Genetic control of compound leaf development in the mungbean(Vigna radiata L.)

Many studies suggest that there are distinct regulatory processes controlling compound leaf development in different clades of legumes.Loss of function of the LEAFY(LFY)orthologs results in a reduction of leaf complexity to different degrees in inverted repeat-lacking clade(IRLC)and non-IRLC species.To further understand the role of LFY orthologs and the molecular mechanism in compound leaf development in non-IRLC plants,we studied leaf development in unifoliate leaf(un)mutant,a classical mutant of mungbean(Vigna radiata L.),which showed a complete conversion of compound leaves into simple leaves.Our analysis revealed that UN encoded the mungbean LFY ortholog(VrLFY)and played a significant role in leaf development.In situ RNA hybridization results showed that STM-like KNOXI genes were expressed in compound leaf primordia in mungbean.Furthermore,increased leaflet number in heptafoliate leaflets1(hel1)mutants was demonstrated to depend on the function of VrLFY and KNOXI genes in mungbean.Our results suggested that HEL1 is a key factor coordinating distinct processes in the control of compound leaf development in mungbean and its related non-IRLC legumes.

The soft glumes of common wheat are sterile-lemmas as determined by the domestication gene Q

The Q gene in common wheat encodes an APETALA2(AP2) transcription factor that causes the free threshing attribute. Wheat spikelets bearing several florets are subtended by a pair of soft glumes that allow free liberation of seeds. In wild species, the glumes are tough and rigid,making threshing difficult. However, the nature of these "soft glumes", caused by the domestication allele Q is not clear. Here, we found that over expression of Q in common wheat leads to homeotic florets at glume positions. We provide phenotypic, microscopy, and marker genes evidence to demonstrate that the soft glumes of common wheat are in fact lemma-like organs, or so-called sterile-lemmas. By comparing the structures subtending spikelets in wheat and other crops such as rice and maize, we found that AP2 genes may play conserved functions in grasses by manipulating vestigial structures, such as floret-derived soft glumes in wheat and empty glumes in rice. Conversion of these seemingly vegetative organs to reproductive organs may be useful in yield improvement of crop species.

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.

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.

Single-Particle Tracking for the Quantification of Membrane Protein Dynamics in Living Plant Cells

The plasma membrane is a sophisticated,organized,and highly heterogeneous structure that compartmentalizes cellular processes.To decipher the biological processes involving membrane proteins,it is necessary to analyze their spatiotemporal dynamics.However,it is difficult to directly assess the dynamics and interactions of biomolecules in living cells using traditional biochemical methods.Singleparticle tracking (SPT)methods for imaging and tracking single particles conjugated with fluorescent probes offer an ideal approach to acquire valuable and complementary information about dynamic intracellular processes.SPT can be used to quantitatively monitor the diverse motions of individual particles in living cells.SPT also provides super-spatiotemporal resolution that allows early-stage or rapid response information to be obtained for a better understanding of molecular basis of associated signal transduction processes.More importantly,SPT can be used to detect the motion paths of individual biomolecules in vivo and in situ,thus unveiling the dynamic behavior of the biomolecules that support developmental processes in living cells.In this review,we give an overview of SPT methods,from image acquisition to the detection of single particles,as well as tracking and data analysis.We also discuss recent applications of SPT methods in the field ofplant biology to reveal the complex biological functions of membrane proteins.

