Transcription factor CsMADS3 coordinately regulates chlorophyll and carotenoid pools in Citrus hesperidium.

Citrus, 1 of the largest fruit crops with global economic and nutritional importance, contains fruit known as hesperidium with unique morphological types. Citrus fruit ripening is accompanied by chlorophyll degradation and carotenoid biosynthesis, which are indispensably linked to color formation and the external appearance of citrus fruits. However, the transcriptional coordination of these metabolites during citrus fruit ripening remains unknown. Here, we identified the MADS-box transcription factor CsMADS3 in Citrus hesperidium that coordinates chlorophyll and carotenoid pools during fruit ripening. CsMADS3 is a nucleus-localized transcriptional activator, and its expression is induced during fruit development and coloration. Overexpression of CsMADS3 in citrus calli, tomato (Solanum lycopersicum), and citrus fruits enhanced carotenoid biosynthesis and upregulated carotenogenic genes while accelerating chlorophyll degradation and upregulating chlorophyll degradation genes. Conversely, the interference of CsMADS3 expression in citrus calli and fruits inhibited carotenoid biosynthesis and chlorophyll degradation and downregulated the transcription of related genes. Further assays confirmed that CsMADS3 directly binds and activates the promoters of phytoene synthase 1 (CsPSY1) and chromoplast-specific lycopene ß-cyclase (CsLCYb2), 2 key genes in the carotenoid biosynthetic pathway, and STAY-GREEN (CsSGR), a critical chlorophyll degradation gene, which explained the expression alterations of CsPSY1, CsLCYb2, and CsSGR in the above transgenic lines. These findings reveal the transcriptional coordination of chlorophyll and carotenoid pools in the unique hesperidium of Citrus and may contribute to citrus crop improvement.

Grape phytochrome-interacting factor VvPIF1 negatively regulates carotenoid biosynthesis by repressing VvPSY expression.

Phytochrome-interacting factors (PIFs) play important roles in light-mediated secondary metabolism; however, the roles of PIFs in grape fruit carotenogenesis remain unclear. Here, by identifying the PIF family genes in grapes, we focused on the role of VvPIF1 in carotenoid metabolism. During grape berry development, VvPIF1 expression was negatively correlated with carotenoid accumulation and the transcription of phytoene synthase 1/2 (VvPSY1/2), which encodes the major flux-controlling enzymes for carotenoid biosynthesis. Light significantly repressed VvPIF1 expression, but induced the expression of carotenogenic genes including VvPSY1/2. VvPIF1 functioned as a nucleus-localized protein and interacted with the light photoreceptor VvphyB. Overexpression of VvPIF1 resulted in the downregulation of the endogenous PIF1 gene, which may unexpectedly induce carotenoid accumulation and PSY expression in tobacco leaves. The transgenic grape leaves and tomato fruits with high VvPIF1 expression produced a significant decrease in carotenoid concentrations, with suppressed transcription of PSY and other carotenogenic genes. Further biochemical assays demonstrated that VvPIF1 bound directly to the promoters of VvPSY1/2 to inhibit their transcription. Collectively, we conclude that VvPIF1 negatively regulates carotenoid biosynthesis by repressing VvPSY expression in grapes. These findings shed light on the role and mode of action of PIFs in the carotenoid regulatory network of grapes.

Jasmonate increases terpene synthase expression, leading to strawberry resistance to Botrytis cinerea infection.

KEY MESSAGE: Jasmonate induced FaTPS1 to produce terpene, and overexpression FaTPS1 led to fruit resistant against B. cinerea infection, FaMYC2 induced FaTPS1 by binding to its promoter that downstream of jasmonate. Jasmonic acid (JA) and its derivatives are associated with plant defence responses against pathogenic organisms. In the present study, a total of 10,631 differentially expressed genes, 239 differentially expressed proteins, and 229 differential metabolites were screened and found to be mainly involved in pathogen perception, hormone biosynthesis and signal transduction, photosynthesis, and secondary metabolism. In strawberry fruits, methyl jasmonate (MeJA) induced FaTPS1 expression and quickly increased the terpene content. Furthermore, FaTPS1 overexpression increased the emission of sesquiterpenes, especially germacrene D, and improved strawberry resistance against Botrytis cinerea infection, although the knockdown of FaTPS1 increased its susceptibility to the same pathogen. Using a yeast one-hybrid assay and transient expression analysis, we demonstrated that FaMYC2 can bind to the G-box element in the promoter region of FaTPS1, thus inducing FaTPS1 expression. MeJA also stimulated FaMYC2 expression and regulated downstream signalling cascades. Moreover, we presented a possible model of the new signalling pathway of MeJA-mediated strawberry resistance to B. cinerea.

