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Carbohydrates such as fructans can be involved in priming or defence stimulation, and hence potentially provide new strategies for crop protection against biotic stress. Chicory (Cichorium intybus) is a model plant for fructan research and is a crop with many known health benefits. Using the chicory-Botrytis cinerea pathosystem, we tested the effectiveness of fructan-induced immunity, focussing on different plant and microbial fructans. Sugar dynamics were followed after priming and subsequent pathogen infection. Our results indicated that many higher plants might detect extracellular levan oligosaccharides (LOS) of microbial origin, while chicory also detects extracellular small inulin-type fructooligosaccharides (FOS) of endogenous origin, thus differing from the findings of previous fructan priming studies. No clear positive effects were observed for inulin or mixed-type fructans. An elicitor-specific burst of reactive oxygen species was observed for sulfated LOS, while FOS and LOS both behaved as genuine priming agents. In addition, a direct antifungal effect was observed for sulfated LOS. Intriguingly, LOS priming led to a temporary increase in apoplastic sugar concentrations, mainly glucose, which could trigger downstream responses. Total sugar and starch contents in total extracts of LOS-primed leaves were higher after leaf detachment, indicating they could maintain their metabolic activity. Our results indicate the importance of balancing intra- and extracellular sugar levels (osmotic balance) in the context of 'sweet immunity' pathways. ispartof: JOURNAL OF EXPERIMENTAL BOTANY vol:73 issue:12 pages:4214-4235 ispartof: location:England status: published

Auxin is a key regulator of many developmental processes in land plants and plays a strikingly similar role in the phylogenetically distant brown seaweeds. Emerging evidence shows that the PIN and PIN-like (PILS) auxin transporter families have preceded the evolution of the canonical auxin response pathway. A wide conservation of PILS-mediated auxin transport, together with reports of auxin function in unicellular algae, would suggest that auxin function preceded the advent of multicellularity. We find that PIN and PILS transporters form two eukaryotic subfamilies within a larger bacterial family. We argue that future functional characterisation of algal PIN and PILS transporters can shed light on a common origin of an auxin function followed by independent co-option in a multicellular context.

Thiamin (or thiamine), known as vitamin B1, represents an indispensable component of human diets, being pivotal in energy metabolism. Thiamin research depends on adequate vitamin quantification in plant tissues. A recently developed quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is able to assess the level of thiamin, its phosphorylated entities and its biosynthetic intermediates in the model plant Arabidopsis thaliana, as well as in rice. However, their implementation requires expensive equipment and substantial technical expertise. Microbiological assays can be useful in deter-mining metabolite levels in plant material and provide an affordable alternative to MS-based analysis. Here, we evaluate, by comparison to the LC-MS/MS reference method, the potential of a carefully chosen panel of yeast assays to estimate levels of total vitamin B1, as well as its biosynthetic intermediates pyrimidine and thiazole in Arabidopsis samples. The examined panel of Saccharomyces cerevisiae mutants was, when implemented in microbiological assays, capable of correctly assigning a series of wild-type and thiamin biofortified Arabidopsis plant samples. The assays provide a readily applicable method allowing rapid screening of vitamin B1 (and its biosynthetic intermediates) content in plant material, which is particularly useful in metabolic engineering approaches and in germplasm screening across or within species.

We present unresolved questions in plant abiotic stress biology as posed by 15 research groups with expertise spanning eco-physiology to cell and molecular biology. We present unresolved questions in plant abiotic stress biology as posed by 15 research groups with expertise spanning eco-physiology to cell and molecular biology. Common themes of these questions include the need to better understand how plants detect water availability, temperature, salinity, and rising carbon dioxide (CO2) levels; how environmental signals interface with endogenous signaling and development (e.g. circadian clock and flowering time); and how this integrated signaling controls downstream responses (e.g. stomatal regulation, proline metabolism, and growth versus defense balance). The plasma membrane comes up frequently as a site of key signaling and transport events (e.g. mechanosensing and lipid-derived signaling, aquaporins). Adaptation to water extremes and rising CO2 affects hydraulic architecture and transpiration, as well as root and shoot growth and morphology, in ways not fully understood. Environmental adaptation involves tradeoffs that limit ecological distribution and crop resilience in the face of changing and increasingly unpredictable environments. Exploration of plant diversity within and among species can help us know which of these tradeoffs represent fundamental limits and which ones can be circumvented by bringing new trait combinations together. Better defining what constitutes beneficial stress resistance in different contexts and making connections between genes and phenotypes, and between laboratory and field observations, are overarching challenges.

