Nucleo-cytoplasmic Partitioning of ARF Proteins Controls Auxin Responses in Arabidopsis thaliana.

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The auxin response factor (ARF) transcription factor family regulates auxin-responsive gene expression and exhibits nuclear localization in regions of high auxin responsiveness. Here we show that the ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness. We found that the intrinsically disordered middle region and the folded PB1 interaction domain of ARFs drive protein assembly formation. Mutation of a single lysine within the PB1 domain abrogates cytoplasmic assemblies, promotes ARF nuclear localization, and results in an altered transcriptome and morphological defects. Our data suggest a model in which ARF nucleo-cytoplasmic partitioning regulates auxin responsiveness, providing a mechanism for cellular competence for auxin signaling.

Arabidopsis HEAT SHOCK FACTOR BINDING PROTEIN is required to limit meiotic crossovers and HEI10 transcription.

The number of meiotic crossovers is tightly controlled and most depend on pro-crossover ZMM proteins, such as the E3 ligase HEI10. Despite the importance of HEI10 dosage for crossover formation, how HEI10 transcription is controlled remains unexplored. In a forward genetic screen using a fluorescent crossover reporter in Arabidopsis thaliana, we identify heat shock factor binding protein (HSBP) as a repressor of HEI10 transcription and crossover numbers. Using genome-wide crossover mapping and cytogenetics, we show that hsbp mutations or meiotic HSBP knockdowns increase ZMM-dependent crossovers toward the telomeres, mirroring the effects of HEI10 overexpression. Through RNA sequencing, DNA methylome, and chromatin immunoprecipitation analysis, we reveal that HSBP is required to repress HEI10 transcription by binding with heat shock factors (HSFs) at the HEI10 promoter and maintaining DNA methylation over the HEI10 5' untranslated region. Our findings provide insights into how the temperature response regulator HSBP restricts meiotic HEI10 transcription and crossover number by attenuating HSF activity.

CPR5-mediated nucleo-cytoplasmic localization of IAA12 and IAA19 controls lateral root development during abiotic stress.

Plasticity of the root system architecture (RSA) is essential in enabling plants to cope with various environmental stresses and is mainly controlled by the phytohormone auxin. Lateral root development is a major determinant of RSA. Abiotic stresses reduce auxin signaling output, inhibiting lateral root development; however, how abiotic stress translates into a lower auxin signaling output is not fully understood. Here, we show that the nucleo-cytoplasmic distribution of the negative regulators of auxin signaling AUXIN/INDOLE-3-ACETIC ACID INDUCIBLE 12 (AUX/IAA12 or IAA12) and IAA19 determines lateral root development under various abiotic stress conditions. The cytoplasmic localization of IAA12 and IAA19 in the root elongation zone enforces auxin signaling output, allowing lateral root development. Among components of the nuclear pore complex, we show that CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES 5 (CPR5) selectively mediates the cytoplasmic translocation of IAA12/19. Under abiotic stress conditions, CPR5 expression is strongly decreased, resulting in the accumulation of nucleus-localized IAA12/19 in the root elongation zone and the suppression of lateral root development, which is reiterated in the cpr5 mutant. This study reveals a regulatory mechanism for auxin signaling whereby the spatial distribution of AUX/IAA regulators is critical for lateral root development, especially in fluctuating environmental conditions.

SnRK1 inhibits anthocyanin biosynthesis through both transcriptional regulation and direct phosphorylation and dissociation of the MYB/bHLH/TTG1 MBW complex.