DNA and Histone Methylation Regulates Different Types of Fruit Ripening by Transcriptome and Proteome Analyses.

Methylation affects different aspects of genetic material stability, gene expression regulation, and histone modification. The previous reports depicted that DNA and histone methylation regulates plant growth and development. In this study, we evaluated the effects of DNA and histone methylation on 'Hongjia' strawberry and 'Lichun' tomato. We investigated the transient transformation system for arginine methyltransferase (FvPRMT1.5) overexpression and interference and assessed the phenotypic appearance and mRNA and protein expression levels. Results depicted that changes in methylation levels caused inhibition of carotenoids and anthocyanins. Furthermore, the profiling of aroma components was altered in response to 5-azacytidine. DNA hypomethylation induced the expression levels of genes involved in photosynthesis, flavonoid biosynthesis, and hormone signal transduction pathways, while the expression levels of related proteins showed a downward trend. Overall, we proposed a model that reveals the possible regulatory effects of DNA and histone methylation during fruit ripening.

Systematic analysis reveals O-methyltransferase gene family members involved in flavonoid biosynthesis in grape.

O-methyltransferases (OMTs) are an important group of enzymes involved in the methylation of various secondary metabolites, including flavonoids. However, the features and functions of OMTs have not been comprehensively studied in grape (Vitis vinifera), a rich source of methylated flavonoids. Here, 47 OMT members were identified in grape genome. They were unevenly distributed on grape chromosomes and some genes were tandem duplicated, indicating the role of duplication processes in the expansion of this gene family. Based on the phylogenetic relationship, these OMTs were clustered into CCoAOMT and COMT subclades, which were further supported by the results of conserved motif and gene structure analysis. Correlation analysis revealed that three members (VvCCoAOMT1, VvCCoAOMT4, and VvCOMT1) were potentially involved in the synthesis of most methylated flavonoids in the berry skins. Expression profiling based on RNA-seq data and qRT-PCR experiments indicated that VvCCoAOMT1 and VvCCoAOMT4 had specific and high expression in berry skins, and responded to abscisic acid and high temperature treatments; and that VvCOMT1 expression was significantly induced during berry development and UVC treatment. Cis-regulatory element analysis suggested important roles of OMTs in growth, development, and defense against stresses. We further demonstrated the transcriptional regulation of VvCCoAOMT4 by VvMYBA1, a master regulator of grape berry anthocyanin, and verified the protein localization of VvCCoAOMT4 in membrane and nucleus. These findings facilitate a better understanding of the characteristics of OMT gene family, especially of the potential members involved in the formation of O-methylated flavonoids in grape.

Targeting cIAPs attenuates CCl4-induced liver fibrosis by increasing MMP9 expression derived from neutrophils.

AIMS: Liver fibrosis is a growing public health concern without effective medical treatment. Recent reports have indicated that inhibitors of apoptosis proteins (IAPs) were potential targets for idiopathic pulmonary fibrosis therapy. However, their roles have not been well identified in liver fibrosis. METHODS: The expression of IAPs were examined in human liver tissue and experimental mouse models. Liver fibrosis in CCl4-induced mouse models were investigated by Sirius red staining, RT-PCR, Western blotting after hepatocytes-specific cIAP2 knockout or IAPs inhibitor APG-1387 treatment. The underlying molecular mechanism of APG-1387 action was explored by apoptosis analysis, matrix metalloprotein 9 (MMP9) inhibition, neutrophils depletion, and CC Motif Chemokine Ligand 5 (CCL5) gene knockout in vitro and in vivo. FINDINGS: Our study showed that increased expression of cIAP2 was associated with liver fibrosis severity in liver tissues. Deletion of cIAP2 from hepatocytes or degrading cIAPs by APG-1387 ameliorated liver fibrosis induced by CCl4. APG-1387 treatment exhibited increased expression of MMP9 and resulted in higher ratio of MMP9 to tissue inhibitor of metalloproteinase-1. MMP9 was mainly derived from CCL5 chemotactic neutrophils. Further, MMP9 inhibition by CTT peptide, neutrophil depletion by Ly6G antibody or CCL5 deficiency blocked the anti-fibrotic effects of APG-1387 in vivo. SIGNIFICANCE: These results suggested that cIAPs, especially cIAP2, might play a novel role in the pathogenesis of liver fibrosis, and targeting cIAPs represented a promising therapeutic strategy for liver fibrosis by increasing MMP9 expression induced by CCL5 chemotactic neutrophils.

