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Intragenomic mutation rates can vary dramatically due to transcription-associated mutagenesis or transcription-coupled repair, which vary based on local epigenomic modifications that are nonuniformly distributed across genomes. One feature associated with decreased mutation is higher expression level, which depends on environmental cues. To understand the magnitude of expression-dependent mutation rate variation, we perturbed expression through a heat treatment in Arabidopsis thaliana. We quantified gene expression to identify differentially expressed genes, which we then targeted for mutation detection using duplex sequencing. This approach provided a highly accurate measurement of the frequency of rare somatic mutations in vegetative plant tissues, which has been a recent source of uncertainty. Somatic mutations in plants may be useful for understanding drivers of DNA damage and repair in the germline since plants experience late germline segregation and both somatic and germline cells share common repair machinery. We included mutant lines lacking mismatch repair (MMR) and base excision repair (BER) capabilities to understand how repair mechanisms may drive biased mutation accumulation. We found wild-type (WT) and BER mutant mutation frequencies to be very low (mean variant frequency 1.8 × 10-8 and 2.6 × 10-8, respectively), while MMR mutant frequencies were significantly elevated (1.13 × 10-6). Interestingly, in the MMR mutant lines, there was no difference in the somatic mutation frequencies between temperature treatments or between highly versus lowly expressed genes. The extremely low somatic variant frequencies in WT plants indicate that larger datasets will be needed to address fundamental evolutionary questions about whether environmental change leads to gene-specific changes in mutation rate.

Plants are colonized by distinct pathogenic and commensal microbiomes across different regions of the globe, but the factors driving their geographic variation are largely unknown. Here, using 16S ribosomal DNA and shotgun sequencing, we characterized the associations of the Arabidopsis thaliana leaf microbiome with host genetics and climate variables from 267 populations in the species native range across Europe. Comparing the distribution of the 575 major bacterial amplicon variants (phylotypes), we discovered that microbiome composition in A. thaliana segregates along a latitudinal gradient. The latitudinal clines in microbiome composition are predicted by metrics of drought, but also by the spatial genetics of the host. To validate the relative effects of drought and host genotype we conducted a common garden field study, finding 10% of the core bacteria to be affected directly by drought and 20% to be affected by host genetic associations with drought. These data provide a valuable resource for the plant microbiome field, with the identified associations suggesting that drought can directly and indirectly shape genetic variation in A. thaliana via the leaf microbiome.

The crosstalk between clathrin-mediated endocytosis (CME) and the autophagy pathway has been reported in mammals; however, the interconnection of CME with autophagy has not been established in plants. Here, we report that the Arabidopsis CLATHRIN LIGHT CHAIN (CLC) subunit 2 and 3 double mutant, clc2-1 clc3-1, phenocopies Arabidopsis AUTOPHAGY-RELATED GENE (ATG) mutants in both autoimmunity and nutrient sensitivity. Accordingly, the autophagy pathway is significantly compromised in the clc2-1 clc3-1 mutant. Interestingly, multiple assays demonstrate that CLC2 directly interacts with ATG8h/ATG8i in a domain-specific manner. As expected, both GFP-ATG8h/GFP-ATG8i and CLC2-GFP are subjected to autophagic degradation, and degradation of GFP-ATG8h is significantly reduced in the clc2-1 clc3-1 mutant. Notably, simultaneous knockout of ATG8h and ATG8i by CRISPR-Cas9 results in enhanced resistance against Golovinomyces cichoracearum, supporting the functional relevance of the CLC2-ATG8h/8i interactions. In conclusion, our results reveal a link between the function of CLCs and the autophagy pathway in Arabidopsis.

The red and far-red light photoreceptor phytochrome B (phyB) transmits light signals following cytosol-to-nuclear translocation to regulate transcriptional networks therein. This necessitates changes in protein-protein interactions of phyB in the cytosol, about which little is presently known. Via introduction of a nucleus-excluding G767R mutation into the dominant, constitutively active phyBY276H (YHB) allele, we explore the functional consequences of expressing a cytosol-localized YHBG767R variant in transgenic Arabidopsis seedlings. We show that YHBG767R elicits selective constitutive photomorphogenic phenotypes in dark-grown phyABCDE null mutants, wild type and other phy-deficient genotypes. These responses include light-independent apical hook opening, cotyledon unfolding, seed germination and agravitropic hypocotyl growth with minimal suppression of hypocotyl elongation. Such phenotypes correlate with reduced PIF3 levels, which implicates cytosolic targeting of PIF3 turnover or PIF3 translational inhibition by YHBG767R. However, as expected for a cytoplasm-tethered phyB, YHBG767R elicits reduced light-mediated signaling activity compared with similarly expressed wild-type phyB in phyABCDE mutant backgrounds. YHBG767R also interferes with wild-type phyB light signaling, presumably by formation of cytosol-retained and/or otherwise inactivated heterodimers. Our results suggest that cytosolic interactions with PIFs play an important role in phyB signaling even under physiological conditions.

Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate cross-linking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect cross-linking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here, we developed a method to generate viable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in callus tissue via CRISPR/Cas9-mediated gene editing. We combined this with a candidate gene approach to characterize the male gametophyte defective 2 (MGP2) gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We showed that in the callus mutant cell walls, RG-II does not cross-link normally because it lacks 3-deoxy-D-manno-octulosonic acid (Kdo) and thus cannot form the α-L-Rhap-(1→5)-α-D-kdop-(1→sidechain). We suggest that MGP2 encodes an inverting RG-II CMP-β-Kdo transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization. Our method also provides a viable strategy for further identifying proteins involved in the biosynthesis of RG-II.

Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the single-celled green alga Chlamydomonas reinhardtii to establish a foundational understanding of osmotic-stress signaling pathways through transcriptomics, phosphoproteomics, and functional genomics approaches. Comparison of pathways identified through these analyses with yeast and Arabidopsis allows us to infer their evolutionary conservation and divergence across these lineages. 76 genes, acting across diverse cellular compartments, were found to be important for osmotic-stress tolerance in Chlamydomonas through their functions in cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs for five of these genes have conserved functions in stress tolerance in Arabidopsis and reveal a novel PROFILIN-dependent stage of acclimation affecting the actin cytoskeleton that ensures tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants, and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway.

We studied the D3-type cyclin function during gynoecium development in Arabidopsis and how they are related to the hormone cytokinin and the transcription factor SPATULA. Growth throughout the life of plants is sustained by cell division and differentiation processes in meristematic tissues. In Arabidopsis, gynoecium development implies a multiphasic process where the tissues required for pollination, fertilization, and seed development form. The Carpel Margin Meristem (CMM) is a mass of undifferentiated cells that gives rise to the gynoecium internal tissues, such as septum, ovules, placenta, funiculus, transmitting tract, style, and stigma. Different genetic and hormonal factors, including cytokinin, control the CMM function. Cytokinin regulates the cell cycle transitions through the activation of cell cycle regulators as cyclin genes. D3-type cyclins are expressed in proliferative tissues, favoring the mitotic cell cycle over the endoreduplication. Though the role of cytokinin in CMM and gynoecium development is highly studied, its specific role in regulating the cell cycle in this tissue remains unclear. Additionally, despite extensive research on the relationship between CYCD3 genes and cytokinin, the regulatory mechanism that connects them remains elusive. Here, we found that D3-type cyclins are expressed in proliferative medial and lateral tissues. Conversely, the depletion of the three CYCD3 genes showed that they are not essential for gynoecium development. However, the addition of exogenous cytokinin showed that they could control the division/differentiation balance in gynoecium internal tissues and outgrowths. Finally, we found that SPATULA can be a mechanistic link between cytokinin and the D3-type cyclins. The data suggest that the role of D3-type cyclins in gynoecium development is related to the cytokinin response, and they might be activated by the transcription factor SPATULA.

