Sclareol and linalyl acetate are produced by glandular trichomes through the MEP pathway

Sclareol,an antifungal specialized metabolite produced by clary sage,Salvia sclarea,is the starting plant natural molecule used for the hemisynthesis of the perfume ingredient ambroxide.Sclareol is mainly produced in clary sage flower calyces;however,the cellular localization of the sclareol biosynthesis remains unknown.To elucidate the site of sclareol biosynthesis,we analyzed its spatial distribution in the clary sage calyx epidermis using laser desorption/ionization mass spectrometry imaging(LDI–FTICR-MSI)and investigated the expression profile of sclareol biosynthesis genes in isolated glandular trichomes(GTs).We showed that sclareol specifically accumulates in GTs’gland cells in which sclareol biosynthesis genes are strongly expressed.We next isolated a glabrous beardless mutant and demonstrate that more than 90%of the sclareol is produced by the large capitate GTs.Feeding experiments,using 1-13 C-glucose,and specific enzyme inhibitors further revealed that the methylerythritol-phosphate(MEP)biosynthetic pathway is the main source of isopentenyl diphosphate(IPP)precursor used for the biosynthesis of sclareol.Our findings demonstrate that sclareol is an MEP-derived diterpene produced by large capitate GTs in clary sage emphasing the role of GTs as biofactories dedicated to the production of specialized metabolites.

Distinctive and complementary roles of E2F transcription factors during plant replication stress responses

Survival of living organisms is fully dependent on their maintenance of genome integrity,being permanently threatened by replication stress in proliferating cells.Although the plant DNA damage response(DDR)regulator SOG1 has been demonstrated to cope with replication defects,accumulating evidence points to other pathways functioning independent of SOG1.Here,we report the roles of the Arabidopsis E2FA and EF2B transcription factors,two well-characterized regulators of DNA replication,in plant response to replication stress.Through a combination of reverse genetics and chromatin immunoprecipitation approaches,we show that E2FA and E2FB share many target genes with SOG1,providing evidence for their involvement in the DDR.Analysis of double-and triple-mutant combinations revealed that E2FB,rather than E2FA,plays the most prominent role in sustaining plant growth in the presence of replication defects,either operating antagonistically or synergistically with SOG1.Conversely,SOG1 aids in overcoming the replication defects of E2FA/E2FB-deficient plants.Collectively,our data reveal a complex transcriptional network controlling the replication stress response in which E2Fs and SOG1 act as key regulatory factors.

The lncRNA APOLO interacts with the transcription factor WRKY42 to trigger root hair cell expansion in response to cold

Plant long noncoding RNAs(lncRNAs)have emerged as important regulators of chromatin dynamics,impacting on transcriptional programs leading to different developmental outputs.The lncRNA AUXIN-REGULATED PROMOTER LOOP(APOLO)directly recognizes multiple independent loci across the Arabidopsis genome and modulates their three-dimensional chromatin conformation,leading to transcriptional shifts.Here,we show that APOLO recognizes the locus encoding the root hair(RH)master regulator ROOT HAIR DEFECTIVE 6(RHD6)and controls RHD6 transcriptional activity,leading to cold-enhanced RH elongation through the consequent activation of the transcription factor gene RHD6-like RSL4.Furthermore,we demonstrate that APOLO interacts with the transcription factor WRKY42 and modulates its binding to the RHD6 promoter.WRKY42 is required for the activation of RHD6 by low temperatures and WRKY42 deregulation impairs cold-induced RH expansion.Collectively,our results indicate that a novel ribonucleoprotein complex with APOLO and WRKY42 forms a regulatory hub to activate RHD6 by shaping its epigenetic environment and integrate signals governing RH growth and development.

New partners for old friends: Plant SWI/SNF complexes

The physical accessibility of specific genomic regions is a chromatin property that regulates gene expression and allows the establishment of an appropriate transcriptional landscape in response to environmental and developmental signals.In eukaryotes,ATP-dependent chromatin remodeling complexes use the energy produced via ATP hydrolysis by an ATPase subunit to perform DNA translocation.These complexes are classified into four subfamilies,based on the domain organization of their catalytic ATPases(Clapier et al.,2017).

The LEAFY floral regulator displays pioneer transcription factor properties

Pioneer transcription factors(TFs)are a special category of TFs with the capacity to bind to closed chromatin regions in which DNA is wrapped around histones and may be highly methylated.Subsequently,pioneer TFs are able to modify the chromatin state to initiate gene expression.In plants,LEAFY(LFY)is a master floral regulator and has been suggested to act as a pioneer TF in Arabidopsis.Here,we demonstrate that LFY is able to bind both methylated and non-methylated DNA using a combination of in vitro genomewide binding experiments and structural modeling.Comparisons between regions bound by LFY in vivo and chromatin accessibility data suggest that a subset of LFY bound regions is occupied by nucleosomes.We confirm that LFY is able to bind nucleosomal DNA in vitro using reconstituted nucleosomes.Finally,we show that constitutive LFY expression in seedling tissues is sufficient to induce chromatin accessibility in the LFY direct target genes APETALA1 and AGAMOUS.Taken together,our study suggests that LFY possesses key pioneer TF features that contribute to launching the floral gene expression program.

