SUMMARYWHIRLY1 belongs to a family of plant‐specific transcription factors capable of binding DNA or RNA in all three plant cell compartments that contain genetic materials. In Arabidopsis thaliana, WHIRLY1 has been studied at the later stages of plant development, including flowering and leaf senescence, as well as in biotic and abiotic stress responses. In this study, WHIRLY1 knockout mutants of A. thaliana were prepared by CRISPR/Cas9‐mediated genome editing to investigate the role of WHIRLY1 during early seedling development. The loss‐of‐function of WHIRLY1 in 5‐day‐old seedlings did not cause differences in the phenotype and the photosynthetic performance of the emerging cotyledons compared with the wild type. Nevertheless, comparative RNA sequencing analysis revealed that the knockout of WHIRLY1 affected the expression of a small but specific set of genes during this critical phase of development. About 110 genes were found to be significantly deregulated in the knockout mutant, wherein several genes involved in the early steps of aliphatic glucosinolate (GSL) biosynthesis were suppressed compared with wild‐type plants. The downregulation of these genes in WHIRLY1 knockout lines led to decreased GSL contents in seedlings and in seeds. Since GSL catabolism mediated by myrosinases was not altered during seed‐to‐seedling transition, the results suggest that AtWHIRLY1 plays a major role in modulation of aliphatic GSL biosynthesis during early seedling development. In addition, phylogenetic analysis revealed a coincidence between the evolution of methionine‐derived aliphatic GSLs and the addition of a new WHIRLY in core families of the plant order Brassicales.

Hyaloperonospora arabidopsidis (Hpa) is an oomycete pathogen that causes downy mildew disease on Arabidopsis. This obligate biotroph manipulates the homeostasis of its host plant by secreting numerous effector proteins, among which are the RxLR effectors. Identifying the host targets of effectors and understanding how their manipulation facilitates colonization of plants are key to improve plant resistance to pathogens. Here we characterize the interaction between the RxLR effector HaRxL106 and BIM1, an Arabidopsis transcription factor (TF) involved in Brassinosteroid (BR) signaling. We report that HaRxL106 interacts with BIM1 in vitro and in planta. BIM1 is required by the effector to increase the host plant susceptibility to (hemi)biotrophic pathogens, and thus can be regarded as a susceptibility factor. Mechanistically, HaRxL106 requires BIM1 to induce the transcriptional activation of BR‐responsive genes and cause alterations in plant growth patterns that phenocopy the shade avoidance syndrome. Our results support previous observations of antagonistic interactions between activation of BR signaling and suppression of plant immune responses and reveal that BIM1, a new player in this crosstalk, is manipulated by the pathogenic effector HaRxL106.

The mature seed in legumes consists of an embryo and seed coat. In contrast to knowledge about the embryo, we know relatively little about the seed coat. We analyzed the gene expression during seed development using a panel of cultivated and wild pea genotypes. Gene co‐expression analysis identified gene modules related to seed development, dormancy, and domestication. Oxidoreductase genes were found to be important components of developmental and domestication processes. Proteomic and metabolomic analysis revealed that domestication favored proteins involved in photosynthesis and protein metabolism at the expense of seed defense. Seed coats of wild peas were rich in cell wall‐bound metabolites and the protective compounds predominated in their seed coats. Altogether, we have shown that domestication altered pea seed development and modified (mostly reduced) the transcripts along with the protein and metabolite composition of the seed coat, especially the content of the compounds involved in defense. We investigated dynamic profiles of selected identified phenolic and flavonoid metabolites across seed development. These compounds usually deteriorated the palatability and processing of the seeds. Our findings further provide resources to study secondary metabolism and strategies for improving the quality of legume seeds which comprise an important part of the human protein diet.

Methylerythritol cyclodiphosphate (MEcPP) is an intermediate in the biosynthesis of isoprenoids in plant plastids and in bacteria, and acts as a stress signal in plants. Here, we show that MEcPP regulates biofilm formation in Escherichia coli K-12 MG1655. Increased MEcPP levels, triggered by genetic manipulation or oxidative stress, inhibit biofilm development and production of fimbriae. Deletion of fimE, encoding a protein known to downregulate production of adhesive fimbriae, restores biofilm formation in cells with elevated MEcPP levels. Limited proteolysis-coupled mass spectrometry (LiP-MS) reveals that MEcPP interacts with the global regulatory protein H-NS, which is known to repress transcription of fimE. MEcPP prevents the binding of H-NS to the fimE promoter. Therefore, our results indicate that MEcPP can regulate biofilm formation by modulating H-NS activity and thus reducing fimbriae production. Further research is needed to test whether MEcPP plays similar regulatory roles in other bacteria.

