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The Agrobacterium-mediated floral dip protocol is the most extensively used transformation method for a model plant Arabidopsis thaliana. Several useful methods for Agrobacterium tumefaciens–mediated transformations of Arabidopsis are existing, but they are time consuming and with low transformation efficiency. Here, we developed a transgenic Arabidopsis lines TET12p::TET12-RFP in a short period of time and enhanced transformation efficiency by using a modified transformation method by applying drought stress after floral dip. In this protocol, Agrobacterium cells carrying TET12p::TET12-RFP recombinant vectors were resuspended in a solution of 5% sucrose, 0.05% (v/v) silwet L-77 to transform female gametes of developing Arabidopsis inflorescences. Treated Arabidopsis were then applied with different levels of drought stresses to stimulate plants for the utilization of maximum plant energy in seed maturation process. The applied stresses achieved the fast maturation of already treated inflorescences while stopped the growing of newly arising untreated inflorescence, thus decreased the chances of wrong collection of untransformed seeds. Consequently, the collected seeds were mostly transgenic with a transformation frequency of at least 10%, thus the screening for positive transformants selection was more advantageous on a selective medium as compared to a classical floral dip method. Within 2-3 months, two hundred of individual transgenic plants were produced from just 10 infiltrated plants. This study concludes that application of drought stresses in a specific stage of plant is a beneficial strategy for achieving the transgenic Arabidopsis in a short period of time with high transformation efficiency. Resumo O protocolo de imersão floral mediado por Agrobacterium é o método de transformação mais amplamente utilizado para uma planta-modelo Arabidopsis thaliana. Existem vários métodos úteis para transformações de Arabidopsis mediados por Agrobacterium tumefaciens, mas são demorados e com baixa eficiência de transformação. Aqui, desenvolvemos uma linha transgênica de Arabidopsis TET12p::TET12-RFP em um curto período de tempo e eficiência de transformação aprimorada usando um método de transformação modificado por meio da aplicação de estresse hídrico após o mergulho floral. Neste protocolo, células de Agrobacterium transportando vetores recombinantes TET12p::TET12-RFP foram ressuspensas em uma solução de 5% de sacarose, 0,05% (v/v) silwet L-77 para transformar gametas femininos de inflorescências de Arabidopsis em desenvolvimento. Arabidopsis tratadas foram então aplicadas com diferentes níveis de estresse hídrico para estimular as plantas a utilizar o máximo de energia da planta no processo de maturação das sementes. Os estresses aplicados alcançaram a rápida maturação das inflorescências já tratadas enquanto paravam o crescimento de inflorescências não tratadas recém-surgidas, diminuindo, assim, as chances de coleta errada de sementes não transformadas. Consequentemente, as sementes coletadas eram principalmente transgênicas, com uma frequência de transformação de pelo menos 10%; portanto, a triagem para seleção dos transformantes positivos foi mais vantajosa em um meio seletivo em comparação com um método clássico de imersão floral. Dentro de 2-3 meses, 200 plantas transgênicas individuais foram produzidas a partir de apenas 10 plantas infiltradas. Este estudo conclui que a aplicação de estresse hídrico em um estágio específico da planta é uma estratégia benéfica para alcançar a Arabidopsis transgênica em um curto período de tempo com alta eficiência de transformação.

Auxin signaling regulates growth and developmental processes in plants. The core of nuclear auxin signaling relies on just three components: TIR1/AFBs, Aux/IAAs, and ARFs. Each component is itself made up of several domains, all of which contribute to the regulation of auxin signaling. Studies of the structural aspects of these three core signaling components have deepened our understanding of auxin signaling dynamics and regulation. In addition to the structured domains of these components, intrinsically disordered regions within the proteins also impact auxin signaling outcomes. New research is beginning to uncover the role intrinsic disorder plays in auxin-regulated degradation and subcellular localization. Structured and intrinsically disordered domains affect auxin perception, protein degradation dynamics, and DNA binding. Taken together, subtle differences within the domains and motifs of each class of auxin signaling component affect signaling outcomes and specificity.

Chloroplasts are a common feature of plant cells and aspects of their metabolism, including photosynthesis, are influenced by low-temperature conditions. Chloroplasts contain a small circular genome that encodes essential components of the photosynthetic apparatus and chloroplast transcription/translation machinery. Here, we show that in Arabidopsis, a nuclear-encoded sigma factor that controls chloroplast transcription (SIGMA FACTOR5) contributes to adaptation to low-temperature conditions. This process involves the regulation of SIGMA FACTOR5 expression in response to cold by the bZIP transcription factors ELONGATED HYPOCOTYL5 and ELONGATED HYPOCOTYL5 HOMOLOG. The response of this pathway to cold is gated by the circadian clock, and it enhances photosynthetic efficiency during long-term cold and freezing exposure. We identify a process that integrates low-temperature and circadian signals, and modulates the response of chloroplasts to low-temperature conditions.

A vast variety of inflorescence architectures have evolved in angiosperms. Here, we analyze the diversity and development of the woodland strawberry (Fragaria vesca) inflorescence. Contrary to historical classifications, we show that it is a closed thyrse: a compound inflorescence with determinate primary monopodial axis and lateral sympodial branches, thus combining features of racemes and cymes. We demonstrate that this architecture is generated by 2 types of inflorescence meristems differing in their geometry. We further show that woodland strawberry homologs of TERMINAL FLOWER 1 (FvTFL1) and FLOWERING LOCUS T (FvFT1) regulate the development of both the racemose and cymose components of the thyrse. Loss of functional FvTFL1 reduces the number of lateral branches of the main axis and iterations in the lateral branches but does not affect their cymose pattern. These changes can be enhanced or compensated by altering FvFT1 expression. We complement our experimental findings with a computational model that captures inflorescence development using a small set of rules. The model highlights the distinct regulation of the fate of the primary and higher-order meristems, and explains the phenotypic diversity among inflorescences in terms of heterochrony resulting from the opposite action of FvTFL1 and FvFT1 within the thyrse framework. Our results represent a detailed analysis of thyrse architecture development at the meristematic and molecular levels.FLOWERING LOCUS T1 and TERMINAL FLOWER 1 control the number of lateral branches and their branching iterations in the thyrse inflorescence of woodland strawberry.

DNA methylation has been utilized for target gene silencing in plants. However, it is not well understood whether other silencing pathways can be also used to manipulate gene expression. Here we performed a gain-of-function screen for proteins that could silence a target gene when fused to an artificial zinc finger. We uncovered many proteins that suppressed gene expression through DNA methylation, histone H3K27me3 deposition, H3K4me3 demethylation, histone deacetylation, inhibition of RNA polymerase II transcription elongation or Ser-5 dephosphorylation. These proteins also silenced many other genes with different efficacies, and a machine learning model could accurately predict the efficacy of each silencer on the basis of various chromatin features of the target loci. Furthermore, some proteins were also able to target gene silencing when used in a dCas9-SunTag system. These results provide a more comprehensive understanding of epigenetic regulatory pathways in plants and provide an armament of tools for targeted gene manipulation.

