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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.

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.

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. [ABSTRACT FROM AUTHOR]

Summary First‐generation polyploids often suffer from more meiotic errors and lower fertility than established wild polyploid populations. One such example is the allopolyploid model species Arabidopsis suecica which originated c. 16 000 generations ago. We present here a comparison of meiosis and its outcomes in naturally evolved and first‐generation ‘synthetic’ A. suecica using a combination of cytological and genomic approaches. We show that while meiosis in natural lines is largely diploid‐like, synthetic lines have high levels of meiotic errors including incomplete synapsis and nonhomologous crossover formation. Whole‐genome re‐sequencing of progeny revealed 20‐fold higher levels of homoeologous exchange and eightfold higher aneuploidy originating from synthetic parents. Homoeologous exchanges showed a strong distal bias and occurred predominantly in genes, regularly generating novel protein variants. We also observed that homoeologous exchanges can generate megabase scale INDELs when occurring in regions of inverted synteny. Finally, we observed evidence of sex‐specific differences in adaptation to polyploidy with higher success in reciprocal crosses to natural lines when synthetic plants were used as the female parent. Our results directly link cytological phenotypes in A. suecica with their genomic outcomes, demonstrating that homoeologous crossovers underlie genomic instability in neo‐allopolyploids and are more distally biased than homologous crossovers. [ABSTRACT FROM AUTHOR]

Plastids are essential, semi-autonomous organelles in plants that carry out a multitude of functions during development. Plastids existing in different subtypes are derived from proplastids progenitors and interconvert in response to environmental and growth cues. Most efforts focus on the differentiation from proplastid to other forms. However, the studies of proplastid development are insufficient and whether proplastid biogenesis affects plant growth is yet to be determined. Arabidopsis TIC236, a translocon component at the inner membrane of the chloroplast envelope, is critical for importing chloroplasttargeted preproteins and chloroplast division. In this study, we uncovered the fundamental influence of proplastid biogenesis on embryo development by exploring the function of TIC236 during embryogenesis. Widespread and strong expression of TIC236 was observed in leaves and embryos. The null mutant tic236 had an embryo-lethal phenotype, with cell division in the mutant embryos delayed starting at the octant stage and arrested at the globular stage. Transmission electron microscopy revealed enlarged proplastids with an aberrant inner structure at the dermatogen and globular stages that ultimately did not differentiate into chloroplasts. Additionally, the fluorescence signal distribution patterns of tic236 embryos carrying the pDR5rev::3xVENUS-N7, pPIN1::PIN1-GFP, pWOX5::GFP, and pSCR::H2B-YFP reporter systems were altered. Together, we provide genetic evidence supporting proplastid biogenesis plays a vital role in embryo development and TIC236 is identified as an indispensable player, ensuring normal proplastid development. [ABSTRACT FROM AUTHOR]

Actin depolymerizing factors (ADFs), as the important actin-binding proteins (ABPs) with depolymerizing/severing actin filaments, play a critical role in plant growth and development, and in response to biotic and abiotic stresses. However, the information and function of the ADF family in melon remains unclear. In this study, 9 melon ADF genes (CmADFs) were identified, distributed in 4 subfamilies, and located on 6 chromosomes respectively. Promoter analysis revealed that the CmADFs contained a large number of cis-acting elements related to hormones and stresses. The similarity of CmADFs with their Arabidopsis homologue AtADFs in sequence, structure, important sites and tissue expression confirmed that ADFs were conserved. Gene expression analysis showed that CmADFs responded to low and high temperature stresses, as well as ABA and SA signals. In particular, CmADF1 was significantly up-regulated under above all stress and hormone treatments, indicating that CmADF1 plays a key role in stress and hormone signaling responses, so CmADF1 was selected to further study the mechanism in plant tolerance low temperature. Under low temperature, virus-induced gene silencing (VIGS) of CmADF1 in oriental melon plants showed increased sensitivity to low temperature stress. Consistently, the stable genetic overexpression of CmADF1 in Arabidopsis improved their low temperature tolerance, possibly due to the role of CmADF1 in the depolymerization of actin filaments. Overall, our findings indicated that CmADF genes, especially CmADF1, function in response to abiotic stresses in melon. [ABSTRACT FROM AUTHOR]

The long-term balancing selection acting on mating types or sex-determining genes is expected to lead to the accumulation of deleterious mutations in the tightly linked chromosomal segments that are locally ‘sheltered’ from purifying selection. However, the factors determining the extent of this accumulation are poorly understood. Here, we took advantage of variations in the intensity of balancing selection along a dominance hierarchy formed by alleles at the sporophytic self-incompatibility system of the Brassicaceae to compare the pace at which linked deleterious mutations accumulate among them. We first experimentally measured the phenotypic manifestation of the linked load at three different levels of the dominance hierarchy. We then sequenced and phased polymorphisms in the chromosomal regions linked to 126 distinct copies of S-alleles in two populations of Arabidopsis halleri and three populations of Arabidopsis lyrata. We find that linkage to the S-locus locally distorts phylogenies over about 10–30 kb along the chromosome. The more intense balancing selection on dominant S-alleles results in greater fixation of linked deleterious mutations, while recessive S-alleles accumulate more linked deleterious mutations that are segregating. Hence, the structure rather than the overall magnitude of the linked genetic load differs between dominant and recessive S-alleles. Our results have consequences for the long-term evolution of new S-alleles, the evolution of dominance modifiers between them, and raise the question of why the non-recombining regions of some sex and mating type chromosomes expand over evolutionary times while others, such as the S-locus of the Brassicaceae, remain restricted to small chromosomal regions. [ABSTRACT FROM AUTHOR]

