Efficacy and Safety of Lianhua Qingke Tablets in the Treatment of Long Coronavirus Disease (COVID) Cough: A Randomized, Double-Blind, Placebo-Controlled, Multicenter Clinical Study

Lianhua Qingke tablets,a patented traditional Chinese medicine that has validated clinical efficacy for treating cough caused by severe acute respiratory syndrome coronavirus 2 infection,lack rigorous evidence-based research evaluating their effect on long coronavirus disease(COVID)cough.A randomized,double-blind,placebo-controlled,multicenter clinical study was conducted among patients with long COVID cough from 19 hospitals and 23 community health centers in China.Patients were randomized 1:1 to receive either Lianhua Qingke tablets or placebo orally for 14 days(four tablets,1.84 g,three times a day).The primary endpoint indicator was the disappearance of cough,with the remission of cough also considered.Among 482 randomized patients,480(full analysis set 480;per-protocol set 470;safety set 480)were included in the primary analysis.According to the full analysis,the time until cough disappearance was significantly shorter in the trial group than in the control group,with a significant increase in the 14-day cough disappearance rate.Accordingly,the time to cough remission was significantly shorter in the trial group than in the control group.The change in the total symptom score was significantly greater in the trial group than in the control group on days 7 and 14,consistent with the results indicated by the visual analog scale(VAS)and cough evaluation test(CET)scores.No serious adverse events were recorded during the study.Lianhua Qingke tablets significantly improved the clinical symptoms of patients with long COVID cough.

Genetic variation in a heat shock transcription factor modulates cold tolerance in maize

Understanding how maize(Zea mays)responds to cold stress is crucial for facilitating breeding programs of cold-tolerant varieties.Despite extensive utilization of the genome-wide association study(GWAs)approach for exploring favorable natural alleles associated with maize cold tolerance,few studies have successfully identified candidate genes that contribute to maize cold tolerance.In this study,we used a diverse panel of inbred maize lines collected from different germplasm sources to perform a GWAS on var-iations in the relative injured area of maize true leaves during cold stress-a trait very closely correlated with maize cold tolerance.We identified HsF21,which encodes a B-class heat shock transcription factor(HSF)that positively regulates cold tolerance at both the seedling and germination stages.Natural varia-tions in the promoter of the cold-tolerant HSF21Hap1 allele led to increased HSF21 expression under cold stress by inhibiting binding of the basic leucine zipper bziP68 transcription factor,a negative regulator of cold tolerance.By integrating transcriptome deep sequencing,DNA affinity purification sequencing,and targeted lipidomic analysis,we revealed the function of HsF21 in regulating lipid metabolism homeo-stasis to modulate cold tolerance in maize.In addition,we found that HsF21 confers maize cold tolerance without incurring yield penalties.Collectively,this study establishes HsF21 as a key regulator that en-hances cold tolerance in maize,providing valuable genetic resources for breeding of cold-tolerant maize varieties.

Integrative regulatory mechanisms of stomatal movements under changing climate

Global climate change-caused drought stress,high temperatures and other extreme weather profoundly impact plant growth and development,restricting sustainable crop production.To cope with various environmental stimuli,plants can optimize the opening and closing of stomata to balance CO_(2)uptake for photosynthesis and water loss from leaves.Guard cells perceive and integrate various signals to adjust stomatal pores through turgor pressure regulation.Molecular mechanisms and signaling networks underlying the stomatal movements in response to environmental stresses have been extensively studied and elucidated.This review focuses on the molecular mechanisms of stomatal movements mediated by abscisic acid,light,CO_(2),reactive oxygen species,pathogens,temperature,and other phytohormones.We discussed the significance of elucidating the integrative mechanisms that regulate stomatal movements in helping design smart crops with enhanced water use efficiency and resilience in a climate-changing world.