As agricultural production is reaching its limits regarding outputs and land use, the need to further improve crop yield is greater than ever. The limited translatability from in vitro lab results into more natural growth conditions in soil remains problematic. Although considerable progress has been made in developing soil-growth assays to tackle this bottleneck, the majority of these assays use pots or whole trays, making them not only space- and resource-intensive, but also hampering the individual treatment of plants. Therefore, we developed a flexible and compact screening system named PhenoWell® in which individual seedlings are grown in wells filled with soil allowing single-plant treatments. The system makes use of an automated image-analysis pipeline that extracts multiple growth parameters from individual seedlings over time, including projected rosette area, relative growth rate, compactness, and stockiness. Macronutrient, hormone, salt, osmotic, and drought stress treatments were tested in the PhenoWell® system. The system is also optimized for maize with results that are consistent with Arabidopsis while different in amplitude. We conclude that the PhenoWell® system enables a high-throughput, precise, and uniform application of a small amount of solution to individually soil-grown plants, which increases the replicability and reduces variability and compound usage.

Throughout the exploration of the soil, roots interact with their environment and adapt to different conditions. Directional root growth is guided by asymmetric molecular patterns but how these become established or are dynamically regulated is poorly understood. Asymmetric gradients of the phytohormone auxin are established during root gravitropism, mainly through directional transport mediated by polarized auxin transporters. Upon gravistimulation, PIN-FORMED2 (PIN2) is differentially distributed and accumulates at the lower root side to facilitate asymmetric auxin transport up to the elongation zone where it inhibits cell elongation. GOLVEN (GLV) peptides function in gravitropism by affecting PIN2 abundance in epidermal cells. In addition, GLV signaling through ROOT GROWTH FACTOR 1 INSENSITIVE (RGI) receptors regulates root apical meristem maintenance. Here, we show that GLV-RGI signaling in these 2 processes in Arabidopsis (Arabidopsis thaliana) can be mapped to different cells in the root tip and that, in the case of gravitropism, it operates mainly in the lateral root cap (LRC) to maintain PIN2 levels at the plasma membrane (PM). Furthermore, we found that GLV signaling upregulates the phosphorylation level of PIN2 in an RGI-dependent manner. In addition, we demonstrated that the RGI5 receptor is asymmetrically distributed in the LRC and accumulates in the lower side of the LRC after gravistimulation. Asymmetric GLV-RGI signaling in the root cap likely accounts for differential PIN2 abundance at the PM to temporarily support auxin transport up to the elongation zone, thereby representing an additional level of control on the asymmetrical auxin flux to mediate differential growth of the root.

International audience; In angiosperms, flower development requires the combined action of the transcription factor LEAFY (LFY) and the ubiquitin ligase adaptor F-box protein, UNUSUAL FLORAL ORGANS (UFO), but the molecular mechanism underlying this synergy has remained unknown. Here we show in transient assays and stable transgenic plants that the connection to ubiquitination pathways suggested by the UFO F-box domain is mostly dispensable. On the basis of biochemical and genome-wide studies, we establish that UFO instead acts by forming an active transcriptional complex with LFY at newly discovered regulatory elements. Structural characterization of the LFY-UFO-DNA complex by cryo-electron microscopy further demonstrates that UFO performs this function by directly interacting with both LFY and DNA. Finally, we propose that this complex might have a deep evolutionary origin, largely predating flowering plants. This work reveals a unique mechanism of an F-box protein directly modulating the DNA binding specificity of a master transcription factor.

Hypoxic stress, caused by the low cellular oxygen in the events of flooding or waterlogging, limits crop productivity in many regions of the world. Hypoxic stress in plants is often dynamic and followed by a reoxygenation process that returns the oxygen level to normal. Although metabolic responses to hypoxia have been studied in many plants, less is known about the recovery processes following stress removal. To better understand the dynamic metabolic shift from a low-oxygen environment to a reoxygenated environment, we performed time-course measurements of metabolites in Arabidopsis seedlings at 0, 6, 12, and 24 h of reoxygenation recovery after 24 h of hypoxia stress (100% N2 environment). Among the 80 metabolic features characterized using GC-MS, 60% of them were significantly changed under hypoxia. The reoxygenation phase was accompanied by progressively fewer metabolic changes. Only 26% significantly changed metabolic features by the 24 h reoxygenation. Hypoxia-induced metabolic changes returned to normal levels at different speeds. For example, hypoxia-induced accumulation of lactate decreased to a basal level after 6 h of reoxygenation, whereas hypoxia-induced accumulation of alanine and GABA showed partial recovery after 24 h of reoxygenation. Some metabolites, such as gluconate, xylose, guanine, and adenosine, constantly increased during hypoxia reoxygenation. These dynamic metabolic changes demonstrate the flexibility and complexity of plant metabolism during hypoxia stress and subsequent reoxygenation recovery.

The interplay between auxin and cytokinins affects facets of plant development as different as ovule formation and lateral root initiation. Moreover, cytokinins favor complexity in the development of Solanum lycopersicum and Cardamine hirsuta compound leaves. Nevertheless, no role has been proposed for cytokinins in patterning the margins of the simple leaves of Arabidopsis thaliana, a process that is assumed to be sufficiently explained by auxin localization. Here, we discuss evidence supporting the hypothesis that cytokinins play a role in simple leaf margin morphogenesis via crosstalk with auxin, as occurs in other plant developmental events. Indeed, mutant or transgenic arabidopsis plants defective in cytokinin biosynthesis or signaling, or with increased cytokinin degradation have leaf margins less serrated than the wild type.

Flowers are composed of organs whose identity is defined by the combinatorial activity of transcription factors (TFs). The interactions between MADS-box TFs and protein complex formation have been schematized in the floral quartet model of flower development. The gynoecium is the flower's female reproductive part, crucial for fruit and seed production and, hence, for reproductive success. After the establishment of carpel identity, many tissues arise to form a mature gynoecium. TFs have been described as regulators of gynoecium development, and some interactions and complexes have been identified. However, broad knowledge about the interactions among these TFs and their participation during development remains scarce. In this study, we used a systems biology approach to understand the formation of a complex reproductive unit-as the gynoecium-by mapping binary interactions between well-characterized TFs. We analyzed almost 4500 combinations and detected more than 250 protein-protein interactions (PPIs), resulting in a process-specific interaction map. Topological analyses suggest hidden functions and novel roles for many TFs. In addition, we observed a close relationship between TFs involved in auxin and cytokinin-signaling pathways and other TFs. Furthermore, we analyzed the network by combining PPI data, expression, and genetic data, which helped us to dissect it into several dynamic spatio-temporal subnetworks related to gynoecium development processes. Finally, we generated an extended PPI network that predicts new players in gynoecium development. Taken together, all these results serve as a valuable resource for the plant community.