Defense and senescence interplay in legume nodules

Immunity and senescence play a crucial role in the functioning of the legume symbiotic nodules.The miss-regulation of one of these processes compromises the symbiosis leading to death of the endosymbiont and the arrest of the nodule functioning.The relationship between immunity and senescence has been exten-sively studied in plant organs where a synergistic response can be observed.However,the interplay be-tween immunity and senescence in the symbiotic organ is poorly discussed in the literature and these phe-nomena are often mixed up.Recent studies revealed that the cooperation between immunity and senescence is not always observed in the nodule,suggesting complex interactions between these two pro-cesses within the symbiotic organ.Here,we discuss recent results on the interplay between immunity and senescence in the nodule and the specificities of this relationship during legume-rhizobium symbiosis.

Phenotyping floral attractiveness to pollinators using volatilomics,3D imaging,and insect monitoring

PLANTS,POLLINATORS,AND BIODIVERSITY:THESTATUSQUO Plant-pollinator coevolution has played a crucial role in shaping the biodiversity of ecosystems that we know today.Moreover,plants and pollinators are key to agriculture,contributing to the production of most fruits and vegetables necessary for healthy human diets.Unfortunately,over the last decades,there is mounting evidence of pollinator decline all over the world,which constitutes a major threat to food security(European Commission,2020).To pollinators,nectar and pollen are the main rewards:pollen is essentially their only source of proteins,lipids,and vitamins,while nectar is a carbohydrate-rich solution that they use to fuel somatic functions(Ollerton,2021).However,nectar is much more than just a sweet solution:it comprises a plethora of secondary metabolites and volatiles of major importance for plant-pollinator communication.Unfortunately,by neglecting these traits and reducing genetic diversity,plant breeding has potentially increased the risk of losing the traits beneficial to pollinators.

Histone Deacetylase AtSRT1 Links Metabolic Flux and Stress Response in Arabidopsis

How plant metabolic flux alters gene expression to optimize plant growth and response to stress remains largely unclear. Here, we report that Arabidopsis thaliana NAD＊-dependent histone deacetylase AtSRT1 negatively regulates plant tolerance to stress and glycolysis but stimulates mitochondrial respiration. We found that AtSRT1 interacts with Arabidopsis cMyc-Binding Protein 1 （AtMBP-1）, a transcriptional repressor produced by alternative translation of the cytosolic glycolytic enolase gene LOS2/EN02. We demonstrated that AtSRT1 could associate with the chromatin of AtMBP-I targets LOS2/EN02 and STZ/ZATIO, both of which encode key stress regulators, and reduce the H3K9ac levels at these genes to repress their transcription. Overexpression of both AtSRT1 and AtMBP-1 had synergistic effects on the expression of glycolytic genes, glycolytic enzymatic activities, and mitochondrial respiration. Furthermore, we found that AtMBP-1 is lysine-acetylated and vulnerable to proteasomal protein degradation, while AtSRT1 could remove its lysine acetylation and significantly enhance its stability in vivo. Taken together, these results indicate that AtSRT1 regulates primary metabolism and stress response by both epigenetic regulation and modulation of AtMBP-1 transcriptional activity in Arabidopsis.

Cell-specific pathways recruited for symbiotic nodulation in the Medicago truncatula legume

Medicago truncatula is a model legume species that has been studied for decades to understand the symbiotic relationship between legumes and soil bacteria collectively named rhizobia.This symbiosis called nodulation is initiated in roots with the infection of root hair cells by the bacteria,as well as the initiation of nodule primordia from root cortical,endodermal,and pericycle cells,leading to the development of a new root organ,the nodule,where bacteria fix and assimilate the atmospheric dinitrogen for the benefit of the plant.Here,we report the isolation and use of the nuclei from mock and rhizobia-inoculated roots for the single nuclei RNA-seq(sNucRNA-seq)profiling to gain a deeper understanding of early responses to rhizobial infection in Medicago roots.A gene expression map of the Medicago root was generated,comprising 25 clusters,which were annotated as specific cell types using 119 Medicago marker genes and orthologs to Arabidopsis cell-type marker genes.A focus on root hair,cortex,endodermis,and pericycle cell types,showing the strongest differential regulation in response to a short-term(48 h)rhizobium inoculation,revealed not only known genes and functional pathways,validating the sNucRNA-seq approach,but also numerous novel genes and pathways,allowing a comprehensive analysis of early root symbiotic responses at a cell type-specific level.