Plants have evolved an extensive specialized secondary metabolism. The colorful flavonoid anthocyanins, for example, not only stimulate flower pollination and seed dispersal, but also protect different tissues against high light, UV and oxidative stress. Their biosynthesis is highly regulated by environmental and developmental cues and induced by high sucrose levels. Expression of the biosynthetic enzymes involved is controlled by a transcriptional MBW complex, comprising (R2R3) MYB- and bHLH-type transcription factors and the WD40 repeat protein TTG1. Anthocyanin biosynthesis is not only useful, but also carbon- and energy-intensive and non-vital. Consistently, the SnRK1 protein kinase, a metabolic sensor activated in carbon- and energy-depleting stress conditions, represses anthocyanin biosynthesis. Here we show that Arabidopsis SnRK1 represses MBW complex activity both at the transcriptional and post-translational level. In addition to repressing expression of the key transcription factor MYB75/PAP1, SnRK1 activity triggers MBW complex dissociation, associated with loss of target promoter binding, MYB75 protein degradation and nuclear export of TTG1. We also provide evidence for direct interaction with and phosphorylation of multiple MBW complex proteins. These results indicate that repression of expensive anthocyanin biosynthesis is an important strategy to save energy and redirect carbon flow to more essential processes for survival in metabolic stress conditions.

JULGI-mediated increment in phloem transport capacity relates to fruit yield in tomato.

The continuous growth of the global population and the increase in the amount of arid land has severely constrained agricultural crop production. To solve this problem, many researchers have attempted to increase productivity through the efficient distribution of energy; however, the direct relationship between the plant vasculature, specifically phloem development, and crop yield is not well established. Here, we demonstrate that an optimum increase in phloem-transportation capacity by reducing SIJUL expression leads to improved sink strength in tomato (Solanum lycopersicum L.). SIJUL, a negative regulator of phloem development, suppresses the translation of a positive regulator of phloem development, SlSMXL5. The suppression of SlJUL increases the number of phloem cells and sucrose transport, but only an optimal reduction of SlJUL function greatly enhances sink strength in tomato, improving fruit setting, and yield contents by 37% and 60%, respectively. We show that the increment in phloem cell number confers spare transport capacity. Our results suggest that the control of phloem-transport capacity within the threshold could enhance the commitment of photosynthates to instigate yield improvement.

CYTOKININ-RESPONSIVE GROWTH REGULATOR regulates cell expansion and cytokinin-mediated cell cycle progression.

The cytokinin (CK) phytohormones have long been known to activate cell proliferation in plants. However, how CKs regulate cell division and cell expansion remains unclear. Here, we reveal that a basic helix-loop-helix transcription factor, CYTOKININ-RESPONSIVE GROWTH REGULATOR (CKG), mediates CK-dependent regulation of cell expansion and cell cycle progression in Arabidopsis thaliana. The overexpression of CKG increased cell size in a ploidy-independent manner and promoted entry into the S phase of the cell cycle, especially at the seedling stage. Furthermore, CKG enhanced organ growth in a pleiotropic fashion, from embryogenesis to reproductive stages, particularly of cotyledons. In contrast, ckg loss-of-function mutants exhibited smaller cotyledons. CKG mainly regulates the expression of genes involved in the regulation of the cell cycle including WEE1. We propose that CKG provides a regulatory module that connects cell cycle progression and organ growth to CK responses.

The CEP5 Peptide Promotes Abiotic Stress Tolerance, As Revealed by Quantitative Proteomics, and Attenuates the AUX/IAA Equilibrium in Arabidopsis.

Peptides derived from non-functional precursors play important roles in various developmental processes, but also in (a)biotic stress signaling. Our (phospho)proteome-wide analyses of C-TERMINALLY ENCODED PEPTIDE 5 (CEP5)-mediated changes revealed an impact on abiotic stress-related processes. Drought has a dramatic impact on plant growth, development and reproduction, and the plant hormone auxin plays a role in drought responses. Our genetic, physiological, biochemical, and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis, and that CEP5 specifically counteracts auxin effects. Specifically, we found that CEP5 signaling stabilizes AUX/IAA transcriptional repressors, suggesting the existence of a novel peptide-dependent control mechanism that tunes auxin signaling. These observations align with the recently described role of AUX/IAAs in stress tolerance and provide a novel role for CEP5 in osmotic and drought stress tolerance.