The growing world population and global increases in the standard of living both result in an increasing demand for food, feed and other plant-derived products. In the coming years, plant-based research will be among the major drivers ensuring food security and the expansion of the bio-based economy. Crop productivity is determined by several factors, including the available physical and agricultural resources, crop management, and the resource use efficiency, quality and intrinsic yield potential of the chosen crop. This review focuses on intrinsic yield potential, since understanding its determinants and their biological basis will allow to maximize the plant's potential in food and energy production. Yield potential is determined by a variety of complex traits that integrate strictly regulated processes and their underlying gene regulatory networks. Due to this inherent complexity, numerous potential targets have been identified that could be exploited to increase crop yield. These encompass diverse metabolic and physical processes at the cellular, organ and canopy level. We present an overview of some of the distinct biological processes considered to be crucial for yield determination that could further be exploited to improve future crop productivity.

The anaphase-promoting complex/cyclosome (APC/C) marks key cell cycle proteins for proteasomal breakdown, thereby ensuring unidirectional progression through the cell cycle. Its target recognition is temporally regulated by activating subunits, one of which is called CELL CYCLE SWITCH 52 A2 (CCS52A2). We sought to expand the knowledge on the APC/C by using the severe growth phenotypes of CCS52A2-deficient Arabidopsis (Arabidopsis thaliana) plants as a readout in a suppressor mutagenesis screen, resulting in the identification of the previously undescribed gene called PIKMIN1 (PKN1). PKN1 deficiency rescues the disorganized root stem cell phenotype of the ccs52a2-1 mutant, whereas an excess of PKN1 inhibits the growth of ccs52a2-1 plants, indicating the need for control of PKN1 abundance for proper development. Accordingly, the lack of PKN1 in a wild-type background negatively impacts cell division, while its systemic overexpression promotes proliferation. PKN1 shows a cell cycle phase-dependent accumulation pattern, localizing to microtubular structures, including the preprophase band, the mitotic spindle, and the phragmoplast. PKN1 is conserved throughout the plant kingdom, with its function in cell division being evolutionarily conserved in the liverwort Marchantia polymorpha. Our data thus demonstrate that PKN1 represents a novel, plant-specific protein with a role in cell division that is likely proteolytically controlled by the CCS52A2-activated APC/C.

Cytokinin and auxin are plant hormones that coordinate many aspects of plant development. Their interactions in plant underground growth are well established, occurring at the levels of metabolism, signaling, and transport. Unlike many plant hormone classes, cytokinins are represented by more than one active molecule. Multiple mutant lines, blocking specific parts of cytokinin biosynthetic pathways, have enabled research in plants with deficiencies in specific cytokinin-types. While most of these mutants have confirmed the impeding effect of cytokinin on root growth, the ipt29 double mutant instead surprisingly exhibits reduced primary root length compared to the wild type. This mutant is impaired in cis-zeatin (cZ) production, a cytokinin-type that had been considered inactive in the past. Here we have further investigated the intriguing ipt29 root phenotype, opposite to known cytokinin functions, and the (bio)activity of cZ. Our data suggest that despite the ipt29 short-root phenotype, cZ application has a negative impact on primary root growth and can activate a cytokinin response in the stele. Grafting experiments revealed that the root phenotype of ipt29 depends mainly on local signaling which does not relate directly to cytokinin levels. Notably, ipt29 displayed increased auxin levels in the root tissue. Moreover, analyses of the differential contributions of ipt2 and ipt9 to the ipt29 short-root phenotype demonstrated that, despite its deficiency on cZ levels, ipt2 does not show any root phenotype or auxin homeostasis variation, while ipt9 mutants were indistinguishable from ipt29. We conclude that IPT9 functions may go beyond cZ biosynthesis, directly or indirectly, implicating effects on auxin homeostasis and therefore influencing plant growth.

Summary Lignins are cell wall‐located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula × P. alba). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR‐Cas9‐generated cse1 and cse2 single mutants had a wild‐type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced‐lignin content translated into a fourfold increase in cellulose‐to‐glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p‐coumaroyl, 5‐hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl‐shikimates, and show that CSE is a promising target to improve plants for the biorefinery.