Brown algae are a group of multicellular, heterokont algae that have convergently evolved developmental complexity that rivals that of embryophytes, animals or fungi. Early in development, brown algal zygotes establish a basal and an apical pole, which will become respectively the basal system (holdfast) and the apical system (thallus) of the adult alga. Brown algae are interesting models for understanding the establishment of cell polarity in a broad evolutionary context, because they exhibit a large diversity of life cycles, reproductive strategies and, importantly, their zygotes are produced in large quantities free of parental tissue, with symmetry breaking and asymmetric division taking place in a highly synchronous manner. This review describes the current knowledge about the establishment of the apical-basal axis in the model brown seaweeds Ectocarpus, Dictyota, Fucus and Saccharina, highlighting the advantages and specific interests of each system. Ectocarpus is a genetic model system that allows access to the molecular basis of early development and life-cycle control over apical-basal polarity. The oogamous brown alga Fucus, together with emerging comparative models Dictyota and Saccharina, emphasize the diversity of strategies of symmetry breaking in determining a cell polarity vector in brown algae. A comparison with symmetry-breaking mechanisms in land plants, animals and fungi, reveals that the one-step zygote polarisation of Fucus compares well to Saccharomyces budding and Arabidopsis stomata development, while the two-phased symmetry breaking in the Dictyota zygote compares to Schizosaccharomyces fission, the Caenorhabditis anterior-posterior zygote polarisation and Arabidopsis prolate pollen polarisation. The apical-basal patterning in Saccharina zygotes on the other hand, may be seen as analogous to that of land plants. Overall, brown algae have the potential to bring exciting new information on how a single cell gives rise to an entire complex body plan.

The phenotypes of plants can be influenced by the environmental conditions experienced by their parents. However, there is still much uncertainty about how common and how predictable such parental environmental effects really are. We carried out a comprehensive experimental test for parental effects, subjecting plants of multiple Arabidopsis thaliana genotypes to 24 different biotic or abiotic stresses, or combinations thereof, and comparing their offspring phenotypes in a common environment. The majority of environmental stresses caused significant parental effects, with -35% to +38% changes in offspring fitness. The expression of parental effects was strongly genotype-dependent, and multiple environmental stresses often acted non-additively when combined. The direction and magnitude of parental effects were unrelated to the direct effects on the parents: some environmental stresses did not affect the parents but caused substantial effects on offspring, while for others the situation was reversed. Our study demonstrates that parental environmental effects are common and often strong in A. thaliana, but they are genotype-dependent, act non-additively, and are difficult to predict. We should thus be cautious with generalizing from simple studies with single plant genotypes and/or only few individual environmental stresses. A thorough and general understanding of parental effects requires large multi-factorial experiments.

The metabolic fluxes throughout the tricarboxylic acid cycle (TCAC) are inhibited in the light by the mitochondrial thioredoxin (TRX) system. However, it is unclear how this system orchestrates the fluxes throughout the TCAC and associated pathways in the dark. Here we carried out a

Aquaporins are water channel proteins that play an essential role in plant growth and development. Despite extensive functional characterization of aquaporins in model plants such as Arabidopsis, their contributions to abiotic stress tolerance in non-model plants are still poorly understood. As a close relative of Arabidopsis thaliana, Eutrema salsugineum is an excellent model for studying salt tolerance. Here, we identified and functionally characterized EsPIP1;4, a gene encoding a plasma membrane intrinsic protein (PIP) aquaporin in E. salsugineum. Overexpression of EsPIP1;4 in Arabidopsis improved seed germination and root growth of transgenic plants under abiotic stress, which was accompanied by an increase in proline accumulation, reduction in MDA, and decrease in the rate of ion leakage. Under abiotic stress, transgenic plants overexpressing EsPIP1;4 also showed increased antioxidant enzyme activity, and enhanced K

Maintaining proper DNA methylation levels in the genome requires active demethylation of DNA. However, removing the methyl group from a modified cytosine is chemically difficult and therefore, the underlying mechanism of demethylation had remained unclear for many years. The discovery of the first eukaryotic DNA demethylase, Arabidopsis thaliana REPRESSOR OF SILENCING 1 (ROS1), led to elucidation of the 5-methylcytosine base excision repair mechanism of active DNA demethylation. In the 20 years since ROS1 was discovered, our understanding of this active DNA demethylation pathway, as well as its regulation and biological functions in plants, has greatly expanded. These exciting developments have laid the groundwork for further dissecting the regulatory mechanisms of active DNA demethylation, with potential applications in epigenome editing to facilitate crop breeding and gene therapy.

Paraquat is one of the most widely used nonselective herbicides in agriculture. Due to its wide use, paraquat resistant weeds have emerged and is becoming a potential threat to agriculture. The molecular mechanisms of paraquat resistance in weeds remain largely unknown. Physiological studies indicated that the impaired translocation of paraquat and enhanced antioxidation could improve paraquat resistance in plants. However, the detoxification of paraquat via active metabolism by plants has not been reported to date. Here we report that an activated expression of At1g01600 encoding the P450 protein CYP86A4 confers paraquat resistance as revealed by the gain-of-function mutant paraquat tolerance 11D (pqt11D), in which a T-DNA with four 35S enhancers was inserted at 1646 bp upstream the ATG of At1g01600. The paraquat resistance can be recapitulated in Arabidopsis wild type by overexpressing AtPQT11 (At1g01600), while its knockout mutant is hypersensitive to paraquat. Moreover, AtPQT11 also confers paraquat resistance in E. coli when overexpressed. We further demonstrate that AtPQT11 has P450 enzyme activity that converts paraquat to N-demethyl paraquat nontoxic to Arabidopsis, therefore detoxifying paraquat in plants. Taken together, our results unequivocally demonstrate that AtPQT11/ CYP86A4 detoxifies paraquat via active metabolism, thus revealing a novel molecular mechanism of paraquat resistance in plants and providing a means potentially enabling crops to resist paraquat.

In plants, transitory starch is synthetized during the day and degraded at night to provide the continuous carbon needed for growth and development. Starch metabolism is highly coordinated, as the starch degradation rate must be coupled to the amount of starch synthetized during the day. Maltose is one of the chloroplastic products obtained from starch degradation, and maltose is exported to the cytosol where disproportionating enzyme-2 (DPE2) is responsible for its metabolism. The amount of DPE2 remained unchanged throughout the day, but its activity notably increased at the end of the day (7 p.m.), suggesting that posttranslational modification drives the mechanism underlying the regulatory activity of this enzyme. Sucrose nonfermenting-related kinase-1 (SnRK1), a protein kinase that controls the activity of several metabolic enzymes, was able to interact and phosphorylate DPE2 at three different residues localized in the α-glucanotransferase domain. This phosphorylation acts as a positive regulator of DPE2, increasing its activity. Complementation of dpe2-deficient mutants with the wild-type (WT) and S786A forms of DPE2 showed that the nonphosphorylated form of DPE2 only partially restored starch degradation, suggesting that phosphorylation at S786 is involved in enzyme regulation.