MiR156 regulates anthocyanin biosynthesis through SPL targets and other microRNAs in poplar

Anthocyanins biosynthesized from the flavonoid pathway are types of pigments that are involved in the protection of poplar from biotic and abiotic stresses.Previous researchers studying anthocyanin-related transcription factors and structural genes in poplar have made significant discoveries.However,little is known about the regulatory role of microRNAs in anthocyanin biosynthesis in poplar.Here,we overexpressed miR156 in poplar to study the comprehensive effects of the miR156-SPL module on the biosynthesis of anthocyanins.Small RNA sequencing analysis revealed 228 microRNAs differentially expressed in transgenic poplar plants with dramatically increased miR156 levels.Furthermore,integrated microRNAomic and transcriptomic analysis suggested that two microRNAs,miR160h,and miR858,have the potential to affect anthocyanin accumulation in poplar by regulating auxin response factors and MYB transcription factors,respectively.Additionally,the accumulation of miR160h and miR858 displayed a positive correlation with miR156 levels,suggesting a possible interaction between the miR156-SPL module and these microRNAs in poplar.Last,metabolomics analysis revealed that the levels of anthocyanins,flavones,and flavonols were substantially elevated in transgenic poplar plants overexpressing miR156 compared with the wild type,whereas the total lignin content was reduced in the transgenic plants.Taken together,our results indicate that miR156 can fine tune the anthocyanin biosynthetic pathway via multiple factors,including microRNAs,transcription factors,and the levels of structural genes,in poplar.This provides additional clues for understanding the complex regulatory network of anthocyanin biosynthesis in woody plants.

RAF22,ABI1 and OST1 form a dynamic interactive network that optimizes plant growth and responses to drought stress in Arabidopsis

Plants adapt to their ever-changing environment via positive and negative signals induced by environmental stimuli.Drought stress,for instance,induces accumulation of the plant hormone abscisic acid(ABA),triggering ABA signal transduction.However,the molecular mechanisms for switching between plant growth promotion and stress response remain poorly understood.Here we report that RAF(rapidly accelerated fibrosarcoma)-LIKE MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 22(RAF22)in Arabidopsis tha/iana physically interacts with ABA INSENSITIVE 1(ABl1)and phosphorylates ABl1 at Ser416 residue to enhance its phosphatase activity.Interestingly,ABl1 can also enhance the activity of RAF22 through dephosphorylation,reciprocally inhibiting ABA signaling and promoting the maintenance of plant growth under normal conditions.Under drought stress,however,the ASA-activated OPEN STOMATA1(OST1)phosphorylates the Ser81 residue of RAF22 and inhibits its kinase activity,restraining its enhancement of ABl1 activity.Taken together,our study reveals that RAF22,ABl1,and OST1 form a dynamic regulatory network that plays crucial roles in optimizing plant growth and environmental adaptation under drought stress.

Future challenges in understanding ROS in plant responses to abiotic stress

Plants must cope with a variety of environmental stresses.Most types of abiotic stresses,such as drought,salinity,flooding,heat and cold stress,disrupt the metabolic balance of cells,resulting in the enhanced production of reactive oxygen species(ROS).While being well-known as a toxic by-product,recent studies about ROS focus on their roles as signaling molecules.It has been reported that ROS functions in plant cell proliferation and cell expansion,root

Phosphorylation of the LCB1 subunit of Arabidopsis serine palmitoyltransferase stimulates its activity and modulates sphingolipid biosynthesis

Sphingolipids are the structural components of membrane lipid bilayers and act as signaling molecules in many cellular processes.Serine palmitoyltransferase(SPT)is the first committed and rate-limiting enzyme in the de novo sphingolipids biosynthetic pathway.The core SPT enzyme is a heterodimer consisting of LONG-CHAIN BASE1(LCB1)and LCB2 subunits.SPT activity is inhibited by orosomucoid proteins and stimulated by small subunits of SPT(ssSPTs).However,whether LCB1 is modified and how such modification might regulate SPT activity have to date been unclear.Here,we show that activation of MITOGEN-ACTIVATED PROTEIN KINASE 3(MPK3)and MPK6 by upstream MKK9 and treatment with Flg22(a pathogen-associated molecular pattern)increases SPT activity and induces the accumulation of sphingosine long-chain base t18:0 in Arabidopsis thaliana,with activated MPK3and MPK6 phosphorylating AtLCB1.Phosphorylation of AtLCB1 strengthened its binding with AtLCB2b,promoted its binding with ssSPTs,and stimulated the formation of higher order oligomeric and active SPT complexes.Our findings therefore suggest a novel regulatory mechanism for SPT activity.