DeepTetrad: high-throughput image analysis of meiotic tetrads by deep learning in Arabidopsis thaliana.

Meiotic crossovers facilitate chromosome segregation and create new combinations of alleles in gametes. Crossover frequency varies along chromosomes and crossover interference limits the coincidence of closely spaced crossovers. Crossovers can be measured by observing the inheritance of linked transgenes expressing different colors of fluorescent protein in Arabidopsis pollen tetrads. Here we establish DeepTetrad, a deep learning-based image recognition package for pollen tetrad analysis that enables high-throughput measurements of crossover frequency and interference in individual plants. DeepTetrad will accelerate the genetic dissection of mechanisms that control meiotic recombination.

Nucleosomes and DNA methylation shape meiotic DSB frequency in Arabidopsis thaliana transposons and gene regulatory regions.

Meiotic recombination initiates from DNA double-strand breaks (DSBs) generated by SPO11 topoisomerase-like complexes. Meiotic DSB frequency varies extensively along eukaryotic chromosomes, with hotspots controlled by chromatin and DNA sequence. To map meiotic DSBs throughout a plant genome, we purified and sequenced Arabidopsis thaliana SPO11-1-oligonucleotides. SPO11-1-oligos are elevated in gene promoters, terminators, and introns, which is driven by AT-sequence richness that excludes nucleosomes and allows SPO11-1 access. A positive relationship was observed between SPO11-1-oligos and crossovers genome-wide, although fine-scale correlations were weaker. This may reflect the influence of interhomolog polymorphism on crossover formation, downstream from DSB formation. Although H3K4me3 is enriched in proximity to SPO11-1-oligo hotspots at gene 5' ends, H3K4me3 levels do not correlate with DSBs. Repetitive transposons are thought to be recombination silenced during meiosis, to prevent nonallelic interactions and genome instability. Unexpectedly, we found high SPO11-1-oligo levels in nucleosome-depleted Helitron/Pogo/Tc1/Mariner DNA transposons, whereas retrotransposons were coldspots. High SPO11-1-oligo transposons are enriched within gene regulatory regions and in proximity to immunity genes, suggesting a role as recombination enhancers. As transposon mobility in plant genomes is restricted by DNA methylation, we used the met1 DNA methyltransferase mutant to investigate the role of heterochromatin in SPO11-1-oligo distributions. Epigenetic activation of meiotic DSBs in proximity to centromeres and transposons occurred in met1 mutants, coincident with reduced nucleosome occupancy, gain of transcription, and H3K4me3. Together, our work reveals a complex relationship between chromatin and meiotic DSBs within A. thaliana genes and transposons, with significance for the diversity and evolution of plant genomes.

Cytokinin-Mediated Regulation of Reactive Oxygen Species Homeostasis Modulates Stomatal Immunity in Arabidopsis.

Stomata play an important role in preinvasive defense responses by limiting pathogen entry into leaves. Although the stress hormones salicylic acid (SA) and abscisic acid (ABA) are known to regulate stomatal immunity, the role of growth promoting hormones is far from understood. Here, we show that in Arabidopsis thaliana, cytokinins (CKs) function in stomatal defense responses. The cytokinin receptor HISTIDINE KINASE3 (AHK3) and RESPONSE REGULATOR2 (ARR2) promote stomatal closure triggered by pathogen-associated molecular pattern (PAMP) and resistance to Pseudomonas syringae pv tomato bacteria. Importantly, the cytokinin trans-zeatin induces stomatal closure and accumulation of reactive oxygen species (ROS) in guard cells through AHK3 and ARR2 in an SA-dependent and ABA-independent manner. Using pharmacological and reverse genetics approaches, we found that CK-mediated stomatal responses involve the apoplastic peroxidases PRX4, PRX33, PRX34, and PRX71, but not the NADPH oxidases RBOHD and RBOHF. Moreover, ARR2 directly activates the expression of PRX33 and PRX34, which are required for SA- and PAMP-triggered ROS production. Thus, the CK signaling pathway regulates ROS homeostasis in guard cells, which leads to enhanced stomatal immunity and plant resistance to bacteria.