Ubiquitination is an important post-translational modification that regulates multiple cellular activities in plants including environmental stress responses. In addition to activity of ubiquitin ligases, the activity of deubiquitinating enzymes (DUBs) is critical for modulating the optimal ubiquitination status of target proteins in response to environmental stimuli. However, while several ubiquitin ligases have been isolated to date, little is known about the DUBs involved in plant stress responses. Here, we report that two DUBs, UBP12 and UBP13, function in response to disrupted carbon (C)/nitrogen (N)-nutrient stress conditions in Arabidopsis. Knockdown of UBP12 and UBP13 expression resulted in hypersensitivity to high C/low N-nutrient stress conditions, whereas overexpression of UBP13 reduced the sensitivity. Additionally, UBP13 physically interacted with and deubiquitinated the ubiquitin ligase ATL31, a key regulator of plant resistance to high C/low N-nutrient stress conditions. Genetic analysis showed that the loss of ATL31 and its homolog ATL6 suppressed the high C/low N-hyposensitivity of UBP13-overexpressing plants, suggesting that ATL31 is epistatic to UBP12 and UBP13. Taken together, our results suggest that the DUBs UBP12 and UBP13 function together with the ubiquitin ligase ATL31 to mediate C/N-nutrient stress responses in plants.

Calcium (Ca) deficiency causes necrotic symptoms of foliar edges known as tipburn; however, the underlying cellular mechanisms have been poorly understood due to the lack of an ideal plant model and research platform. Using a phenotyping system that quantitates growth and tipburn traits in the model bryophyte Marchantia polymorpha, we evaluated metabolic compounds and the Gβ-null mutant (gpb1) that modulate the occurrence and expansion of the tipburn. Transcriptomic comparisons between wild-type and gpb1 plants revealed the phenylalanine/phenylpropanoid biosynthesis pathway and reactive oxygen species (ROS) important for Ca deficiency responses. gpb1 plants reduced ROS production possibly through transcriptomic regulations of class III peroxidases and induced the expression of phenylpropanoid pathway enzymes without changing downstream lignin contents. Supplementation of intermediate metabolites and chemical inhibitors further confirmed the cell-protective mechanisms of the phenylpropanoid and ROS pathways. Marchantia polymorpha, Arabidopsis thaliana, and Lactuca sativa showed comparable transcriptomic changes where genes related to phenylpropanoid and ROS pathways were enriched in response to Ca deficiency. In conclusion, our study demonstrated unresolved signaling and metabolic pathways of Ca deficiency response. The phenotyping platform can speed up the discovery of chemical and genetic pathways, which could be widely conserved between M. polymorpha and angiosperms.

Intracellular immune receptor nucleotide-binding leucine-rich repeats (NLRs) are highly regulated transcriptionally and post-transcriptionally for balanced plant defense and growth. NLR genes often exist in gene clusters and are usually co-expressed under various conditions. Despite intensive studies of the regulation of NLR proteins, cis-acting elements for NLR gene induction, repression or co-expression are largely unknown due to a larger than usual cis-region for their expression regulation. Here we used the CRISPR/Cas9 genome editing technology to generate a series of in situ deletions at the endogenous location of an NLR gene SNC1 residing in the RPP5 gene cluster. These deletions that made in the wild type and the SNC1 constitutive expressing autoimmune mutant bon1 revealed both positive and negative cis-acting elements for SNC1 expression. Two transcription factors that could bind to these elements were found to have an impact on the expression of SNC1. In addition, co-expression of two genes with SNC1 in the same cluster is found to be mostly dependent on the SNC1 function. Therefore, SNC1 expression is under complex local regulation involving multiple cis-elements and SNC1 itself is a critical regulator of gene expression of other NLR genes in the same gene cluster.

The basic helix-loop-helix (bHLH) family, as one of the largest families of transcription factors (TFs) in plants, plays crucial roles in regulating growth, development, and abiotic stress responses. However, studies on the association of the bHLH genes with apple iron (Fe) deficiency are limited. Here, multiple bHLH genes that responded to Fe deficiency were selected for quantitative real-time PCR in Malus halliana. The results showed that the expression of HEC2-like gene exerted more values compared to other genes under Fe deficiency stress, but the mechanism by which it regulates Fe deficiency stress is unclear. Subsequently, MhHEC2-like gene (ID: 103,455,961) was cloned from M. halliana for functional identification. We found that both transgenic Arabidopsis thaliana and tobacco displayed less chlorosis and more robust growth than wild-type (WT) controls under Fe deficiency stress. At the same time, the overexpressed apple calli grew prominently larger and better under Fe deficiency compared to the wild type. On the other hand, physiological index measurements revealed that overexpressed MhHEC2-like gene enhanced tolerance to Fe deficiency stress in A. thaliana and tobacco by promoting the synthesis of photosynthetic pigments, improving antioxidant enzyme activity, and enhancing Fe reduction, and strengthened tolerance to Fe deficiency stress in apple calli by reducing pH, boosting Fe reduction, and increasing antioxidant enzyme activity. To sum up, the overexpression of MhHEC2-like gene strengthened tolerance to Fe deficiency stress in Arabidopsis thaliana, tobacco, and apple calli.

Osmotic stress imposed by drought and high salinity inhibits plant growth and crop yield. However, our current knowledge on the mechanism by which plants sense osmotic stress is still limited. Here, we identify the transcriptional regulator SEUSS (SEU) as a key player in hyperosmotic stress response in Arabidopsis. SEU rapidly coalesces into liquid-like nuclear condensates when extracellular osmolarity increases. The intrinsically disordered region 1 (IDR1) of SEU is responsible for its condensation. IDR1 undergoes conformational changes to adopt more compact states after an increase in molecular crowding both in vitro and in cells, and two predicted α-helical peptides are required. SEU condensation is indispensable for osmotic stress tolerance, and loss of SEU dramatically compromises the expression of stress tolerance genes. Our work uncovers a critical role of biomolecular condensates in cellular stress perception and response and expands our understanding of the osmotic stress pathway.

Expression of the VaRPP13 in Arabidopsis and tobacco enhanced resistance to oomycete pathogens, and this enhancement is closely related to the activation of salicylic acid (SA) signaling pathway. Resistance (R) genes, which usually contain a nucleotide-binding site and a leucine-rich repeat (NBS-LRR) domain, play crucial roles in disease resistance. In this study, we cloned a CC-NBS-LRR gene VaRPP13 from Vitis amurensis 'Shuang Hong' grapevine, and investigated its function on disease resistance. VaRPP13 expression was induced by Plasmopara viticola, an oomycetes pathogen causing downy mildew disease in grapevine. Heterologous expression VaRPP13 could also enhance resistance to Hyaloperonospora arabidopsidis in Arabidopsis thaliana and Phytophthora capsici in Nicotiana benthamiana, both oomycete pathogens. Further study indicated that VaRPP13 could enhance the expression of genes in SA signal pathway, while exogenous SA could also induce the expression of VaRPP13. In conclusion, our studies demonstrated that VaRPP13 contributes to a broad-spectrum resistance to oomycetes via activating SA signaling pathway.

