMBD) and IQ motifs. Overexpression in tobacco showed that PpCAMTA1/2/3 were located in the nucleus, while PpCAMTA4 and PpCAMTA5 were located in the plasma membrane. Increased expression levels were observed for PpCAMTA1 and PpCAMTA3 during peach fruit ripening. Expression of PpCAMTA1 was induced by cold treatment and was inhibited by ultraviolet B irradiation (UV-B). Driven by AtCAMTA3 promoter, PpCAMTA1/2/3 were overexpressed in Arabidopsis mutant. Here, we characterized peach PpCAMTA1, representing an ortholog of AtCAMTA3. PpCAMTA1 expression in Arabidopsis complements the developmental deficiencies of the camta2,3 mutant, and restored the plant size to the wild type level. Moreover, overexpressing PpCAMTA1 in camta2,3 mutant inhibited salicylic acid (SA) biosynthesis and expression of SA-related genes, resulting in a susceptibility phenotype to Pst DC3000. Taken together, our results provide new insights for CAMTAs in peach fruit and indicate that PpCAMTA1 is associated with response to stresses during development.

Contrasting Roles of Ethylene Response Factors in Pathogen Response and Ripening in Fleshy Fruit.

Fleshy fruits are generally hard and unpalatable when unripe; however, as they mature, their quality is transformed by the complex and dynamic genetic and biochemical process of ripening, which affects all cell compartments. Ripening fruits are enriched with nutrients such as acids, sugars, vitamins, attractive volatiles and pigments and develop a pleasant taste and texture and become attractive to eat. Ripening also increases sensitivity to pathogens, and this presents a crucial problem for fruit postharvest transport and storage: how to enhance pathogen resistance while maintaining ripening quality. Fruit development and ripening involve many changes in gene expression regulated by transcription factors (TFs), some of which respond to hormones such as auxin, abscisic acid (ABA) and ethylene. Ethylene response factor (ERF) TFs regulate both fruit ripening and resistance to pathogen stresses. Different ERFs regulate fruit ripening and/or pathogen responses in both fleshy climacteric and non-climacteric fruits and function cooperatively or independently of other TFs. In this review, we summarize the current status of studies on ERFs that regulate fruit ripening and responses to infection by several fungal pathogens, including a systematic ERF transcriptome analysis of fungal grey mould infection of tomato caused by Botrytis cinerea. This deepening understanding of the function of ERFs in fruit ripening and pathogen responses may identify novel approaches for engineering transcriptional regulation to improve fruit quality and pathogen resistance.

Facile microfluidic fabrication and characterization of ethyl cellulose/PVP films with neatly arranged fibers.

Much attention and endeavor have been paid to developing biocompatible food packaging films. Here, ethyl cellulose (EC) and polyvinylpyrrolidone (PVP) were fabricated into films through a facile method, microfluidic spinning. Morphology observations showed that the fibers were neatly arranged with an average diameter of 1-4 µm. FTIR and X-ray diffraction analysis suggested the existence of good compatibility and interaction between EC and PVP. Thermogravimetric analysis demonstrated that PVP ameliorates the thermal properties; moreover, the tensile properties were improved, with tensile strength (TS) and Young's modulus up to 11.10 ± 1.04 MPa and 350.16 ± 45.46 MPa, respectively. The optimal formula was EC/PVP (2:3), of which the film displayed an enhanced TS of 4.61 ± 1.15 MPa and a modified water contact angle of 61.8 ± 4.4°, showing fine tensile and hydrophilic performance. This study provides a facile and green film fabrication method promising to be used for food wrapping.

Unravelling the consecutive glycosylation and methylation of flavonols in peach in response to UV-B irradiation.