Ca^(2+)-independent ZmCPK2 is inhibited by Ca^(2+)-dependent ZmCPK17 during drought response in maize

Calcium oscillations are induced by different stresses.Calcium-dependent protein kinases(CDPKs/CPKs)are one major group of the plant calcium decoders that are involved in various processes including drought response.Some CPKs are calcium-independent.Here,we identified ZmCPK2 as a negative regulator of drought resistance by screening an overexpression transgenic maize pool.We found that ZmCPK2 does not bind calcium,and its activity is mainly inhibited during short term abscisic acid(ABA)treatment,and dynamically changed in prolonged treatment.Interestingly,ZmCPK2 interacts with and is inhibited by calcium-dependent ZmCPK17,a positive regulator of drought resistance,which is activated by ABA.ZmCPK17 could prevent the nuclear localization of ZmCPK2 through phosphorylation of ZmCPK2T60.ZmCPK2 interacts with and phosphorylates and activates ZmYAB15,a negative transcriptional factor for drought resistance.Our results suggest that drought stress-induced Ca^(2+)can be decoded directly by ZmCPK17 that inhibits ZmCPK2,thereby promoting plant adaptation to water deficit.

Phosphorylation of ZmAL14 by ZmSnRK2.2 regulates drought resistance through derepressing ZmROP8 expression

Drought stress has negative effects on crop growth and production.Characterization of transcription factors that regulate the expression of drought-responsive genes is critical for understanding the transcriptional regulatory networks in response to drought,which facilitates the improvement of crop drought tolerance.Here,we identified an Alfin-like(AL)family gene ZmAL14 that negatively regulates drought resistance.Overexpression of ZmAL14 exhibits susceptibility to drought while mutation of ZmAL14 enhances drought resistance.An abscisic acid(ABA)-activated protein kinase ZmSnRK2.2 interacts and phosphorylates ZmAL14 at T38 residue.Knockout of ZmSnRK2.2 gene decreases drought resistance of maize.A dehydration-induced Rho-like small guanosine triphosphatase gene ZmROP8 is directly targeted and repressed by ZmAL14.Phosphorylation of ZmAL14 by ZmSnRK2.2 prevents its binding to the ZmROP8 promoter,thereby releasing the repression of ZmROP8 transcription.Overexpression of ZmROP8 stimulates peroxidase activity and reduces hydrogen peroxide accumulation after drought treatment.Collectively,our study indicates that ZmAL14 is a negative regulator of drought resistance,which can be phosphorylated by ZmSnRK2.2 through the ABA signaling pathway,thus preventing its suppression on ZmROP8 transcription during drought stress response.

Coronatine promotes maize water uptake by directly binding to the aquaporin ZmPIP2;5 and enhancing its activity

Water uptake is crucial for crop growth and development and drought stress tolerance. The water channel aquaporins(AQP) play important roles in plant water uptake. Here, we discovered that a jasmonic acid analog, coronatine(COR), enhanced maize(Zea mays) root water uptake capacity under artificial water deficiency conditions. COR treatment induced the expression of the AQP gene Plasma membrane intrinsic protein 2;5(ZmPIP2;5).In vivo and in vitro experiments indicated that COR also directly acts on ZmPIP2;5 to improve water uptake in maize and Xenopus oocytes. The leaf water potential and hydraulic conductivity of roots growing under hyperosmotic conditions were higher in ZmPIP2;5-overexpression lines and lower in the zmpip2;5 knockout mutant, compared to wild-type plants. Based on a comparison between ZmPIP2;5 and other PIP2s, we predicted that COR may bind to the functional site in loop E of ZmPIP2;5. We confirmed this prediction by surface plasmon resonance technology and a microscale thermophoresis assay, and showed that deleting the binding motif greatly reduced COR binding. We identified the N241 residue as the COR-specific binding site, which may activate the channel of the AQP tetramer and increase water transport activity,which may facilitate water uptake under hyperosmotic stress.