Summary Activity‐based sensing probes are powerful tools for monitoring enzymatic activities in complex biological samples such as cellular and live animals; however, their application in plants remains challenging. Herein, fourteen activity‐based fluorescent probes were assayed against Arabidopsis O‐methyltransferases (AtOMTs). One probe, 3‐BTD, displayed a high selectivity, reactivity, and fluorescence response toward AtOMTs especially the isoform AtCCoAOMT. We further characterized the features of this probe and explored whether it could be used to detect OMT activities in living plant cells. Our results show that 3‐BTD can be used to visualize OMT activity in Arabidopsis, and no fluorescent signal was observed in the comt/ccoaomt double mutant, indicating that it has good specificity. Interestingly, in contrast to the observation that AtCCoAOMT‐YFP accumulated in both cytoplasm and nucleus, OMT enzymatic activity tracked by 3‐BTD probe was found only in the cytoplasm. This underscores the importance of activity‐based sensing in studying protein function. Moreover, 3‐BTD can be successfully applied in OMT visualization of different plants. This study indicates that 3‐BTD can serve as a potential probe for in situ monitoring the real activity of OMT in multiple plants and provides a strategy for visualizing the activity of other enzymes in plants. [ABSTRACT FROM AUTHOR]

The developmental plasticity of the root system plays an essential role in the adaptation of plants to the environment. Among many other signals, auxin and its directional, intercellular transport are critical in regulating root growth and development. In particular, the PIN-FORMED2 (PIN2) auxin exporter acts as a key regulator of root gravitropic growth. Multiple regulators have been reported to be involved in PIN2-mediated root growth; however, our information remains incomplete. Here, we identified ROWY Bro1-domain proteins as important regulators of PIN2 sorting control. Genetic analysis revealed that Arabidopsis rowy1 single mutants and higher-order rowy1 rowy2 rowy3 triple mutants presented a wavy root growth phenotype. Cell biological experiments revealed that ROWY1 and PIN2 colocalized to the apical side of the plasma membrane in the root epidermis and that ROWYs are required for correct PM targeting of PIN2. In addition, ROWYs also affected PIN3 protein abundance in the stele, suggesting the potential involvement of additional PIN transporters as well as other proteins. A global transcriptome analysis revealed that ROWY genes are involved in the Fe2+ availability perception pathway. This work establishes ROWYs as important novel regulators of root gravitropic growth by connecting micronutrient availability to the proper subcellular targeting of PIN auxin transporters. A study on the PIN2 auxin exporter identifies the ROWY BRO1-domain proteins as important regulators of PIN2 sorting control as well as PIN3 abundance, which both affect auxin transport. [ABSTRACT FROM AUTHOR]

Key message: GPI anchor addition is important for JAGGER localization and in vivo function. Loss of correct GPI anchor addition in JAGGER, negatively affects its localization and function. In flowering plants, successful double fertilization requires the correct delivery of two sperm cells to the female gametophyte inside the ovule. The delivery of a single pair of sperm cells is achieved by the entrance of a single pollen tube into one female gametophyte. To prevent polyspermy, Arabidopsis ovules avoid the attraction of multiple pollen tubes to one ovule–polytubey block. In Arabidopsisjagger mutants, a significant number of ovules attract more than one pollen tube to an ovule due to an impairment in synergid degeneration. JAGGER encodes a putative arabinogalactan protein which is predicted to be anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Here, we show that JAGGER fused to citrine yellow fluorescent protein (JAGGER-cYFP) is functional and localizes mostly to the periphery of ovule integuments and transmitting tract cells. We further investigated the importance of GPI-anchor addition domains for JAGGER localization and function. Different JAGGER proteins with deletions in predicted ω-site regions and GPI attachment signal domain, expected to compromise the addition of the GPI anchor, led to disruption of JAGGER localization in the cell periphery. All JAGGER proteins with disrupted localization were also not able to rescue the polytubey phenotype, pointing to the importance of GPI-anchor addition to in vivo function of the JAGGER protein. [ABSTRACT FROM AUTHOR]

Key message: The VAMP721, VAMP722, SYP121, SYP122 and SNAP33 SNAREs are required in the Arabidopsis stigma for pollen hydration, further supporting a role for vesicle trafficking in the stigma's pollen responses. In the Brassicaceae, the process of accepting compatible pollen is a key step in successful reproduction and highly regulated following interactions between the pollen and the stigma. Central to this is the initiation of secretion in the stigma, which is proposed to provide resources to the pollen for hydration and germination and pollen tube growth. Previously, the eight exocyst subunit genes were shown to be required in the Arabidopsis stigma to support these pollen responses. One of the roles of the exocyst is to tether secretory vesicles at the plasma membrane for membrane fusion by the SNARE complex to enable vesicle cargo release. Here, we investigate the role of Arabidopsis SNARE genes in the stigma for pollen responses. Using a combination of different knockout and knockdown SNARE mutant lines, we show that VAMP721, VAMP722, SYP121, SYP122 and SNAP33 are involved in this process. Significant disruptions in pollen hydration were observed following pollination of wildtype pollen on the mutant SNARE stigmas. Overall, these results place the Arabidopsis SNARE complex as a contributor in the stigma for pollen responses and reaffirm the significance of secretion in the stigma to support the pollen–stigma interactions. [ABSTRACT FROM AUTHOR]