Cell specialization and coordination in Arabidopsis leaves upon pathogenic attack revealed by scRNA-seq

Plant defense responses involve several biological processes that allow plants to fight against pathogenic attacks.How these different processes are orchestrated within organs and depend on specific cell types is poorly known.Here,using single-cell RNA sequencing(scRNA-seq)technology on three independent biological replicates,we identified several cell populations representing the core transcriptional responses of wild-type Arabidopsis leaves inoculated with the bacterial pathogen Pseudomonas syringae DC3000.Among these populations,we retrieved major cell types of the leaves(mesophyll,guard,epidermal,companion,and vascular S cells)with which we could associate characteristic transcriptional reprogramming and regulators,thereby specifying different cell-type responses to the pathogen.Further analyses of transcriptional dynamics,on the basis of inference of cell trajectories,indicated that the different cell types,in addition to their characteristic defense responses,can also share similar modules of gene reprogramming,uncovering a ubiquitous antagonism between immune and susceptible processes.Moreover,it appears that the defense responses of vascular S cells,epidermal cells,and mesophyll cells can evolve along two separate paths,one converging toward an identical cell fate,characterized mostly by lignification and detoxification functions.As this divergence does not correspond to the differentiation between immune and susceptible cells,we speculate that this might reflect the discrimination between cellautonomous and non-cell-autonomous responses.Altogether our data provide an upgraded framework to describe,explore,and explain the specialization and the coordination of plant cell responses upon pathogenic challenge.

The lncRNA MARS modulates the epigenetic reprogramming of the marneral cluster in response to ABA

Clustered organization of biosynthetic non-homologous genes is emerging as a characteristic feature of plant genomes.The co-regulation of clustered genes seems to largely depend on epigenetic reprogram-ming and three-dimensional chromatin conformation.In this study,we identified the long non-coding RNA(lncRNA)MARneral Silencing(MARS),localized inside the Arabidopsis marneral cluster,which con-trols the local epigenetic activation of its surrounding region in response to abscisic acid(ABA).MARS modulates the POLYCOMB REPRESSIVE COMPLEX 1(PRC1)component LIKE HETEROCHROMATIN PROTEIN 1(LHP1)binding throughout the cluster in a dose-dependent manner,determining H3K27me3 deposition and chromatin condensation.In response to ABA,MARS decoys LHP1 away from the cluster and promotes the formation of a chromatin loop bringing together the MARNERAL SYNTHASE 1(MRN1)locus and a distal ABA-responsive enhancer.The enrichment of co-regulated lncRNAs in clustered meta-bolic genes in Arabidopsis suggests that the acquisition of novel non-coding transcriptional units may constitute an additional regulatory layer driving the evolution of biosynthetic pathways.

The F-box protein SHORT PRIMARY ROOT modulates primary root meristem activity by targeting SEUSS-LIKE protein for degradation in rice

Root meristem activity is essential for root morphogenesis and adaptation,but the molecular mechanism regulating root meristem activity is not fully understood.Here,we identify an F-box family E3 ubiquitin ligase named SHORT PRIMARY ROOT(SHPR) that regulates primary root(PR)meristem activity and cell proliferation in rice.SHPR loss-of-function mutations impair PR elongation in rice.SHPR is involved in the formation of an SCF complex with the Oryza sativa SKP1-like protein OSK1/20.We show that SHPR interacts with Oryza sativa SEUSS-LIKE(OsSLK) in the nucleus and is required for OsSLK polyubiquitination and degradation by the ubiquitin 26S-proteasome system(UPS).Transgenic plants overexpressing OsSLK display a shorter PR phenotype,which is similar to the SHPR loss-of-function mutants.Genetic analysis suggests that SHPR promotes PR elongation in an OsSLK-dependent manner.Collectively,our study establishes SHPR as an E3 ubiquitin ligase that targets OsSLK for degradation,and uncovers a protein ubiquitination pathway as a mechanism for modulating root meristem activity in rice.

Ustilaginoidea virens modulates lysine 2-hydroxyisobutyrylation in rice flowers during infection