Transcriptomic and Metabolomic Profiling Reveals the Effect of LED Light Quality on Fruit Ripening and Anthocyanin Accumulation in Cabernet Sauvignon Grape.

Different light qualities have various impacts on the formation of fruit quality. The present study explored the influence of different visible light spectra (red, green, blue, and white) on the formation of quality traits and their metabolic pathways in grape berries. We found that blue light and red light had different effects on the berries. Compared with white light, blue light significantly increased the anthocyanins (malvidin-3-O-glucoside and peonidin-3-O-glucoside), volatile substances (alcohols and phenols), and soluble sugars (glucose and fructose), reduced the organic acids (citric acid and malic acid), whereas red light achieved the opposite effect. Transcriptomics and metabolomics analyses revealed that 2707, 2547, 2145, and 2583 differentially expressed genes (DEGs) and (221, 19), (254, 22), (189, 17), and (234, 80) significantly changed metabolites (SCMs) were filtered in the dark vs. blue light, green light, red light, and white light, respectively. According to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, most of the DEGs identified were involved in photosynthesis and biosynthesis of flavonoids and flavonols. Using weighted gene co-expression network analysis (WGCNA) of 23410 highly expressed genes, two modules significantly related to anthocyanins and soluble sugars were screened out. The anthocyanins accumulation is significantly associated with increased expression of transcription factors (VvHY5, VvMYB90, VvMYB86) and anthocyanin structural genes (VvC4H, Vv4CL, VvCHS3, VvCHI1, VvCHI2, VvDFR), while significantly negatively correlated with VvPIF4. VvISA1, VvISA2, VvAMY1, VvCWINV, VvßGLU12, and VvFK12 were all related to starch and sucrose metabolism. These findings help elucidate the characteristics of different light qualities on the formation of plant traits and can inform the use of supplemental light in the field and after harvest to improve the overall quality of fruit.

Integrative Analyses of Metabolomes and Transcriptomes Provide Insights into Flavonoid Variation in Grape Berries.

Flavonoids in grapes contribute the quality of the berry, but the flavonoid diversity and the regulatory networks underlying the variation require a further investigation. In this study, we integrated multi-omics data to systematically explore the global metabolic and transcriptional profiles in the skins and pulps of three grape cultivars. The results revealed large-scale differences involved in the flavonoid metabolic pathway. A total of 133 flavonoids, including flavone and flavone C-glycosides, were identified. Beyond the visible differences of anthocyanins, there was large variation in other sub-branched flavonoids, most of which were positively correlated with anthocyanins in grapes. The expressions of most flavonoid biosynthetic genes and the major regulators MYBA1 were strongly consistent with the changes in flavonoids. Integrative analysis identified two novel transcription factors (MYB24 and MADS5) and two ubiquitin proteins (RHA2) as promising regulatory candidates for flavonoid biosynthesis in grapes. Further verification in various grape accessions indicated that five major genes including flavonol 3'5'-hydroxylase (F3'5'H), UDP-glucose:flavonoid 3-O-glycosyl-transferase, anthocyanin O-methyltransferase, acyltransferase (3AT), and glutathione S-transferase (GST4) controlled flavonoid variation in grape berries. These findings provide valuable information for understanding the mechanism of flavonoid biosynthesis in grape berries and the further development of grape health products.