Tobacco mild green mosaic virus (TMGMV)-like nanocarriers were designed for gene delivery to plant cells. High aspect ratio TMGMVs were coated with a polycationic biopolymer, poly(allylamine) hydrochloride (PAH), to generate highly charged nanomaterials (TMGMV-PAH; 56.20 ± 4.7 mV) that efficiently load (1:6 TMGMV:DNA mass ratio) and deliver single-stranded and plasmid DNA to plant cells. The TMGMV-PAH were taken up through energy-independent mechanisms in Arabidopsis protoplasts. TMGMV-PAH delivered a plasmid DNA encoding a green fluorescent protein (GFP) to the protoplast nucleus (70% viability), as evidenced by GFP expression using confocal microscopy and Western blot analysis. TMGMV-PAH were inactivated (iTMGMV-PAH) using UV cross-linking to prevent systemic infection in intact plants. Inactivated iTMGMV-PAH-mediated pDNA delivery and gene expression of GFP in vivo was determined using confocal microscopy and RT-qPCR. Virus-like nanocarrier-mediated gene delivery can act as a facile and biocompatible tool for advancing genetic engineering in plants.

Transcription factors can promote gene expression through activation domains. Whole-genome screens have systematically mapped activation domains in transcription factors but not in non-transcription factor proteins (e.g., chromatin regulators and coactivators). To fill this knowledge gap, we employed the activation domain predictor PADDLE to analyze the proteomes of Arabidopsis thaliana and Saccharomyces cerevisiae. We screened 18,000 predicted activation domains from >800 non-transcription factor genes in both species, confirming that 89% of candidate proteins contain active fragments. Our work enables the annotation of hundreds of nuclear proteins as putative coactivators, many of which have never been ascribed any function in plants. Analysis of peptide sequence compositions reveals how the distribution of key amino acids dictates activity. Finally, we validated short, "universal" activation domains with comparable performance to state-of-the-art activation domains used for genome engineering. Our approach enables the genome-wide discovery and annotation of activation domains that can function across diverse eukaryotes.

Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodelling, and timely deposition of polysaccharides, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM), we studied cell plate development in four dimensions, through the behavior of yellow fluorescent protein (YFP)-tagged cytokinesis-specific GTPase RABA2a vesicles. We monitored the entire duration of cell plate development, from its first emergence, with the aid of YFP-RABA2a, in both the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process.

The α-Aurora kinase is a crucial regulator of spindle microtubule organization during mitosis in plants. Here, we report a post-mitotic role for α-Aurora in reorganizing the phragmoplast microtubule array. In Arabidopsis thaliana, α-Aurora relocated from spindle poles to the phragmoplast midzone, where it interacted with the microtubule cross-linker MAP65-3. In a hypomorphic α-Aurora mutant, MAP65-3 was detected on spindle microtubules, followed by a diffuse association pattern across the phragmoplast midzone. Simultaneously, phragmoplast microtubules remained belatedly in a solid disk array before transitioning to a ring shape. Microtubules at the leading edge of the matured phragmoplast were often disengaged, accompanied by conspicuous retentions of MAP65-3 at the phragmoplast interior edge. Specifically, α-Aurora phosphorylated two residues towards the C-terminus of MAP65-3. Mutation of these residues to alanines resulted in an increased association of MAP65-3 with microtubules within the phragmoplast. Consequently, the expansion of the phragmoplast was notably slower compared to wild-type cells or cells expressing a phospho-mimetic variant of MAP65-3. Moreover, mimicking phosphorylation reinstated disrupted MAP65-3 behaviors in plants with compromised α-Aurora function. Overall, our findings reveal a mechanism in which α-Aurora facilitates cytokinesis progression through phosphorylation-dependent restriction of MAP65-3 associating with microtubules at the phragmoplast midzone.

Plants rely on Nucleotide-binding, Leucine-rich repeat Receptors (NLRs) for pathogen recognition. Highly variable NLRs (hvNLRs) show remarkable intraspecies diversity, while their low-variability paralogs (non-hvNLRs) are conserved between ecotypes. At a population level, hvNLRs provide new pathogen-recognition specificities, but the association between allelic diversity and genomic and epigenomic features has not been established. Our investigation of NLRs in Arabidopsis Col-0 has revealed that hvNLRs show higher expression, less gene body cytosine methylation, and closer proximity to transposable elements than non-hvNLRs. hvNLRs show elevated synonymous and nonsynonymous nucleotide diversity and are in chromatin states associated with an increased probability of mutation. Diversifying selection maintains variability at a subset of codons of hvNLRs, while purifying selection maintains conservation at non-hvNLRs. How these features are established and maintained, and whether they contribute to the observed diversity of hvNLRs is key to understanding the evolution of plant innate immune receptors.

Photobodies (PBs) are membraneless subnuclear organelles that self-assemble via concentration-dependent liquid-liquid phase separation (LLPS) of the plant photoreceptor and thermosensor phytochrome B (PHYB). The current PHYB LLPS model posits that PHYB phase separates randomly in the nucleoplasm regardless of the cellular or nuclear context. Here, we established a robust Oligopaints method in Arabidopsis to determine the positioning of individual PBs. We show surprisingly that even in PHYB overexpression lines - where PHYB condensation would be more likely to occur randomly - PBs positioned at twelve distinct subnuclear locations distinguishable by chromocenter and nucleolus landmarks, suggesting that PHYB condensation occurs nonrandomly at preferred seeding sites. Intriguingly, warm temperatures reduce PB number by inducing the disappearance of specific thermo-sensitive PBs, demonstrating that individual PBs possess different thermosensitivities. These results reveal a nonrandom PB nucleation model, which provides the framework for the biogenesis of spatially distinct individual PBs with diverse environmental sensitivities within a single plant nucleus.

Photoactivation of the plant photoreceptor and thermosensor phytochrome B (PHYB) triggers its condensation into subnuclear membraneless organelles named photobodies (PBs). However, the function of PBs in PHYB signaling remains frustratingly elusive. Here, we found that PHYB recruits PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) to PBs. Surprisingly, PHYB exerts opposing roles in degrading and stabilizing PIF5. Perturbing PB size by overproducing PHYB provoked a biphasic PIF5 response: while a moderate increase in PHYB enhanced PIF5 degradation, further elevating the PHYB level stabilized PIF5 by retaining more of it in enlarged PBs. Conversely, reducing PB size by dim light, which enhanced PB dynamics and nucleoplasmic PHYB and PIF5, switched the balance towards PIF5 degradation. Together, these results reveal that PB formation spatially segregates two antagonistic PHYB signaling actions - PIF5 stabilization in PBs and PIF5 degradation in the surrounding nucleoplasm - which could enable an environmentally sensitive, counterbalancing mechanism to titrate nucleoplasmic PIF5 and environmental responses.

Plants shed organs such as leaves, petals, or fruits through the process of abscission. Monitoring cues such as age, resource availability, and biotic and abiotic stresses allow plants to abscise organs in a timely manner. How these signals are integrated into the molecular pathways that drive abscission is largely unknown. The INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) gene is one of the main drivers of floral organ abscission in Arabidopsis and is known to transcriptionally respond to most abscission-regulating cues. By interrogating the IDA promoter in silico and in vitro, we identified transcription factors that could potentially modulate IDA expression. We probed the importance of ERF- and WRKY-binding sites for IDA expression during floral organ abscission, with WRKYs being of special relevance to mediate IDA up-regulation in response to biotic stress in tissues destined for separation. We further characterized WRKY57 as a positive regulator of IDA and IDA-like gene expression in abscission zones. Our findings highlight the promise of promoter element-targeted approaches to modulate the responsiveness of the IDA signaling pathway to harness controlled abscission timing for improved crop productivity.