AtMCM10 promotes DNA replication-coupled nucleosome assembly in Arabidopsis

Minichromosome Maintenance protein 10(MCM10)is essential for DNA replication initiation and DNA elongation in yeasts and animals.Although the functions of MCM10 in DNA replication and repair have been well documented,the detailed mechanisms for MCM10 in these processes are not well known.Here,we identified AtMCM10 gene through a forward genetic screening for releasing a silenced marker gene.Although plant MCM10 possesses a similar crystal structure as animal MCM10,AtMCM10 is not essential for plant growth or development in Arabidopsis.AtMCM10 can directly bind to histone H3-H4 and promotes nucleosome assembly in vitro.The nucleosome density is decreased in Atmcm10,and most of the nucleosome density decreased regions in Atmcm10 are also regulated by newly synthesized histone chaperone Chromatin Assembly Factor-1(CAF-1).Loss of both AtMCM10 and CAF-1 is embryo lethal,indicating that AtM CM10 and CAF-1 are indispensable for replication-coupled nucleosome assembly.AtMCM10 interacts with both new and parental histones.Atmcm10 mutants have lower H3.1abundance and reduced H3K27me1/3 levels with releasing some silenced transposons.We propose that AtM CM10 deposits new and parental histones during nucleosome assembly,maintaining proper epigenetic modifications and genome stability during DNA replication.

The Antagonistic Action of Abscisic Acid and Cytokinin Signaling Mediates Drought Stress Response in Arabidopsis

As sessile organisms, plants encounter a variety of environmental stresses and must optimize their growth for survival. Abscisic acid （ABA） and cytokinin antagonistically regulate many developmental processes and environmental stress responses in plants. However, the molecular mechanism underlying this antag- onism remains poorly defined. In this study, we demonstrated that Sucrose nonfermentingl-related kinases SnRK2.2, SnRK2.3, and SnRK2.6, the key kinases of the ABA signaling pathway, directly interact with and phosphorylate type-A response regulator 5 （ARR5）, a negative regulator of cytokinin signaling. The phosphorylation of ARR5 Ser residues by SnRK2s enhanced ARR5 protein stability. Accord- ingly, plants overexpressing ARR5 showed ABA hypersensitivity and drought tolerance, and these pheno- types could not be recapitulated by overexpressing a non-phosphorylated ARR5 mimic. Moreover, the type-B ARRs, ARR1, ARR11 and ARR12, physically interacted with SnRK2s and repressed the kinase activ- ity of SnRK2.6. The arrl, 11,12 triple mutant exhibited hypersensitivity to ABA. Genetic analysis demon- strated that SnRK2s act upstream of ARR5 but downstream of ARR1, ARR11 and ARR12 in mediating ABA response and drought tolerance. Taken together, this study unravels the antagonistic actions of several molecular components of the ABA and cytokinin signaling pathways in mediates drought stress response, providing significant insights into how plants coordinate growth and drought stress response by integrating multiple hormone pathways.

Plant abiotic stress response and nutrient use efficiency

Abiotic stresses and soil nutrient limitations are major environmental conditions that reduce plant growth,productivity and quality.Plants have evolved mechanisms to perceive these environmental challenges,transmit the stress signals within cells as well as between cells and tissues,and make appropriate adjustments in their growth and development in order to survive and reproduce.In recent years,significant progress has been made on many fronts of the stress signaling research,particularly in understanding the downstream signaling events that culminate at the activation of stress-and nutrient limitation-responsive genes,cellular ion homeostasis,and growth adjustment.However,the revelation of the early events of stress signaling,particularly the identification of primary stress sensors,still lags behind.In this review,we summarize recent work on the genetic and molecular mechanisms of plant abiotic stress and nutrient limitation sensing and signaling and discuss new directions for future studies.

Protein kinases in plant responses to drought, salt,and cold stress

Protein kinases are major players in various signal transduction pathways. Understanding the molecular mechanisms behind plant responses to biotic and abiotic stresses has become critical for developing and breeding climate-resilient crops. In this review,we summarize recent progress on understanding plant drought, salt, and cold stress responses, with a focus on signal perception and transduction by different protein kinases, especially sucrose nonfermenting1(SNF1)-related protein kinases(Sn RKs),mitogen-activated protein kinase(MAPK) cascades,calcium-dependent protein kinases(CDPKs/CPKs),and receptor-like kinases(RLKs). We also discuss future challenges in these research fields.

