The BTB-BACK-TAZ domain protein MdBT2 reduces drought resistance by weakening the positive regulatory effect of MdHDZ27 on apple drought tolerance via ubiquitination.

Drought stress is one of the dominating challenges to the growth and productivity in crop plants. Elucidating the molecular mechanisms of plants responses to drought stress is fundamental to improve fruit quality. However, such molecular mechanisms are poorly understood in apple (Malus domestica Borkh.). In this study, we explored that the BTB-BACK-TAZ protein, MdBT2, negatively modulates the drought tolerance of apple plantlets. Moreover, we identified a novel Homeodomain-leucine zipper (HD-Zip) transcription factor, MdHDZ27, using a yeast two-hybrid (Y2H) screen with MdBT2 as the bait. Overexpression of MdHDZ27 in apple plantlets, calli, and tomato plantlets enhanced their drought tolerance by promoting the expression of drought tolerance-related genes [responsive to dehydration 29A (MdRD29A) and MdRD29B]. Biochemical analyses demonstrated that MdHDZ27 directly binds to and activates the promoters of MdRD29A and MdRD29B. Furthermore, in vitro and in vivo assays indicate that MdBT2 interacts with and ubiquitinates MdHDZ27, via the ubiquitin/26S proteasome pathway. This ubiquitination results in the degradation of MdHDZ27 and weakens the transcriptional activation of MdHDZ27 on MdRD29A and MdRD29B. Finally, a series of transgenic analyses in apple plantlets further clarified the role of the relationship between MdBT2 and MdHDZ27, as well as the effect of their interaction on drought resistance in apple plantlets. Collectively, our findings reveal a novel mechanism by which the MdBT2-MdHDZ27 regulatory module controls drought tolerance, which is of great significance for enhancing the drought resistance of apple and other plants.

Recent Advances in Enterovirus A71 Infection and Antiviral Agents.

Enterovirus A71 (EV-A71) is one of the major causative agents of hand, foot, and mouth disease (HFMD) that majorly affects children. Most of the time, HFMD is a mild disease but can progress to severe complications, such as meningitis, brain stem encephalitis, acute flaccid paralysis, and even death. HFMD caused by EV-A71 has emerged as an acutely infectious disease of highly pathogenic potential in the Asia-Pacific region. In this review, we introduced the properties and life cycle of EV-A71, and the pathogenesis and the pathophysiology of EV-A71 infection, including tissue tropism and host range of virus infection, the diseases caused by the virus, as well as the genes and host cell immune mechanisms of major diseases caused by enterovirus 71 (EV-A71) infection, such as encephalitis and neurologic pulmonary edema. At the same time, clinicopathologic characteristics of EV-A71 infection were introduced. There is currently no specific medication for EV-A71 infection, highlighting the urgency and significance of developing suitable anti-EV-A71 agents. This overview also summarizes the targets of existing anti-EV-A71 agents, including virus entry, translation, polyprotein processing, replication, assembly and release; interferons; interleukins; the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase B signaling pathways; the oxidative stress pathway; the ubiquitin-proteasome system; and so on. Furthermore, it overviews the effects of natural products, monoclonal antibodies, and RNA interference against EV-A71. It also discusses issues limiting the research of antiviral drugs. This review is a systematic and comprehensive summary of the mechanism and pathological characteristics of EV-A71 infection, the latest progress of existing anti-EV-A71 agents. It would provide better understanding and guidance for the research and application of EV-A71 infection and antiviral inhibitors.

MdPRX34L, a class III peroxidase gene, activates the immune response in apple to the fungal pathogen Botryosphaeria dothidea.

MAIN CONCLUSION: MdPRX34L enhanced resistance to Botryosphaeria dothidea by increasing salicylic acid (SA) and abscisic acid (ABA) content as well as the expression of related defense genes. The class III peroxidase (PRX) multigene family is involved in complex biological processes. However, the molecular mechanism of PRXs in the pathogen defense of plants against Botryosphaeria dothidea (B. dothidea) remains unclear. Here, we cloned the PRX gene MdPRX34L, which was identified as a positive regulator of the defense response to B. dothidea, from the apple cultivar 'Royal Gala.' Overexpression of MdPRX34L in apple calli decreased sensitivity to salicylic acid (SA) and abscisic acid（ABA). Subsequently, overexpression of MdPRX34L in apple calli increased resistance to B. dothidea infection. In addition, SA contents and the expression levels of genes related to SA synthesis and signaling in apple calli overexpressing MdPRX34L were higher than those in the control after inoculation, suggesting that MdPRX34L enhances resistance to B. dothidea via the SA pathway. Interestingly, infections in apple calli by B. dothidea caused an increase in endogenous levels of ABA followed by induction of ABA-related genes expression. These findings suggest a potential mechanism by which MdPRX34L enhances plant-pathogen defense against B. dothidea by regulating the SA and ABA pathways.