Flavonol glycosides are bioactive compounds important for plant defence and human nutrition. Glycosylation and methylation play an important role in enriching the diversity of flavonols in response to the environment. Peach flowers and fruit are rich in flavonol diglycosides such as isorhamnetin 3-O-rutinoside (I3Rut), kaempferol 3-O-rutinoside and quercetin 3-O-rutinoside, and flavonol monoglycosides such as I 3-O-glucoside and Q 3-O-galactoside. UV-B irradiation of fruit significantly induced accumulation of all these flavonol glycosides. Candidate biosynthetic genes induced by UV-B were identified by genome homology searches and the in vitro catalytic activities of purified recombinant proteins determined. PpUGT78T3 and PpUGT78A2 were identified as flavonol 3-O-glucosyltransferase and 3-O-galactosyltransferase, respectively. PpUGT91AK6 was identified as flavonol 1,6-rhamnosyl trasferase catalysing the formation of flavonol rutinosides and PpFOMT1 was identified as a flavonol O-methyltransferase that methylated Q at the 3'-OH-OH to form isorhamnetin derivatives. Transient expression in Nicotiana benthamiana confirmed the specificity of PpUGT78T3 as a flavonol 3-O-glucosyltransferase, PpUGT78A2 as a 3-O-galactosyltransferase, PpUGT91AK6 as a 1,6-rhamnosyltrasferase and PpFOMT1 as an O-methyltransferase. This study provides new insights into the mechanisms of glycosylation and methylation of flavonols, especially the formation of flavonol diglycosides such as I3Rut, and will also be useful for future potential metabolic engineering of complex flavonols.

Hydroxylation decoration patterns of flavonoids in horticultural crops: chemistry, bioactivity and biosynthesis.

Flavonoids are the most widespread polyphenolic compounds and are important dietary constituents present in horticultural crops such as fruits, vegetables, and tea. Natural flavonoids are responsible for important quality traits, such as food colors and beneficial dietary antioxidants and numerous investigations have shown that intake of flavonoids can reduce the incidence of various non-communicable diseases (NCDs). Analysis of the thousands of flavonoids reported so far has shown that different hydroxylation modifications affect their chemical properties and nutritional values. These diverse flavonoids can be classified based on different hydroxylation patterns in the B, C, A rings and multiple structure-activity analyses have shown that hydroxylation decoration at specific positions markedly enhances their bioactivities. This review focuses on current knowledge concerning hydroxylation of flavonoids catalyzed by several different types of hydroxylase enzymes. Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3'5'-hydroxylase (F3'5'H) are important enzymes for the hydroxylation of the B ring of flavonoids. Flavanone 3-hydroxylase (F3H) is key for the hydroxylation of the C ring, while flavone 6-hydroxylase (F6H) and flavone 8-hydroxylase (F8H) are key enzymes for hydroxylation of the A ring. These key hydroxylases in the flavonoid biosynthesis pathway are promising targets for the future bioengineering of plants and mass production of flavonoids with designated hydroxylation patterns of high nutritional importance. In addition, hydroxylation in key places on the ring may help render flavonoids ready for degradation, the catabolic turnover of which may open the door for new lines of inquiry.

Cyanidin-3-O-Glucoside improves the viability of human islet cells treated with amylin or Aß1-42 in vitro.

Islet transplantation is being considered as an alternative treatment for type 1 diabetes. Despite recent progress, transplant recipients continue to experience progressive loss of insulin independence. Cyanidin-3-O-Glucoside (C3G) has shown to be protective against damage that may lead to post-transplant islet loss. In this study, human islets cultured with or without C3G were treated with human amylin, Aß1-42, H2O2, or rapamycin to mimic stresses encountered in the post-transplant environment. Samples of these islets were collected and assayed to determine C3G's effect on cell viability and function, reactive oxygen species (ROS), oxidative stress, amyloid formation, and the presence of inflammatory as well as autophagic markers. C3G treatment of human islets exposed to either amylin or Aß1-42 increased cell viability (p<0.01) and inhibited amyloid formation (p<0.01). A reduction in ROS and an increase in HO-1 gene expression as well as in vitro islet function were also observed in C3G-treated islets exposed to amylin or Aß1-42, although not significantly. Additionally, treatment with C3G resulted in a significant reduction in the protein expression of inflammatory markers IL-1ß and NLRP3 (p<0.01) as well as an increase in LC3 autophagic marker (p<0.05) in human islets treated with amylin, Aß1-42, rapamycin, or H2O2. Thus, C3G appears to have a multi-faceted protective effect on human islets in vitro, possibly through its anti-oxidant property and alteration of inflammatory as well as autophagic pathways.

