Expansin SlExp1 and endoglucanase SlCel2 synergistically promote fruit softening and cell wall disassembly in tomato.

Fruit softening, an irreversible process that occurs during fruit ripening, can lead to losses and waste during postharvest transportation and storage. Cell wall disassembly is the main factor leading to loss of fruit firmness, and several ripening-associated cell wall genes have been targeted for genetic modification, particularly pectin modifiers. However, individual knockdown of most cell wall-related genes has had minimal influence on cell wall integrity and fruit firmness, with the notable exception of pectate lyase. Compared to pectin disassembly, studies of the cell wall matrix, the xyloglucan-cellulose framework, and underlying mechanisms during fruit softening are limited. Here, a tomato (Solanum lycopersicum) fruit ripening-associated α-expansin (SlExpansin1/SlExp1) and an endoglucanase (SlCellulase2/SlCel2), which function in the cell wall matrix, were knocked out individually and together using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9-mediated genome editing. Simultaneous knockout of SlExp1 and SlCel2 enhanced fruit firmness, reduced depolymerization of homogalacturonan-type pectin and xyloglucan, and increased cell adhesion. In contrast, single knockouts of either SlExp1 or SlCel2 did not substantially change fruit firmness, while simultaneous overexpression of SlExp1 and SlCel2 promoted early fruit softening. Collectively, our results demonstrate that SlExp1 and SlCel2 synergistically regulate cell wall disassembly and fruit softening in tomato.

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.

FMO2 ameliorates nonalcoholic fatty liver disease by suppressing ER-to-Golgi transport of SREBP1.

BACKGROUND AND AIMS: NAFLD comprises a spectrum of liver disorders with the initial abnormal accumulation of lipids in hepatocytes called NAFL, progressing to the more serious NASH in a subset of individuals. Our previous study revealed that global flavin-containing monooxygenase 2 (FMO2) knockout causes higher liver weight in rats. However, the role of FMO2 in NAFLD remains unclear. Herein, we aimed to determine the function and mechanism of FMO2 in liver steatosis and steatohepatitis. APPROACH AND RESULTS: The expression of FMO2 was significantly downregulated in patients with NAFL/NASH and mouse models. Both global and hepatocyte-specific knockout of FMO2 resulted in increased lipogenesis and severe hepatic steatosis, inflammation, and fibrosis, whereas FMO2 overexpression in mice improved NAFL/NASH. RNA sequencing showed that hepatic FMO2 deficiency is associated with impaired lipogenesis in response to metabolic challenges. Mechanistically, FMO2 directly interacts with SREBP1 at amino acids 217-296 competitively with SREBP cleavage-activating protein (SCAP) and inhibits SREBP1 translocation from the endoplasmic reticulum (ER) to the Golgi apparatus and its subsequent activation, thus suppressing de novo lipogenesis (DNL) and improving NAFL/NASH. CONCLUSIONS: In hepatocytes, FMO2 is a novel molecule that protects against the progression of NAFL/NASH independent of enzyme activity. FMO2 impairs lipogenesis in high-fat diet-induced or choline-deficient, methionine-deficient, amino acid-defined high-fat diet-induced steatosis, inflammation, and fibrosis by directly binding to SREBP1 and preventing its organelle translocation and subsequent activation. FMO2 thus is a promising molecule for targeting the activation of SREBP1 and for the treatment of NAFL/NASH.

Insights into cell wall changes during fruit softening from transgenic and naturally occurring mutants.

Excessive softening during fleshy fruit ripening leads to physical damage and infection that reduce quality and cause massive supply chain losses. Changes in cell wall (CW) metabolism, involving loosening and disassembly of the constituent macromolecules, are the main cause of softening. Several genes encoding CW metabolizing enzymes have been targeted for genetic modification to attenuate softening. At least 9 genes encoding CW-modifying proteins have increased expression during ripening. Any alteration of these genes could modify CW structure and properties and contribute to softening, but evidence for their relative importance is sparse. The results of studies with transgenic tomato (Solanum lycopersicum), the model for fleshy fruit ripening, investigations with strawberry (Fragaria spp.) and apple (Malus domestica), and results from naturally occurring textural mutants provide direct evidence of gene function and the contribution of CW biochemical modifications to fruit softening. Here we review the revised CW structure model and biochemical and structural changes in CW components during fruit softening and then focus on and integrate the results of changes in CW characteristics derived from studies on transgenic fruits and mutants. Potential strategies and future research directions to understand and control the rate of fruit softening are also discussed.