Citrus transcription factor CsHB5 regulates abscisic acid biosynthetic genes and promotes senescence.

Senescence is a gradual physiological process involving the integration of numerous internal and environmental signals. Abscisic acid (ABA) is a well-known inducer of senescence. However, the regulatory mechanisms underlying ABA-mediated senescence remain largely unknown. Here, we report that the citrus homeodomain leucine zipper I (HD-ZIP I) transcription factor CsHB5 functions as a regulator of ABA-triggered senescence. CsHB5 acts as a nucleus-localized transcriptional activator, the expression of which appeared to be closely associated with citrus senescence. Overexpression of CsHB5 in citrus calli upregulated the expression of ABA- and reactive oxygen species (ROS)-related genes, and significantly increased the content of ABA and hydrogen peroxide (H2 O2 ), whereas silencing CsHB5 in citrus calli downregulated the expression of ABA-related genes. Additionally, heterogenous overexpression of CsHB5 in Solanum lycopersicum (tomato) and Arabidopsis thaliana (Arabidopsis) leads to early leaf yellowing under dark-induced senescence conditions. Meanwhile, the levels of ABA and H2 O2 in transgenic tomatoes increased significantly and the lycopene content decreased. Transcriptome analysis of CsHB5-overexpressing citrus calli and tomato showed that CsHB5 was involved in multiple senescence-associated processes, including chlorophyll degradation, nutrient compound biosynthesis and transport, as well as ABA and ROS signal transduction. The results of yeast one-hybrid assays, electrophoretic mobility shift assays and dual luciferase assays indicated that CsHB5 directly binds to the promoters of ABA biosynthetic genes, including ß-carotene hydroxylase 1 (BCH1) and 9-cis-epoxycarotenoid dioxygenase 2 (NCED2), thereby activating their transcription. Our findings revealed that CsHB5 participates in senescence, at least partly, by directly controlling ABA accumulation. Our work provides insight into the regulatory mechanisms underlying ABA-mediated senescence.

A fruit ripening-associated transcription factor CsMADS5 positively regulates carotenoid biosynthesis in citrus.

Carotenoids in citrus contribute to the quality of the fruit, but the mechanism of its transcriptional regulation is fairly unknown. Here, we characterized a citrus FRUITFULL sub-clade MADS gene, CsMADS5, that was ripening-inducible and acted as a nucleus-localized trans-activator. Transient overexpression of CsMADS5 in citrus induced fruit coloration and enhanced carotenoid concentrations. The expression of carotenogenic genes including phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene ß-cyclase 1 (LCYb1) was increased in the peels of fruits overexpressing CsMADS5. Similar results were observed from stable overexpression of CsMADS5 in tomato fruits and citrus calli, even though the effect of CsMADS5 on carotenoid metabolism in transgenic citrus calli was limited. Further biochemical analyses demonstrated that CsMADS5 activated the transcription of PSY, PDS, and LCYb1 by directly binding to their promoters. We concluded that CsMADS5 positively regulates carotenoid biosynthesis in fruits by directly activating the transcription of carotenogenic genes. Moreover, CsMADS5 physically interacted with a positive regulator CsMADS6, indicating that CsMADS5 may form an enhancer complex with CsMADS6 to synergistically promote carotenoid accumulation. These findings expand our understanding of the complex transcriptional regulatory hierarchy of carotenoid biosynthesis during fruit ripening.

Ethylene activation of carotenoid biosynthesis by a novel transcription factor CsERF061.

Chromoplast-specific lycopene ß-cyclase (LCYb2) is a critical carotenogenic enzyme, which controls the massive accumulation of downstream carotenoids, especially provitamin A carotenoids, in citrus. Its regulatory metabolism is largely unknown. Here, we identified a group I ethylene response factor, CsERF061, in citrus by yeast one-hybrid screen with the promoter of LCYb2. The expression of CsERF061 was induced by ethylene. Transcript and protein levels of CsERF061 were increased during fruit development and coloration. CsERF061 is a nucleus-localized transcriptional activator, which directly binds to the promoter of LCYb2 and activates its expression. Overexpression of CsERF061 in citrus calli and tomato fruits enhanced carotenoid accumulation by increasing the expression of key carotenoid pathway genes, and increased the number of chromoplasts needed to sequester the elevated concentrations of carotenoids, which was accompanied by changes in the concentrations of abscisic acid and gibberellin. Electrophoretic mobility shift and dual-luciferase assays verified that CsERF061 activates the promoters of nine other key carotenoid pathway genes, PSY1, PDS, CRTISO, LCYb1, BCH, ZEP, NCED3, CCD1, and CCD4, revealing the multitargeted regulation of CsERF061. Collectively, our findings decipher a novel regulatory network of carotenoid enhancement by CsERF061, induced by ethylene, which will be useful for manipulating carotenoid accumulation in citrus and other plants.