Arabidopsis primary root growth response to phosphate (Pi) deficiency is mainly controlled by changes in apoplastic iron (Fe). Upon Pi deficiency, apoplastic Fe deposition in the root apical meristem activates pathways leading to the arrest of meristem maintenance and inhibition of cell elongation. Here, we report that a member of the uncharacterized cytochrome b561 and DOMON domain (CYBDOM) protein family, named CRR, promotes iron reduction in an ascorbate-dependent manner and controls apoplastic iron deposition. Under low Pi, the crr mutant shows an enhanced reduction of primary root growth associated with increased apoplastic Fe in the root meristem and a reduction in meristematic cell division. Conversely, CRR overexpression abolishes apoplastic Fe deposition rendering primary root growth insensitive to low Pi. The crr single mutant and crr hyp1 double mutant, harboring a null allele in another member of the CYDOM family, shows increased tolerance to high-Fe stress upon germination and seedling growth. Conversely, CRR overexpression is associated with increased uptake and translocation of Fe to the shoot and results in plants highly sensitive to Fe excess. Our results identify a ferric reductase implicated in Fe homeostasis and developmental responses to abiotic stress, and reveal a biological role for CYBDOM proteins in plants.

The genomes of many plants, animals, and fungi frequently comprise dispensable B chromosomes that rely upon various chromosomal drive mechanisms to counteract the tendency of non-essential genetic elements to be purged over time. The B chromosome of rye – a model system for nearly a century – undergoes targeted nondisjunction during first pollen mitosis, favouring segregation into the generative nucleus, thus increasing their numbers over generations. However, the genetic mechanisms underlying this process are poorly understood. Here, using a newly-assembled, ~430 Mb-long rye B chromosome pseudomolecule, we identify five candidate genes whose role as trans-acting moderators of the chromosomal drive is supported by karyotyping, chromosome drive analysis and comparative RNA-seq. Among them, we identify DCR28, coding a microtubule-associated protein related to cell division, and detect this gene also in the B chromosome of Aegilops speltoides. The DCR28 gene family is neo-functionalised and serially-duplicated with 15 B chromosome-located copies that are uniquely highly expressed in the first pollen mitosis of rye.

RNA‐guided endonucleases originating from the bacterial CRISPR/Cas system are a versatile tool for targeted gene editing. To determine the functional relevance of a gene of interest, deletion of the entire open reading frame (ORF) by two independent double‐strand breaks (DSBs) is particularly attractive. This strategy greatly benefits from high editing efficiency, which is strongly influenced by the Cas endonuclease version used. We developed two reporter switch‐on assays, for quantitative comparison and optimization of Cas constructs. The assays are based on four components: (i) A reporter gene, the mRNA of which carries a hairpin (HP) loop targeted by (ii) the endoribonuclease Csy4. Cleavage of the mRNA at the HP loop by Csy4 abolishes the translation of the reporter. Csy4 was used as the target for full deletion. (iii) A Cas system targeting sites flanking the Csy4 ORF with a 20‐bp spacer either side to preferentially detect full‐deletion events. Loss of functional Csy4 would lead to reporter gene expression, allowing indirect quantification of Cas‐mediated deletion events. (iv) A reference gene for normalization. We tested these assays on Nicotiana benthamiana leaves and Lotus japonicus calli induced on hypocotyl sections, using Firefly luciferase and mCitrine as reporter genes and Renilla luciferase and hygromycin phosphotransferase II as reference genes, respectively. We observed a >90% correlation between reporter expression and full Csy4 deletion events, demonstrating the validity of these assays. The principle of using the Csy4–HP module as Cas target should be applicable to other editing goals including single DSBs in all organisms.