The Jasmine(Jasminum sambac)Genome Provides Insight into the Biosynthesis of Flower Fragrances and Jasmonates

Jasminum sambac(jasmine flower),a world-renowned plant appreciated for its exceptional flower fragrance,is of cultural and economic importance.However,the genetic basis of its fragrance is largely unknown.Here,we present the first de novo genome assembly of J.sambac with 550.12 Mb(scaffold N50=40.10 Mb)assembled into 13 pseudochromosomes.Terpene synthase(TPS)genes associated with flower fragrance are considerably amplified in the form of gene clusters through tandem duplications in the genome.Gene clusters within the salicylic acid/benzoic acid/theobromine(SABATH)and benzylalcohol O-acetyltransferase/anthocyanin O-hydroxycinnamoyltransferases/anthranilate N-hydroxycinnamoyl/benzoyltransferase/deacetylvindoline 4-O-acetyltransferase(BAHD)superfamilies were identified to be related to the biosynthesis of phenylpropanoid/benzenoid compounds.Several key genes involved in jasmonate biosynthesis were duplicated,causing an increase in copy numbers.In addition,multi-omics analyses identified various aromatic compounds and many genes involved in fragrance biosynthesis pathways.Furthermore,the roles of JsTPS3 in b-ocimene biosynthesis,as well as JsAOC1 and JsAOS in jasmonic acid biosynthesis,were functionally validated.The genome assembled in this study for J.sambac offers a basic genetic resource for studying floral scent and jasmonate biosynthesis,and provides a foundation for functional genomic research and variety improvements in Jasminum.

Modulation of Guard Cell Turgor and Drought Folerance by a Peroxisomal Acetate-Malate Shunt

In plants, stomatal movements are tightly controlled by changes in cellular turgor pressure. Carbohydrates produced by glycolysis and the tricarboxylic acid cycle play an important role in regulating turgor pressure. Here, we describe anArabidopsis mutant, bzul, isolated in a screen for elevated leaf temperature in response to drought stress, which displays smaller stomatal pores and higher drought resistance than wild-type plants. BZU1 encodes a known acetyl-coenzyme A synthetase, ACN1, which acts in the first step of a metabolic pathway converting acetate to malate in peroxisomes. We showed that BZUl/ACNl-mediated acetate-to-malate conversion provides a shunt that plays an important role in osmoregulation of stomatal turgor. We found that the smaller stomatal pores in the bzul mutant are a consequence of reduced accumu- lation of malate, which acts as an osmoticum and/or a signaling molecule in the control of turgor pressure within guard cells, and these results provided new genetic evidence for malate-regulated stomatal movement. Collectively, our results indicate that a peroxisomal BZUl/ACNl-mediated acetate--malate shunt regulates drought resistance by controlling the turgor pressure of guard cells in Arabidopsis.

Arabidopsis calcium-dependent protein kinase CPK28 is potentially involved in the response to osmotic stress

As calcium sensors, plant calcium-dependent protein kinases(CDPKs) play important roles in plants' responses to various abiotic stresses. Here, we report the functional characterization of CPK28, a member of the CDPK family in Arabidopsis, in response to osmotic stress.The cpk28 mutant, a loss-of-function mutant, exhibited an NaCl- and mannitol-sensitive phenotype in green cotyledons, while CPK28-overexpressing plants displayed stronger tolerance to NaCl and mannitol stresses than wildtype plants. Reverse transcriptase-polymerase chain reaction and beta-glucuronidase staining assays showed that NaCl and mannitol stresses induced CPK28. CPK28-overexpressing lines accumulated significantly more proline relative to wild-type plants and mutant plants under NaCl and mannitol stresses. Transient expression of CPK28-GFP in mesophyll cell protoplasts, as well as stable transgenic lines expressing CPK28-GFP, showed that CPK28 was localized in the plasma membrane. Expression levels of known stress-responsive genes were not significantly altered in the null mutant and overexpression lines,suggesting that CPK28 possibly mediated the stress response via the regulation of target proteins rather than via regulation at the level of transcription. Meanwhile, CPK28could autophosphorylate. Taken together, these data demonstrated that CPK28, a potential positive regulator, is involved in the response to osmotic stress in Arabidopsis.