MdWRKY31-MdNAC7 regulatory network: orchestrating fruit softening by modulating cell wall-modifying enzyme MdXTH2 in response to ethylene signalling.

Softening in fruit adversely impacts their edible quality and commercial value, leading to substantial economic losses during fruit ripening, long-term storage, long-distance transportation, and marketing. As the apple fruit demonstrates climacteric respiration, its firmness decreases with increasing ethylene release rate during fruit ripening and postharvest storage. However, the molecular mechanisms underlying ethylene-mediated regulation of fruit softening in apple remain poorly understood. In this study, we identified a WRKY transcription factor (TF) MdWRKY31, which is repressed by ethylene treatment. Using transgenic approaches, we found that overexpression of MdWRKY31 delays softening by negatively regulating xyloglucan endotransglucosylase/hydrolases 2 (MdXTH2) expression. Yeast one-hybrid (Y1H), electrophoretic mobility shift (EMSA), and dual-luciferase assays further suggested that MdWRKY31 directly binds to the MdXTH2 promoter via a W-box element and represses its transcription. Transient overexpression of ethylene-induced MdNAC7, a NAC TF, in apple fruit promoted softening by decreasing cellulose content and increasing water-soluble pectin content in fruit. MdNAC7 interacted with MdWRKY31 to form a protein complex, and their interaction decreased the transcriptional repression of MdWRKY31 on MdXTH2. Furthermore, MdNAC7 does not directly regulate MdXTH2 expression, but the protein complex formed with MdWRKY31 hinders MdWRKY31 from binding to the promoter of MdXTH2. Our findings underscore the significance of the regulatory complex NAC7-WRKY31 in ethylene-responsive signalling, connecting the ethylene signal to XTH2 expression to promote fruit softening. This sheds light on the intricate mechanisms governing apple fruit firmness and opens avenues for enhancing fruit quality and reducing economic losses associated with softening.

The OsZIP2 transporter is involved in root-to-shoot translocation and intervascular transfer of cadmium in rice.

Cadmium (Cd) is a toxic metal that poses serious threats to human health. Rice is a major source of dietary Cd but how rice plants transport Cd to the grain is not fully understood. Here, we characterize the function of the ZIP (ZRT, IRT-like protein) family protein, OsZIP2, in the root-to-shoot translocation of Cd and intervascular transfer of Cd in nodes. OsZIP2 is localized at the plasma membrane and exhibited Cd2+ transport activity when heterologously expressed in yeast. OsZIP2 is strongly expressed in xylem parenchyma cells in roots and in enlarged vascular bundles in nodes. Knockout of OsZIP2 significantly enhanced root-to-shoot translocation of Cd and alleviated the inhibition of root elongation by excess Cd stress; whereas overexpression of OsZIP2 decreased Cd translocation to shoots and resulted in Cd sensitivity. Knockout of OsZIP2 increased Cd allocation to the flag leaf but decreased Cd allocation to the panicle and grain. We further reveal that the variation of OsZIP2 expression level contributes to grain Cd concentration among rice germplasms. Our results demonstrate that OsZIP2 functions in root-to-shoot translocation of Cd in roots and intervascular transfer of Cd in nodes, which can be used for breeding low Cd rice varieties.

MdABCI17 acts as a positive regulator to enhance apple resistance to Botryosphaeria dothidea.