Genome-Wide Analysis of MYB Gene Family in Chinese Bayberry (Morella rubra) and Identification of Members Regulating Flavonoid Biosynthesis.

Chinese bayberry (Morella rubra), the most economically important fruit tree in the Myricaceae family, is a rich source of natural flavonoids. Recently the Chinese bayberry genome has been sequenced, and this provides an opportunity to investigate the organization and evolutionary characteristics of MrMYB genes from a whole genome view. In the present study, we performed the genome-wide analysis of MYB genes in Chinese bayberry and identified 174 MrMYB transcription factors (TFs), including 122 R2R3-MYBs, 43 1R-MYBs, two 3R-MYBs, one 4R-MYB, and six atypical MYBs. Collinearity analysis indicated that both syntenic and tandem duplications contributed to expansion of the MrMYB gene family. Analysis of transcript levels revealed the distinct expression patterns of different MrMYB genes, and those which may play important roles in leaf and flower development. Through phylogenetic analysis and correlation analyses, nine MrMYB TFs were selected as candidates regulating flavonoid biosynthesis. By using dual-luciferase assays, MrMYB12 was shown to trans-activate the MrFLS1 promoter, and MrMYB39 and MrMYB58a trans-activated the MrLAR1 promoter. In addition, overexpression of 35S:MrMYB12 caused a significant increase in flavonol contents and induced the expression of NtCHS, NtF3H, and NtFLS in transgenic tobacco leaves and flowers and significantly reduced anthocyanin accumulation, resulting in pale-pink or pure white flowers. This indicates that MrMYB12 redirected the flux away from anthocyanin biosynthesis resulting in higher flavonol content. The present study provides valuable information for understanding the classification, gene and motif structure, evolution and predicted functions of the MrMYB gene family and identifies MYBs regulating different aspects of flavonoid biosynthesis in Chinese bayberry.

Elucidation of myricetin biosynthesis in Morella rubra of the Myricaceae.

Flavonols are health-promoting bioactive compounds important for plant defense and human nutrition. Quercetin (Q) and kaempferol (K) biosynthesis have been studied extensively while little is known about myricetin (M) biosynthesis. The roles of flavonol synthases (FLSs) and flavonoid 3',5'-hydroxylase (F3'5'H) in M biosynthesis in Morella rubra, a member of the Myricaceae rich in M-based flavonols, were investigated. The level of MrFLS transcripts alone did not correlate well with the accumulation of M-based flavonols. However, combined transcript data for MrFLS1 and MrF3'5'H showed a good correlation with the accumulation of M-based flavonols in different tissues of M. rubra. Recombinant MrFLS1 and MrFLS2 proteins showed strong activity with dihydroquercetin (DHQ), dihydrokaempferol (DHK), and dihydromyricetin (DHM) as substrates, while recombinant MrF3'5'H protein preferred converting K to M, amongst a range of substrates. Tobacco (Nicotiana tabacum) overexpressing 35S::MrFLSs produced elevated levels of K-based and Q-based flavonols without affecting M-based flavonol levels, while tobacco overexpressing 35S::MrF3'5'H accumulated significantly higher levels of M-based flavonols. We conclude that M accumulation in M. rubra is affected by gene expression and enzyme specificity of FLS and F3'5'H as well as substrate availability. In the metabolic grid of flavonol biosynthesis, the strong activity of MrF3'5'H with K as substrate additionally promotes metabolic flux towards M in M. rubra.

Citrus CitERF6 Contributes to Citric Acid Degradation via Upregulation of CitAclα1, Encoding ATP-Citrate Lyase Subunit α.