Integration of multiple signaling pathways shapes the auxin response.

The phytohormone auxin is a pivotal signaling molecule that functions throughout the plant lifecycle. Proper regulation of the auxin response is critical for optimizing plant growth under ever-changing environmental conditions. Recent studies have demonstrated that the signaling components that modulate auxin sensitivity and responses are functionally and mechanically diverse. In addition to auxin itself, various environmental and hormonal signals are integrated to modulate the auxin response through directly controlling auxin signaling components. This review explores the non-canonical mechanisms that modulate auxin signaling components, including transcriptional, translational, and post-translational regulation. All of these contribute to the wide range in sensitivity and complexity in auxin responses to various signaling cues.

Overexpression of INCREASED CAMBIAL ACTIVITY, a putative methyltransferase, increases cambial activity and plant growth.

Cambial activity is a prerequisite for secondary growth in plants; however, regulatory factors controlling the activity of the secondary meristem in radial growth remain elusive. Here, we identified INCREASED CAMBIAL ACTIVITY (ICA), a gene encoding a putative pectin methyltransferase, which could function as a modulator for the meristematic activity of fascicular and interfascicular cambium in Arabidopsis. An overexpressing transgenic line, 35S::ICA, showed accelerated stem elongation and radial thickening, resulting in increased accumulation of biomass, and increased levels of cytokinins (CKs) and gibberellins (GAs). Expression of genes encoding pectin methylesterases involved in pectin modification together with pectin methyltransferases was highly induced in 35S::ICA, which might contribute to an increase of methanol emission as a byproduct in 35S::ICA. Methanol treatment induced the expression of GA- or CK-responsive genes and stimulated plant growth. Overall, we propose that ectopic expression of ICA increases cambial activity by regulating CK and GA homeostasis, and methanol emission, eventually leading to stem elongation and radial growth in the inflorescence stem.

Functional water flow pathways and hydraulic regulation in the xylem network of Arabidopsis.

In vascular plants, the xylem network constitutes a complex microfluidic system. The relationship between vascular network architecture and functional hydraulic regulation during actual water flow remains unexplored. Here, we developed a method to visualize individual xylem vessels of the 3D xylem network of Arabidopsis thaliana, and to analyze the functional activities of these vessels using synchrotron X-ray computed tomography with hydrophilic gold nanoparticles as flow tracers. We show how the organization of the xylem network changes dynamically throughout the plant, and reveal how the elementary units of this transport system are organized to ensure both long-distance axial water transport and local lateral water transport. Xylem vessels form distinct clusters that operate as functional units, and the activity of these units, which determines water flow pathways, is modulated not only by varying the number and size of xylem vessels, but also by altering their interconnectivity and spatial arrangement. Based on these findings, we propose a regulatory model of water transport that ensures hydraulic efficiency and safety.

BLH1 and KNAT3 modulate ABA responses during germination and early seedling development in Arabidopsis.

The signal transduction pathway governed by the phytohormone abscisic acid (ABA) regulates not only abiotic stress responses but also early developmental programs such as seed dormancy, germination and seedling growth in response to environmental signals. Optimal plant growth and development depend on the integration of environmental stimuli and intrinsic developmental programs. Here, we show that the homeodomain transcription factors BLH1 and KNAT3, previously implicated in embryo sac development, have additional functions in ABA-mediated seed dormancy and early seedling development. The ABA-dependent induction of BLH1 and KNAT3 expression required the presence of functional PYR/PYL/RCAR receptors. The blh1 and knat3 mutants were less sensitive than the wild-type to ABA or salinity exposure during seed germination and early seedling development. In contrast, BLH1 over-expressing lines were hypersensitive to ABA and salinity, and exhibited increased expression of ABA-responsive genes, such as ABI3 and ABI5. BLH1 interacted with KNAT3 and enhanced the retention of KNAT3 in the nucleus. BLH1 and KNAT3 synergistically increased the ABA responses by binding to and subsequently activating the ABI3 promoter. Taken together, we propose that BLH1 and KNAT3 together modulate seed germination and early seedling development by directly regulating ABI3 expression.