Abscisic Acid-mediated Epigenetic Processes in Plant Development and Stress Responses

Abscisic acid （ABA） regulates diverse plant processes, growth and development under non-stress conditions and plays a pivotal role in abiotic stress tolerance. Although ABA-regulated genetic processes are well known, recent discoveries reveal that epigenetic processes are an integral part of ABA-regulated processes. Epigenetic mechanisms, namely, histone modifications and cytosine DNA methylation-induced modification of genome give rise to epigenomes, which add diversity and complexity to the genome of organisms. Histone monoubiquitination appears to regulate ABA levels in developing seeds through histone H2B monoubiquitination. ABA and H2B ubiquitination dependent chromatin remodeling regulate seed dormancy. Transcription factor networks necessary for seed maturation are repressed by histone deacetylases （HDACs）-dependent and PICKLE chromatin remodeling complexes （CRCs）, whereas ABA induces the expression of these genes directly or through repression of HDACs. Abiotic stress-induced ABA regulates stomatal response and stress- responsive gene expression through HDACs and HOS15-dependent histone deacetylation, as well as through the ATP- dependent SWITCH/SUCROSE NONFERMENTING CRC. ABA also probably regulates the abiotic stress response through DNA methylation and short interfering RNA pathways. Further studies on ABA-regulated epigenome will be of immense use to understand the plant development, stress adaptation and stress memory.

Reactive oxygen species signaling and stomatal movement in plant responses to drought stress and pathogen attack

Stomata, the pores formed by a pair of guard cells, are the main gateways for water transpiration and photosynthetic CO2 exchange, as well as pathogen invasion in land plants. Guard cell movement is regulated by a combination of environmental factors, including water status, light, CO2 levels and pathogen attack, as well as endogenous signals, such as abscisic acid and apoplastic reactive oxygen species （ROS）. Under abiotic and biotic stress conditions, extracellular ROS are mainly produced by plasma membrane-localized NADPH oxidases, whereas intracellular ROS are produced in multiple organelles. These ROS form a sophisticated cellular signaling network, with the accumulation of apoplastic ROS an early hallmark of stomatal movement. Here, we review recent progress in understanding the molecular mechanisms of the ROS signaling network, primarily during drought stress and pathogen attack. We summarize the roles of apoplastic ROS in regulating stomatal movement, ABA and CO2 signaling, and immunity responses. Finally, we discuss ROS accumulation and communication between organelles and cells. This information provides a conceptual framework for understanding how ROS signaling is integrated with various signaling pathways during plant responses to abiotic and biotic stress stimuli.

Cold-Induced CBF–PIF3 Interaction Enhances Freezing Tolerance by Stabilizing the phyB Thermosensor in Arabidopsis

Growth inhibition and cold-acclimation strategies help plants withstand cold stress,which adversely affects growth and survival.PHYTOCHROME B(phyB)regulates plant growth through perceiving both light and ambient temperature signals.However,the mechanism by which phyB mediates the plant response to cold stress remains elusive.Here,we show that the key transcription factors mediating cold acclimation,C-REPEAT BINDING FACTORs(CBFs),interact with PHYTOCHROME-INTERACTING FACTOR 3(PIF3)under cold stress,thus attenuating the mutually assured destruction of PIF3–phyB.Cold-stabilized phyB acts downstream of CBFs to positively regulate freezing tolerance by modulating the expression of stress-responsive and growth-related genes.Consistent with this,phyB mutants exhibited a freezing-sensitive phenotype,whereas phyB-overexpression transgenic plants displayed enhanced freezing tolerance.Further analysis showed that the PIF1,PIF4,and PIF5 proteins,all of which negatively regulate plant freezing tolerance,were destabilized by cold stress in a phytochrome-dependent manner.Collectively,our study reveals that CBFs–PIF3–phyB serves as an important regulatory module for modulating plant response to cold stress.