A LysM Receptor Heteromer Mediates Perception of Arbuscular Mycorrhizal Symbiotic Signal in Rice

Symbiotic microorganisms improve nutrient uptake by plants.To initiate mutualistic symbiosis with arbus-cular mycorrhizal(AM)fungi,plants perceive Myc factors,including lipochitooligosaccharides(LCOs)and short-chain chitooligosaccharides(CO4/CO5),secreted by AM fungi.However,the molecular mechanism of Myc factor perception remains elusive.In this study,we identified a heteromer of LysM receptor-like kinases consisting of OsMYR1/OsLYK2 and OsCERK1 that mediates the perception of AM fungi in rice.CO4 directly binds to OsMYR1,promoting the dimerization and phosphorylation of this receptor complex.Compared with control plants,Osmyr1 and Oscerk1 mutant rice plants are less sensitive to Myc factors and show decreased AM colonization.We engineered transgenic rice by expressing chimeric receptors that respectively replaced the ectodomains of OsMYR1 and OsCERK1 with those from the homologous Nod factor receptors MtNFP and MtL YK3 of Medicago truncatula.Transgenic plants displayed increased cal-cium oscillations in response to Nod factors compared with control rice.Our study provides significant mechanistic insights into AM symbiotic signal perception in rice.Expression of chimeric Nod/Myc recep-tors achieves a potentially important step toward generating cereals that host nitrogen-fixing bacteria.

Medicago AP2-Domain Transcription Factor WRI5a Is a Master Regulator of Lipid Biosynthesis and Transfer during Mycorrhizal Symbiosis

Most land plants have evolved a mutualistic symbiosis with arbuscular mycorrhiza (AM)fungi that improve nutrient acquisition from the soil.In return,up to 20% of host plant photosynthate is transferred to the mycorrhizal fungus in the form of lipids and sugar.Nutrient exchange must be regulated by both partners in order to maintain a reliable symbiotic relationship.However,the mechanisms underlying the regulation of lipid transfer from the plantto the AM fungus remain elusive.Here,we show that the Medicago truncatula AP2/EREBP transcription factor WRI5a,and likely its two homologs WRI5b/Erfl and WRI5c,are master regulators of AM symbiosis Controlling lipid transfer and periarbuscular membrane formation.We found that WRI5a binds AW-box cis-regulatory elements in the promoters of M.truncatula STR,which encodes a periarbuscular membrane-localized ABC transporter required for lipid transfer from the plant to the AM fungus, and MtPT4,whichr encodes a phosphate transporter required for phosphate transfer from the AM fungus to the plant.The hairy roots of the M.truncatula wti5a mutant and RNAi composite plants displayed impaired arbuscule formation,whereas overexpression of WRI5a resulted in enhanced expression of STR and MtPT4,suggesting that WRI5a regulates bidirectional symbiotic nutrient exchange.Moreover,we found that WRI5a and RAM1(Required for Arbuscular Mycorrhization symbiosis 1),which encodes a GRASdomain transcription factor,regulate each other at the transcriptional level,forming a positive feedback loop for regulatingAM symbiosis.Collectively,our data suggest a role for WRI5a in controlling bidirectional nutrient exchange and periarbuscular membrane formation via the regulation of genes involved in the biosynthesis of fatty acids and phosphate uptake in arbuscule-containing cells.