The post-translational modification lysine 2-hydroxyisobutyrylation(K_(hib))plays an important role in gene transcription,metabolism,and enzymatic activity.K_(hib)sites have been identified in rice(Oryza sativa).However,the K_(hib)status of proteins in rice flowers during pathogen infection remains unclear.Here,we report a comprehensive identification of K_(hib)-modified proteins in rice flowers,and the changes in these proteins during infection with the fungal pathogen Ustilaginoidea virens.By using a tandem mass tag-based quantitative proteomics approach,we identified 2,891 K_(hib)sites on 964 proteins in rice flowers.Our data demonstrated that 2-hydroxyisobutyrylated proteins are involved in diverse biological processes.K_(hib)levels were substantially reduced upon infection with U.virens.Chromatin immunoprecipitation polymerase chain reaction(PCR)and reverse transcription quantitative PCR analyses revealed that histone K_(hib)is involved in the expression of disease-resistance genes.More importantly,most quantified sites on core histones H3 were downregulated upon U.virens infection.In addition,the histone deacetylases HDA705,HDA716,SRT1,and SRT2 are involved in the removal of K_(hib)marks in rice.HDA705 was further confirmed to negatively regulate rice disease resistance to pathogens U.virens,Magnaporthe oryzae,and Xanthomonas oryzae pv.oryzae(Xoo).Our data suggest that U.virens could modulate K_(hib)in rice flowers during infection.

Utility of Triti-Map for bulk-segregated mapping of causal genes and regulatory elements in Triticeae

Triticeae species,including wheat,barley,and rye,are critical for global food security.Mapping agronomically important genes is crucial for elucidating molecular mechanisms and improving crops.However,Triticeae includes many wild relatives with desirable agronomic traits,and frequent introgressions occurred during Triticeae evolution and domestication.Thus,Triticeae genomes are generally large and complex,making the localization of genes or functional elements that control agronomic traits challenging.Here,we developed Triti-Map,which contains a suite of user-friendly computational packages specifically designed and optimized to overcome the obstacles of gene mapping in Triticeae,as well as a web interface integrating multi-omics data from Triticeae for the efficient mining of genes or functional elements that control particular traits.The Triti-Map pipeline accepts bothDNA and RNAbulk-segregated sequencing data as well as traditional QTL data as inputs for locating genes and elucidating their functions.We illustrate the usage of Triti-Map with a combination of bulk-segregated ChIP-seq data to detect a wheat disease-resistance gene with its promoter sequence that is absent from the reference genome and clarify its evolutionary process.We hope that Triti-Map will facilitate gene isolation and accelerate Triticeae breeding.

A chromosome-level,haplotype-phased Vanilla planifolia genome highlights the challenge of partial endoreplication for accurate wholegenome assembly

Vanilla planifolia, the species cultivated to produce one of the world’s most popular flavors, is highly proneto partial genome endoreplication, which leads to highly unbalanced DNA content in cells. We report herethe first molecular evidence of partial endoreplication at the chromosome scale by the assembly and annotation of an accurate haplotype-phased genome of V. planifolia. Cytogenetic data demonstrated that thediploid genome size is 4.09 Gb, with 16 chromosome pairs, although aneuploid cells are frequentlyobserved. Using PacBio HiFi and optical mapping, we assembled and phased a diploid genome of 3.4 Gbwith a scaffold N50 of 1.2 Mb and 59 128 predicted protein-coding genes. The atypical k-mer frequenciesand the uneven sequencing depth observed agreed with our expectation of unbalanced genome representation. Sixty-seven percent of the genes were scattered over only 30% of the genome, putatively linkinggene-rich regions and the endoreplication phenomenon. By contrast, low-coverage regions (non-endoreplicated) were rich in repeated elements but also contained 33% of the annotated genes. Furthermore, this assembly showed distinct haplotype-specific sequencing depth variation patterns, suggesting complexmolecular regulation of endoreplication along the chromosomes. This high-quality, anchored assemblyrepresents 83% of the estimated V. planifolia genome. It provides a significant step toward the elucidationof this complex genome. To support post-genomics efforts, we developed the Vanilla Genome Hub, a userfriendly integrated web portal that enables centralized access to high-throughput genomic and other omicsdata and interoperable use of bioinformatics tools.

An enhanced network of energy metabolism,lysine acetylation,and growth-promoting protein accumulation is associated with heterosis in elite hybrid rice

Heterosis refers to the superior performance of a hybrid compared with its parental lines.Although several genetic and molecular models have been proposed to explain heterosis,it remains unclear how hybrid cells integrate complementary gene expression or activity to drive heterotic growth.In this work,we show that accumulation of growth-promoting and energy metabolism proteins,enhanced energy metabolism activities,and increased protein lysine acetylation were associated with superior growth of the panicle meristem in the elite hybrid rice Shanyou 63 relative to its parental varieties.Metabolism of nuclear/cytosolic acetylcoenzyme A was also enhanced in the hybrid,which paralleled increases in histone H3 acetylation to selectively target the expression of growth-promoting and metabolic genes.Lysine acetylation of cellular proteins,including TARGET OF RAPAMYCIN complex 1,ribosomal proteins,and energy metabolism enzymes,was also augmented and/or remodeled to modulate their activities.The data indicate that an enhanced network of energy-producing metabolic activity and growth-promoting histone acetylation/gene expression in the hybrid could contribute to its superior growth rate and may constitute a model to explain heterosis.