Citric acid is the most abundant organic acid in citrus fruit, and the acetyl-CoA pathway potentially plays an important role in citric acid degradation, which occurs during fruit ripening. Analysis of transcripts during fruit development of key genes in the acetyl-CoA pathway and transient overexpression assay in citrus leaves indicated that CitAclα1 could be a potential target gene involved in citrate degradation. In order to understand more about CitAclα1, 23 transcription factors coexpressed with CitAclα1 in citrus fruit were identified by RNA-seq. Using dual-luciferase assays, CitERF6 was shown to trans-activate the promoter of CitAclα1 and electrophoretic mobility shift assays (EMSAs) showed that CitERF6 directly bound to a 5'-CAACA-3' motif in the CitAclα1 promoter. Furthermore, citric acid content was significantly reduced when CitERF6 was overexpressed in transgenic tobacco leaves. Taken together, these results indicate an important role for CitERF6 in transcriptional regulation of CitAclα1 and control of citrate degradation.

ETHYLENE RESPONSE FACTOR39-MYB8 complex regulates low-temperature-induced lignification of loquat fruit.

Flesh lignification is a specific chilling response that causes deterioration in the quality of stored red-fleshed loquat fruit (Eribotrya japonica) and is one aspect of wider chilling injury. APETALA2/ETHLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors are important regulators of plant low-temperature responses and lignin biosynthesis. In this study, the expression and action of 27 AP2/ERF genes from the red-fleshed loquat cultivar 'Luoyangqing' were investigated in order to identify transcription factors regulating low-temperature-induced lignification. EjERF27, EjERF30, EjERF36, and EjERF39 were significantly induced by storage at 0 °C but inhibited by a low-temperature conditioning treatment (pre-storage at 5 °C for 6 days before storage at 0 °C, which reduces low-temperature-induced lignification), and their transcript levels positively correlated with flesh lignification. A dual-luciferase assay indicated that EjERF39 could transactivate the promoter of the lignin biosynthetic gene Ej4CL1, and an electrophoretic mobility shift assay confirmed that EjERF39 recognizes the DRE element in the promoter region of Ej4CL1. Furthermore, the combination of EjERF39 and the previously characterized EjMYB8 synergistically transactivated the Ej4CL1 promoter, and both transcription factors showed expression patterns correlated with lignification in postharvest treatments and red-fleshed 'Luoyangqing' and white-fleshed 'Ninghaibai' cultivars with different lignification responses. Bimolecular fluorescence complementation and luciferase complementation imaging assays confirmed direct protein-protein interaction between EjERF39 and EjMYB8. These data indicate that EjERF39 is a novel cold-responsive transcriptional activator of Ej4CL1 that forms a synergistic activator complex with EjMYB8 and contributes to loquat fruit lignification at low temperatures.

Roles of RIN and ethylene in tomato fruit ripening and ripening-associated traits.

RIPENING INHIBITOR (RIN)-deficient fruits generated by CRISPR/Cas9 initiated partial ripening at a similar time to wild-type (WT) fruits but only 10% WT concentrations of carotenoids and ethylene (ET) were synthesized. RIN-deficient fruit never ripened completely, even when supplied with exogenous ET. The low amount of endogenous ET that they did produce was sufficient to enable ripening initiation and this could be suppressed by the ET perception inhibitor 1-MCP. The reduced ET production by RIN-deficient tomatoes was due to an inability to induce autocatalytic system-2 ET synthesis, a characteristic feature of climacteric ripening. Production of volatiles and transcripts of key volatile biosynthetic genes also were greatly reduced in the absence of RIN. By contrast, the initial extent and rates of softening in the absence of RIN were similar to WT fruits, although detailed analysis showed that the expression of some cell wall-modifying enzymes was delayed and others increased in the absence of RIN. These results support a model where RIN and ET, via ERFs, are required for full expression of ripening genes. Ethylene initiates ripening of mature green fruit, upregulates RIN expression and other changes, including system-2 ET production. RIN, ET and other factors are required for completion of the full fruit-ripening programme.