Fusarium graminearum causes Fusarium head blight (FHB) disease in wheat worldwide. Although F. graminearum is reported to secrete several effectors, their role in virulence and pathogenicity is unknown. The study aimed at identifying candidate genes with a role in pathogenicity and virulence using two different host systems, Arabidopsis thaliana and wheat, challenged with F. graminearum TN01. Detached leaf assay and histological studies revealed the virulent nature of TN01. A genome-wide in silico search revealed several candidate genes, of which 23 genes were selected based on reproducibility. Gene expression studies by reverse transcriptase–polymerase chain reaction (RT-PCR) in leaf tissues of Arabidopsis and the two wheat genotypes, the susceptible (Sonalika) and the resistant (Nobeoka Bozu/Nobeoka), compared with mock-treated controls in a time-course study using fungal- and plant-specific genes as internal controls revealed that these genes were differentially regulated. Further, expression of these candidates in F. graminearum–inoculated Sonalika and Nobeoka spikes compared with mock-treated controls revealed their role in pathogenicity and virulence. Gene ontology studies revealed that some of these secretory proteins possessed a role in apoptosis and ceratoplatanin and KP4 killer toxin syntheses. A three-dimensional protein configuration was performed by homology modeling using trRosetta. Further, real-time quantitative PCR (RT-qPCR) studies in F. graminearum–inoculated Arabidopsis and wheat at early time points of inoculation revealed an increased expression of the majority of these genes in Sonalika, suggesting their possible role in pathogenicity, whereas low mRNA abundance was observed for 11 of these genes in the resistant genotype, Nobeoka, compared with Sonalika, indicating their role in virulence of F. graminearum. [ABSTRACT FROM AUTHOR]

Summary: Vascular bundles transport water and photosynthate to all organs, and increased bundle number contributes to crop lodging resistance. However, the regulation of vascular bundle formation is poorly understood in the Arabidopsis stem.We report a novel semi‐dominant mutant with high vascular activity, hva‐d, showing increased vascular bundle number and enhanced cambium proliferation in the stem. The activation of a C2H2 zinc finger transcription factor, AT5G27880/HVA, is responsible for the hva‐d phenotype. Genetic, biochemical, and fluorescent microscopic analyses were used to dissect the functions of HVA.HVA functions as a repressor and interacts with TOPLESS via the conserved Ethylene‐responsive element binding factor‐associated Amphiphilic Repression motif. In contrast to the HVA activation line, knockout of HVA function with a CRISPR‐Cas9 approach or expression of HVA fused with an activation domain VP16 (HVA‐VP16) resulted in fewer vascular bundles. Further, HVA directly regulates the expression of the auxin transport efflux facilitator PIN1, as a result affecting auxin accumulation. Genetics analysis demonstrated that PIN1 is epistatic to HVA in controlling bundle number.This research identifies HVA as a positive regulator of vascular initiation through negatively modulating auxin transport and sheds new light on the mechanism of bundle formation in the stem. [ABSTRACT FROM AUTHOR]

Summary: Boron (B) is crucial for plant growth and development. B deficiency can impair numerous physiological and metabolic processes, particularly in root development and pollen germination, seriously impeding crop growth and yield. However, the molecular mechanism underlying boron signal perception and signal transduction is rather limited.In this study, we discovered that CPK10, a calcium‐dependent protein kinase in the CPK family, has the strongest interaction with the boron transporter BOR1. Mutations in CPK10 led to growth and root development defects under B‐deficiency conditions, while constitutively active CPK10 enhanced plant tolerance to B deficiency.Furthermore, we found that CPK10 interacted with and phosphorylated BOR1 at the Ser689 residue. Through various biochemical analyses and complementation of B transport in yeast and plants, we revealed that Ser689 of BOR1 is important for its transport activity.In summary, these findings highlight the significance of the CPK10‐BOR1 signaling pathway in maintaining B homeostasis in plants and provide targets for the genetic improvement of crop tolerance to B‐deficiency stress. See also the Commentary on this article by Huang, 243: 1633–1635. [ABSTRACT FROM AUTHOR]