DNA damage response (DDR) in eukaryotes is essential for the maintenance of genome integrity in challenging environments. The regulatory mechanisms of DDR have been well-established in yeast and humans. However, increasing evidence supports the idea that plants seem to employ different signaling pathways that remain largely unknown. Here, we report the role of MODIFIER OF SNC1, 4-ASSOCIATED COMPLEX SUBUNIT 5A (MAC5A) in DDR in Arabidopsis (Arabidopsis thaliana). Lack of MAC5A in mac5a mutants causes hypersensitive phenotypes to methyl methanesulfonate (MMS), a DNA damage inducer. Consistent with this observation, MAC5A can regulate alternative splicing of DDR genes to maintain the proper response to genotoxic stress. Interestingly, MAC5A interacts with the 26S proteasome (26SP) and is required for its proteasome activity. MAC core subunits are also involved in MMS-induced DDR. Moreover, we find that MAC5A, the MAC core subunits, and 26SP may act collaboratively to mediate high-boron-induced growth repression through DDR. Collectively, our findings uncover the crucial role of MAC in MMS-induced DDR in orchestrating growth and stress adaptation in plants.

Sulfur (S) is an essential macronutrient for plants and a signaling molecule in abiotic stress responses. It is known that S availability modulates root system architecture; however, the underlying molecular mechanisms are largely unknown. We previously reported an Arabidopsis gain-of-function mutant sulfate utilization efficiency4 (sue4) that could tolerate S deficiency during germination and early seedling growth with faster primary root elongation. Here, we report that SUE4, a novel plasma membrane-localized protein, interacts with the polar auxin transporter PIN1, resulting in reduced PIN1 protein levels and thus decreasing auxin transport to the root tips, which promotes primary root elongation. Moreover, SUE4 is induced by sulfate deficiency, consistent with its role in root elongation. Further analyses showed that the SUE4-PIN1 interaction decreased PIN1 levels, possibly through 26 S proteasome-mediated degradation. Taken together, our finding of SUE4-mediated root elongation is consistent with root adaptation to highly mobile sulfate in soil, thus revealing a novel component in the adaptive response of roots to S deficiency.

Sperm chromatin is typically transformed by protamines into a compact and transcriptionally inactive state1,2. Sperm cells of flowering plants lack protamines, yet they have small, transcriptionally active nuclei with chromatin condensed through an unknown mechanism3,4. Here we show that a histone variant, H2B.8, mediates sperm chromatin and nuclear condensation in Arabidopsis thaliana. Loss of H2B.8 causes enlarged sperm nuclei with dispersed chromatin, whereas ectopic expression in somatic cells produces smaller nuclei with aggregated chromatin. This result demonstrates that H2B.8 is sufficient for chromatin condensation. H2B.8 aggregates transcriptionally inactive AT-rich chromatin into phase-separated condensates, which facilitates nuclear compaction without reducing transcription. Reciprocal crosses show that mutation of h2b.8 reduces male transmission, which suggests that H2B.8-mediated sperm compaction is important for fertility. Altogether, our results reveal a new mechanism of nuclear compaction through global aggregation of unexpressed chromatin. We propose that H2B.8 is an evolutionary innovation of flowering plants that achieves nuclear condensation compatible with active transcription.

Germinated plants grow in darkness until they emerge above the soil. To help the seedling penetrate the soil, most dicot seedlings develop an etiolated apical structure consisting of an apical hook and folded, unexpanded cotyledons atop a rapidly elongating hypocotyl. Brassinosteroids (BRs) are necessary for etiolated apical development, but their precise role and mechanisms remain unclear. Arabidopsis thaliana SMALL AUXIN UP RNA17 (SAUR17) is an apical-organ-specific regulator that promotes production of an apical hook and closed cotyledons. In darkness, ethylene and BRs stimulate SAUR17 expression by transcription factor complexes containing PHYTOCHROME-INTERACTING FACTORs (PIFs), ETHYLENE INSENSITIVE 3 (EIN3), and its homolog EIN3-LIKE 1 (EIL1), and BRASSINAZOLE RESISTANT1 (BZR1). BZR1 requires EIN3 and PIFs for enhanced DNA-binding and transcriptional activation of the SAUR17 promoter; while EIN3, PIF3, and PIF4 stability depends on BR signaling. BZR1 transcriptionally downregulates EIN3-BINDING F-BOX 1 and 2 (EBF1 and EBF2), which encode ubiquitin ligases mediating EIN3 and PIF3 protein degradation. By modulating the EBF-EIN3/PIF protein-stability circuit, BRs induce EIN3 and PIF3 accumulation, which underlies BR-responsive expression of SAUR17 and HOOKLESS1 and ultimately apical hook development. We suggest that in the etiolated development of apical structures, BRs primarily modulate plant sensitivity to darkness and ethylene.

RNA degradation is critical for control of gene expression, and endonucleolytic cleavage– dependent RNA degradation is conserved among eukaryotes. Some cleavage sites are secondarily capped in the cytoplasm and identified using the CAGE method. Although uncapped cleavage sites are widespread in eukaryotes, comparatively little information has been obtained about these sites using CAGE-based degradome analysis. Previously, we developed the truncated RNA-end sequencing (TREseq) method in plant species and used it to acquire comprehensive information about uncapped cleavage sites; we observed G-rich sequences near cleavage sites. However, it remains unclear whether this finding is general to other eukaryotes. In this study, we conducted TREseq analyses in fruit flies (Drosophila melanogaster) and budding yeast (Saccharomyces cerevisiae). The results revealed specific sequence features related to RNA cleavage in D. melanogaster and S. cerevisiae that were similar to sequence patterns in Arabidopsis thaliana. Although previous studies suggest that ribosome movements are important for determining cleavage position, feature selection using a random forest classifier showed that sequences around cleavage sites were major determinant for cleaved or uncleaved sites. Together, our results suggest that sequence features around cleavage sites are critical for determining cleavage position, and that sequence-specific endonucleolytic cleavage–dependent RNA degradation is highly conserved across eukaryotes.

Cucumber is one of the most widely consumed vegetables worldwide, and the fruit spine is an important fruit quality trait. Expansins play critical roles in fruit development; however, the regulation of expansins in cucumber fruit spine development has not been reported. In this study, 33 expansin genes were identified in the cucumber genome V3; additionally, expansin genes in Citrullus lanatus, Cucumis melo, Cucurbita maxima, Lagenaria siceraria, and Benincasa hispida were also identified. Phylogenetic analysis of expansin proteins in Cucurbitaceae and Arabidopsis showed that they evolved separately in each plant species. Phylogenetic analysis showed that C. maxima was derived earlier than the other five Cucurbitaceae species. The expression of CsEXPA2, CsEXPA14, and CsEXLA3 varied in cucumber lines with different fruit spine densities. A yeast two-hybrid assay showed that a putative auxin transporter encoded by numerous spine gene (ns) interacts with CsEXLA2, which may be involved in the development of the numerous spines in cucumber. These results provide novel insights into the expansins related to plant development and fruit spine development in cucumber.