Arabidopsis U-box E3 ubiquitin ligase PUB11 negatively regulates drought tolerance by degrading the receptor-like protein kinases LRR1 and KIN7

Both plant receptor-like protein kinases(RLKs)and ubiquitin-mediated proteolysis play crucial roles in plant responses to drought stress.However,the mechanism by which E3 ubiquitin ligases modulate RLKs is poorly understood.In this study,we showed that Arabidopsis PLANT U-BOX PROTEIN 11(PUB11),an E3 ubiquitin ligase,negatively regulates abscisic acid(ABA)-mediated drought responses.PUB11 interacts with and ubiquitinates two receptor-like protein kinases,LEUCINE RICH REPEAT PROTEIN 1(LRR1)and KINASE 7(KIN7),and mediates their degradation during plant responses to drought stress in vitro and in vivo.pub11 mutants were more tolerant,whereas Irr1 and kin7 mutants were more sensitive,to drought stress than the wild type.Genetic analyses show that the pub11 Irr1 kin7 triple mutant exhibited similar drought sensitivity as the Irr1 kin7 double mutant,placing PUB11 upstream of the two RLKs.Abscisic acid and drought treatment promoted the accumulation of PUB11,which likely accelerates LRR1 and KIN7 degradation.Together,our results reveal that PUB11 negatively regulates plant responses to drought stress by destabilizing the LRR1 and KIN7 RLKs.

Natural variations of ZmSRO1d modulate the trade-off between drought resistance and yield by affecting ZmRBOHC-mediated stomatal ROS production in maize

While crop yields have historically increased,drought resistance has become a major concern in the context of global climate change.The trade-off between crop yield and drought resistance is a common phenomenon;however,the underlying molecular modulators remain undetermined.Through genome-wide association study,we revealed that three non-synonymous variants in a drought-resistant allele of ZmSRO1d-R resulted in plasma membrane localization and enhanced mono-ADP-ribosyltransferase activity of ZmSRO1d toward ZmRBOHC,which increased reactive oxygen species(ROS)levels in guard cells and promoted stomatal closure.ZmSRO1d-R enhanced plant drought resilience and protected grain yields under drought conditions,but it led to yield drag under favorable conditions.In contrast,loss-of-function mutants of ZmRBOHC showed remarkably increased yields under well-watered conditions,whereas they showed compromised drought resistance.Interestingly,by analyzing 189 teosinte accessions,we found that the ZmSRO1d-R allele was present in teosinte but was selected against during maize domestication and modern breeding.Collectively,our work suggests that the allele frequency reduction of ZmSRO1d-R in breeding programs may have compromised maize drought resistance while increased yields.Therefore,introduction of the ZmSRO1d-R allele into modern maize cultivars would contribute to food security under drought stress caused by global climate change.

The direct targets of CBFs:In cold stress response and beyond

Cold acclimation in Arabidopsis thaliana triggers a significant transcriptional reprogramming altering the expression patterns of thousands of cold-responsive(COR) genes. Essential to this process is the C-repeat binding factor(CBF)-dependent pathway, involving the activity of AP2/ERF(APETALA2/ethylene-responsive factor)-type CBF transcription factors required for plant cold acclimation. In this study, we performed chromatin immunoprecipitation assays followed by deep sequencing(ChIP-seq) to determine the genomewide binding sites of the CBF transcription factors. Cold-induced CBF proteins specifically bind to the conserved C-repeat(CRT)/dehydrationresponsive elements(CRT/DRE;G/ACCGAC) of their target genes. A Gene Ontology enrichment analysis showed that 1,012 genes are targeted by all three CBFs. Combined with a transcriptional analysis of the cbf1,2,3 triple mutant, we define 146 CBF regulons as direct CBF targets. In addition, the CBF-target genes are significantly enriched in functions associated with hormone, light,and circadian rhythm signaling, suggesting that the CBFs act as key integrators of endogenous and external environmental cues. Our findings not only define the genome-wide binding patterns of the CBFs during the early cold response, but also provide insights into the role of the CBFs in regulating multiple biological processes of plants.