In plants, exposure to high light irradiation induces various stress responses, which entail complex metabolic rearrangements. To explore these dynamics, we conducted time‐course experiments spanning 2 min to 72 h with Arabidopsis thaliana under high and control light. Comparative metabolomics, transcriptomics, redox proteomics, and stable isotope labeling on leaf rosettes identified a series of synchronous and successive responses that provide a deeper insight into well‐orchestrated mechanisms contributing to high‐light acclimation. We observed transient transcriptome downregulation related to light harvesting and electron flow before the profound remodeling of the photosynthetic apparatus. Throughout the entire time course, redox homeostasis is tightly balanced between downregulation of production and enhanced transformation of NADPH accompanied by redistribution of reducing equivalents across several subcellular compartments. In both light conditions, C4 acids such as malate and fumarate are produced via anaplerosis. In carbon units, their accumulation in vacuoles surpasses plastidic levels of starch and intensifies notably under high light. In parallel, citrate synthesis from pyruvate is significantly hindered diurnally. Isotopic labeling in 2‐oxoglutarate and glutamate suggests a moderate de novo synthesis of C5 acids from a vacuolar citrate reservoir during the light phase while they are largely renewed during the night. In the absence of a diurnal clockwise flow through the tricarboxylic acid (TCA) cycle, increased oxidation of photorespiratory glycine takes over as a source of reductants to fuel mitochondrial ATP production. These findings, along with previous research, contribute to a model integrating redox balance and linking increased carbon assimilation and nitrogen metabolism, especially in the context of an incomplete TCA cycle.

The unsaturation of phospholipids influences the function of membranes. In Arabidopsis thaliana, the oleoyl Δ12‐desaturase FAD2 converts oleic (18:1Δ9) to linoleic acid (18:2Δ9,12) and influences phospholipid unsaturation in different cellular membranes. Despite its importance, the precise localization of Arabidopsis FAD2 has not been unambiguously described. As FAD2 is thought to modify phospholipid‐associated fatty acids at the endoplasmic reticulum (ER), from where unsaturates are distributed to other cellular sites, we hypothesized that FAD2 locates to ER subdomains enabling trafficking of lipid intermediates through the secretory pathway. Fluorescent FAD2 fusions used to test this hypothesis were first assessed for functionality by heterologous expression in yeast (Saccharomyces cerevisiae), and in planta by Arabidopsis fad2 mutant rescue upon ectopic expression from an intrinsic FAD2 promoter fragment. Light sheet fluorescence, laser scanning confocal or spinning disc microscopy of roots, leaves, or mesophyll protoplasts showed the functional fluorescence‐tagged FAD2 variants in flattened donut‐shaped structures of ~0.5–1 μm diameter, in a pattern not resembling mere ER association. High‐resolution imaging of coexpressed organellar markers showed fluorescence‐tagged FAD2 in a ring‐shaped pattern surrounding ER‐proximal Golgi particles, colocalizing with pre‐cis‐Golgi markers. This localization required the unusual C‐terminal retention signal of FAD2, and deletion or substitutions in this protein region resulted in relaxed distribution and diffuse association with the ER. The distinct association of FAD2 with pre‐cis‐Golgi stacks in Arabidopsis root and leaf tissue is consistent with a contribution of FAD2 to membrane lipid homeostasis through the secretory pathway, as verified by an increased plasma membrane liquid phase order in the fad2 mutant.

Wind, rain, herbivores, obstacles, neighbouring plants, etc. provide important mechanical cues to steerplant growth and survival. Mechanostimulation to stimulate yield and stress resistance of crops is of signifi-cant research interest, yet a molecular understanding of transcriptional responses to touch is largely absentin cereals. To address this, we performed whole-genome transcriptomics following mechanostimulation ofwheat, barley, and the recent genome-sequenced oat. The largest transcriptome changes occurred 25 minafter touching, with most of the genes being upregulated. While most genes returned to basal expressionlevel by 1–2 h in oat, many genes retained high expression even 4 h post-treatment in barley and wheat.Functional categories such as transcription factors, kinases, phytohormones, and Ca2+regulation wereaffected. In addition, cell wall-related genes involved in (hemi)cellulose, lignin, suberin, and callose biosyn-thesis were touch-responsive, providing molecular insight into mechanically induced changes in cell wallcomposition. Furthermore, several cereal-specific transcriptomic footprints were identified that were notobserved in Arabidopsis. In oat and barley, we found evidence for systemic spreading of touch-induced sig-nalling. Finally, we provide evidence that both the jasmonic acid-dependent and the jasmonic acid-independent pathways underlie touch-signalling in cereals, providing a detailed framework and markergenes for further study of (a)biotic stress responses in cereals.