Reprogramming of mature plant cells requires histone acetylation-mediated activation of auxin biosynthesis to reinitiate the mitotic cell cycle. Plant cells exhibit remarkable plasticity of their differentiation states, enabling regeneration of whole plants from differentiated somatic cells. How they revert cell fate and express pluripotency, however, remains unclear. In this study, we demonstrate that transcriptional activation of auxin biosynthesis is crucial for reprogramming differentiated Arabidopsis (Arabidopsis thaliana) leaf cells. Our data show that interfering with the activity of histone acetyltransferases dramatically reduces callus formation from leaf mesophyll protoplasts. Histone acetylation permits transcriptional activation of PLETHORAs, leading to the induction of their downstream YUCCA1 gene encoding an enzyme for auxin biosynthesis. Auxin biosynthesis is in turn required to accomplish initial cell division through the activation of G2/M phase genes mediated by MYB DOMAIN PROTEIN 3-RELATED (MYB3Rs). We further show that the AUXIN RESPONSE FACTOR 7 (ARF7)/ARF19 and INDOLE-3-ACETIC ACID INDUCIBLE 3 (IAA3)/IAA18-mediated auxin signaling pathway is responsible for cell cycle reactivation by transcriptionally upregulating MYB3R4. These findings provide a mechanistic model of how differentiated plant cells revert their fate and reinitiate the cell cycle to become pluripotent.

Vascular cambium contains bifacial stem cells, which produce secondary xylem to one side and secondary phloem to the other. However, how these fate decisions are regulated is unknown. Here we show that the positioning of an auxin signalling maximum within the cambium determines the fate of stem cell daughters. The position is modulated by gibberellin-regulated, PIN1-dependent polar auxin transport. Gibberellin treatment broadens auxin maximum from the xylem side of the cambium towards the phloem. As a result, xylem-side stem cell daughter preferentially differentiates into xylem, while phloem-side daughter retains stem cell identity. Occasionally, this broadening leads to direct specification of both daughters as xylem, and consequently, adjacent phloem-identity cell reverts to being stem cell. Conversely, reduced gibberellin levels favour specification of phloem-side stem cell daughter as phloem. Together, our data provide a mechanism by which gibberellin regulates the ratio of xylem and phloem production.Auxin is a key regulator in vascular cambium development. This study shows that gibberellins promote polar auxin transport along the root, which leads to broadening of high auxin signalling domain in cambium, and thus, to increased xylem formation.

Due to the presence of a transmembrane domain, the subcellular mobility plan of membrane-bound or membrane-tethered transcription factors (MB-TFs) differs from that of their cytosolic counterparts. The MB-TFs are mostly locked in (sub)cellular membranes, until they are released by a proteolytic cleavage event or when the transmembrane domain (TMD) is omitted from the transcript due to alternative splicing. Here, we review the current knowledge on the proteolytic activation mechanisms of MB-TFs in plants, with a particular focus on regulated intramembrane proteolysis (RIP), and discuss the analogy with the proteolytic cleavage of MB-TFs in animal systems. We present a comprehensive inventory of all known and predicted MB-TFs in the model plant Arabidopsis thaliana and examine their experimentally determined or anticipated subcellular localizations and membrane topologies. We predict proteolytically activated MB-TFs by the mapping of protease recognition sequences and structural features that facilitate RIP in and around the TMD, based on data from metazoan intramembrane proteases. Finally, the MB-TF functions in plant responses to environmental stresses and in plant development are considered and novel functions for still uncharacterized MB-TFs are forecasted by means of a regulatory network-based approach.

Plasma membrane aquaporin AtPIP2;5 is permeable to CO2 and contributes to mesophyll conductance of CO2 in leaves., The roles of different plasma membrane aquaporins (PIPs) in leaf-level gas exchange of Arabidopsis thaliana were examined using knockout mutants. Since multiple Arabidopsis PIPs are implicated in CO2 transport across cell membranes, we focused on identifying the effects of the knockout mutations on photosynthesis, and whether they are mediated through the control of stomatal conductance of water vapour (gs), mesophyll conductance of CO2 (gm), or both. We grew Arabidopsis plants in low and high humidity environments and found that the contribution of PIPs to gs was larger under low air humidity when the evaporative demand was high, whereas any effect of a lack of PIP function was minimal under higher humidity. The pip2;4 knockout mutant had 44% higher gs than wild-type plants under low humidity, which in turn resulted in an increased net photosynthetic rate (Anet). We also observed a 23% increase in whole-plant transpiration (E) for this knockout mutant. The lack of functional plasma membrane aquaporin AtPIP2;5 did not affect gs or E, but resulted in homeostasis of gm despite changes in humidity, indicating a possible role in regulating CO2 membrane permeability. CO2 transport measurements in yeast expressing AtPIP2;5 confirmed that this aquaporin is indeed permeable to CO2.