BAT1, a putative acyltransferase, modulates brassinosteroid levels in Arabidopsis.

Brassinosteroids (BRs) are essential for various aspects of plant development. Cellular BR homeostasis is critical for proper growth and development of plants; however, its regulatory mechanism remains largely unknown. BAT1 (BR-related acyltransferase 1), a gene encoding a putative acyltransferase, was found to be involved in vascular bundle development in a full-length cDNA over-expressor (FOX) screen. Over-expression of BAT1 resulted in typical BR-deficient phenotypes, which were rescued by exogenously applied castasterone and brassinolide. Analyses of BR profiles demonstrated that BAT1 alters levels of several brassinolide biosynthetic intermediates, including 6-deoxotyphasterol, typhasterol and 6-deoxocastasterone. BAT1 is mainly localized in the endoplasmic reticulum. BAT1 is highly expressed in young tissues and vascular bundles, and its expression is induced by auxin. These data suggest that BAT1 is involved in BR homeostasis, probably by conversion of brassinolide intermediates into acylated BR conjugates.

Investigating water transport through the xylem network in vascular plants.

Our understanding of physical and physiological mechanisms depends on the development of advanced technologies and tools to prove or re-evaluate established theories, and test new hypotheses. Water flow in land plants is a fascinating phenomenon, a vital component of the water cycle, and essential for life on Earth. The cohesion-tension theory (CTT), formulated more than a century ago and based on the physical properties of water, laid the foundation for our understanding of water transport in vascular plants. Numerous experimental tools have since been developed to evaluate various aspects of the CTT, such as the existence of negative hydrostatic pressure. This review focuses on the evolution of the experimental methods used to study water transport in plants, and summarizes the different ways to investigate the diversity of the xylem network structure and sap flow dynamics in various species. As water transport is documented at different scales, from the level of single conduits to entire plants, it is critical that new results be subjected to systematic cross-validation and that findings based on different organs be integrated at the whole-plant level. We also discuss the functional trade-offs between optimizing hydraulic efficiency and maintaining the safety of the entire transport system. Furthermore, we evaluate future directions in sap flow research and highlight the importance of integrating the combined effects of various levels of hydraulic regulation.

Control of plant germline proliferation by SCF(FBL17) degradation of cell cycle inhibitors.

Flowering plants possess a unique reproductive strategy, involving double fertilization by twin sperm cells. Unlike animal germ lines, the male germ cell lineage in plants only forms after meiosis and involves asymmetric division of haploid microspores, to produce a large, non-germline vegetative cell and a germ cell that undergoes one further division to produce the twin sperm cells. Although this switch in cell cycle control is critical for sperm cell production and delivery, the underlying molecular mechanisms are unknown. Here we identify a novel F-box protein of Arabidopsis thaliana, designated FBL17 (F-box-like 17), that enables this switch by targeting the degradation of cyclin-dependent kinase A;1 inhibitors specifically in male germ cells. We show that FBL17 is transiently expressed in the male germ line after asymmetric division and forms an SKP1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complex (SCF(FBL17)) that targets the cyclin-dependent kinase inhibitors KRP6 and KRP7 for proteasome-dependent degradation. Accordingly, the loss of FBL17 function leads to the stabilization of KRP6 and inhibition of germ cell cycle progression. Our results identify SCF(FBL17) as an essential male germ cell proliferation complex that promotes twin sperm cell production and double fertilization in flowering plants.

Control of meiotic crossover interference by a proteolytic chaperone network.