The ATP-binding cassette (ABC) superfamily is involved in numerous complex biological processes. However, the understanding of ABCs in plant pathogen defense, particularly against Botryosphaeria dothidea, remains limited. In this study, we identified MdABCI17 that plays a positive role in apple resistance to B. dothidea. Overexpression of MdABCI17 significantly enhanced the resistance of apple calli and fruits to B. dothidea. Our findings revealed that the jasmonic acid (JA) content and the expression of genes associated with JA biosynthesis and signal transduction were higher in stable MdABCI17-overexpressing apple calli than that of wild-type after inoculation with B. dothidea. Similar results were obtained for apple fruits with transient overexpression of MdABCI17. Our research indicates that MdABCI17 enhances apple resistance to B. dothidea through the JA signaling pathway. We further determined that MdABCI17 plays a crucial role in the apple's response to JA signaling. Moreover, exogenous methyl jasmonate (MeJA) treatment significantly enhanced the effectiveness of MdABCI17 in boosting apple resistance to B. dothidea. We proposed a positive feedback regulatory loop between MdABCI17-mediated apple resistance to B. dothidea and JA signal. In summary, our study offers new insights into the role of ABC superfamily members in the control of plant disease resistance. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01501-9.

The BTB/TAZ domain-containing protein CmBT1-mediated CmANR1 ubiquitination negatively regulates root development in chrysanthemum.

Roots are fundamental for plants to adapt to variable environmental conditions. The development of a robust root system is orchestrated by numerous genetic determinants and, among them, the MADS-box gene ANR1 has garnered substantial attention. Prior research has demonstrated that, in chrysanthemum, CmANR1 positively regulates root system development. Nevertheless, the upstream regulators involved in the CmANR1-mediated regulation of root development remain unidentified. In this study, we successfully identified bric-a-brac, tramtrack and broad (BTB) and transcription adapter putative zinc finger (TAZ) domain protein CmBT1 as the interacting partner of CmANR1 through a yeast-two-hybrid (Y2H) screening library. Furthermore, we validated this physical interaction through bimolecular fluorescence complementation and pull-down assays. Functional assays revealed that CmBT1 exerted a negative influence on root development in chrysanthemum. In both in vitro and in vivo assays, it was evident that CmBT1 mediated the ubiquitination of CmANR1 through the ubiquitin/26S proteasome pathway. This ubiquitination subsequently led to the degradation of the CmANR1 protein and a reduction in the transcription of CmANR1-targeted gene CmPIN2, which was crucial for root development in chrysanthemum. Genetic analysis suggested that CmBT1 modulated root development, at least in part, by regulating the level of CmANR1 protein. Collectively, these findings shed new light on the regulatory role of CmBT1 in degrading CmANR1 through ubiquitination, thereby repressing the expression of its targeted gene and inhibiting root development in chrysanthemum.

Streptococcus suis prophage lysin as a new strategy for combating streptococci-induced mastitis and Streptococcus suis infection.

OBJECTIVES: The genus Streptococcus contains species of important zoonotic pathogens such as those that cause bovine mastitis. Unfortunately, many Streptococcus species have developed antibiotic resistance. Phage lysins are considered promising alternatives to antibiotics because it is difficult for bacteria to develop lysin resistance. However, there remains a lack of phage lysin resources for the treatment of streptococci-induced mastitis. METHODS: We identified the prophage lysin Lys0859 from the genome of the Streptococcus suis SS0859 strain. Lys0859 was subsequently characterized to determine its host range, MIC, bactericidal activity in milk, and ability to clear biofilms in vitro. Finally, to determine the effects of Lys0859 on the treatment of both bovine mastitis and S. suis infection in vivo, we established models of Streptococcus agalactiae ATCC 13813-induced mastitis and S. suis serotype 2 SC19 systemic infection. RESULTS: Our results demonstrate that Lys0859 possesses broad-spectrum lytic activity against Streptococcus and Staphylococcus species isolated from animals with bovine mastitis and 15 serotypes of S. suis isolated from swine. Intramammary and intramuscular injection of Lys0859 reduced the number of bacteria in mammary tissue by 3.75 and 1.45 logs compared with the PBS group, respectively. Furthermore, 100 µg/mouse of Lys0859 administered intraperitoneally at 1 h post-infection protected 83.3% (5/6) of mice from a lethal dose of S. suis infection. CONCLUSIONS: Overall, our results enhance the understanding and development of new strategies to combat both streptococci-induced mastitis and S. suis infection.

Ethylene inhibits malate accumulation in apple by transcriptional repression of aluminum-activated malate transporter 9 via the WRKY31-ERF72 network.