A Citrus Phosphate Starvation Response Factor CsPHL3 Negatively Regulates Carotenoid Metabolism.

Carotenoids provide precursors for the biosynthesis of strigolactones, which are a new class of hormones that are essential in phosphate (Pi) signaling during plant development. Carotenoid metabolism is a finely tuned pathway, but our understanding of the regulation mechanisms is still limited. In this study, we isolated a protein designated as CsPHL3 from citrus. CsPHL3 belonged to the Pi starvation response factor (PHR)-like subclade and was upregulated by low Pi. Acting as a nucleus-localized protein with transactivation activity, CsPHL3 bound directly to activate the promoter of a key metabolic gene, lycopene ß-cyclase1 (LCYb1). Transgenic analysis revealed that the CsPHL3-overexpressing tomato plants exhibited abnormal growth, like the plants grew under limited Pi conditions. The transgenic lines showed reduced carotenoid contents and elevated expression of LCYb genes but downregulation of other key carotenogenic genes, including phytoene synthase (PSY). Moreover, CsPHL3 induced anthocyanin biosynthesis and affected Pi signaling in the transgenic plants. We further demonstrated that the expression of PSY was negatively regulated by CsPHL3 and high Pi. It is concluded that CsPHL3 is a Pi starvation response factor that negatively regulates carotenoid metabolism by modulating the expression of carotenogenic genes. Establishment of the CsPHL3-CsLCYb1 network provides new valuable knowledge of the function and underlying mechanism of PHR transcription factors and expands our understanding of the complex regulation mechanisms of carotenoid biosynthesis.

Comparative Analysis of Cuticular Wax in Various Grape Cultivars During Berry Development and After Storage.

Cuticular wax covering the surface of fleshy fruit is closely related to fruit glossiness, development, and post-harvest storage quality. However, the information about formation characteristics and molecular mechanisms of cuticular wax in grape berry is limited. In this study, crystal morphology, chemical composition, and gene expression of cuticular wax in grape berry were comprehensively investigated. Morphological analysis revealed high density of irregular lamellar crystal structures, which were correlated with the glaucous appearances of grape berry. Compositional analysis showed that the dominant wax compounds were triterpenoids, while the most diverse were alkanes. The amounts of triterpenoids declined sharply after véraison, while those of other compounds maintained nearly constant throughout the berry development. The amounts of each wax compounds varied among different cultivars and showed no correlation with berry skin colors. Moreover, the expression profiles of related genes were in accordance with the accumulation of wax compounds. Further investigation revealed the contribution of cuticular wax to the water preservation capacity during storage. These findings not only facilitate a better understanding of the characteristics of cuticular wax, but also shed light on the molecular basis of wax biosynthesis in grape.

Viral and Antibody Kinetics of COVID-19 Patients with Different Disease Severities in Acute and Convalescent Phases: A 6-Month Follow-Up Study.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread rapidly around the world, posing a major threat to human health and the economy. Currently, long-term data on viral shedding and the serum antibody responses in COVID-19 patients are still limited. Herein, we report the clinical features, viral RNA loads, and serum antibody levels in a cohort of 112 COVID-19 patients admitted to the Honghu People's Hospital, Hubei Province, China. Overall, 5.36% (6/112) of patients showed persistent viral RNA shedding (> 45 days). The peak viral load was higher in the severe disease group than in the mild group (median cycle threshold value, 36.4 versus 31.5; P = 0.002). For most patients the disappearance of IgM antibodies occurred approximately 4-6 weeks after symptoms onset, while IgG persisted for over 194 days after the onset of symptoms, although patients showed a 46% reduction in antibodies titres against SARS-CoV-2 nucleocapsid protein compared with the acute phase. We also studied 18 asymptomatic individuals with RT-qPCR confirmed SARS-CoV-2 infection together with 17 symptomatic patients, and the asymptomatic individuals were the close contacts of these symptomatic cases. Delayed IgG seroconversion and lower IgM seropositive rates were observed in asymptomatic individuals. These data indicate that higher viral loads and stronger antibody responses are related to more severe disease status in patients with SARS-CoV-2 infection, and the antibodies persisted in the recovered patient for more than 6 months so that the vaccine may provide protection against SARS-CoV-2 infection.