BACKGROUND: Sulfur (S) is a mineral nutrient essential for plant growth and development, which is incorporated into diverse molecules fundamental for primary and secondary metabolism, plant defense, signaling, and maintaining cellular homeostasis. Although, S starvation response is well documented in the dicot model Arabidopsis thaliana, it is not clear if the same transcriptional networks control the response also in the monocots. RESULTS: We performed series of physiological, expression, and metabolite analyses in two model monocot species, one representing the C3 plants, Oryza sativa cv. kitaake, and second representing the C4 plants, Setaria viridis. Our comprehensive transcriptomic analysis revealed twice as many differentially expressed genes (DEGs) in S. viridis than in O. sativa under S-deficiency, consistent with a greater loss of sulfur and S-containing metabolites under these conditions. Surprisingly, most of the DEGs and enriched gene ontology terms were species-specific, with an intersect of only 58 common DEGs. The transcriptional networks were different in roots and shoots of both species, in particular no genes were down-regulated by S-deficiency in the roots of both species. CONCLUSIONS: Our analysis shows that S-deficiency seems to have different physiological consequences in the two monocot species and their nutrient homeostasis might be under distinct control mechanisms.

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during cell division by monitoring kinetochore-microtubule attachment. Plants produce both sequence-conserved and diverged SAC components, and it has been largely unknown how SAC activation leads to the assembly of these proteins at unattached kinetochores to prevent cells from entering anaphase. In Arabidopsis thaliana, the noncanonical BUB3.3 protein was detected at kinetochores throughout mitosis, unlike MAD1 and the plant-specific BUB1/MAD3 family protein BMF3 that associated with unattached chromosomes only. When BUB3.3 was lost by a genetic mutation, mitotic cells often entered anaphase with misaligned chromosomes and presented lagging chromosomes after they were challenged by low doses of the microtubule depolymerizing agent oryzalin, resulting in the formation of micronuclei. Surprisingly, BUB3.3 was not required for the kinetochore localization of other SAC proteins or vice versa. Instead, BUB3.3 specifically bound to BMF3 through two internal repeat motifs that were not required for BMF3 kinetochore localization. This interaction enabled BMF3 to recruit CDC20, a downstream SAC target, to unattached kinetochores. Taken together, our findings demonstrate that plant SAC utilizes unconventional protein interactions for arresting mitosis, with BUB3.3 directing BMF3's role in CDC20 recruitment, rather than the recruitment of BUB1/MAD3 proteins observed in fungi and animals. This distinct mechanism highlights how plants adapted divergent versions of conserved cell cycle machinery to achieve specialized SAC control.

The kinetochore scaffold 1 (KNL1) protein recruits spindle assembly checkpoint (SAC) proteins to ensure accurate chromosome segregation during mitosis. Despite such a conserved function among eukaryotic organisms, its molecular architectures have rapidly evolved so that the functional mode of plant KNL1 is largely unknown. To understand how SAC signaling is regulated at kinetochores, we characterized the function of the KNL1 gene in Arabidopsis thaliana. The KNL1 protein was detected at kinetochores throughout the mitotic cell cycle, and null knl1 mutants were viable and fertile but exhibited severe vegetative and reproductive defects. The mutant cells showed serious impairments of chromosome congression and segregation, that resulted in the formation of micronuclei. In the absence of KNL1, core SAC proteins were no longer detected at the kinetochores, and the SAC was not activated by unattached or misaligned chromosomes. Arabidopsis KNL1 interacted with SAC essential proteins BUB3.3 and BMF3 through specific regions that were not found in known KNL1 proteins of other species, and recruited them independently to kinetochores. Furthermore, we demonstrated that upon ectopic expression, the KNL1 homolog from the dicot tomato was able to functionally substitute KNL1 in A. thaliana, while others from the monocot rice or moss associated with kinetochores but were not functional, as reflected by sequence variations of the kinetochore proteins in different plant lineages. Our results brought insights into understanding the rapid evolution and lineage-specific connection between KNL1 and the SAC signaling molecules.

Phytohormone levels are regulated through specialized enzymes, participating not only in their biosynthesis but also in post-signaling processes for signal inactivation and cue depletion. Arabidopsis thaliana (At) carboxylesterase 15 (CXE15) and carboxylesterase 20 (CXE20) have been shown to deplete strigolactones (SLs) that coordinate various growth and developmental processes and function as signaling molecules in the rhizosphere. Here, we elucidate the X-ray crystal structures of AtCXE15 (both apo and SL intermediate bound) and AtCXE20, revealing insights into the mechanisms of SL binding and catabolism. The N-terminal regions of CXE15 and CXE20 exhibit distinct secondary structures, with CXE15 characterized by an alpha helix and CXE20 by an alpha/beta fold. These structural differences play pivotal roles in regulating variable SL hydrolysis rates. Our findings, both in vitro and in planta, indicate that a transition of the N-terminal helix domain of CXE15 between open and closed forms facilitates robust SL hydrolysis. The results not only illuminate the distinctive process of phytohormone breakdown but also uncover a molecular architecture and mode of plasticity within a specific class of carboxylesterases.

Plants sense abscisic acid (ABA) using chemical-induced dimerization (CID) modules, including the receptor PYR1 and HAB1, a phosphatase inhibited by ligand-activated PYR1. This system is unique because of the relative ease with which ligand recognition can be reprogrammed. To expand the PYR1 system, we designed an orthogonal * module, which harbors a dimer interface salt bridge; X-ray crystallographic, biochemical and in vivo analyses confirm its orthogonality. We used this module to create PYR1*MANDI/HAB1* and PYR1*AZIN/HAB1*, which possess nanomolar sensitivities to their activating ligands mandipropamid and azinphos-ethyl. Experiments in Arabidopsis thaliana and Saccharomyces cerevisiae demonstrate the sensitive detection of banned organophosphate contaminants using living biosensors and the construction of multi-input/output genetic circuits. Our new modules enable ligand-programmable multi-channel CID systems for plant and eukaryotic synthetic biology that can empower new plant-based and microbe-based sensing modalities.

Plant roots integrate environmental signals with development using exquisite spatiotemporal control. This is apparent in the deposition of suberin, an apoplastic diffusion barrier, which regulates flow of water, solutes and gases, and is environmentally plastic. Suberin is considered a hallmark of endodermal differentiation but is absent in the tomato endodermis. Instead, suberin is present in the exodermis, a cell type that is absent in the model organism Arabidopsis thaliana. Here we demonstrate that the suberin regulatory network has the same parts driving suberin production in the tomato exodermis and the Arabidopsis endodermis. Despite this co-option of network components, the network has undergone rewiring to drive distinct spatial expression and with distinct contributions of specific genes. Functional genetic analyses of the tomato MYB92 transcription factor and ASFT enzyme demonstrate the importance of exodermal suberin for a plant water-deficit response and that the exodermal barrier serves an equivalent function to that of the endodermis and can act in its place.

The root system of a plant is essential for plant growth and development because it absorbs nutrients and water from the soil. The root stem cells are maintained and replenished by the quiescent center and are essential for the formation of the specific root structure. In Arabidopsis , the WUSCHEL-RELATED HOMEOBOX 5 (WOX5) plays a key role in regulating root stem cell fate. However, which factor can directly modulate the stability of WOX5 protein remains totally unknown. Here, we report that the peptidase DA1 (LARGE IN CHINESE) interacts with and cleaves WOX5, resulting in the destabilization of WOX5. Genetic analyses support that DA1 acts through WOX5 to regulate root stem cell function. We further demonstrate that cytokinin (CK) signaling induces the accumulation of DA1 protein, thereby decreasing the abundance of WOX5 protein in the root. Consistent with these results, the mutation in DA1 increases the layers of columella stem cells and influences CK-induced differentiation of columella stem cells. Thus, our results reveal a previously unrecognized mechanism by which the cleavage of WOX5 by DA1 connects CK signaling and root stem cell function in Arabidopsis. [Display omitted] • DA1 interacts with and cleaves WOX5, resulting in the destabilization of WOX5 • DA1 acts through WOX5 to regulate root stem cell function • Cytokinin signaling induces the accumulation of DA1 protein • The cleavage of WOX5 by DA1 connects cytokinin signaling and root stem cell function The transcription factor WOX5 plays a key role in regulating root stem cell fate. Cui et al. find that the peptidase DA1 regulates root stem cell function by interacting with and cleaving WOX5. They further demonstrate that the cleavage of WOX5 by DA1 connects cytokinin signaling and root stem cell function in Arabidopsis. [ABSTRACT FROM AUTHOR]