Tonoplast-associated calcium signaling regulates manganese homeostasis in Arabidopsis

Manganese(Mn)is an essential micronutrient in plants.However,excessive Mn absorption in acidic and waterlogged soils can lead to Mn toxicity.Despite their essential roles in Mn homeostasis,transcriptional and post-transcriptional modifications of Mn transporters remain poorly understood.Here,we demonstrated that high-Mn stress induces an obvious Ca^(2+) signature in Arabidopsis.We identified four calcium-dependent protein kinases,CPK4/5/6/11,that interact with the tonoplast-localized Mn and iron(Fe)transporter MTP8 in vitro and in vivo.The cpk4/5/6/11 quadruple mutant displayed a dramatic high-Mn-sensitive phenotype similar to that of the mtp8 mutant.CPKs phosphorylated the N-terminal domain of MTP8 primarily at the Ser31 and Ser32 residues.Transport assays combined with multiple physiological experiments on phospho-dead variant MTP8^(S31/32A)and phospho-mimetic variant MTP8^(S31/32D)plants under different Mn and Fe conditions suggested that Ser31 and Ser32 are crucial for MTP8 function.In addition,genetic analysis showed that CPKs functioned upstream of MTP8.In summary,we identified a tonoplast-associated calcium signaling cascade that orchestrates Mn homeostasis and links Mn toxicity,Ca^(2+) signaling,and Mn transporters.These findings provide new insight into Mn homeostasis mechanisms and Ca^(2+) signaling pathways in plants,providing potential targets for engineering heavy metal toxicity-tolerant plants.

Rheostatic Control of ABA Signaling through HOS15-Mediated OST1 Degradation

Dehydrating stresses trigger the accumulation of abscisic acid(ABA),a key plant stress-signaling hormone that activates Snf1-Related Kinases(SnRK2s)to mount adaptive responses.However,the regulatory circuits that terminate the SnRK2s signal relay after acclimation or post-stress conditions remain to be defined.Here,we show that the desensitization of the ABA signal is achieved by the regulation of OST1(SnRK2.6)protein stability via the E3-ubiquitin ligase HOS15.Upon ABA signal,HOS15-induced degradation of OST1 is inhibited and stabilized OST1 promotes the stress response.When the ABA signal terminates,protein phosphatases ABI1/2 promote rapid degradation of OST1 via HOS15.Notably,we found that even in the presence of ABA,OST1 levels are also depleted within hours of ABA signal onset.The unexpected dynamics of OST1 abundance are then resolved by systematic mathematical modeling,demonstrating a desensitizing feedback loop by which OST1-induced upregulation of ABI1/2 leads to the degradation of OST1.This model illustrates the complex rheostat dynamics underlying the ABA-induced stress response and desensitization.

Flowering phenology as a core domestication trait in soybean

Flowering time variation in soybean is well characterized within domesticated germplasms and is critical for modern production,but its importance during domestication is unclear.Recently,Lu et al.(Nature Genetics,2020)reported that two homeologous pseudo-response-regulator genes,Tof12 and Tof11,were sequentially selected in early soybean evolution for ancient flowering time adaptation and intensifcation of crop cultivation.

PHYTOCHROME-INTERACTING FACTORS Interact with the ABA Receptors PYL8 and PYL9 to Orchestrate ABA Signaling in Darkness

PHYTOCHROME-INTERACTING FACTORS(PIFs)are a group of basic helix-loop-helix transcription factors that can physically interact with photoreceptors,including phytochromes and cryptochromes.It was previously demonstrated that PIFs accumulated in darkness and repressed seedling photomorphogenesis,and that PIFs linked different photosensory and hormonal pathways to control plant growth and development.In this study,we show that PIFs positively regulate the ABA signaling pathway during the seedling stage specifically in darkness.We found that PIFs positively regulate ABI5 transcript and protein levels in darkness in response to exogenous ABA treatment by binding directly to the G-box motifs in the ABI5 promoter.Consistently,PIFs and the G-box motifs in the ABI5 promoter determine ABI5 expression in darkness,and overexpression of ABI5 could rescue the ABA-insensitive phenotypes of pifq mutants in the dark.Moreover,we discovered that PIFs can physically interact with the ABA receptors PYL8 and PYL9,and that this interaction is not regulated by ABA.Further analyses showed that PYL8 and PYL9 promote PIF4 protein accumulation in the dark and enhance PIF4 binding to the ABI5 promoter,but negatively regulate PIF4-mediated ABI5 activation.Taken together,our data demonstrate that PIFs interact with ABA receptors to orchestrate ABA signaling in darkness by controlling ABI5 expression,providing new insights into the pivotal roles of PIFs as signal integrators in regulating plant growth and development.