Calmodulin (CaM) and calmodulin-like (CML) proteins are the most prominent calcium (Ca

Strigolactones (SLs) play prominent roles in regulating shoot branching and root architecture in model plants. However, their roles in non-model (particularly woody) plants remain unclear. Liriodendron chinense is a timber tree species widely planted in southern China. The outturn percentage and wood quality of L. chinense are greatly affected by the branching characteristics of its shoot, and the rooting ability of the cuttings is key for its vegetative propagation. Here, we isolated and analyzed the function of the MORE AXILLARY GROWTH 1 (LcMAX1) gene, which is involved in L. chinense SL biosynthesis. RT-qPCR showed that LcMAX1 was highly expressed in the roots and axillary buds. LcMAX1 was located in the endoplasmic reticulum (ER) and nucleus. LcMAX1 ectopic expression promoted primary root growth, whereas there were no phenotypic differences in shoot branching between transgenic and wild-type (WT) A. thaliana plants. LcMAX1 overexpression in the max1 mutant restored them to the WT A. thaliana phenotypes. Additionally, AtPIN1, AtPIN2, and AtBRC1 expressions were significantly upregulated in transgenic A. thaliana and the max1 mutant. It was therefore speculated that LcMAX1 promotes primary root growth by regulating expression of auxin transport-related genes in A. thaliana, and LcMAX1 inhibits shoot branching by upregulating expression of AtBRC1 in the max1 mutant. Altogether, these results demonstrated that the root development and shoot branching functions of LcMAX1 were similar to those of AtMAX1. Our findings provide a foundation for obtaining further insights into root and branch development in L. chinense.

Temperature is an important environmental cue that affects flowering time in plants. The MADS-box transcription factor FLOWERING LOCUS M (FLM) forms a heterodimeric complex with SHORT VEGETATIVE PHASE (SVP) and controls ambient temperature-responsive flowering in Arabidopsis. FLM-β and FLM-δ, two major splice variants produced from the FLM locus, exert opposite effects on flowering, but the molecular mechanism by which the interaction between FLM isoforms and SVP affects temperature-responsive flowering remains poorly understood. Here, we show that FLM-β and FLM-δ play important roles in modulating the temperature-dependent behavior, conformation, and stability of SVP. Nuclear localization of SVP decreases as temperature increases. FLM-β is required for SVP nuclear translocation at low temperature, whereas SVP interacts with FLM-δ mainly in the cytoplasm at high temperature. SVP preferentially binds to FLM-β at low temperature in tobacco leaf cells. SVP shows high binding affinity to FLM-β at low temperature and to FLM-δ at high temperature. SVP undergoes similar structural changes in the interactions with FLM-β and FLM-δ; however, FLM-δ likely causes more pronounced conformational changes in the SVP structure. FLM-δ causes rapid degradation of SVP at high temperature, compared with FLM-β, possibly via ubiquitination. Mutation of lysine 53 or lysine 165 in SVP causes increased abundance of SVP due to reduced ubiquitination of SVP and thus delays flowering at high temperature. Our findings suggest that temperature-dependent differential interactions between SVP and FLM isoforms modulate the temperature-responsive induction of flowering in Arabidopsis.

Ribonuclease P (RNase P) enzymes are responsible for the 5′ processing of tRNA precursors. In addition to the well-characterised ribozyme-based RNase P enzymes, an evolutionarily distinct group of protein-only RNase Ps exists. These proteinaceous RNase Ps (PRORPs) can be found in all three domains of life and can be divided into two structurally different types: eukaryotic and prokaryotic. Recent structural studies on members of both families reveal a surprising diversity of molecular architectures, but also highlight conceptual and mechanistic similarities. Here, we provide a comparison between the different types of PRORP enzymes and review how the combination of structural, biochemical, and biophysical studies has led to a molecular picture of protein-mediated tRNA processing.

Increases in recorded high temperatures around the world are causing plant thermomorphogenesis and decreasing crop productivity. PHYTOCHROME INTERACTING FACTOR 4 (PIF4) is a central positive regulator of plant thermomorphogenesis. However, the molecular mechanisms underlying PIF4-regulated thermomorphogenesis remain largely unclear. In this study, we identified ABNORMAL THERMOMORPHOGENESIS 1 (ABT1) as an important negative regulator of PIF4 and plant thermomorphogenesis. Overexpression of ABT1 in the activation tagging mutant abt1-D caused shorter hypocotyls and petioles under moderately high temperature (HT). ABT1 encodes WRKY14, which belongs to subgroup II of the WRKY transcription factors. Overexpression of ABT1/WRKY14 or its close homologs, including ABT2/WRKY35, ABT3/WRKY65, and ABT4/WRKY69in transgenic plants caused insensitivity to HT, whereas the quadruple mutant abt1 abt2 abt3 abt4 exhibited greater sensitivity to HT. ABTs were expressed in hypocotyls, cotyledons, shoot apical meristems, and leaves, but their expression were suppressed by HT. Biochemical assays showed that ABT1 can interact with TCP5, a known positive regulator of PIF4, and interrupt the formation of the TCP5-PIF4 complex and repress its transcriptional activation activity. Genetic analysis showed that ABT1 functioned antagonistically with TCP5, BZR1, and PIF4 in plant thermomorphogenesis. Taken together, our results identify ABT1/WRKY14 as a critical repressor of plant thermomorphogenesis and suggest that ABT1/WRKY14, TCP5, and PIF4 may form a sophisticated regulatory module to fine-tune PIF4 activity and temperature-dependent plant growth.

Double fertilization is an innovative phenomenon in angiosperms, in which one sperm cell first fuses with the egg cell to produce the embryo, and then the other sperm fuses with the central cell to produce the endosperm. However, the molecular mechanism of the preferential fertilization of egg cells is poorly understood. In this study, we report that two egg cell-secreted aspartic proteases, ECS1 and ECS2, play an important role in promoting preferential fertilization of egg cells in Arabidopsis. We show that simultaneous loss of ECS1 and ECS2 function resulted in an approximately 20% reduction in fertility, which can be complemented by the full-length ECS1/2 but not by corresponding active site mutants or by secretion-defective versions of ECS1/2. Detailed phenotypic analysis revealed that the egg cell-sperm cell attachment was compromised in ecs1 ecs2 siliques. Limited pollination assays with cyclin-dependent kinase a1 (cdka;1) pollen showed that preferential egg cell fertilization was impaired in the ecs1 ecs2 mutant. Taken together, these results demonstrate that egg cells secret two aspartic proteases, ECS1 and ECS2, to facilitate the attachment of sperm cells to egg cells so that preferential fertilization of egg cells is achieved. This study reveals the molecular mechanism of preferential fertilization in Arabidopsis thaliana.