Root hairs (RHs) are an innovation of vascular plants whose development is coordinated by endogenous and environmental cues, such as ethylene and light conditions. However, the potential crosstalk between ethylene and light conditions in RH development is unclear. We report that Arabidopsis constitutive photomorphogenic 1 (COP1) integrates ethylene and light signaling to mediate RH development. Darkness suppresses RH development largely through COP1. COP1 inhibits both cell fate determination of trichoblast and tip growth of RHs based on pharmacological, genetic, and physiological analyses. Indeed, COP1 interacts with and catalyzes the ubiquitination of ACS2 and ACS6. COP1- or darkness-promoted proteasome-dependent degradation of ACS2/6 leads to a low ethylene level in underground tissues. The negative role of COP1 in RH development by downregulating ethylene signaling may be coordinated with the positive role of COP1 in hypocotyl elongation by upregulating ethylene signaling, providing an evolutionary advantage for seedling fitness. [Display omitted] • Root hairs respond to light or ethylene by increasing both density and length • Arabidopsis COP1, the light responsive factor, inhibits root hair development • COP1 catalyzes the ubiquitination of type I ACSs to reduce ethylene responses • Dark-induced 26S-proteasome-mediated degradation of type I ACSs largely depends on COP1 Yin et al. report that Arabidopsis constitutive photomorphogenic 1 (COP1), a light response factor, controls root hair development by catalyzing the ubiquitination of type I ACSs and degradation to inhibit ethylene biosynthesis. The negative role of COP1 in root hairs may coordinate with its positive role in hypocotyl elongation to promote seedling fitness. [ABSTRACT FROM AUTHOR]

The heat shock transcription factor (HSF) family is one of the most widely studied transcription factor families in plants; HSFs can participate in the response to various stressors, such as heat stress, high salt, and drought stress. Based on garlic transcriptome data, we screened and identified 22 garlic HSFs. The HSF proteins of garlic and Arabidopsis can be divided into three (A, B, C) subfamilies. The phylogenetic relationship, chromosome localization, sequence characteristics, conserved motifs, and promoter analysis of the HSF family were analyzed through bioinformatics methods. RT-qPCR analysis showed that the nine selected genes had different degrees of response to heat stress. In addition, we isolated and identified a class B HSF gene, AsHSFB1, from garlic variety 'Xusuan No.6'. Subsequently, the AsHSFB1 gene was overexpressed in Arabidopsis thaliana. Under heat stress, the germination rate and growth of wild-type plants were better than that of transgenic plants. Moreover, after heat treatment, the contents of peroxidase, catalase, and chlorophyll a and b of transgenic plants were lower, but the contents of malondialdehyde (MDA) and leaf conductivity were higher. Nitroblue tetrazolium (NBT) staining showed that the stained area of transgenic plant leaves was larger than that of the wild type. Further studies showed that AsHSFB1 overexpression inhibited the expression of related reverse resistance genes. These results indicate that AsHSFB1 might play a negative regulatory role in garlic resistance under high stress. Altogether, these findings provide valuable data for revealing the function of HSF genes and lay a foundation for the subsequent selection of heat-resistant garlic varieties. [ABSTRACT FROM AUTHOR]

SUMMARY Calmodulin7 (CAM7) is a key transcription factor of Arabidopsis seedling development. CAM7 works together with HY5 bZIP protein to promote photomorphogenesis at various wavelengths of light. In this study, we show that AtMYB4, identified from a yeast two‐hybrid screen, physically interacts with CAM7 and works as a positive regulator of photomorphogenesis at various wavelengths of light. CAM7 and HY5 directly bind to the promoter of AtMYB4 to promote its expression for photomorphogenic growth. On the other hand, ARA4, identified from the same yeast two‐hybrid screen, works as a negative regulator of photomorphogenic growth specifically in white light. The double mutant analysis reveals that the altered hypocotyl elongation of atmyb4 and ara4 is either partly or completely suppressed by additional loss of function of CAM7. Furthermore, ARA4 genetically interacts with AtMYB4 in an antagonistic manner to suppress the elongated hypocotyl phenotype of atmyb4. The transactivation studies reveal that while CAM7 activates the promoter of AtMYB4 in association with HY5, ARA4 negatively regulates AtMYB4 expression. Taken together, these results demonstrate that working as a negative regulator of photomorphogenesis, ARA4 plays a balancing act on CAM7 and HY5‐mediated regulation of AtMYB4. [ABSTRACT FROM AUTHOR]

Summary Sun‐loving plants undergo shade avoidance syndrome (SAS) to compete with their neighbors for sunlight in shade conditions. Phytochrome B (phyB) plays a dominant role in sensing the shading signals (low red to far‐red ratios) and triggering SAS. Shade drives phyB conversion to inactive form, consequently leading to the accumulation of PHYTOCHROMEINTERACTING FACTOR 4 (PIF4) that promotes plant growth. Here, we show B‐box PROTEIN 7 (BBX7)/BBX8 and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) positively regulate the low R : FR‐induced PIF4 expression and promote the low R : FR‐triggered hypocotyl growth in Arabidopsis. Shade interferes the interactions of phyB with BBX7 or BBX8 and triggers the accumulation of BBX7 and BBX8 independent of phyB. BBX7 and BBX8 associate with CCA1 and LHY to activate their transcription, the gene produces of which subsequently upregulate the expression of PIF4 in shade. Genetically, BBX7 and BBX8 act upstream of CCA1, LHY, and PIF4 with respect to hypocotyl growth in shade conditions. Our study reveals the BBX7/8‐CCA1/LHY transcription factor cascade upregulates PIF4 expression and increases its abundance to promote plant growth and development in response to shade. [ABSTRACT FROM AUTHOR]

Summary The endoplasmic reticulum‐associated degradation (ERAD) system eliminates misfolded and short‐lived proteins to maintain physiological homeostasis in the cell. We have previously reported that ERAD is involved in salt tolerance in Arabidopsis. Given the central role of the phytohormone abscisic acid (ABA) in plant stress responses, we sought to identify potential intersections between the ABA and the ERAD pathways in plant stress response. By screening for the ABA response of a wide array of ERAD mutants, we isolated a gain‐of‐function mutant, doa10a‐1, which conferred ABA hypersensitivity to seedlings. Genetic and biochemical assays showed that DOA10A is a functional E3 ubiquitin ligase which, by acting in concert with specific E2 enzymes, mediates mono‐ubiquitination of the ABA receptor, followed by their relocalization to the plasma membrane. This in turn leads to enhanced ABA perception. In summary, we report here the identification of a novel RING‐type E3 ligase, DOA10A, which regulates ABA perception by affecting the localization and the activity of ABA receptors through their mono‐ubiquitination. [ABSTRACT FROM AUTHOR]

SUMMARY Tartary buckwheat is known for its ability to adapt to intricate growth conditions and to possess robust stress‐resistant properties. Nevertheless, it remains vulnerable to drought stress, which can lead to reduced crop yield. To identify potential genes involved in drought resistance, a genome‐wide association study on drought tolerance in Tartary buckwheat germplasm was conducted. A gene encoding pectin methylesterase inhibitors protein (FtPMEI13) was identified, which is not only associated with drought tolerance but also showed induction during drought stress and abscisic acid (ABA) treatment. Further analysis revealed that overexpression of FtPMEI13 leads to improved drought tolerance by altering the activities of antioxidant enzymes and the levels of osmotically active metabolites. Additionally, FtPMEI13 interacts with pectin methylesterase (PME) and inhibits PME activity in response to drought stress. Our results suggest that FtPMEI13 may inhibit the activity of FtPME44/FtPME61, thereby affecting pectin methylesterification in the cell wall and modulating stomatal closure in response to drought stress. Yeast one‐hybrid, dual‐luciferase assays, and electrophoretic mobility shift assays demonstrated that an ABA‐responsive transcription factor FtbZIP46, could bind to the FtPMEI13 promoter, enhancing FtPMEI13 expression. Further analysis indicated that Tartary buckwheat accessions with the genotype resulting in higher FtPMEI13 and FtbZIP46 expression exhibited higher drought tolerance compared to the others. This suggests that this genotype has potential for application in Tartary buckwheat breeding. Furthermore, the natural variation of FtPMEI13 was responsible for decreased drought tolerance during Tartary buckwheat domestication. Taken together, these results provide basic support for Tartary buckwheat breeding for drought tolerance. [ABSTRACT FROM AUTHOR]