[Display omitted] • Phytochrome B variant phyB[G515E] with a slow dark reversion was generated. • phyB[G515E] forms warm temperature-insensitive nuclear photobodies. • PHYB[G515E] expression inhibited the phytochrome interacting factor4-auxin pathway. • PHYB[G515E] transgenic Arabidopsis and rice showed warming-insensitive growth. • phyB[G515E] is a powerful tool for engineering warm temperature-insensitive crops. Ambient temperature-induced hypocotyl elongation in Arabidopsis seedlings is sensed by the epidermis-localized phytochrome B (phyB) and transduced into auxin biosynthesis via a basic helix-loop-helix transcription factor, phytochrome-interacting factor 4 (PIF4). Once synthesized, auxin travels down from the cotyledons to the hypocotyl, triggering hypocotyl cell elongation. Thus, the phyB–PIF4 module involved in thermosensing and signal transduction is a potential genetic target for engineering warm temperature-insensitive plants. This study aims to manipulate warm temperature-induced elongation of plants at the post-translational level using phyB variants with dark reversion, the expression of which is subjected to heat stress. The thermosensitive growth response of Arabidopsis was manipulated by expressing the single amino acid substitution variant of phyB (phyB[G515E]), which exhibited a lower dark reversion rate than wild-type phyB. Other variants with slow (phyB[G564E]) or rapid (phyB[S584F]) dark reversion or light insensitivity (phyB[G767R]) were also included in this study for comparison. Warming-induced transient expression of phyB variants was achieved using heat shock-inducible promoters. Arabidopsis PHYB[G515E] and PHYB[G564E] were also constitutively expressed in rice in an attempt to manipulate the heat sensitivity of a monocotyledonous plant species. At an elevated temperature, Arabidopsis seedlings transiently expressing PHYB[G515E] under the control of a heat shock-inducible promoter exhibited shorter hypocotyls than those expressing PHYB and other PHYB variant genes. This warm temperature-insensitive growth was related to the lowered PIF4 and auxin responses. In addition, transgenic rice seedlings expressing Arabidopsis PHYB[G515E] and PHYB[G564E] showed warm temperature-insensitive shoot growth. Transient expression of phyB variants with altered dark reversion rates could serve as an effective optogenetic technique for manipulating PIF4–auxin-mediated thermomorphogenic responses in plants. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) is the largest organelle in terms of membrane content, occupying the entire cytoplasmic volume. It is tethered to the cell cortex through ER‐plasma membrane contact sites (EPCS). Previous studies have shown that EPCSs labelled by VAP27 align with cortical microtubules, and that ER tubules elongate along microtubules. Here, we addressed the question whether this relationship is bidirectional, with EPCSs influencing microtubule organisation. Using TIRF microscopy to track EPCSs and microtubule dynamics simultaneously, we demonstrate that while EPCSs remain stable, microtubules are highly dynamic and can adjust their positioning based on nearby EPCS in Arabidopsis cotyledon epidermis. In lobes of epidermal cells enclosed by two indentations, where microtubules bundle together, EPCSs flank the bundles and exhibit a distinctive arrangement, forming symmetric arcs in relation to the lobe axis. In guard cells, transversely oriented ER tubules co‐align with microtubules. Disrupting microtubules with the drug oryzalin leads to transient guard cells‐ER remodelling, followed by its reorganisation into transverse tubules before microtubule recovery. Taken together our observations suggest, that the positioning of EPCSs and cortical microtubules, can affect each other and the organisation of cortical ER. [ABSTRACT FROM AUTHOR]

Cytokinin oxidase/dehydrogenase (CKX) inhibitors reduce the degradation of cytokinins in plants and thereby may improve the efficiency of agriculture and plant tissue culture-based practices. Here, we report a synthesis and structure–activity relationship study of novel urea derivatives concerning their CKX inhibitory activity. The most active compounds showed sub-nanomolar IC50 values with maize ZmCKX1, the lowest value yet documented. Other CKX isoforms of maize and Arabidopsis were also inhibited very effectively. The binding mode of four compounds was characterized based on high-resolution crystal complex structures. Using the soil nematode Caenorhabditis elegans , and human skin fibroblasts, key CKX inhibitors with low toxicity were identified. These compounds enhanced the shoot regeneration of Lobelia , Drosera , and Plectranthus , as well as the growth of Arabidopsis and Brassica napus. At the same time, a key compound (identified as 82) activated a cytokinin primary response gene, ARR5:GUS , and a cytokinin sensor, TCSv2:GUS , without activating the Arabidopsis cytokinin receptors AHK3 and AHK4. This strongly implies that the effect of compound 82 is due to the up-regulation of cytokinin signalling. Overall, this study identifies highly effective and easily prepared CKX inhibitors with a low risk of environmental toxicity for further investigation of their potential in agriculture and biotechnology. [ABSTRACT FROM AUTHOR]

Phosphorus nutrition has been known for a long time to influence floral transition in plants, but the underlying mechanism is unclear. Arabidopsis phosphate transporter PHOSPHATE1 (PHO1) plays a critical role in phosphate translocation from roots to shoots, but whether and how it regulates floral transition is unknown. Here, we show that knockout mutation of PHO1 delays flowering under both long- and short-day conditions. The late flowering of pho1 mutants can be partially rescued by Pi supplementation in rosettes or shoot apices. Grafting assay indicates that the late flowering of pho1 mutants is a result of impaired phosphate translocation from roots to shoots. Knockout mutation of SPX1 and SPX2 , two negative regulators of the phosphate starvation response, partially rescues the late flowering of pho1 mutants. PHO1 is epistatic to PHO2, a negative regulator of PHO1, in flowering time regulation. Loss of PHO1 represses the expression of some floral activators, including FT encoding florigen, and induces the expression of some floral repressors in shoots. Genetic analyses indicate that at least jasmonic acid signaling is partially responsible for the late flowering of pho1 mutants. In addition, we find that rice PHO1;2 , the homolog of PHO1 , plays a similar role in floral transition. These results suggest that PHO1 integrates phosphorus nutrition and flowering time, and could be used as a potential target in modulating phosphorus nutrition-mediated flowering time in plants. [ABSTRACT FROM AUTHOR]