Metaproteomics is a powerful tool for the characterization of metabolism, physiology, and functional interactions in microbial communities, including plant-associated microbiota. However, the metaproteomic methods that have been used to study plant-associated microbiota are very laborious and require large amounts of plant tissue, hindering wider application of these methods. We optimized and evaluated different protein extraction methods for metaproteomics of plant-associated microbiota in two different plant species ( Arabidopsis and maize). Our main goal was to identify a method that would work with low amounts of input material (40 to 70 mg) and that would maximize the number of identified microbial proteins. We tested eight protocols, each comprising a different combination of physical lysis method, extraction buffer, and cell-enrichment method on roots from plants grown with synthetic microbial communities. We assessed the performance of the extraction protocols by liquid chromatography-tandem mass spectrometry–based metaproteomics and found that the optimal extraction method differed between the two species. For Arabidopsis roots, protein extraction by beating whole roots with small beads provided the greatest number of identified microbial proteins and improved the identification of proteins from gram-positive bacteria. For maize, vortexing root pieces in the presence of large glass beads yielded the greatest number of microbial proteins identified. Based on these data, we recommend the use of these two methods for metaproteomics with Arabidopsis and maize. Furthermore, detailed descriptions of the eight tested protocols will enable future optimization of protein extraction for metaproteomics in other dicot and monocot plants. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Mitochondrial alternative oxidase is an important protein involved in maintaining cellular metabolic and energy balance, especially under stress conditions. AOX genes knockout is aimed at revealing the functions of AOX genes. Under unfavourable conditions, AOX-suppressed plants (mainly based on Arabidopsis AOX1a-knockout lines) usually experience strong oxidative stress. However, a compensation effect, which consists of the absence of AOX1a leading to an increase in defence response mechanisms, concomitant with a decrease in ROS content, has also been demonstrated. This review briefly describes the possible mechanisms underlying the compensation effect upon the suppression of AOX1a. Information about mitochondrial retrograde regulation of AOX is given. The importance of ROS and mitochondrial membrane potential in triggering the signal transmission from mitochondria in the absence of AOX or disturbance of mitochondrial electron transport chain functions is indicated. The few available data on the response of the cell to the absence of AOX at the level of changes in the hormonal balance and the reactions of chloroplasts are presented. The decrease in the relative amount of reduced ascorbate at stable ROS levels as a result of compensation in AOX1a-suppressed plants is proposed as a sign of stress development. Obtaining direct evidence on the mechanisms and signalling pathways involved in AOX modulation in the genome should facilitate a deeper understanding of the role of AOX in the integration of cellular signalling pathways.

Repression of embryonic traits during the seed-to-seedling phase transition requires the inactivation of master transcription factors associated with embryogenesis. How the timing of such inactivation is controlled is unclear. Here, we report on a novel transcriptional co-repressor, Arabidopsis thaliana SDR4L, that forms a feedback inhibition loop with the master transcription factors LEC1 and ABI3 to repress embryonic traits post-imbibition. LEC1 and ABI3 regulate their own expression by inducing AtSDR4L during mid to late embryogenesis. AtSDR4L binds to sites upstream of LEC1 and ABI4, and these transcripts are upregulated in Atsdr4l seedlings. Atsdr4l seedlings phenocopy a LEC1 overexpressor. The embryonic traits of Atsdr4l can be partially rescued by impairing LEC1 or ABI3. The penetrance and expressivity of the Atsdr4l phenotypes depend on both developmental and external cues, demonstrating the importance of AtSDR4L in seedling establishment under suboptimal conditions.

The phytohormone auxin is the major coordinative signal in plant development

Phosphoglucose isomerase (PGI) catalyzes the interconversion of fructose-6-phosphate and glucose-6-phosphate, which impacts cell carbon metabolic flow. Arabidopsis (Arabidopsis thaliana) contains two nuclear PGI genes respectively encoding plastidial PGI1 and cytosolic PGI (cPGI). The loss of PGI1 impairs the conversion of F6P of the Calvin–Benson cycle to G6P for the synthesis of transitory starch in leaf chloroplasts. Since cpgi knockout mutants have not yet been obtained, they are thought to be lethal. The cpgi lethality can be rescued by expressing CaMV 35S promoter (p35S)-driven cPGI; however, the complemented line is completely sterile due to pollen degeneration. Here, we generated a cpgi mutant expressing p35S::cPGI-YFP in which YFP fluorescence in developing anthers was undetectable specifically in the tapetum and in pollen, which could be associated with male sterility. We also generated RNAi-cPGI knockdown lines with strong cPGI repression in floral buds that exhibited reduced male fertility due to the degeneration of most pollen. Histological analyses indicated that the synthesis of intersporal callose walls was impaired, causing microsporocytes to fail to separate haploid daughter nuclei to form tetrads, which might be responsible for subsequent pollen degeneration. We successfully isolated cpgi knockout mutants in the progeny of a heterozygous cpgi mutant floral-dipped with sugar solutions. The rescued cpgi mutants exhibited diminished young vegetative growth, reduced female fertility, and impaired intersporal callose wall formation in a meiocyte, and, thus, male sterility. Collectively, our data suggest that cPGI plays a vital role in carbohydrate partitioning, which is indispensable for microsporogenesis and early embryogenesis.

DNA methylation, a conserved epigenetic mark, is critical for tuning temporal and spatial gene expression. The Arabidopsis thaliana DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required to prevent DNA hypermethylation at thousands of genomic loci. However, how ROS1 is recruited to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor that is required to prevent gene silencing and DNA hypermethylation. AGDP3 binds to H3K9me2 marks in its target DNA via its AGD12 cassette. Analysis of the crystal structure of the AGD12 cassette of AGDP3 in complex with an H3K9me2 peptide revealed that dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. A histidine residue located in the methyllysine binding aromatic cage provides AGDP3 with pH-dependent H3K9me2 binding capacity. Our results uncover a molecular mechanism for the regulation of DNA demethylation by the gene silencing mark H3K9me2.

RNA-binding proteins (RBPs) participate in a diverse set of biological processes in plants, but their functions and underlying mechanisms in plant–pathogen interactions are largely unknown. We previously showed that Arabidopsis thaliana BPA1-LIKE PROTEIN3 (BPL3) belongs to a conserved plant RBP family and negatively regulates reactive oxygen species (ROS) accumulation and cell death under biotic stress. In this study, we demonstrate that BPL3 suppresses FORKED-LIKE7 (FL7) transcript accumulation and raises levels of the cis-natural antisense long non-coding RNA (lncRNA) of FL7 (nalncFL7). FL7 positively regulated plant immunity to Phytophthora capsici while nalncFL7 negatively regulated resistance. We also showed that BPL3 directly binds to and stabilizes nalncFL7. Moreover, nalncFL7 suppressed accumulation of FL7 transcripts. Furthermore, FL7 interacted with HIGHLY ABA-INDUCED PP2C1 (HAI1), a type 2C protein phosphatase, and inhibited HAI1 phosphatase activity. By suppressing HAI1 activity, FL7 increased the phosphorylation levels of MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3) and MPK6, thus enhancing immunity responses. BPL3 and FL7 are conserved in all plant species tested, but the BPL3–nalncFL7–FL7 cascade was specific to the Brassicaceae. Thus, we identified a conserved BPL3–nalncFL7–FL7 cascade that coordinates plant immunity.

Nitrogen (N) availability is a major limiting factor for plant growth and agricultural productivity. Although the gene regulation network in response to N starvation has been extensively studied, it remains unknown whether N starvation has an impact on the activity of transposable elements (TEs). Here, we report that TEs can be transcriptionally activated in Arabidopsis under N starvation conditions. Through genetic screening of idm1-14 suppressors, we cloned GLU1, which encodes a glutamate synthase that catalyzes the synthesis of glutamate in the primary N assimilation pathway. We found that glutamate synthase 1 (GLU1) and its functional homologs GLU2 and glutamate transport 1 (GLT1) are redundantly required for TE silencing, suggesting that N metabolism can regulate TE activity. Transcriptome and methylome analyses revealed that N starvation results in genome-wide TE activation without inducing obvious alteration of DNA methylation. Genetic analysis indicated that N starvation-induced TE activation is also independent of other well-established epigenetic mechanisms, including histone methylation and heterochromatin decondensation. Our results provide new insights into the regulation of TE activity under stressful environments in planta.