The ubiquitin receptors RPN10 and RPN13 harbor multiple activities including ubiquitin binding; however, solid evidence connecting a particular activity to specific in vivo functions is scarce. Through complementation, the ubiquitin-binding site-truncated Arabidopsis RPN10 (N215) rescued the growth defects of rpn10-2, supporting the idea that the ubiquitin-binding ability of RPN10 is dispensable and N215, which harbors a vWA domain, is fully functional. Instead, a structural role played by RPN10 in the 26S proteasomes is likely vital in vivo. A site-specific variant, RPN10-11A, that likely has a destabilized vWA domain could partially rescue the rpn10-2 growth defects and is not integrated into 26S proteasomes. Native polyacrylamide gel electrophoresis and mass spectrometry with rpn10-2 26S proteasomes showed that the loss of RPN10 reduced the abundance of double-capped proteasomes, induced the integration of specific subunit paralogues, and increased the association of ECM29, a well-known factor critical for quality checkpoints by binding and inhibiting aberrant proteasomes. Extensive Y2H and GST-pulldown analyses identified RPN2-binding residues on RPN13 that overlapped with ubiquitin-binding and UCH2-binding sites in the RPN13 C-terminus (246–254). Interestingly, an analysis of homozygous rpn10-2 segregation in a rpn13-1 background harboring RPN13 variants defective for ubiquitin binding and/or RPN2 binding supports the criticality of the RPN13–RPN2 association in vivo. [ABSTRACT FROM AUTHOR]

SUMMARY: Wheat (Triticum aestivum L.) is a globally staple crop vulnerable to various fungal diseases, significantly impacting its yield. Plant cell surface receptors play a crucial role in recognizing pathogen‐associated molecular patterns (PAMPs) and activating PAMP‐triggered immunity, boosting resistance against a wide range of plant diseases. Although the role of plant chitin receptor CERK1 in immune recognition and defense has been established in Arabidopsis and rice, its function and potential agricultural applications in enhancing resistance to crop diseases remain largely unexplored. Here, we identify and characterize TaCERK1 in Triticeae crop wheat, uncovering its involvement in chitin recognition, immune regulation, and resistance to fungal diseases. By a comparative analysis of CERK1 homologs in Arabidopsis and monocot crops, we demonstrate that AtCERK1 in Arabidopsis elicits the most robust immune response. Moreover, we show that overexpressing TaCERK1 and AtCERK1 in wheat confers resistance to multiple fungal diseases, including Fusarium head blight, stripe rust, and powdery mildew. Notably, transgenic wheat lines with moderately expressed AtCERK1 display superior disease resistance and heightened immune responses without adversely affecting growth and yield, compared to TaCERK1 overexpression transgenics. Our findings highlight the significance of plant chitin receptors across diverse plant species and suggest potential strategies for bolstering crop resistance against broad‐spectrum diseases in agricultural production through the utilization of plant immune receptors. Significance Statement: CERK1 acts as a chitin immune receptor for plant defense and survival, but its role and applied potential in Triticeae crops remain unknown. We identify TaCERK1 in wheat, revealing distinct characteristics of CERK1 homologs in chitin recognition and immune activation between cereals and the dicot Arabidopsis. Notably, overexpressing CERK1s from both cereal and Arabidopsis in wheat confers broad‐spectrum resistance to fungal diseases. Our findings highlight the potential of plant immune receptors in controlling crop diseases. [ABSTRACT FROM AUTHOR]

SUMMARY: Changes in plant morphology due to mechanical stimulation are known as thigmo responses. As climbing organs in plants, tendrils can sense mechanical stimulation after attaching to a support and then change their morphology within a short time. Here, the thigmo responses of cucumber tendril were investigated. Our results showed that mechanical stimulation stopped tendril elongation and that tendril length was determined by the distance from the support in cucumber. The mimicry touch treatment indicated that mechanical stimulation stopped tendril elongation by inhibiting cell expansion. RNA‐seq data showed that three gibberellin (GA) metabolic genes (CsGA2ox3, CsCYP714A2, and CsCYP714A3) were upregulated in mechanically stimulated tendrils, and a major endogenous bio‐active GA (GA4) was reduced in mechanically stimulated tendrils. The roles of CsGA2ox3, CsCYP714A2, and CsCYP714A3 in GA deactivation were confirmed by their overexpression in transgenic Arabidopsis. Moreover, exogenous GA treatment recovered tendril elongation under mechanical stimulation, whereas exogenous uniconazole treatment inhibited tendril elongation without mechanical stimulation, suggesting that mechanical stimulation stopped tendril elongation, depending on GA deactivation. In summary, our results suggest that GA deactivation plays an important role in tendril thigmo response, ensuring that tendrils obtain a suitable final length according to their distance from the support in cucumber. [ABSTRACT FROM AUTHOR]

SUMMARY: Hydathodes are small organs found on the leaf margins of vascular plants which release excess xylem sap through a process called guttation. While previous studies have hinted at additional functions of hydathode in metabolite transport or auxin metabolism, experimental support is limited. We conducted comprehensive transcriptomic, metabolomic and physiological analyses of mature Arabidopsis hydathodes. This study identified 1460 genes differentially expressed in hydathodes compared to leaf blades, indicating higher expression of most genes associated with auxin metabolism, metabolite transport, stress response, DNA, RNA or microRNA processes, plant cell wall dynamics and wax metabolism. Notably, we observed differential expression of genes encoding auxin‐related transcriptional regulators, biosynthetic processes, transport and vacuolar storage supported by the measured accumulation of free and conjugated auxin in hydathodes. We also showed that 78% of the total content of 52 xylem metabolites was removed from guttation fluid at hydathodes. We demonstrate that NRT2.1 and PHT1;4 transporters capture nitrate and inorganic phosphate in guttation fluid, respectively, thus limiting the loss of nutrients during this process. Our transcriptomic and metabolomic analyses unveil an organ with its specific physiological and biological identity. Significance Statement: Hydathodes are vascular discontinuities at leaf margins of all vascular plants which allow the release of excess water during a process known as guttation. The physiology of this organ is poorly characterised. We conducted comprehensive transcriptomic, metabolomic and physiological analyses of mature hydathodes of Arabidopsis and demonstrated that those organs are sites of auxin accumulation and active nutrient scavenging. [ABSTRACT FROM AUTHOR]

SUMMARY Rhythmic oscillation of biological processes helps organisms adapt their physiological responses to the most appropriate time of the day. Chromatin remodeling has been described as one of the molecular mechanisms controlling these oscillations. The importance of these changes in transcriptional activation as well as in the maintenance of heterochromatic regions has been widely demonstrated. However, little is still known on how diurnal changes can impact the global status of chromatin modifications and, hence, control gene expression. In plants, the repressive mark H3K27me1, deposited by ARABIDOPSIS TRITHORAX‐RELATED PROTEIN 5 and 6 (ATXR5 and 6) methyltransferases, is largely associated with transposable elements but also covers lowly expressed genes. Here we show that this histone modification is preferentially deposited during the night. In euchromatic regions, it is found along the bodies of DNA damage response genes (DDR), where it is needed for their proper expression. The absence of H3K27me1 translates into an enhanced expression of DDR genes that follows a rhythmic oscillation pattern. This evidences a link between chromatin modifications and their synchronization with the diurnal cycle in order to accurately modulate the activation of biological processes to the most appropriate time of the day. [ABSTRACT FROM AUTHOR]