Phot2-regulated relocation of NPH_3 mediates phototropic response to high-intensity blue light in Arabidopsis thaliana

Two redundant blue-light receptors, known as phototropins （phot1 and phot2）, influence a variety of physiological responses, including phototropism, chloroplast positioning, and stomatal opening in Arabidopsis thaliana. Whereas photl functions in both low- and high-intensity blue light （HBL）, phot2 functions primarily in HBL. Here, we aimed to elucidate phot2-specific functions by screening for HBL- insensitive mutants among mutagenized Arabidopsis photl mutants. One of the resulting phot2 signaling associated （p2sa） double mutants, phot1 p2sa2, exhibited phototropic defects that could be restored by constitutively expressing NON-PHOTOTROPIC HYPOCOTYL 3 （NPH3）, indicating that P2SA2 was allelic to NPH3. It was observed that NPH3-GFP signal mainly localized to and clustered on the plasma membrane in darkness. This NPH3 clustering on the plasmamembrane was not affected by mutations in genes encoding proteins that interact with N PH3, including PHOT11, PHOT2 and ROOTPHOTOTROPISM 2 （RPT2）. However, the HBL irradiation- mediated release of NPH3 proteins into the cytoplasm was inhibited in photl mutants and enhanced in phot2 and rpt2-2 mutants. Furthermore, HBL-induced hypocotyl phototropism was enhanced in photl mutants and inhibited in the phot2 and rpt2-2 mutants. Our findings indicate that photl regulates the dissociation of NPH3 from the plasma membrane, whereas phot2 mediates the stabilization and relocation of NPH3 to the plasma membrane to acclimate to HBL.

Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS

Dear Editor,Prime editing is a novel and universal CRISPR-Cas-derived precise genome-editing technology and has great potentials for applications in basic plant research and crop molecular breeding(Anzalone et al.,2019).Although low efficiency has restrained the original prime editors(PEs)from being used as a routine tool for precise genome editing in plants,an iterative update of the PEs is removing this obstacle(Lin et al.,2021;Xu et al.,2022).Recently,the Liu group reported three optimization strategies for improving prime editing efficiency(Chen et al.,2021;Nelson et al.,2022).The first strategy is based on engineered prime editing guide RNAs(epegRNAs),which were generated by incorporating structured RNA motifs to the 3′terminus of pegRNAs.This strategy enhances pegRNA stability and prevents degradation of the 3′extension(Nelson et al.,2022).The second strategy is based on the optimized PE2 protein(PEmax),which harbors a SpCas9 variant with increased nuclease activity,an additional nuclear localization signal(NLS)sequences,and a new linker between nCas9 and reverse transcriptase(Chen et al.,2021).The third strategy is based on inhibition of DNA mismatch repair(MMR)in cells(Chen et al.,2021).In this work,we tested the optimized PEs generated with these three strategies in rice,demonstrating that the optimized PEs greatly improved prime editing efficiency in rice.We named the two optimized PEs ePE3max and ePE5max:the former is comprised of the PEmax protein,an epegRNA with evopreQ1 appended to its 3′end,and a nicking sgRNA;the latter is comprised of the ePE3max system and a dominant negative OsMLH1 variant for inhibiting MMR.Using the two optimized PEs,we efficiently generated homozygous and heterozygous T173I,A174V,and P177S(TAP-IVS)mutation in EPSPS in rice,which lays a solid foundation for rice non-transgenic glyphosate-resistance breeding.