Malic acid accumulation in the vacuole largely determines acidity and perception of sweetness of apple. It has long been observed that reduction in malate level is associated with increase in ethylene production during the ripening process of climacteric fruits, but the molecular mechanism linking ethylene to malate reduction is unclear. Here, we show that ethylene-modulated WRKY transcription factor 31 (WRKY31)-Ethylene Response Factor 72 (ERF72)-ALUMINUM ACTIVATED MALATE TRANSPORTER 9 (Ma1) network regulates malate accumulation in apple fruit. ERF72 binds to the promoter of ALMT9, a key tonoplast transporter for malate accumulation of apple, transcriptionally repressing ALMT9 expression in response to ethylene. WRKY31 interacts with ERF72, suppressing its transcriptional inhibition activity on ALMT9. In addition, WRKY31 directly binds to the promoters of ERF72 and ALMT9, transcriptionally repressing and activating ERF72 and ALMT9, respectively. The expression of WRKY31 decreases in response to ethylene, lowering the transcription of ALMT9 directly and via its interactions with ERF72. These findings reveal that the regulatory complex WRKY31 forms with ERF72 responds to ethylene, linking the ethylene signal to ALMT9 expression in reducing malate transport into the vacuole during fruit ripening.

Effect of Clostridium butyricum on intestinal microbiota and resistance to Vibrio alginolyticus of Penaeus vannamei.

In order to evaluate the effect of Clostridium butyricum (C. butyricum) feeding on intestinal microorganisms and protection against infection by Vibrio alginolyticus (V. alginolyticus) in Penaeus vannamei (P. vannamei). We set up two groups, CG30 (fed normal feed) and CB30 (fed feed supplemented with C. butyricum), for the 30d C. butyricum feeding test, and four groups, CG (CG30 group injected with PBS), CB (CB30 group injected with PBS), VACG (CG30 group injected with V. alginolyticus), and VACB (CB30 group injected with V. alginolyticus), for the 24 h infection test. The protective effect of C. butyricum against acute V. alginolyticus infection in P. vannamei was explained in terms of survival, histopathology, changes in enzyme activity, transcriptome analysis, and immune-related genes. We found that feeding C. butyricum significantly altered intestinal microbial populations' abundance and significantly reduced Vibrio spp. In the V. alginolyticus stress test, C. butyricum improved the survival rate and alleviated pathological changes in hepatopancreatic tissues, alleviated the reduction of superoxide dismutase (SOD) and phenoloxidase (PO) activity caused by infection, and increased the lysozyme content in P. vannamei. VACB group compared with the VACG group, 1730 up-regulated differentially expressed genes (DEGs) and 2029 down-regulated DEGs were screened. Quantitative real-time PCR (qRT-PCR) showed that dietary supplementation with C. butyricum suppressed the upregulation of alkaline phosphatase (AKP) transcription factors and the downregulation of prophenoloxidase (proPO), alpha-2-macroglobulin (A2M), and anti-lipopolysaccharide factor (ALF) induced by V. alginolyticus infection. In conclusion, feed supplementation with C. butyricum changed P. vannamei's population ratio of intestinal microorganisms. Moreover, C. butyricum has the potential to act as an inhibitor of V. alginolyticus infection and enhance the resistance of P. vannamei to V. alginolyticus infection.

[Mechanism of Zhongfeng Xingnao Decoction in improving microcirculatory disorders in cerebral hemorrhage based on network pharmacology and molecular docking techniques].