To cope with environmental stresses and ensure maximal reproductive success, plants have developed strategies to adjust the timing of their transition to reproductive growth. This has a substantial impact on the stress resilience of crops and ultimately on agricultural productivity. Here, we report a previously uncharacterized, plant-specific gene family designated as Regulator of Flowering and Stress (RFS). Overexpression of the BdRFS gene in Brachypodium distachyon delayed flowering, increased biomass accumulation, and promoted drought tolerance, whereas clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated knockout mutants exhibited opposite phenotypes. A double T-DNA insertional mutant in the two Arabidopsis (Arabidopsis thaliana) homologs replicated the effects on flowering and water deprivation seen in the B. distachyon CRISPR knockout lines, highlighting the functional conservation of the family between monocots and dicots. Lipid analysis of B. distachyon and Arabidopsis revealed that digalactosyldiacylglycerol (DGDG) and phosphatidylcholine (PC) contents were significantly, and reciprocally, altered in overexpressor and knockout mutants. Importantly, alteration of C16:0-containing PC, a Flowering Locus T-interacting lipid, associated with flowering phenotype, with elevated levels corresponding to earlier flowering. Co-immunoprecipitation analysis suggested that BdRFS interacts with phospholipase Dα1 as well as several other abscisic acid-related proteins. Furthermore, reduction of C18:3 fatty acids in DGDG corresponded with reduced jasmonic acid metabolites in CRISPR mutants. Collectively, we suggest that stress-inducible RFS proteins represent a regulatory component of lipid metabolism that impacts several agronomic traits of biotechnological importance.

International audience; Emergence of secondary roots through parental tissue is a highly controlled developmental process. Although the model plant Arabidopsis has been useful to uncover the predominant role of auxin in this process, its simple root structure is not representative of how emergence takes place in most plants, which display more complex root anatomy. White lupin is a legume crop producing structures called cluster roots, where closely spaced rootlets emerge synchronously. Rootlet primordia push their way through several cortical cell layers while maintaining the parent root integrity, reflecting more generally the lateral root emergence process in most multilayered species. In this study, we showed that lupin rootlet emergence is associated with an upregulation of cell wall pectin modifying and degrading genes under the active control of auxin. Among them, we identified LaPG3, a polygalacturonase gene typically expressed in cells surrounding the rootlet primordium and we showed that its downregulation delays emergence. Immunolabeling of pectin epitopes and their quantification uncovered a gradual pectin demethylesterification in the emergence zone, which was further enhanced by auxin treatment, revealing a direct hormonal control of cell wall properties. We also report rhamnogalacturonan-I modifications affecting cortical cells that undergo separation as a consequence of primordium outgrowth. In conclusion, we describe a model of how external tissues in front of rootlet primordia display cell wall modifications to allow for the passage of newly formed rootlets.

Chloroplast ATP synthases consist of a membrane-spanning coupling factor (CFO) and a soluble coupling factor (CF1). It was previously demonstrated that CONSERVED ONLY IN THE GREEN LINEAGE160 (CGL160) promotes the formation of plant CFO and performs a similar function in the assembly of its c-ring to that of the distantly related bacterial Atp1/UncI protein. Here, we show that in Arabidopsis (Arabidopsis thaliana) the N-terminal portion of CGL160 (AtCGL160N) is required for late steps in CF1-CFO assembly. In plants that lacked AtCGL160N, CF1-CFO content, photosynthesis, and chloroplast development were impaired. Loss of AtCGL160N did not perturb c-ring formation, but led to a 10-fold increase in the numbers of stromal CF1 subcomplexes relative to that in the wild type. Co-immunoprecipitation and protein crosslinking assays revealed an association of AtCGL160 with CF1 subunits. Yeast two-hybrid assays localized the interaction to a stretch of AtCGL160N that binds to the DELSEED-containing CF1-β subdomain. Since Atp1 of Synechocystis (Synechocystis sp. PCC 6803) could functionally replace the membrane domain of AtCGL160 in Arabidopsis, we propose that CGL160 evolved from a cyanobacterial ancestor and acquired an additional function in the recruitment of a soluble CF1 subcomplex, which is critical for the modulation of CF1-CFO activity and photosynthesis.

Key message Elevated methylglyoxal levels contribute to ammonium-induced growth disorders in Arabidopsis thaliana. Methylglyoxal detoxification pathway limitation, mainly the glyoxalase I activity, leads to enhanced sensitivity of plants to ammonium nutrition. Abstract Ammonium applied to plants as the exclusive source of nitrogen often triggers multiple phenotypic effects, with severe growth inhibition being the most prominent symptom. Glycolytic flux increase, leading to overproduction of its toxic by-product methylglyoxal (MG), is one of the major metabolic consequences of long-term ammonium nutrition. This study aimed to evaluate the influence of MG metabolism on ammonium-dependent growth restriction in Arabidopsis thaliana plants. As the level of MG in plant cells is maintained by the glyoxalase (GLX) system, we analyzed MG-related metabolism in plants with a dysfunctional glyoxalase pathway. We report that MG detoxification, based on glutathione-dependent glyoxalases, is crucial for plants exposed to ammonium nutrition, and its essential role in ammonium sensitivity relays on glyoxalase I (GLXI) activity. Our results indicated that the accumulation of MG-derived advanced glycation end products significantly contributes to the incidence of ammonium toxicity symptoms. Using A. thaliana frostbite1 as a model plant that overcomes growth repression on ammonium, we have shown that its resistance to enhanced MG levels is based on increased GLXI activity and tolerance to elevated MG-derived advanced glycation end-product (MAGE) levels. Furthermore, our results show that glyoxalase pathway activity strongly affects cellular antioxidative systems. Under stress conditions, the disruption of the MG detoxification pathway limits the functioning of antioxidant defense. However, under optimal growth conditions, a defect in the MG detoxification route results in the activation of antioxidative systems.

Encasements formed around haustoria and biotrophic hyphae as well as hypersensitive reaction (HR) cell death are essential plant immune responses to filamentous pathogens. In this study we examine the components that may contribute to the absence of these responses in susceptible barley attacked by the powdery mildew fungus. We find that the effector CSEP0162 from this pathogen targets plant MONENSIN SENSITIVITY1 (MON1), which is important for the fusion of multivesicular bodies to their target membranes. Overexpression of CSEP0162 and silencing of barley MON1 both inhibit encasement formation. We find that the Arabidopsis ecotype No-0 has resistance to powdery mildew, and that this is partially dependent on MON1. Surprisingly, we find the MON1-dependent resistance in No-0 not only includes an encasement response, but also an effective HR. Similarly, silencing of MON1 in barley also blocks Mla3-mediated HR-based powdery mildew resistance. Our results indicate that MON1 is a vital plant immunity component, and we speculate that the barley powdery mildew fungus introduces the effector CSEP0162 to target MON1 and hence reduce encasement formation and HR.