The transcription factor CitZAT5 modifies sugar accumulation and hexose proportion in citrus fruit.

Sugars are fundamental to plant developmental processes. For fruits, the accumulation and proportion of sugars play crucial roles in the development of quality and attractiveness. In citrus (Citrus reticulata Blanco.), we found that the difference in sweetness between mature fruits of "Gongchuan" and its bud sport "Youliang" is related to hexose contents. Expression of a SuS (sucrose synthase) gene CitSUS5 and a SWEET (sugars will eventually be exported transporter) gene CitSWEET6, characterized by transcriptome analysis at different developmental stages of these 2 varieties, revealed higher expression levels in "Youliang" fruit. The roles of CitSUS5 and CitSWEET6 were investigated by enzyme activity and transient assays. CitSUS5 promoted the cleavage of sucrose to hexoses, and CitSWEET6 was identified as a fructose transporter. Further investigation identified the transcription factor CitZAT5 (ZINC FINGER OF ARABIDOPSIS THALIANA) that contributes to sucrose metabolism and fructose transportation by positively regulating CitSUS5 and CitSWEET6. The role of CitZAT5 in fruit sugar accumulation and hexose proportion was investigated by homologous transient CitZAT5 overexpression, -VIGS, and -RNAi. CitZAT5 modulates the hexose proportion in citrus by mediating CitSUS5 and CitSWEET6 expression, and the molecular mechanism explained the differences in sugar composition of "Youliang" and "Gongchuan" fruit.

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.

Human amniotic epithelial stem cell is a cell therapy candidate for preventing acute graft-versus-host disease.

Graft-versus-host disease (GVHD), an immunological disorder that arises from donor T cell activation through recognition of host alloantigens, is the major limitation in the application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Traditional immunosuppressive agents can relieve GVHD, but they induce serious side effects. It is highly required to explore alternative therapeutic strategy. Human amniotic epithelial stem cells (hAESCs) were recently considered as an ideal source for cell therapy with special immune regulatory property. In this study, we evaluated the therapeutic role of hAESCs in the treatment of GVHD, based on our previous developed cGMP-grade hAESCs product. Humanized mouse model of acute GVHD (aGVHD) was established by injection of huPBMCs via the tail vein. For prevention or treatment of aGVHD, hAESCs were injected to the mice on day -1 or on day 7 post-PBMC infusion, respectively. We showed that hAESCs infusion significantly alleviated the disease phenotype, increased the survival rate of aGVHD mice, and ameliorated pathological injuries in aGVHD target organs. We demonstrated that hAESCs directly induced CD4+ T cell polarization, in which Th1 and Th17 subsets were downregulated, and Treg subset was elevated. Correspondingly, the levels of a series of pro-inflammatory cytokines were reduced while the levels of the anti-inflammatory cytokines were upregulated in the presence of hAESCs. We found that hAESCs regulated CD4+ subset polarization in a paracrine mode, in which TGFß and PGE2 were selectively secreted to mediate Treg elevation and Th1/Th17 inhibition, respectively. In addition, transplanted hAESCs preserved the graft-versus-leukemia (GVL) effect by inhibiting leukemia cell growth. More intriguingly, hAESCs infusion in HSCT patients displayed potential anti-GVHD effect with no safety concerns and confirmed the immunoregulatory mechanisms in the preclinical study. We conclude that hAESCs infusion is a promising therapeutic strategy for post-HSCT GVHD without compromising the GVL effect. The clinical trial was registered at www.clinicaltrials.gov as #NCT03764228.

A tomato LATERAL ORGAN BOUNDARIES transcription factor, SlLOB1, predominantly regulates cell wall and softening components of ripening.