SUMMARY Among the three active aldehyde oxidases in Arabidopsis thaliana leaves (AAO1‐3), AAO3, which catalyzes the oxidation of abscisic‐aldehyde to abscisic‐acid, was shown recently to function as a reactive aldehyde detoxifier. Notably, aao2KO mutants exhibited less senescence symptoms and lower aldehyde accumulation, such as acrolein, benzaldehyde, and 4‐hydroxyl‐2‐nonenal (HNE) than in wild‐type leaves exposed to UV‐C or Rose‐Bengal. The effect of AAO2 expression absence on aldehyde detoxification by AAO3 and/or AAO1 was studied by comparing the response of wild‐type plants to the response of single‐functioning aao1 mutant (aao1S), aao2KO mutants, and single‐functioning aao3 mutants (aao3Ss). Notably, aao3Ss exhibited similar aldehyde accumulation and chlorophyll content to aao2KO treated with UV‐C or Rose‐Bengal. In contrast, wild‐type and aao1S exhibited higher aldehyde accumulation that resulted in lower remaining chlorophyll than in aao2KO leaves, indicating that the absence of active AAO2 enhanced AAO3 detoxification activity in aao2KO mutants. In support of this notion, employing abscisic‐aldehyde as a specific substrate marker for AAO3 activity revealed enhanced AAO3 activity in aao2KO and aao3Ss leaves compared to wild‐type treated with UV‐C or Rose‐Bengal. The similar abscisic‐acid level accumulated in leaves of unstressed or stressed genotypes indicates that aldehyde detoxification by AAO3 is the cause for better stress resistance in aao2KO mutants. Employing the sulfuration process (known to activate aldehyde oxidases) in wild‐type, aao2KO, and molybdenum‐cofactor sulfurase (aba3‐1) mutant plants revealed that the active AAO2 in WT employs sulfuration processes essential for AAO3 activity level, resulting in the lower AAO3 activity in WT than AAO3 activity in aao2KO. [ABSTRACT FROM AUTHOR]

Summary Aberrant RNA modifications can lead to dysregulated gene expression and impeded growth in plants. Ribosomal RNA (rRNA) constitutes a substantial portion of total RNA, while the precise functions and molecular mechanisms underlying rRNA modifications in plants remain largely elusive. Here, we elucidated the exclusive occurrence of the canonical RNA modification N6‐methyladenosine (m6A) solely 18S rRNA, but not 25S rRNA. We identified a completely uncharacterized protein, ATMETTL5, as an Arabidopsis m6A methyltransferase responsible for installing m6A methylation at the 1771 site of the 18S rRNA. ATMETTL5 is ubiquitously expressed and localized in both nucleus and cytoplasm, mediating rRNA m6A methylation. Mechanistically, the loss of ATMETTL5‐mediated methylation results in attenuated translation. Furthermore, we uncovered the role of ATMETTL5‐mediated methylation in coordinating blue light‐mediated hypocotyl growth by regulating the translation of blue light‐related messenger RNAs (mRNAs), specifically HYH and PRR9. Our findings provide mechanistic insights into how rRNA modification regulates ribosome function in mRNA translation and the response to blue light, thereby advancing our understanding of the role of epigenetic modifications in precisely regulating mRNA translation in plants. [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 Calcium (Ca2+) is essential for plant growth and cellular homeostasis, with cation exchangers (CAXs) regulating Ca2+ transport into plant vacuoles. In Arabidopsis, multiple CAXs feature a common structural arrangement, comprising an N‐terminal autoinhibitory domain followed by two pseudosymmetrical modules. Mutations in CAX1 enhance stress tolerance, notably tolerance to anoxia (a condition marked by oxygen depletion), crucial for flood resilience. Here we engineered a dominant‐negative CAX1 variant, named ½N‐CAX1, incorporating the autoinhibitory domain and the N‐terminal pseudosymmetrical module, which, when expressed in wild‐type Arabidopsis plants, phenocopied the anoxia tolerance of cax1. Physiological evaluations, yeast assays, and calcium imaging demonstrated that wild‐type plants expressing ½N‐CAX1 have phenotypes consistent with inhibition of CAX1, which is likely through direct interaction of ½N‐CAX1 with CAX1. Eliminating segments within the N‐terminal pseudosymmetrical module, as well as incorporating modules from other plant CAXs and expressing these variants into wild‐type plants, failed to produce anoxia tolerance. This underscores the requirement for both the CAX1 autoinhibitory domain and the intact pseudosymmetrical module to produce the dominant‐negative phenotype. Our study elucidates the interaction of this ½N‐CAX1 variant with CAX1 and its impact on anoxia tolerance, offering insights into further approaches for engineering plant stress tolerance. [ABSTRACT FROM AUTHOR]

