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.

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.

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.

Natural antisense long noncoding RNAs (lncNATs) are involved in the regulation of gene expression in plants, modulating different relevant developmental processes and responses to various stimuli. We have identified and characterized two lncNATs (NAT1UGT73C6 and NAT2UGT73C6, collectively NATsUGT73C6) from Arabidopsis thaliana that are transcribed from gene fully overlapping UGT73C6, a member of the UGT73C subfamily of genes encoding UDP-glycosyltransferases (UGTs). Expression of both NATsUGT73C6 is developmentally controlled and occurs independently of the transcription of UGT73C6 in cis. Downregulation of NATsUGT73C6 levels through artificial microRNAs results in a reduction of the rosette area, while constitutive overexpression of NAT1UGT73C6 or NAT2UGT73C6 leads to the opposite phenotype, an increase in rosette size. This activity of NATsUGT73C6 relies on its RNA sequence, and, although modulation of UGT73C6 in cis cannot be excluded, the observed phenotypes are not a consequence of the regulation of UGT73C6 in trans. The NATsUGT73C6 levels were shown to affect cell proliferation and thus individual leaf size. Consistent with this concept, our data suggest that the NATsUGT73C6 influence the expression levels of key transcription factors involved in regulating leaf growth by modulating cell proliferation. These findings thus reveal an additional regulatory layer on the process of leaf growth.

Several commercially important secondary metabolites are produced and accumulated in high amounts by glandular trichomes, giving the prospect of using them as metabolic cell factories. Due to extremely high metabolic fluxes through glandular trichomes, previous research focused on how such flows are achieved. The question regarding their bioenergetics became even more interesting with the discovery of photosynthetic activity in some glandular trichomes. Despite recent advances, how primary metabolism contributes to the high metabolic fluxes in glandular trichomes is still not fully elucidated. Using computational methods and available multi-omics data, we first developed a quantitative framework to investigate the possible role of photosynthetic energy supply in terpenoid production and next tested experimentally the simulation-driven hypothesis. With this work, we provide the first reconstruction of specialised metabolism in Type-VI photosynthetic glandular trichomes of Solanum lycopersicum. Our model predicted that increasing light intensities results in a shift of carbon partitioning from catabolic to anabolic reactions driven by the energy availability of the cell. Moreover, we show the benefit of shifting between isoprenoid pathways under different light regimes, leading to a production of different classes of terpenes. Our computational predictions were confirmed in vivo, demonstrating a significant increase in production of monoterpenoids while the sesquiterpenes remained unchanged under higher light intensities. The outcomes of this research provide quantitative measures to assess the beneficial role of chloroplast in glandular trichomes for enhanced production of secondary metabolites and can guide the design of new experiments that aim at modulating terpenoid production.

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.

Piperine (1-piperoyl piperidine) is responsible for the pungent perception of dried black pepper (Pipernigrum) fruits and essentially contributes to the aromatic properties of this spice in combination with ablend of terpenoids. The final step in piperine biosynthesis involves piperine synthase (PS), which catalyzesthe reaction of piperoyl CoA and piperidine to the biologically active and pungent amide. Nevertheless, experimental data on the cellular localization of piperine and the complete biosynthetic pathway are missing. Not only co-localization of enzymes and products, but also potential transport of piperamides to thesink organs is a possible alternative. This work, which includes purification of the native enzyme, immunolocalization, laser microdissection, fluorescence microscopy, and electron microscopy combinedwith liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), providesexperimental evidence that piperine and PS are co-localized in specialized cells of the black pepper fruit peri-sperm. PS accumulates during early stages of fruit development and its level declines before the fruits arefully mature. The product piperine is co-localized to PS and can be monitored at the cellular level by itsstrong bluish fluorescence. Rising piperine levels during fruit maturation are consistent with the increasingnumbers of fluorescent cells within the perisperm. Signal intensities of individual laser-dissected cells whenmonitored by LC-ESI-MS/MS indicate molar concentrations of this alkaloid. Significant levels of piperineand additional piperamides were also detected in cells distributed in the cortex of black pepper roots. Insummary, the data provide comprehensive experimental evidence of and insights into cell-specific biosyn-thesis and storage of piperidine alkaloids, specific and characteristic for the Piperaceae. By a combination offluorescence microscopy and LC-MS/MS analysis we localized the major piperidine alkaloids to specific cellsof the fruit perisperm and the root cortex. Immunolocalization of native piperine and piperamide synthasesshows that enzymes are co-localized with high concentrations of products in these idioblasts.