• A machine learning model, AtML, was constructed to predict the stages of Arabidopsis root cells and identify biomarkers. • Performance testing revealed that AtML achieved very high accuracy and recall. • The AtML model identified 160 important marker genes for annotating cell type. Arabidopsis thaliana synthesizes various medicinal compounds, and serves as a model plant for medicinal plant research. Single-cell transcriptomics technologies are essential for understanding the developmental trajectory of plant roots, facilitating the analysis of synthesis and accumulation patterns of medicinal compounds in different cell subpopulations. Although methods for interpreting single-cell transcriptomics data are rapidly advancing in Arabidopsis, challenges remain in precisely annotating cell identity due to the lack of marker genes for certain cell types. In this work, we trained a machine learning system, AtML, using sequencing datasets from six cell subpopulations, comprising a total of 6000 cells, to predict Arabidopsis root cell stages and identify biomarkers through complete model interpretability. Performance testing using an external dataset revealed that AtML achieved 96.50% accuracy and 96.51% recall. Through the interpretability provided by AtML, our model identified 160 important marker genes, contributing to the understanding of cell type annotations. In conclusion, we trained AtML to efficiently identify Arabidopsis root cell stages, providing a new tool for elucidating the mechanisms of medicinal compound accumulation in Arabidopsis roots. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (H2O2) is naturally produced by plant cells during normal development and serves as a messenger that regulates cell metabolism. Despite its importance, the relationship between hydrogen peroxide and the target of rapamycin (TOR) pathway, as well as its impact on cell division, has been poorly analyzed. In this study, we explore the interaction of H2O2 with TOR, a serine/threonine protein kinase that plays a central role in controlling cell growth, size, and metabolism in Arabidopsis thaliana. By applying two concentrations of H2O2 exogenously (0.5 and 1 mM), we could correlate developmental traits, such as primary root growth, lateral root formation, and fresh weight, with the expression of the cell cycle gene CYCB1;1, as well as TOR expression. When assessing the expression of the ribosome biogenesis-related gene RPS27B, an increase of 94.34% was noted following exposure to 1 mM H2O2 treatment. This increase was suppressed by the TOR inhibitor torin 2. The elimination of H2O2 accumulation with ascorbic acid (AA) resulted in decreased cell division as well as TOR expression. The potential molecular mechanisms associated with the effects of H2O2 on the cell cycle and TOR expression in roots are discussed in the context of the results. [ABSTRACT FROM AUTHOR]

Summary: Leaf senescence is a developmental program regulated by both endogenous and environmental cues. Abiotic stresses such as nutrient deprivation can induce premature leaf senescence, which profoundly impacts plant growth and crop yield. However, the molecular mechanisms underlying stress‐induced senescence are not fully understood.In this work, employing a carbon deprivation (C‐deprivation)‐induced senescence assay in Arabidopsis seedlings, we identified PLEIOTROPIC REGULATORY LOCUS 1 (PRL1), a component of the NineTeen Complex, as a negative regulator of C‐deprivation‐induced senescence.Furthermore, we demonstrated that PRL1 directly interacts with the RPA2A subunit of the single‐stranded DNA‐binding Replication Protein A (RPA) complex. Consistently, the loss of RPA2A leads to premature senescence, while increased expression of RPA2A inhibits senescence. Moreover, overexpression of RPA2A reverses the accelerated senescence in prl1 mutants, and the interaction with PRL1 stabilizes RPA2A under C‐deprivation.In summary, our findings reveal the involvement of the PRL1‐RPA2A functional module in C‐deprivation‐induced plant senescence. [ABSTRACT FROM AUTHOR]

Summary: In Arabidopsis, the enzymatically active lysin motif‐containing receptor‐like kinase (LysM‐RLK) CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) and the pseudokinases LYSIN MOTIF‐CONTAINING RECEPTOR‐LIKE KINASE 5 (LYK5) and LYK4 are the core components of the canonical chitin receptor complex. CERK1 dimerizes and autophosphorylates upon chitin binding, resulting in activation of chitin signaling.In this study, we clarified and further elucidated the individual contributions of LYK4 and LYK5 to chitin‐dependent signaling using mutant (combination)s and stably transformed Arabidopsis plants expressing fluorescence‐tagged LYK5 and LYK4 variants from their endogenous promoters.Our analyses revealed that LYK5 interacts with CERK1 upon chitin treatment, independently of LYK4 and vice versa. We show that chitin‐induced autophosphorylation of CERK1 is predominantly dependent on LYK5, whereas chitin‐triggered ROS generation is almost exclusively mediated by LYK4. This suggests specific signaling functions of these two co‐receptor proteins apart from their redundant function in mitogen‐activated protein kinase (MAPK) signaling and transcriptional reprogramming.Moreover, we demonstrate that LYK5 is subject to chitin‐induced and CERK1‐dependent ubiquitination, which serves as a signal for chitin‐induced internalization of LYK5. Our experiments provide evidence that a combination of phosphorylation and ubiquitination events controls LYK5 removal from the plasma membrane via endocytosis, which likely contributes to receptor complex desensitization. [ABSTRACT FROM AUTHOR]

Summary: Arabidopsis PSEUDORESPONSE REGULATOR7 (PRR7) is a core component of the circadian oscillator which also plays a crucial role in freezing tolerance. PRR7 undergoes proteasome‐dependent degradation to discretely phase maximal expression in early evening. While its repressive activity on downstream genes is integral to cold regulation, the mechanism of the conditional regulation of the PRR7 abundance is unknown.We used mutant analysis, protein interaction and ubiquitylation assays to establish that the ubiquitin ligase adaptor, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), controls the protein accumulation pattern of PRR7 through direct protein–protein interactions at low temperatures. Freezing tolerance and electrolyte leakage assays show that PRR7 enhances cold temperature sensitivity, supported by ChIP‐qPCR at C‐REPEAT BINDING FACTOR1 (CBF1) and COLD‐REGULATED 15A (COR15A) promoters where PRR7 levels were higher in hos15 mutants.HOS15 mediates PRR7 turnover through enhanced ubiquitylation at low temperature in the dark. Under the same conditions, increased PRR7 association with the promoters of CBFs and COR15A in hos15 correlates with decreased CBF1 and COR15A transcription and enhanced freezing sensitivity.We propose a novel mechanism whereby HOS15‐mediated degradation of PRR7 provides an intersection between the circadian system and other cold acclimation pathways that lead to increased freezing tolerance. [ABSTRACT FROM AUTHOR]

Flooding events are highly detrimental to most terrestrial plant species. However, there is an impressive diversity of plant species that thrive in flood‐prone regions and represent a treasure trove of unexplored flood‐resilience mechanisms. Here we surveyed a panel of four species from the Cardamineae tribe representing a broad tolerance range. This included the flood‐tolerant Cardamine pratensis, Rorippa sylvestris and Rorippa palustris and the flood‐sensitive species Cardamine hirsuta. All four species displayed a quiescent strategy, evidenced by the repression of shoot growth underwater. Comparative transcriptomics analyses between the four species and the sensitive model species Arabidopsis thaliana were facilitated via de novo transcriptome assembly and identification of 16 902 universal orthogroups at a high resolution. Our results suggest that tolerance likely evolved separately in the Cardamine and Rorippa species. While the Rorippa response was marked by a strong downregulation of cell‐cycle genes, Cardamine minimized overall transcriptional regulation. However, a weak starvation response was a universal trait of tolerant species, potentially achieved in multiple ways. It could result from a strong decline in cell‐cycle activity, but is also intertwined with autophagy, senescence, day‐time photosynthesis and night‐time fermentation capacity. Our data set provides a rich source to study adaptational mechanisms of flooding tolerance. Summary Statement: This collection of submergence‐induced transcriptome responses of Brassicaceae species with strong variation in tolerance provides a treasure trove for the study of adaptational mechanisms of flooding tolerance. [ABSTRACT FROM AUTHOR]