This study aimed to explore the mechanism of Zhongfeng Xingnao Decoction(ZFXN) in intervening microcirculatory di-sorders in cerebral hemorrhage by network pharmacology and molecular docking techniques. The information on the components of ZFXN was obtained through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) database, and the predicted targets of chemical components were obtained from PubChem and SwissTargetPrediction. The relevant targets of cerebral hemorrhage and microcirculatory disorders were collected from the GeneCards database, and the common targets of the components and diseases were analyzed by the Database for Annotation, Visualization, and Integrated Discovery(DAVID) for Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses. Visualization of the correlation network was carried out using Cytoscape software to further screen important chemical components for molecular docking prediction with disease targets. The animal experiment validation was performed using modified neurological severity score(mNSS), enzyme-linked immunosorbent assay(ELISA), quantitative real-time polymerase chain reaction(qRT-PCR), immunofluorescence, and Western blot to detect the effects of ZFXN intervention in mice with cerebral hemorrhage. The results showed that there were 31 chemical components and 856 targets in the four drugs contained in ZFXN, 173 targets for microcirculatory disorders in cerebral hemorrhage, and 57 common targets for diseases and components. The enrichment analysis showed that common targets were mainly involved in biological processes, such as cell proliferation and apoptosis, and signaling pathways, such as tumor pathway, viral infection, phosphoinositide-3-kinase/protein kinase B(PI3K/AKT) signaling pathway, and mitogen-activated protein kinase(MAPK) signaling pathway. Molecular docking results revealed that the common components ß-sitosterol of Rhei Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Ginseng Radix et Rhizoma Rubra showed good docking with proto-oncogene tyrosine-protein kinase(SRC), signal transducer and activator of transcription 3(STAT3), phosphoinositide-3-kinase catalytic alpha polypeptide gene(PIK3CA), recombinant protein tyrosine phosphatase non receptor type 11(PTPN11), AKT1, epidermal growth factor receptor(EGFR), calcium adhesion-associated protein beta 1(CTNNB1), vascular endothelial growth factor A(VEGFA), and tumor protein p53(TP53). Moreover, sennoside E of Rhei Radix et Rhizoma showed good docking with MAPK1. The results revealed that the ZFXN relieved the neural injury in mice with cerebral hemorrhage, decreased the expression of S100 calcium-binding protein B(S100ß), neuron specific enolase(NSE), matrix metalloproteinase 9(MMP9), tumor necrosis factor α(TNF-α), interleukin 1ß(IL-1ß), SRC, EGFR, CTNNB1, VEGFA, TP53, glial fibrillary acidic protein(GFAP), and leukocyte differentiation antigen 86(CD86), and increased the expression of p-PI3K, p-AKT, and zona occludens 1(ZO-1). The results indicate that ZFXN may inhibit neuronal apoptosis and inflammatory response through PI3K/AKT/p53 pathway to protect the blood-brain barrier, thereby slowing down microcirculatory impairment in cerebral hemorrhage.

Molybdenum: More than an essential element.

Molybdenum (Mo) is an essential element for almost all living organisms. After being taken up into the cells as molybdate, it is incorporated into the molybdenum cofactor, which functions as the active site of several molybdenum-requiring enzymes and thus plays crucial roles in multiple biological processes. The uptake and transport of molybdate is mainly mediated by two types of molybdate transporters. The homeostasis of Mo in plant cells is tightly controlled, and such homeostasis likely plays vital roles in plant adaptation to local environments. Recent evidence suggests that Mo is more than an essential element required for plant growth and development; it is also involved in local adaptation to coastal salinity. In this review, we summarize recent research progress on molybdate uptake and transport, molybdenum homeostasis network in plants, and discuss the potential roles of the molybdate transporter in plant adaptation to their local environment.

Glucose sensor MdHXK1 activates an immune response to the fungal pathogen Botryosphaeria dothidea in apple.

Sugars are essential regulatory molecules involved in plant growth and development and defense response. Although the relationship between sugars and disease resistance has been widely discussed, the underlying molecular mechanisms remain unexplored. Ring rot caused by Botryosphaeria dothidea (B. dothidea), which severely affects fruit quality and yield, is a destructive disease of apples (Malus domestica Borkh.). The present study found that the degree of disease resistance in apple fruit was closely related to glucose content. Therefore, the gene encoding a hexokinase, MdHXK1, was isolated from the apple cultivar 'Gala', and characterized during the defense response. Overexpression of MdHXK1 enhanced disease resistance in apple calli, leaves and fruits by increasing the expression levels of genes related to salicylate (SA) synthesis (PHYTOALEXIN DEFICIENT 4, PAD4; PHENYLALANINE AMMONIA-LYASE, PAL; and ENHANCED DISEASE SUSCEPTIBILITY 1, EDS1) and signaling (PR1; PR5; and NONEXPRESSER OF PR GENES 1, NPR1) as well as increasing the superoxide (O2- ) production rate and the hydrogen peroxide (H2 O2 ) content. Overall, the study provides new insights into the MdHXK1-mediated molecular mechanisms by which glucose signaling regulates apple ring rot resistance.

Metal-free visible-light-driven cascade cyclization reaction to synthesize 2-oxindoles via benzoyl and phenylsulfinyl radicals with acrylamide derivatives.