The Citrus Transcription Factor CsMADS6 Modulates Carotenoid Metabolism by Directly Regulating Carotenogenic Genes.

Although remarkable progress has been made toward understanding carotenoid biosynthesis, the mechanisms that regulate the transcription of carotenogenic genes remain poorly understood. Lycopene ß-cyclases (LCYb) are critical enzymes located at the branch point of the carotenoid biosynthetic pathway. Here, we used the promoter sequence of LCYb1 as bait in a yeast one-hybrid screen for promoter-binding proteins from sweet orange (Citrus sinensis). This screen identified a MADS transcription factor, CsMADS6, that was coordinately expressed with fruit development and coloration. Acting as a nucleus-localized transcriptional activator, CsMADS6 directly bound the promoter of LCYb1 and activated its expression. Overexpression of CsMADS6 in citrus calli increased carotenoid contents and induced the expression of LCYb1 and other carotenogenic genes, including phytoene synthase (PSY), phytoene desaturase (PDS), and carotenoid cleavage dioxygenase1 (CCD1). CsMADS6 up-regulated the expression of PSY, PDS, and CCD1 by directly binding to their promoters, which suggested the multitargeted regulation of carotenoid metabolism by CsMADS6. In addition, the ectopic expression of CsMADS6 in tomato (Solanum lycopersicum) affected carotenoid contents and the expression of carotenogenic genes. The sepals of CsMADS6-overexpressing tomato lines exhibited dramatic changes in carotenoid profiles, accompanied by changes in plastid ultrastructure. Global transcriptome analysis of transgenic sepals revealed that CsMADS6 regulates a series of pathways that promote increases in flux through the carotenoid pathway. Overall, these findings establish that CsMADS6 directly regulates LCYb1 and other carotenogenic genes to coordinately and positively modulate carotenoid metabolism in plants, which may provide strategies to improve the nutritional quality of crops.

Isolation and Functional Characterization of a Lycopene ß-cyclase Gene Promoter from Citrus.

Lycopene ß-cyclases are key enzymes located at the branch point of the carotenoid biosynthesis pathway. However, the transcriptional regulatory mechanisms of LCYb1 in citrus with abundant carotenoid accumulation are still unclear. To understand the molecular basis of CsLCYb1 expression, we isolated and functionally characterized the 5' upstream sequences of CsLCYb1 from citrus. The full-length CsLCYb1 promoter and a series of its 5' deletions were fused to the ß-glucuronidase (GUS) reporter gene and transferred into different plants (tomato, Arabidopsis and citrus callus) to test the promoter activities. The results of all transgenic species showed that the 1584 bp upstream region from the translational start site displayed maximal promoter activity, and the minimal promoter containing 746 bp upstream sequences was sufficient for strong basal promoter activity. Furthermore, the CsLCYb1 promoter activity was developmentally and tissue-specially regulated in transgenic Arabidopsis, and it was affected by multiple hormones and environmental cues in transgenic citrus callus under various treatments. Finer deletion analysis identified an enhancer element existing as a tandem repeat in the promoter region between -574 to -513 bp and conferring strong promoter activity. The copy numbers of the enhancer element differed among various citrus species, leading to the development of a derived simple sequence repeat marker to distinguish different species. In conclusion, this study elucidates the expression characteristics of the LCYb1 promoter from citrus and further identifies a novel enhancer element required for the promoter activity. The characterized promoter fragment would be an ideal candidate for genetic engineering and seeking of upstream trans-acting elements.