Presence of cadmium in soil affects the growth status of plants; research on the underlying mechanism has focused on its lethal and sublethal consequences in plants, ignoring the impacts of soil organisms. Therefore, this study designed a microorganism – earthworm – plant ecosystem, investigated the influence of Cd (0, 0.3, 1.0, and 3.0 mg kg−1) on the growth of Arabidopsis thaliana in the presence of earthworms (Eisenia fetida), and revealed the mechanisms from the perspective of altered relationships among different soil organisms. In this study, there was no significant difference in the death rate of earthworms with low Cd addition concentration, but the microorganisms in earthworms and soil microorganisms changed, abundance of Actinomycetes in soil increased, while Planctomycetes decreased, Mycobacterium in the gut of Eisenia fetida decreased under Cd stress. Microorganisms affect soil pH, which in turn affects soil physicochemical properties and the form of cadmium in soil. Compared with the control, soil pH significantly decreases under cadmium stress, Under 0.3 mg kg−1 treatment, soil total nitrogen, available phosphorus and total potassium are 181.11, 5.09, 51.90, and 7.55 mg kg−1, respectively, They were significantly higher than control group. Improved the growth environment of Arabidopsis thaliana and promoted the absorption of Cd in soil which affected the proportion of available Cd in the soil (when Cd was added at 1.0 mg kg−1, the proportion of available Cd in the soil decreased significantly from 95.05% to 49.40%). A. thaliana also absorbed more Cd, which increased Cd toxicity, and the reproductive growth of A. thaliana was enhanced, while vegetative growth was weakened.Compared with the control group (278.11 mg), the biomass of A. thaliana decreased significantly to 189.83 mg when Cd was supplemented at 0.3 mg kg−1. Therefore, the activities of and interactions among earthworms, plants, and microorganisms play important roles in reducing soil heavy metals and improving soil properties. HIGHLIGHTS: Under cadmium (Cd) stress, the relative abundance of Actinomycetes in soil increased, while Planctomycetes decreased. The relative abundance of Mycobacterium in the gut of Eisenia fetida decreased under Cd stress. Microbial changes and E. fetida activity changed soil physical and chemical properties and reduced the ratio of soil available Cd. Microorganisms and E. fetida enhanced the reproductive growth and weakened the vegetative growth of Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

Main conclusion: PATOL1 contributes to increasing biomass not only by effective stomatal movement but also by root meristematic activity. PATROL1 (PROTON ATPase TRANSLOCATION CONTROL 1), a protein with a MUN domain, is involved in the intercellular trafficking of AHA1 H+-ATPase to the plasma membrane in guard cells. This allows for larger stomatal opening and more efficient photosynthesis, leading to increased biomass. Although PATROL1 is expressed not only in stomata but also in other tissues of the shoot and root, the role in other tissues than stomata has not been determined yet. Here, we investigated PATROL1 functions in roots using a loss-of-function mutant and an overexpressor. Cytological observations revealed that root meristematic size was significantly smaller in the mutant resulting in the short primary root. Grafting experiments showed that the shoot biomass of the mutant scion was increased when it grafted onto wild-type or overexpressor rootstocks. Conversely, grafting of the overexpressor scion shoot enhanced the growth of the mutant rootstock. The leaf temperatures of the grafted plants were consistent with those of their respective genotypes, indicating cell-autonomous behavior of stomatal movement and independent roles of PATROL1 in plant growth. Moreover, plasma membrane localization of AHA1 was not altered in root epidermal cells in the patrol1 mutant implying existence of a different mode of PATROL1 action in roots. Thus PATROL1 plays a role in root meristem and contributes to increase shoot biomass. [ABSTRACT FROM AUTHOR]

Phytochrome B (phyB) and phytochrome-interacting factors (PIFs) constitute a well-established signaling module critical for plants adapting to ambient light. However, mechanisms underlying phyB photoactivation and PIF binding for signal transduction remain elusive. Here, we report the cryo-electron microscopy (cryo-EM) structures of the photoactivated phyB or the constitutively active phyBY276H mutant in complex with PIF6, revealing a similar trimer. The light-induced configuration switch of the chromophore drives a conformational transition of the nearby tongue signature within the phytochrome-specific (PHY) domain of phyB. The resulting α-helical PHY tongue further disrupts the head-to-tail dimer of phyB in the dark-adapted state. These structural remodelings of phyB facilitate the induced-fit recognition of PIF6, consequently stabilizing the N-terminal extension domain and a head-to-head dimer of activated phyB. Interestingly, the phyB dimer exhibits slight asymmetry, resulting in the binding of only one PIF6 molecule. Overall, our findings solve a key question with respect to how light-induced remodeling of phyB enables PIF signaling in phytochrome research. [Display omitted] • Red light activates phyB by remodeling the head-to-tail dimer in the dark-adapted state • PIF6-APB binding stabilizes the NTE domain and a head-to-head dimer of activated phyB • The asymmetric phyB dimer can accommodate only one PIF6-APB molecule • The constitutively active phyBY276H assembles a similar trimeric complex with PIF6-APB Cryo-EM structures of Arabidopsis photoactivated phyB and its constitutively active phyBY276H mutant in complex with the N terminus of PIF6 provide insights into light-induced photoreceptor remodeling and signal transduction. [ABSTRACT FROM AUTHOR]

Stomatal pores in leaves mediate CO2 uptake into the plant and water loss via transpiration. Most plants are hypostomatous with stomata present only in the lower leaf surface (abaxial epidermis). Many herbs, including the model plant Arabidopsis, have substantial numbers of stomata also on the upper (adaxial) leaf surface. Studies of stomatal development have mostly focused on abaxial stomata and very little is known of adaxial stomatal formation. We analysed the role of leaf number in determining stomatal density and stomatal ratio, and studied adaxial and abaxial stomatal patterns in Arabidopsis mutants deficient in known abaxial stomatal development regulators. We found that stomatal density in some genetic backgrounds varies between different fully expanded leaves, and thus we recommend using defined leaves for analyses of stomatal patterning. Our results indicate that stomatal development is at least partly independently regulated in adaxial and abaxial epidermis, as (i) plants deficient in ABA biosynthesis and perception have increased stomatal ratios, (ii) the epf1epf2 , tmm , and sdd1 mutants have reduced stomatal ratios, (iii) erl2 mutants have increased adaxial but not abaxial stomatal index, and (iv) stomatal precursors preferentially occur in abaxial epidermis. Further studies of adaxial stomata can reveal new insights into stomatal form and function. [ABSTRACT FROM AUTHOR]

Both the pollen tube and hyphae of filamentous pathogens penetrate the outer layer of the host and then grow within host tissues. Early epidermal responses are decisive for the outcome of these two-cell interaction processes. We identified a single cell type, the papilla in the stigma of Arabidospis, as a tool to conduct a comprehensive comparative analysis on how an epidermal cell responds to the invasion of an unwanted pathogen or a welcome pollen tube. We showed that Phytophtora parasitica , a root oomycete, effectively breaches the stigmatic cell wall and develops as a biotroph within the papilla cytoplasm. These invasive features resemble the behaviour exhibited by the pathogen within its natural host cell, but diverge from the manner in which the pollen tube progresses, being engulfed within the papilla cell wall. Quantitative analysis revealed that both invaders trigger reorganization of the stigmatic endomembrane system and the actin cytoskeleton. While some remodelling processes are shared between the two interactions, others appear more specific towards the respective invader. These findings underscore the remarkable ability of an epidermal cell to differentiate between two types of invaders, thereby enabling it to trigger the most suitable response during the onset of invasion. [ABSTRACT FROM AUTHOR]