A metal-free visible-light-driven cascade cyclization reaction to synthesize 3-methyl-3-acetophenone-2-oxindoles and 3-methyl-3-(methylsulfonyl)benzene-2-oxindoles in yields up to 96% and 99%, via benzoyl and phenylsulfinyl radicals with acrylamide derivatives is reported, respectively. Extensive studies, including gram-scale, radical capture and isotope experiments, were performed to indicate that the reaction may involve a radical process.

Nicotinamide adenine dinucleotide attenuates acetaminophen-induced acute liver injury via activation of PARP1, Sirt1, and Nrf2 in mice.

The aim of this study was to investigate the protective effect of nicotinamide adenine dinucleotide (NAD+) against acute liver injury (ALI) induced by acetaminophen (APAP) overdose in mice. First, serum transaminases were used to assess the protective effect of NAD+, and the data revealed that NAD+ mitigated the APAP-induced ALI in a dose-dependent manner. Then, we performed hematoxylin-eosin staining of liver tissues and found that NAD+ alleviated the abnormalities of histopathology. Meanwhile, increase in the malondialdehyde content and decrease in glutathione, superoxide dismutase (SOD), and glutathione peroxidase were identified in the APAP group, which were partially prevented by the NAD+ pretreatment. Moreover, compared with the mice treated with APAP only, the expression of poly ADP-ribose polymerase 1 (PARP1), Sirtuin1 (Sirt1), SOD2, nuclear factor erythroid 2-related factor 2 (Nrf2), and hemoxygenase-1 was upregulated, while Kelch-like ECH-associated protein 1 and histone H2AX phosphorylated on Ser-139 were downregulated by NAD+ in NAD+ + APAP group. Conversely, NAD+ could not correct the elevated expression of phospho-Jun N-terminal kinase and phospho-extracellular signal-regulated kinase induced by APAP. Taken together, these findings suggest that NAD+ confers an anti-ALI effect to enhance the expression of PARP1 and Sirt1, and to simultaneously stimulate the Nrf2 anti-oxidant signaling pathway.

MdbHLH3 modulates apple soluble sugar content by activating phosphofructokinase gene expression.

Sugars are involved in plant growth, fruit quality, and signaling perception. Therefore, understanding the mechanisms involved in soluble sugar accumulation is essential to understand fruit development. Here, we report that MdPFPß, a pyrophosphate-dependent phosphofructokinase gene, regulates soluble sugar accumulation by enhancing the photosynthetic performance and sugar-metabolizing enzyme activities in apple (Malus domestica Borkh.). Biochemical analysis revealed that a basic helix-loop-helix (bHLH) transcription factor, MdbHLH3, binds to the MdPFPß promoter and activates its expression, thus promoting soluble sugar accumulation in apple fruit. In addition, MdPFPß overexpression in tomato influenced photosynthesis and carbon metabolism in the plant. Furthermore, we determined that MdbHLH3 increases photosynthetic rates and soluble sugar accumulation in apple by activating MdPFPß expression. Our results thus shed light on the mechanism of soluble sugar accumulation in apple leaves and fruit: MdbHLH3 regulates soluble sugar accumulation by activating MdPFPß gene expression and coordinating carbohydrate allocation.

New insights into the role of MADS-box transcription factor gene CmANR1 on root and shoot development in chrysanthemum (Chrysanthemum morifolium).

BACKGROUND: MADS-box transcription factors (TFs) are the key regulators of multiple developmental processes in plants; among them, a chrysanthemum MADS-box TF CmANR1 has been isolated and described as functioning in root development in response to high nitrate concentration signals. However, how CmANR1 affects root and shoot development remains unclear. RESULTS: We report that CmANR1 plays a positive role in root system development in chrysanthemum throughout the developmental stages of in vitro tissue cultures. Metabolomics combined with transcriptomics assays show that CmANR1 promotes robust root system development by facilitating nitrate assimilation, and influencing the metabolic pathways of amino acid, glycolysis, and the tricarboxylic acid cycle (TCA) cycle. Also, we found that the expression levels of TFs associated with the nitrate signaling pathways, such as AGL8, AGL21, and LBD29, are significantly up-regulated in CmANR1-transgenic plants relative to the wild-type (WT) control; by contrast, the expression levels of RHD3-LIKE, LBD37, and GATA23 were significantly down-regulated. These results suggest that these nitrate signaling associated TFs are involved in CmANR1-modulated control of root development. In addition, CmANR1 also acts as a positive regulator to control shoot growth and development. CONCLUSIONS: These findings provide potential mechanisms of MADS-box TF CmANR1 modulation of root and shoot development, which occurs by regulating a series of nitrate signaling associated TFs, and influencing the metabolic pathways of amino acid and glycolysis, as well as TCA cycle and nitrate assimilation.