SUMMARY Cotton fiber (Gossypium hirsutum) serves as an ideal model for investigating the molecular mechanisms of plant cell elongation at the single‐cell level. Brassinosteroids (BRs) play a crucial role in regulating plant growth and development. However, the mechanism by which BR influences cotton fiber elongation remains incompletely understood. In this study, we identified EXORDIUM‐like (GhEXL3) through transcriptome analysis of fibers from BR‐deficient cotton mutant pagoda 1 (pag1) and BRI1‐EMS‐SUPPRESSOR 1 (GhBES1.4, encoding a central transcription factor of BR signaling) overexpression cotton lines. Knockout of GhEXL3 using CRISPR/Cas9 was found to impede cotton fiber elongation, while its overexpression promoted fiber elongation, suggesting a positive regulatory function for GhEXL3 in fiber elongation. Furthermore, in vitro ovule culture experiments revealed that the overexpression of GhEXL3 partially counteracted the inhibitory effects of brassinazole (BRZ) on cotton fiber elongation, providing additional evidence of GhEXL3 involvement in BR signaling pathways. Moreover, our findings demonstrate that GhBES1.4 directly binds to the E‐box (CACGTG) motif in the GhEXL3 promoter region and enhances its transcription. RNA‐seq analysis revealed that overexpression of GhEXL3 upregulated the expression of EXPs, XTHs, and other genes associated with fiber cell elongation. Overall, our study contributes to understanding the mechanism by which BR regulates the elongation of cotton fibers through the direct modulation of GhEXL3 expression by GhBES1.4. [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]

Summary Strengthening future food security through the application of unsustainable levels of inorganic nitrogen (N) fertilizers to crop fields may exacerbate environmental damage. Coordination of N‐use efficiency (NUE) and plant growth is, therefore, crucial for sustainable agriculture. Auxin plays pivotal roles in developmental and signaling responses that affect NUE. Hence, a better understanding of these processes provides great potential to improve crop NUE. This review summarizes the effects of auxin on N‐related and root developmental processes that either directly or indirectly affect NUE in the model plant Arabidopsis and major crop species to highlight the potential of fostering sustainable agricultural development in the future through modulating auxin‐related processes. [ABSTRACT FROM AUTHOR]

SUMMARY Plants synthesize hundreds of small secretory peptides, which are perceived by the receptor‐like kinase (RLK) family at the cell surface. Various signaling peptide‐RLK pairs ensure plant adaptation to distinct environmental conditions. Here, we report that SERINE RICH ENDOGENOUS PEPTIDE (SCOOP) immune peptides modulate root growth and development by regulating PIN‐FORMED (PIN)‐regulated polar auxin transport in Arabidopsis. The SCOOP4 and SCOOP12 treatments impaired root gravitropic growth, auxin redistribution in response to gravistimulation, and PIN abundance in the PM. Furthermore, genetic and cell biological analyses revealed that these physiological and cellular effects of SCOOP4 and SCOOP12 peptides are mediated by the receptor MALE DISCOVERER1‐INTERACTING RECEPTOR LIKE KINASE2 (MIK2) and the downstream mitogen‐activated kinase MPK6. Biochemical evidence indicates that MPK6 directly phosphorylates the cytosolic loop of PIN proteins. Our work established a link between the immune signaling peptide SCOOPs and root growth pathways, providing insights into the molecular mechanisms underlying plant root adaptive growth in the defense response. [ABSTRACT FROM AUTHOR]

VQ1 and VQ10 are largely unstructured homologous proteins with a significant potential for protein–protein interactions. Yeast two-hybrid (Y2H) analysis confirmed that both proteins interact not only with themselves and each other but also with other VQ and WRKY proteins. Screening an Arabidopsis Y2H library with VQ1 as bait identified 287 interacting proteins. Validation of the screening confirmed that interactions with VQ1 also occurred with VQ10, supporting their functional homology. Although VQ1 or VQ10 proteins do not localize in plastids, 47 VQ1-targets were found to be plastidial proteins. In planta interaction with the isoprenoid biosynthetic enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS) was confirmed by co-immunoprecipitation. DXS oligomerizes through redox-regulated intermolecular disulfide bond formation, and the interaction with VQ1 or VQ10 do not involve their unique C residues. The VQ-DXS protein interaction did not alter plastid DXS localization or its oligomerization state. Although plants with enhanced or reduced VQ1 and VQ10 expression did not exhibit significantly altered levels of isoprenoids compared to wild-type plants, they did display significantly improved or diminished photosynthesis efficiency, respectively. [ABSTRACT FROM AUTHOR]

Heat stress (HS) impacts the nuclear proteome and, subsequently, protein activities in different nuclear compartments. In Arabidopsis thaliana, a short exposure to 37 °C leads to loss of the standard tripartite architecture of the nucleolus, the most prominent nuclear substructure, and, consequently, affects the assembly of ribosomes. Here, we report a quantitative label-free LC‒MS/MS (Liquid Chromatography coupled to tandem Mass Spectrometry) analysis to determine the nuclear proteome of Arabidopsis at 22 °C, HS (37 °C for 4 and 24 h), and a recovery phase. This analysis identified ten distinct groups of proteins based on relative abundance changes in the nucleus before, during and after HS: Early, Late, Transient, Early Persistent, Late Persistent, Recovery, Early-Like, Late-Like, Transient-Like and Continuous Groups (EG, LG, TG, EPG, LPG, RG, ELG, LLG, TLG and CG, respectively). Interestingly, the RNA polymerase I subunit NRPA3 and other main nucleolar proteins, including NUCLEOLIN 1 and FIBRILLARIN 1 and 2, were detected in RG and CG, suggesting that plants require increased nucleolar activity and likely ribosome assembly to restore protein synthesis after HS. [ABSTRACT FROM AUTHOR]