The four-carbon non-proteinogenic amino acid γ-aminobutyric acid (GABA) accumulates to high levels in plants in response to various abiotic and biotic stress stimuli, and plays a role in C:N balance, signaling, and as a transport regulator. Expression in Xenopus oocytes and voltage-clamping allowed the characterization of Arabidopsis GAT2 (At5g41800) as a low affinity GABA transporter with a K 0.5 G A B A ~8 mM. l -Alanine and butylamine represented additional substrates. GABA-induced currents were strongly dependent on the membrane potential, reaching the highest affinity and highest transport rates at strongly negative membrane potentials. Mutation of Ser17, previously reported to be phosphorylated in planta, did not result in altered affinity. In a short-term stress experiment, AtGAT2 mRNA levels were up-regulated at low water potential and under osmotic stress (polyethylene glycol and mannitol). Furthermore, AtGAT2 promoter activity was detected in vascular tissues, maturating pollen, and the phloem unloading region of young seeds. Even though this suggested a role for AtGAT2 in long-distance transport and loading of sink organs, under the conditions tested neither AtGAT2 -overexpressing plants, atgat2 or atgat1 T-DNA insertion lines, nor atgat1 atgat2 doubleknockout mutants differed from wild-type plants in growth on GABA, amino acid levels, or resistance to salt and osmotic stress. [ABSTRACT FROM AUTHOR]

SUMMARY Land plants have evolved a hydrophobic cuticle on the surface of aerial organs as an adaptation to ensure survival in terrestrial environments. Cuticle is mainly composed of lipids, namely cutin and intracuticular wax, with epicuticular wax deposited on plant surface. The composition and permeability of cuticle have a large influence on its ability to protect plants against drought stress. However, the regulatory mechanisms underlying cuticular wax biosynthesis in response to drought stress have not been fully elucidated. Here, we identified three AP2/ERF transcription factors (DREB26/ERF12, ERF13 and ERF14) involved in the regulation of water permeability of the plant surface. Transmission electron microscopy revealed thicker cuticle on the leaves of DREB26‐overexpressing (DREB26OX) plants, and thinner cuticle on the leaves of transgenic plants expressing SRDX repression domain‐fused DREB26 (DREB26SR). Genes involved in cuticular wax formation were upregulated in DREB26OX and downregulated in DREB26SR. The levels of very‐long chain (VLC) alkanes, which are a major wax component, increased in DREB26OX leaves and decreased in DREB26SR leaves. Under dehydration stress, water loss was reduced in DREB26OX and increased in DREB26SR. The erf12/13/14 triple mutant showed delayed growth, decreased leaf water content, and reduced drought‐inducible VLC alkane accumulation. Taken together, our results indicate that the DREB26/ERF12 and its closed family members, ERF13 and ERF14, play an important role in cuticular wax biosynthesis in response to drought stress. The complex transcriptional cascade involved in the regulation of cuticular wax biosynthesis under drought stress conditions is discussed. [ABSTRACT FROM AUTHOR]

ABSTRACT Nitrate (NO3−) deficiency decreases root water uptake and root hydraulic conductance. This adaptive response is correlated with reduced abundance and activity of plasma membrane intrinsic protein (PIP) aquaporins. We therefore screened changes in the root architecture of a complete set of Arabidopsis pip loss‐of‐function mutants grown under NO3− deficiency to systematically approach the impact of PIPs under these conditions. NO3− deprivation led to attenuated responses of specific pip single mutants compared to the strongly altered LR parameters of wild‐type plants. In particular, pip1;1 exhibited a lower relative reduction in LR length and LR density, revealing that PIP1;1 represses LR development when NO3− is scarce. Indeed, PIP1;1 compromises root and shoot NO3− accumulation during early developmental stages. A fluorescent VENUS‐PIP1;1 fusion revealed that PIP1;1 is specifically repressed in the pericycle, endodermis and at the flanks of emerging LRs upon NO3− deficiency. Thus, LR plasticity and NO3− uptake are affected by an interactive mechanism involving aquaporins (PIP1;1) and nitrate accumulation during seedling development under NO3−‐deficient conditions. [ABSTRACT FROM AUTHOR]

The addition of non-templated nucleotides at the 3' terminus of RNA is a pervasive and evolutionarily conserved posttranscriptional modification in eukaryotes. Apart from canonical poly(A) polymerases (PAPs), which are responsible for catalyzing polyadenylation of messenger RNAs in the nucleus, a distinct group of non-canonical PAPs (ncPAPs), also known as nucleotidyl transferase proteins (NTPs), mediate the addition of uridine and adenosine or of more intricate combinations of nucleotides. Among these, HEN1 SUPPRESSOR 1 (HESO1) and UTP: RNA URIDYLYLTRANSFERASE (URT1) are the two most extensively studied NTPs responsible for the addition of uridine to the 3' ends of RNAs (RNA uridylation). Recent discoveries have improved our understanding of the functions and mechanisms of uridylation mediated by HESO1 and URT1 in RNA metabolism. Furthermore, more NTPs have been identified to function in the 3' tailing of RNA and not solely through uridylation. Accumulating evidence indicates that RNA tailing plays important roles in plant growth and development, stress responses, and disease resistance. In this review, we examined the latest developments in RNA tailing by NTPs, with a focus on RNA uridylation and metabolism in plants. We also discussed the essential aspects for future research in this field. [ABSTRACT FROM AUTHOR]

Developmental reprogramming allows for flexibility in growth and adaptation to changing environmental conditions. In plants, wounding events can result in new stem cell niches and lateral organs. Adventitious roots develop from aerial parts of the plant and are regulated by multiple stimuli, including wounding. Here, we find that Arabidopsis thaliana seedlings wounded at the hypocotyl-root junction reprogram certain pericycle cells to produce adventitious roots proximal to the wound site. We have determined that competence for this reprogramming is controlled; basal cells close to the wound site can produce adventitious roots, whereas cells distal from the wound site mostly cannot. We found that altering cytokinin response or indole-3-butyric acid (IBA)-to-(indole-3-acetic acid) IAA conversion resulted in an expanded adventitious root competence zone and delineated the connection between these pathways. Our work highlights the importance of endogenous IBA-derived auxin and its interaction with cytokinin in adventitious root formation and the regenerative properties of plants. [Display omitted] • Excision-induced adventitious roots (ARs) form proximal to the wound site • Endogenous IBA-derived auxin promotes AR formation • IBA-derived auxin regulates the zone of AR formation • Cytokinin regulates IBA-derived auxin to control the AR competence zone Damodaran and Strader determine that, in Arabidopsis, adventitious roots created in response to wounding occur proximal to the wound site, suggesting that the wound site sets a zone of competence for cellular reprogramming. Cytokinin and IBA-derived auxin dictate this competence zone. [ABSTRACT FROM AUTHOR]

N 6-Methyladenosine (m6A) prevalently occurs on cellular RNA across almost all kingdoms of life. It governs RNA fate and is essential for development and stress responses. However, the dynamic, context-dependent m6A methylomes across tissues and in response to various stimuli remain largely unknown in multicellular organisms. Here, we generate a comprehensive census that identifies m6A methylomes in 100 samples during development or following exposure to various external conditions in Arabidopsis thaliana. We demonstrate that m6A is a suitable biomarker to reflect the developmental lineage, and that various stimuli rapidly affect m6A methylomes that constitute the regulatory network required for an effective response to the stimuli. Integrative analyses of the census and its correlation with m6A regulators identify multiple layers of regulation on highly context-dependent m6A modification in response to diverse developmental and environmental stimuli, providing insights into m6A modification dynamics in the myriad contexts of multicellular organisms. [Display omitted] • m6A dynamics of Arabidopsis development and response to stimuli are surveyed • m6A is a suitable biomarker to reflect the developmental lineage • Reversible reprogramming of m6A occurs during stress treatment and recovery • Multiple layers of regulation determine highly context-dependent functions of m6A N 6-Methyladenosine (m6A) prevalently occurs on cellular RNA across almost all kingdoms of life. Zhang et al. report a comprehensive m6A census of Arabidopsis development and response to stimuli, which offers insights into the highly context-dependent nature of m6A signatures and their underlying modulatory mechanisms in multicellular organisms. [ABSTRACT FROM AUTHOR]