The apple bHLH transcription factor MdbHLH3 functions in determining the fruit carbohydrates and malate.

Changes in carbohydrates and organic acids largely determine the palatability of edible tissues of horticulture crops. Elucidating the potential molecular mechanisms involved in the change in carbohydrates and organic acids, and their temporal and spatial crosstalk are key steps in understanding fruit developmental processes. Here, we used apple (Malus domestica Borkh.) as research materials and found that MdbHLH3, a basic helix-loop-helix transcription factor (bHLH TF), modulates the accumulation of malate and carbohydrates. Biochemical analyses demonstrated that MdbHLH3 directly binds to the promoter of MdcyMDH that encodes an apple cytosolic NAD-dependent malate dehydrogenase, activating its transcriptional expression, thereby promoting malate accumulation in apple fruits. Additionally, MdbHLH3 overexpression increased the photosynthetic capacity and carbohydrate levels in apple leaves and also enhanced the carbohydrate accumulation in fruits by adjusting carbohydrate allocation from sources to sinks. Overall, our findings provide new insights into the mechanism of how the bHLH TF MdbHLH3 modulates the fruit quality. It directly regulates the expression of cytosolic malate dehydrogenase MdcyMDH to coordinate carbohydrate allocation and malate accumulation in apple.

BTB-TAZ Domain Protein MdBT2 Modulates Malate Accumulation and Vacuolar Acidification in Response to Nitrate.

Excessive application of nitrate, an essential macronutrient and a signal regulating diverse physiological processes, decreases malate accumulation in apple (Malus domestica) fruit, but the underlying mechanism remains poorly understood. Here, we show that an apple BTB/TAZ protein, MdBT2, is involved in regulating malate accumulation and vacuolar pH in response to nitrate. In vitro and in vivo assays indicate that MdBT2 interacts directly with and ubiquitinates a bHLH transcription factor, MdCIbHLH1, via the ubiquitin/26S proteasome pathway in response to nitrate. This ubiquitination results in the degradation of MdCIbHLH1 protein and reduces the transcription of MdCIbHLH1-targeted genes involved in malate accumulation and vacuolar acidification, including MdVHA-A, which encodes a vacuolar H+-ATPase, and MdVHP1, which encodes a vacuolar H+-pyrophosphatase, as well as MdALMT9, which encodes an aluminum-activated malate transporter. A series of transgenic analyses in apple materials including fruits, plantlets, and calli demonstrate that MdBT2 controls nitrate-mediated malate accumulation and vacuolar pH at least partially, if not completely, via regulating the MdCIbHLH1 protein level. Taken together, these findings reveal that MdBT2 regulates the stability of MdCIbHLH1 via ubiquitination in response to nitrate, which in succession transcriptionally reduces the expression of malate-associated genes, thereby controlling malate accumulation and vacuolar acidification in apples under high nitrate supply.

Integrated environment-occupant-pathogen information modeling to assess and communicate room-level outbreak risks of infectious diseases.

Microbial pathogen transmission within built environments is a main public health concern. The pandemic of coronavirus disease 2019 (COVID-19) adds to the urgency of developing effective means to reduce pathogen transmission in mass-gathering public buildings such as schools, hospitals, and airports. To inform occupants and guide facility managers to prevent and respond to infectious disease outbreaks, this study proposed a framework to assess room-level outbreak risks in buildings by modeling built environment characteristics, occupancy information, and pathogen transmission. Building information modeling (BIM) is exploited to automatically retrieve building parameters and possible occupant interactions that are relevant to pathogen transmission. The extracted information is fed into an environment pathogen transmission model to derive the basic reproduction numbers for different pathogens, which serve as proxies of outbreak potentials in rooms. A web-based system is developed to provide timely information regarding outbreak risks to occupants and facility managers. The efficacy of the proposed method was demonstrated by a case study, in which building characteristics, occupancy schedules, pathogen parameters, as well as hygiene and cleaning practices are considered for outbreak risk assessment. This study contributes to the body of knowledge by computationally integrating building, occupant, and pathogen information modeling for infectious disease outbreak assessment, and communicating actionable information for built environment management.