Plant senescence is a highly coordinated process that is intricately regulated by numerous endogenous and environmental signals. The involvement of phytic acid in various cell signaling and plant processes has been recognized, but the specific roles of phytic acid metabolism in Arabidopsis leaf senescence remain unclear. Here, we demonstrate that in Arabidopsis thaliana the multiple inositol phosphate phosphatase (AtMINPP) gene, encoding an enzyme with phytase activity, plays a crucial role in regulating leaf senescence by coordinating the ethylene signal transduction pathway. Through overexpressing AtMINPP (AtMINPP–OE), we observed early leaf senescence and reduced chlorophyll contents. Conversely, a loss-of-function heterozygous mutant (atminpp/+) exhibited the opposite phenotype. Correspondingly, the expression of senescence-associated genes (SAGs) was significantly upregulated in AtMINPP–OE but markedly decreased in atminpp/+. Yeast one-hybrid and chromatin immunoprecipitation assays indicated that the EIN3 transcription factor directly binds to the promoter of AtMINPP. Genetic analysis further revealed that AtMINPP–OE could accelerate the senescence of ein3–1eil1–3 mutants. These findings elucidate the mechanism by which AtMINPP regulates ethylene-induced leaf senescence in Arabidopsis, providing insights into the genetic manipulation of leaf senescence and plant growth. [ABSTRACT FROM AUTHOR]

Potassium (K) is an essential nutrient for the growth and development of plants. Root hairs are the main parts of plants that absorb K+. The regulation of plant root hair growth in response to a wide range of environmental stresses is crucially associated with the dynamics of actin filaments, and the thick actin bundles at the apical and sub-apical regions are essential for terminating the rapid elongation of root hair cells. However, the dynamics and roles of actin filaments in root hair growth in plants' response to low K+ stress are not fully understood. Here, we revealed that root hairs grow faster and longer under low K+ stress than the control conditions. Compared to control conditions, the actin filaments in the sub-apex of fast-growing wild-type root hairs were longer and more parallel under low K+ stress, which correlates with an increased root hair growth rate under low K+ stress; the finer actin filaments in the sub-apex of the early fully grown Col-0 root hairs under low K+ stress, which is associated with low K+ stress-induced root hair growth time. Further, Arabidopsis thaliana actin bundling protein Villin1 (VLN1) and Villin4 (VLN4) was inhibited and induced under low K+ stress, respectively. Low K+ stress-inhibited VLN1 led to decreased bundling rate and thick bundle formation in the early fully grown phase. Low K+ stress-induced VLN4 functioned in keeping long filaments in the fast-growing phase. Furthermore, the analysis of genetics pointed out the involvement of VLN1 and VLN4 in the growth of root hairs under the stress of low potassium levels in plants. Our results provide a basis for the dynamics of actin filaments and their molecular regulation mechanisms in root hair growth in response to low K+ stress. [ABSTRACT FROM AUTHOR]

Selective macroautophagy/autophagy in metazoans involves the conserved receptors NBR1 and SQSTM1/p62. Both autophagy receptors manage ubiquitinated cargo recognition, while SQSTM1 has an additional, distinct role of facilitating liquid-liquid phase separation (LLPS) during autophagy. Given that plants lack SQSTM1, it is postulated that plant NBR1 may combine activities of both metazoan NBR1 and SQSTM1. However, the precise mechanism by which plant NBR1 recognizes non-ubiquitinated substrates and its ability to undergo LLPS during selective autophagy remain elusive. Here, we implicate both the ZZ-type zinc finger motif and the four-tryptophan domain of Arabidopsis NBR1 (AtNBR1) in the recognition of non-ubiquitinated cargo proteins. Additionally, we reveal that AtNBR1 indeed undergoes LLPS prior to ATG8-mediated autophagosome formation, crucial for heat stress resistance in Arabidopsis. Our findings unveil the dual roles of AtNBR1 in both cargo recognition and LLPS during plant autophagy and advance our understanding of NBR1-mediated autophagy in plants compared to metazoans.Abbreviations: ATG8: autophagy 8; Co-IP: co-immunoprecipitation; EXO70E2: exocyst subunit EXO70 family protein E2; FRAP: fluorescence recovery after photobleaching; FW domain: four-tryptophan domain; GFP: green fluorescent protein; HS: heat stress; LLPS: liquid-liquid phase separation; LIR: LC3-interacting region; NBR1: next to BRCA1 gene 1; PAS: phagophore assembly site; PB1 domain: Phox and Bem1 domain; RFP: red fluorescent protein; ROF1: rotamase FKBP 1; SARs: selective autophagy receptors; UBA domain: ubiquitin-associated domain; Y2H: yeast two-hybrid; ZZ domain: ZZ-type zinc finger motif domain [ABSTRACT FROM AUTHOR]