Meiosis is a specialized eukaryotic division that produces genetically diverse gametes for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal exchanges, called crossovers, which recombine genetic variation. Meiotic crossovers are stringently controlled with at least one obligate exchange forming per chromosome pair, while closely spaced crossovers are inhibited by interference. In Arabidopsis, crossover positions can be explained by a diffusion-mediated coarsening model, in which large, approximately evenly spaced foci of the pro-crossover E3 ligase HEI10 grow at the expense of smaller, closely spaced clusters. However, the mechanisms that control HEI10 dynamics during meiosis remain unclear. Here, through a forward genetic screen in Arabidopsis, we identified high crossover rate3 (hcr3), a dominant-negative mutant that reduces crossover interference and increases crossovers genome-wide. HCR3 encodes J3, a co-chaperone related to HSP40, which acts to target protein aggregates and biomolecular condensates to the disassembly chaperone HSP70, thereby promoting proteasomal degradation. Consistently, we show that a network of HCR3 and HSP70 chaperones facilitates proteolysis of HEI10, thereby regulating interference and the recombination landscape. These results reveal a new role for the HSP40/J3-HSP70 chaperones in regulating chromosome-wide dynamics of recombination via control of HEI10 proteolysis.

Cytokinin-facilitated proteolysis of ARABIDOPSIS RESPONSE REGULATOR 2 attenuates signaling output in two-component circuitry.

Cytokinins propagate signals via multiple phosphorelays in a mechanism similar to bacterial two-component systems. In Arabidopsis, signal outputs are determined by the activation state of transcription factors termed type-B Arabidopsis response regulators (ARRs); however, their regulatory mechanisms are largely unknown. In this study, we demonstrate that the proteolysis of ARR2, a type-B ARR, modulates cytokinin signaling outputs. ARR2-hemagglutinin (HA) is rapidly degraded by cytokinin treatment, but other type-B ARRs, such as ARR1-HA, ARR10-HA, ARR12-HA and ARR18-HA, are not. ARR2 degradation is mediated by the 26S proteasome pathway, and requires cytokinin-induced phosphorylation of Asp80 residue in the receiver domain. Through mutational analysis of amino acid residues in the receiver domain, we found that substitution of Lys90 with Gly inhibits ARR2 degradation. ARR2(K90G) -HA in transgenic Arabidopsis conferred enhanced cytokinin sensitivity in various developmental processes, including primary root elongation, callus induction, leaf senescence and hypocotyl growth. ARR2(K90G) -HA increased the expression of type-A ARRs, primary cytokinin-responsive genes and indicators of signaling output in two-component circuits. Expression of ARR2(K90G) -HA from the native ARR2 promoter in the arr2-4 knock-out mutant also increased cytokinin sensitivity. In conclusion, ARR2 proteolysis is involved in the maintenance of the primary signaling output for normal developmental processes mediated by cytokinin in Arabidopsis.

The LBD11-ROS feedback regulatory loop modulates vascular cambium proliferation and secondary growth in Arabidopsis.

Vascular cambium produces the phloem and xylem, vascular tissues that transport resources and provide mechanical support, making it an ideal target for crop improvement. However, much remains unknown about how vascular cambium proliferates. In this study, through pharmaceutical and genetic manipulation of reactive oxygen species (ROS) maxima, we demonstrate a direct link between levels of ROS and activity of LATERAL ORGAN BOUNDARIES DOMAIN 11 (LBD11) in maintaining vascular cambium activity. LBD11 activates the transcription of several key ROS metabolic genes, including PEROXIDASE 71 and RESPIRATORY BURST OXIDASE HOMOLOGS D and F, to generate local ROS maxima in cambium, which in turn enhance the proliferation of cambial cells. In a negative feedback mechanism, higher ROS levels then repress LBD11 expression and maintain the balance of cambial cell proliferation. Our findings thus reveal the role of a novel LBD11/ROS-dependent feedback regulatory system in maintaining vascular cambium-specific redox homeostasis and radial growth in plants.