Fruit softening is a key component of the irreversible ripening program, contributing to the palatability necessary for frugivore-mediated seed dispersal. The underlying textural changes are complex and result from cell wall remodeling and changes in both cell adhesion and turgor. While a number of transcription factors (TFs) that regulate ripening have been identified, these affect most canonical ripening-related physiological processes. Here, we show that a tomato fruit ripening-specific LATERAL ORGAN BOUNDRIES (LOB) TF, SlLOB1, up-regulates a suite of cell wall-associated genes during late maturation and ripening of locule and pericarp tissues. SlLOB1 repression in transgenic fruit impedes softening, while overexpression throughout the plant under the direction of the 35s promoter confers precocious induction of cell wall gene expression and premature softening. Transcript and protein levels of the wall-loosening protein EXPANSIN1 (EXP1) are strongly suppressed in SlLOB1 RNA interference lines, while EXP1 is induced in SlLOB1-overexpressing transgenic leaves and fruit. In contrast to the role of ethylene and previously characterized ripening TFs, which are comprehensive facilitators of ripening phenomena including softening, SlLOB1 participates in a regulatory subcircuit predominant to cell wall dynamics and softening.

DNA methylation controlling abscisic acid catabolism responds to light to mediate strawberry fruit ripening.

Phytohormones, epigenetic regulation and environmental factors regulate fruit ripening but their interplay during strawberry fruit ripening remains to be determined. In this study, bagged strawberry fruit exhibited delayed ripening compared with fruit grown in normal light, correlating with reduced abscisic acid (ABA) accumulation. Transcription of the key ABA catabolism gene, ABA 8'-hydroxylase FaCYP707A4, was induced in bagged fruit. With light exclusion whole genome DNA methylation levels were up-regulated, corresponding to a delayed ripening process, while DNA methylation levels in the promoter of FaCYP707A4 were suppressed, correlating with increases in transcript and decreased ABA content. Experiments indicated FaCRY1, a blue light receptor repressed in bagged fruit and FaAGO4, a key protein involved in RNA-directed DNA methylation, could bind to the promoter of FaCYP707A4. The interaction between FaCRY1 and FaAGO4, and an increased enrichment of FaAGO4 directed to the FaCYP707A4 promoter in fruit grown under light suggests FaCRY1 may influence FaAGO4 to modulate the DNA methylation status of the FaCYP707A4 promoter. Furthermore, transient overexpression of FaCRY1, or an increase in FaCRY1 transcription by blue light treatment, increases the methylation level of the FaCYP707A4 promoter, while transient RNA interference of FaCRY1 displayed opposite phenotypes. These findings reveal a mechanism by which DNA methylation influences ABA catabolism, and participates in light-mediated strawberry ripening.

Quantitative N-Glycomic and N-Glycoproteomic Profiling of Peach [Prunus persica (L.) Batsch] during Fruit Ripening.

Being part of the human diet, peach is an important fruit consumed worldwide. In the present study, a systematic first insight into the N-glycosylation of peach fruit during ripening was provided. First, N-glycome by reactive matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated that 6 of 24 N-glycans of peach were differentially expressed. Second, a comparative N-glycoproteome was characterized via 18O-tagged N-glycosylation site labeling followed by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS). Totally 1464 N-glycosites on 881 N-glycoproteins were identified, among which 291 N-glycosites on 237 N-glycoproteins were expressed differentially with a fold change value of 1.5 or 0.67. The enrichment analysis of GO and KEGG revealed that four pathways including other glycan degradation, phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, and protein processing in endoplasmic reticulum were mainly enriched, in which several important N-glycoproteins with dynamic change during fruit ripening were further screened out. Our findings on a large scale for N-glycosylation analysis of peach fruit during ripening may provide new molecular insights for comprehending N-glycoprotein functions, which should be of great interest to both glycobiologists and analytical chemists.

An EjbHLH14-EjHB1-EjPRX12 module is involved in methyl jasmonate alleviation of chilling-induced lignin deposition in loquat fruit.