Effect of Ethylene on Cell Wall and Lipid Metabolism during Alleviation of Postharvest Chilling Injury in Peach.

Peach is prone to postharvest chilling injury (CI). Here it was found that exogenous ethylene alleviated CI, accompanied by an increased endogenous ethylene production. Ethylene treatment resulted in a moderately more rapid flesh softening as a result of stronger expression of genes encoding expansin and cell wall hydrolases, especially xylosidase and galactosidase. Ethylene treatment alleviated internal browning, accompanied by changes in expression of polyphenol oxidase, peroxidase and lipoxygenases. An enhanced content of phospholipids and glycerolipids and a reduced content of ceramide were observed in ethylene-treated fruit, and these were associated with up-regulation of lipid phosphate phosphatase, fatty acid alpha-hydroxylase, and golgi-localized nucleotide sugar transporter, as well as down-regulation of aminoalcoholphosphotransferases. Expression of two ethylene response factors (ERFs), ESE3 and ABR1, was highly correlated with that of genes involved in cell wall metabolism and lipid metabolism, respectively. Furthermore, the expression of these two ERFs was strongly regulated by ethylene treatment and the temperature changes during transfer of fruit into or out of cold storage. It is proposed that ERFs fulfill roles as crucial integrators between cell wall modifications and lipid metabolism involved in CI processes ameliorated by exogenous ethylene.

Auto- and mutual-regulation between two CitERFs contribute to ethylene-induced citrus fruit degreening.

Citrus fruit postharvest degreening is a critical stage in marketing, carried out by exposure to ethylene or ethephon. Genome-wide screening of the AP2/ERF superfamily indicated that a novel ERF-II (CitERF6) was shown to trans-activate the CitPPH promoter. Expression of CitERF6 is associated with both developmental and postharvest degreening in citrus fruit. Transient and stable over-expression of CitERF6 in Nicotiana tabacum leaves and 'Ponkan' fruit also results in rapid chlorophyll degradation. Auto- and mutual-regulation was also found between CitERF6 and the previously characterized CitERF13 using the dual-luciferase and yeast one-hybrid assays. Moreover, substitution of the 35S promoter for endogenous promoters showed that both pCitERF6::CitERF6 and pCitERF13::CitERF13 were effective in trans-activating their promoters or triggering chlorophyll degradation. It is proposed that ethylene is one of the triggers activating promoters of CitERF6 and CitERF13, and subsequent auto- and mutual-regulation between CitERF6 and CitERF13 might facilitate the effect of ethylene, leading to fruit degreening.

Combination Strategy of Reactive and Catalytic Matrices for Qualitative and Quantitative Profiling of N-Glycans in MALDI-MS.

Qualitative and quantitative analysis of N-glycans by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is hampered mainly by the low ionization efficiency of analytes and their poor cocrystallization with traditional organic acid matrices. In the present study, a combination strategy of reactive and catalytic matrices (CSRCM) was proposed for the on-target derivatization and detection of reducing N-glycans: a novel reactive matrix, i.e., 2,5-dihydroxybenzohydrazide (DHBH), having a skeleton structure similar to that of DHB, was designed and synthesized, and this reactive matrix was mixed with catalytic matrix DHB to form a rationally combinatorial matrix (DHB/DHBH). Qualitatively, DHB/DHBH could improve the ionization efficiency of reducing carbohydrates significantly. Quantitatively, the acid-base chemistry of DHB/DHBH leads to a uniform cocrystallization of analytes-matrix mixtures. Consequently, CSRCM provides accurate quantitation for N-glycans with high derivatization efficiency and good linearity (R2 > 0.99) within 2 orders of magnitude on the basis of an internal standard method. Furthermore, the CSRCM was successfully applied to evaluating N-glycan in serum samples of colorectal cancer patients, thus showing potential in clinical applications for biomarker discovery.