Plants maintain iron (Fe) homeostasis under varying environmental conditions by balancing processes such as Fe uptake, transport and storage. In Arabidopsis, POPEYE (PYE), a basic helix‐loop‐helix transcription factor (TF), has been shown to play a crucial role in regulating this balance. In recent years, the mechanisms regulating Fe uptake have been well established but the upstream transcriptional regulators of Fe transport and storage are still poorly understood. In this study, we report that ELONGATED HYPOCOTYL5 (HY5), a basic leucine zipper (bZIP) TF which has recently been shown to play a crucial role in Fe homeostasis, interacts with PYE. Molecular, genetic and biochemical approaches revealed that PYE and HY5 have overlapping as well as some distinct roles in the regulation of Fe deficiency response. We found that HY5 and PYE both act as a repressor of Fe transport genes such as YSL3, FRD3, NPF5.9, YSL2, NAS4 and OPT3. HY5 was found to directly bind on the promoter of these genes and regulate intercellular Fe transport. Further analysis revealed that HY5 and PYE directly interact at the same region on PYE and NAS4 promoter. Overall, this study revealed that HY5 regulates Fe homeostasis by physically interacting with PYE as well as independently. [ABSTRACT FROM AUTHOR]

Plants, being sessile organisms, constantly need to respond to environmental stresses, often leading to the accumulation of reactive oxygen species (ROS). While ROS can be harmful, they also act as second messengers guiding plant growth and stress responses. Because chloroplasts are sensitive to environmental changes and are both a source and a target of ROS during stress conditions, they are important in conveying environmental changes to the nucleus, where acclimation responses are coordinated to maintain organellar and overall cellular homeostasis. ANAC102 has previously been established as a regulator of β-cyclocitral-mediated chloroplast-to-nucleus signaling, protecting plants against photooxidative stress. However, debates persist about where ANAC102 is located—in chloroplasts or in the nucleus. Our study, utilizing the genomic ANAC102 sequence driven by its native promoter, establishes ANAC102 primarily as a nuclear protein, lacking a complete N-terminal chloroplast-targeting peptide. Moreover, our research reveals the sensitivity of plants overexpressing ANAC102 to severe superoxide-induced chloroplast oxidative stress. Transcriptome analysis unraveled a dual role of ANAC102 in negatively and positively regulating genome-wide transcriptional responses to chloroplast oxidative stress. Through the integration of published data and our own study, we constructed a comprehensive transcriptional network, which suggests that ANAC102 exerts direct and indirect control over transcriptional responses through downstream transcription factor networks, providing deeper insights into the ANAC102-mediated regulatory landscape during oxidative stress. [ABSTRACT FROM AUTHOR]

SUMMARY Maintaining an optimal redox status is essential for plant growth and development, particularly when the plants are under stress. AT‐hook motif nuclear localized (AHL) proteins are evolutionarily conserved transcription factors in plants. Much of our understanding about this gene family has been derived from studies on clade A members. To elucidate the functions of clade B genes, we first analyzed their spatial expression patterns using transgenic plants expressing a nuclear localized GFP under the control of their promoter sequences. AHL1, 2, 6, 7, and 10 were further functionally characterized owing to their high expression in the root apical meristem. Through mutant analyses and transgenic studies, we showed that these genes have the ability to promote root growth. Using yeast one‐hybrid and dual luciferase assays, we demonstrated that AHL1, 2, 6, 7, and 10 are transcription regulators and this activity is required for their roles in root growth. Although mutants for these genes did not showed obvious defects in root growth, transgenic plants expressing their fusion proteins with the SRDX repressor motif exhibited a short‐root phenotype. Through transcriptome analysis, histochemical staining and molecular genetics experiments, we found that AHL10 maintains redox homeostasis via direct regulation of glutathione transferase (GST) genes. When the transcript level of GSTF2, a top‐ranked target of AHL10, was reduced by RNAi, the short‐root phenotype in the AHL10‐SRDX expressing plant was largely rescued. These results together suggest that AHL genes function redundantly in promoting root growth through direct regulation of redox homeostasis. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase (MAPK) cascades have been discovered to play a fundamental role in regulating organ abscission. However, the identity of protein substrates targeted by MAPK cascades, as well as whether the role of MAPK protein cascades in the abscission process is conserved across different plant species, remain unknown. Here, the role of homologs of MPK3 and MPK6 in regulating fruit abscission were characterized in litchi. Ectopic expression of LcMPK3 or LcMPK6 in Arabidopsis mpk3 mpk6 mutant rescued the deficiency in floral organ abscission, while silencing of LcMPK3 or LcMPK6 in litchi significantly decreased fruitlet abscission. Importantly, a total of 49 proteins interacting with LcMPK3 were identified through yeast two-hybrid screening, including two components of the MAPK signaling cascade, five transcription factors, and two aquaporins. Furthermore, the interaction between LcMPK3/6 with LcBZR1/2, core components in brassinosteroids signaling that suppress litchi fruitlet abscission, was confirmed using in vitro and in vivo assays. Moreover, phos-tag assays demonstrated that LcMPK3/6 could phosphorylate LcBZR1/2, with several phosphorylation residues identified. Together, our findings suggest that LcMPK3 and LcMPK6 play a positive regulatory role in fruitlet abscission in litchi, and offer crucial information for the investigation of mechanisms underlying MPK3/6-mediated organ abscission in plants. [ABSTRACT FROM AUTHOR]