Loquat fruit are susceptible to chilling injuries induced by postharvest storage at low temperature. The major symptoms are increased lignin content and flesh firmness, which cause a leathery texture. Pretreatment with methyl jasmonate (MeJA) can alleviate this low-temperature-induced lignification, but the mechanism is not understood. In this study, we characterized a novel class III peroxidase, EjPRX12, and studied its relationship to lignification. Transcript levels of EjPRX12 were attenuated following MeJA pretreatment, consistent with the reduced lignin content in fruit. In vitro enzyme activity assay indicated that EjPRX12 polymerized sinapyl alcohol, and overexpression of EjPRX12 in Arabidopsis promoted lignin accumulation, indicating that it plays a functional role in lignin polymerization. We also identified an HD-ZIP transcription factor, EjHB1, repressed by MeJA pretreatment, which directly bound to and significantly activated the EjPRX12 promoter. Overexpression of EjHB1 in Arabidopsis promoted lignin accumulation with induced expression of lignin-related genes, especially AtPRX64. Furthermore, a JAZ-interacting repressor, EjbHLH14, was characterized, and it is proposed that MeJA pretreatment caused EjbHLH14 to be released to repress the expression of EjHB1. These results identified a novel regulatory pathway involving EjbHLH14-EjHB1-EjPRX12 and revealed the molecular mechanism whereby MeJA alleviated lignification of loquat fruit at low temperature.

Transcriptional regulation of fleshy fruit texture.

Fleshy fruit texture is a critically important quality characteristic of ripe fruit. Softening is an irreversible process which operates in most fleshy fruits during ripening which, together with changes in color and taste, contributes to improvements in mouthfeel and general attractiveness. Softening results mainly from the expression of genes encoding enzymes responsible for cell wall modifications but starch degradation and high levels of flavonoids can also contribute to texture change. Some fleshy fruit undergo lignification during development and post-harvest, which negatively affects eating quality. Excessive softening can also lead to physical damage and infection, particularly during transport and storage which causes severe supply chain losses. Many transcription factors (TFs) that regulate fruit texture by controlling the expression of genes involved in cell wall and starch metabolism have been characterized. Some TFs directly regulate cell wall targets, while others act as part of a broader regulatory program governing several aspects of the ripening process. In this review, we focus on advances in our understanding of the transcriptional regulatory mechanisms governing fruit textural change during fruit development, ripening and post-harvest. Potential targets for breeding and future research directions for the control of texture and quality improvement are discussed.

Comparative Transcriptome Analysis Revealed Two Alternative Splicing bHLHs Account for Flower Color Alteration in Chrysanthemum.

'Jimba' is a white chrysanthemum cultivar, which occasionally and spontaneously produces red flower petals under natural cultivation due to cyanidin-based anthocyanin accumulation. To investigate the underlying mechanism of this process, a comparative transcriptome was analyzed between white and turning red 'Jimba'. The structural and regulatory genes of anthocyanin pathway were significantly up-regulated in turning red 'Jimba'. Among them, two alternative splicings, CmbHLH2 and CmbHLH2.1, showed the most significantly up-regulated in turning red tissue. Transiently over-expressed 35S::CmMYB6-CmbHLH2 strongly induced anthocyanin accumulation in 'Jimba' flower petals, while moderate amount of anthocyanin was detected when over-expressed 35S::CmMYB6-CmbHLH2.1. Both CmbHLH2 and CmbHLH2.1 could interact with CmMYB6 to activate CmDFR promoter according to Yeast two-hybrid and dual-luciferase assay. Moreover, CmMYB6-CmbHLH2 but not CmMYB6-CmbHLH2.1 could activate the CmbHLH2 promoter to provide positive feedback loop regulation. Taken together, it suggested that both CmbHLH2 and CmbHLH2.1 involved in regulation flower color alteration in turning red 'Jimba', and CmbHLH2 played a predominant role in this process.

The strawberry transcription factor FaRAV1 positively regulates anthocyanin accumulation by activation of FaMYB10 and anthocyanin pathway genes.

The RAV (related to ABI3/viviparous 1) group of transcription factors (TFs) play multifaceted roles in plant development and stress responses. Here, we show that strawberry (Fragaria × ananassa) FaRAV1 positively regulates anthocyanin accumulation during fruit ripening via a hierarchy of activation processes. Dual-luciferase assay screening of all fruit-expressed AP2/ERFs showed FaRAV1 had the highest transcriptional activation of the promoter of FaMYB10, a key activator of anthocyanin biosynthesis. Yeast one-hybrid and electrophoretic mobility shift assays indicated that FaRAV1 could directly bind to the promoter of FaMYB10. Transient overexpression of FaRAV1 in strawberry fruit increased FaMYB10 expression and anthocyanin production significantly. Correspondingly, transient RNA interference-induced silencing of FaRAV1 led to decreases in FaMYB10 expression and anthocyanin content. Transcriptome analysis of FaRAV1-overexpressing strawberry fruit revealed that transcripts of phenylpropanoid and flavonoid biosynthesis pathway genes were up-regulated. Luciferase assays showed that FaRAV1 could also activate the promoters of strawberry anthocyanin biosynthetic genes directly, revealing a second level of FaRAV1 action in promoting anthocyanin accumulation. These results show that FaRAV1 stimulates anthocyanin accumulation in strawberry both by direct activation of anthocyanin pathway gene promoters and by up-regulation of FaMYB10, which also positively regulates these genes.