Chromosome-level genome assembly of the diploid blueberry Vaccinium darrowii provides insights into its subtropical adaptation and cuticle synthesis

Vaccinium darrowii is a subtropical wild blueberry species that has been used to breed economically important southern highbush cultivars.The adaptive traits of V.darrowii to subtropical climates can provide valuable information for breeding blueberry and perhaps other plants,especially against the background of global warming.Here,we assembled the V.darrowii genome into 12 pseudochromosomes using Oxford Nanopore long reads complemented with Hi-C scaffolding technologies,and we predicted 41815 genes using RNA-sequencing evidence.Syntenic analysis across three Vaccinium species revealed a highly conserved genome structure,with the highest collinearity between V.darrowii and Vaccinium corymbosum.This conserved genome structure may explain the high fertility observed during crossbreeding of V.darrowii with other blueberry cultivars.Analysis of gene expansion and tandem duplication indicated possible roles for defense-and flowering-associated genes in the adaptation of V.darrowii to the subtropics.Putative SOC1 genes in V.darrowii were identified based on phylogeny and expression analysis.Blueberries are covered in a thick cuticle layer and contain anthocyanins,which confer their powdery blue color.Using RNA sequencing,we delineated the cuticle biosynthesis pathways of Vaccinium species in V.darrowii.This result can serve as a reference for breeding berries whose colors are appealing to customers.The V.darrowii reference genome,together with the unique traits of this species,including its diploid genome,short vegetative phase,and high compatibility in hybridization with other blueberries,make V.darrowii a potential research model for blueberry species.

Biological function analysis of the phosphorylation sites for Arabidopsis CAP1

Cells need to respond successfully to ever-changing environmental conditions to maintain normal growth.This is achieved through various signal transduction cascades.Receptor-like kinases(RLKs)are involved in many aspects of the growth and development of plants.More than 600 RLKs have been identified and that are involved in various biological processes in

Na_(2)CO_(3)-responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by Phosphoproteomics

Alkali-salinity exerts severe osmotic,ionic,and high-p H stresses to plants.To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species(ROS)homeostasis,physiological and diverse quantitative proteomics analyses of alkaligrass(Puccinellia tenuiflora)under Na_(2)CO_(3)stress were conducted.In addition,Western blot,real-time PCR,and transgenic techniques were applied to validate the proteomic results and test the functions of the Na_(2)CO_(3)-responsive proteins.A total of 104 and 102 Na_(2)CO_(3)-responsive proteins were identified in leaves and chloroplasts,respectively.In addition,84 Na_(2)CO_(3)-responsive phosphoproteins were identified,including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts,which are crucial for the regulation of photosynthesis,ion transport,signal transduction,and energy homeostasis.A full-length Pt FBA encoding an alkaligrass chloroplastic fructosebisphosphate aldolase(FBA)was overexpressed in wild-type cells of cyanobacterium Synechocystis sp.Strain PCC 6803,leading to enhanced Na_(2)CO_(3)tolerance.All these results indicate that thermal dissipation,state transition,cyclic electron transport,photorespiration,repair of photosystem(PS)Ⅱ,PSI activity,and ROS homeostasis were altered in response to Na_(2)CO_(3)stress,which help to improve our understanding of the Na_(2)CO_(3)-responsive mechanisms in halophytes.

Optimized prime editing efficiently generates heritable mutations in maize

Low efficiency is the main obstacle to using prime editing in maize(Zea mays).Recently,prime-editing efficiency was greatly improved in mammalian cells and rice(Oryza sativa)plants by engineering primeediting guide RNAs(pegRNAs),optimizing the prime editor(PE)protein,and manipulating cellular determinants of prime editing.In this study,we tested PEs optimized via these three strategies in maize.We demonstrated that the ePE5max system,composed of PEmax,epegRNAs(pegRNA-evopreQ.1),nicking single guide RNAs(sgRNAs),and MLH1dn,efficiently generated heritable mutations that conferred resistance to herbicides that inhibit 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS),acetolactate synthase(ALS),or acetyl CoA carboxylase(ACCase)activity.Collectively,we demonstrate that the ePE5max system has sufficient efficiency to generate heritable(homozygous or heterozygous)mutations in maize target genes and that the main obstacle to using PEs in maize has thus been removed.