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.

SUMOylation Negatively Regulates Angiogenesis by Targeting Endothelial NOTCH Signaling.

RATIONALE: The highly conserved NOTCH (neurogenic locus notch homolog protein) signaling pathway functions as a key cell-cell interaction mechanism controlling cell fate and tissue patterning, whereas its dysregulation is implicated in a variety of developmental disorders and cancers. The pivotal role of endothelial NOTCH in regulation of angiogenesis is widely appreciated; however, little is known about what controls its signal transduction. Our previous study indicated the potential role of post-translational SUMO (small ubiquitin-like modifier) modification (SUMOylation) in vascular disorders. OBJECTIVE: The aim of this study was to investigate the role of SUMOylation in endothelial NOTCH signaling and angiogenesis. METHODS AND RESULTS: Endothelial SENP1 (sentrin-specific protease 1) deletion, in newly generated endothelial SENP1 (the major protease of the SUMO system)-deficient mice, significantly delayed retinal vascularization by maintaining prolonged NOTCH1 signaling, as confirmed in cultured endothelial cells. An in vitro SUMOylation assay and immunoprecipitation revealed that when SENP1 associated with N1ICD (NOTCH1 intracellular domain), it functions as a deSUMOylase of N1ICD SUMOylation on conserved lysines. Immunoblot and immunoprecipitation analyses and dual-luciferase assays of natural and SUMO-conjugated/nonconjugated NOTCH1 forms demonstrated that SUMO conjugation facilitated NOTCH1 cleavage. This released N1ICD from the membrane and stabilized it for translocation to the nucleus where it functions as a cotranscriptional factor. Functionally, SENP1-mediated NOTCH1 deSUMOylation was required for NOTCH signal activation in response to DLL4 (Delta-like 4) stimulation. This in turn suppressed VEGF (vascular endothelial growth factor) receptor signaling and angiogenesis, as evidenced by immunoblotted signaling molecules and in vitro angiogenesis assays. CONCLUSIONS: These results establish reversible NOTCH1 SUMOylation as a regulatory mechanism in coordinating endothelial angiogenic signaling; SENP1 acts as a critical intrinsic mediator of this process. These findings may apply to NOTCH-regulated biological events in nonvascular tissues and provide a novel therapeutic strategy for vascular diseases and tumors.

The calcium-mediated homogalacturonan pectin complexation in cell walls contributes the firmness increase in loquat fruit during postharvest storage.

INTRODUCTION: Postharvest textural changes in fruit are mainly divided into softening and lignification. Loquat fruit could have severe lignification with increased firmness during postharvest storage. Pectin is mainly associated with the postharvest softening of fruit, but some studies also found that pectin could be involved in strengthening the mechanical properties of the plant. OBJECTIVES: This study focused on characterizing the dynamics of pectin and its complexation in the cell wall of lignified loquat fruit during postharvest storage, and how these changes could influence fruit firmness. METHODS: The homogalacturonan (HG) pectin in the cell wall of loquat fruit was identified using monoclonal antibodies. An oligogalacturonide (OG) probe was used to label the egg-box structure formed by Ca2+ cross-linking with low-methylesterified HG. An exogenous injection was used to verify the role of egg-box structures in the firmness increase in loquat fruit. RESULTS: The JIM5 antibody revealed that low-methylesterified HG accumulated in the tricellular junctions and middle lamella of loquat fruit that had severe lignification symptoms. The pectin methylesterase (PME) activity increased during the early stages of storage at 0 °C, and the calcium-pectate content and flesh firmness constantly increased during storage. The OG probe demonstrated the accumulation of egg-box structures at the cellular level. The exogenous injection of PME and Ca2+ into the loquat flesh led to an increase in firmness with more low-methylesterified HG and egg-box structure signals. CONCLUSION: PME-mediated demethylesterification generated large amounts of low-methylesterified HG in the cell wall. This low-methylesterified HG further cross-linked with Ca2+ to form egg-box structures. The pectin-involved complexations then contributed to the increased firmness in loquat fruit. Overall, besides being involved in fruit softening, pectin could also be involved in strengthening the mechanical properties of postharvest fruit. This study provides new ideas for obtaining a better texture of postharvest loquat fruits based on pectin regulation.

DNA methylation mediated by RdDM pathway and demethylation affects furanone accumulation through regulation of QUINONE OXIDOREDUCTASE in strawberry.

Recently, increasing evidence suggests that DNA methylation plays a crucial role in fruit ripening. However, the role of DNA methylation in regulating specific traits, such as flavor, remains unclear. Here, we report a role of DNA methylation in affecting furanone biosynthesis in strawberry. Strawberry quinone oxidoreductase (FaQR) is a key enzyme in furanone biosynthesis. There are four FaQR homologs in strawberry cultivar 'Yuexin', and one of them, FaQR3, contributes ~50% of FaQR transcripts, indicating a major role of FaQR3 in furanone biosynthesis. Through characterization of levels of DNA methylation and FaQR3 transcript and furanone contents during fruit ripening and after the application of DNA methylation inhibitor, we found that the DNA methylation level of the FaQR3 promoter was negatively correlated with FaQR3 expression and furanone accumulation, suggesting that DNA methylation may be involved in furanone biosynthesis through adjusting FaQR3 expression, and responded to different temperatures consistently. In addition, transient expression of a gene in the RNA-directed DNA methylation (RdDM) pathway, FaAGO4, and enrichment analysis of the 24-nucleotide siRNAs suggested that DNA methylation in the FaQR3 promoter is mediated by the RdDM pathway. Transient RNA interference (RNAi) of FaDML indicated that the demethylation pathway may be involved in regulating furanone accumulation. These findings provide new insights into the role of DNA methylation and demethylation in affecting flavor quality in strawberry during fruit ripening.

Abscisic acid mediated strawberry receptacle ripening involves the interplay of multiple phytohormone signaling networks.

As a canonical non-climacteric fruit, strawberry (Fragaria spp.) ripening is mainly mediated by abscisic acid (ABA), which involves multiple other phytohormone signalings. Many details of these complex associations are not well understood. We present an coexpression network, involving ABA and other phytohormone signalings, based on weighted gene coexpression network analysis of spatiotemporally resolved transcriptome data and phenotypic changes of strawberry receptacles during development and following various treatments. This coexpression network consists of 18,998 transcripts and includes transcripts related to phytohormone signaling pathways, MADS and NAC family transcription factors and biosynthetic pathways associated with fruit quality. Members of eight phytohormone signaling pathways are predicted to participate in ripening and fruit quality attributes mediated by ABA, of which 43 transcripts were screened to consist of the hub phytohormone signalings. In addition to using several genes reported from previous studies to verify the reliability and accuracy of this network, we explored the role of two hub signalings, small auxin up-regulated RNA 1 and 2 in receptacle ripening mediated by ABA, which are also predicted to contribute to fruit quality. These results and publicly accessible datasets provide a valuable resource to elucidate ripening and quality formation mediated by ABA and involves multiple other phytohormone signalings in strawberry receptacle and serve as a model for other non-climacteric fruits.

Expression of ethylene response genes during persimmon fruit astringency removal.

Thirteen ethylene signaling related genes were isolated and studied during ripening of non-astringent 'Yangfeng' and astringent 'Mopan' persimmon fruit. Some of these genes were characterized as ethylene responsive. Treatments, including ethylene and CO(2), had different effects on persimmon ripening, but overlapping roles in astringency removal, such as increasing the reduction in levels of soluble tannins. DkERS1, DkETR2, and DkERF8, may participate in persimmon fruit ripening and softening. The expression patterns of DkETR2, DkERF4, and DkERF5 had significant correlations with decreases in soluble tannins in 'Mopan' persimmon fruit, suggesting that these genes might be key components in persimmon fruit astringency removal and be the linkage between different treatments, while DkERF1 and DkERF6 may be specifically involved in CO(2) induced astringency removal. The possible roles of ethylene signaling genes in persimmon fruit astringency removal are discussed.