Clubroot of Brassicaceae, an economically important soil borne disease, is caused by Plasmodiophora brassicae Woronin, an obligate, biotrophic protist. This disease poses a serious threat to canola and related crops in Canada and around the globe causing significant losses. The pathogen is continuously evolving and new pathotypes are emerging, which necessitates the development of novel resistant canola cultivars to manage the disease. Proteins play a crucial role in many biological functions and the identification of differentially abundant proteins (DAP) using proteomics is a suitable approach to understand plant–pathogen interactions to assist in the development of gene specific markers for developing clubroot resistant (CR) cultivars. In this study, P. brassicae pathotype 3 (P3H) was used to challenge CR and clubroot susceptible (CS) canola lines. Root samples were collected at three distinct stages of pathogenesis, 7−, 14−, and 21-days post inoculation (DPI), protein samples were isolated, digested with trypsin and subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. A total of 937 proteins demonstrated a significant (q-value < 0.05) change in abundance in at least in one of the time points when compared between control and inoculated CR-parent, CR-progeny, CS-parent, CS-progeny and 784 proteins were significantly (q < 0.05) changed in abundance in at least in one of the time points when compared between the inoculated- CR and CS root proteomes of parent and progeny across the three time points tested. Functional annotation of differentially abundant proteins (DAPs) revealed several proteins related to calcium dependent signaling pathways. In addition, proteins related to reactive oxygen species (ROS) biochemistry, dehydrins, lignin, thaumatin, and phytohormones were identified. Among the DAPs, 73 putative proteins orthologous to CR proteins and quantitative trait loci (QTL) associated with eight CR loci in different chromosomes including chromosomes A3 and A8 were identified. Proteins including BnaA02T0335400WE, BnaA03T0374600WE, BnaA03T0262200WE, and BnaA03T0464700WE are orthologous to identified CR loci with possible roles in mediating clubroot responses. In conclusion, these results have contributed to an improved understanding of the mechanisms involved in mediating response to P. brassicae in canola at the protein level.

Increasing crop productivity is our major challenge if we are to meet global needs for food, fodder and fuel. Controlling the content of the plant hormone cytokinin is a method of improving plant productivity. Cytokinin oxidase/dehydrogenase (CKO/CKX) is a major target in this regard because it degrades cytokinins. Here, we describe the synthesis and biological activities of new CKX inhibitors derived mainly from diphenylurea. They were tested on four CKX isoforms from maize and Arabidopsis, where the best compounds showed IC50 values in the 10–8 M concentration range. The binding mode of the most efficient inhibitors was characterized from high-resolution crystal complexed structures. Although these compounds do not possess intrinsic cytokinin activity, we have demonstrated their tremendous potential for use in the plant tissue culture industry as well as in agriculture. We have identified a key substance, compound 19, which not only increases stress resistance and seed yield in Arabidopsis, but also improves the yield of wheat, barley and rapeseed grains under field conditions. Our findings reveal that modulation of cytokinin levels via CKX inhibition can positively affect plant growth, development and yield, and prove that CKX inhibitors can be an attractive target in plant biotechnology and agriculture.

Plants stand out among eukaryotes due to the large variety of sterols and sterol derivatives that they can produce. These metabolites not only serve as critical determinants of membrane structures, but also act as signaling molecules, as growth-regulating hormones, or as modulators of enzyme activities. Therefore, it is critical to understand the wiring of the biosynthetic pathways by which plants generate these distinct sterols, to allow their manipulation and to dissect their precise physiological roles. Here, we review the complexity and variation of the biosynthetic routes of the most abundant phytosterols and cholesterol in the green lineage and how different enzymes in these pathways are conserved and diverged from humans, yeast, and even bacteria. Many enzymatic steps show a deep evolutionary conservation, while others are executed by completely different enzymes. This has important implications for the use and specificity of available human and yeast sterol biosynthesis inhibitors in plants, and argues for the development of plant-tailored inhibitors of sterol biosynthesis.

Leaf respiration in the dark (R-dark) is often measured at a single time during the day, with hot-acclimation lowering R-dark at a common measuring temperature. However, it is unclear whether the diel cycle influences the extent of thermal acclimation of R-dark, or how temperature and time of day interact to influence respiratory metabolites. To examine these issues, we grew rice under 25 degrees C : 20 degrees C, 30 degrees C : 25 degrees C and 40 degrees C : 35 degrees C day : night cycles, measuring R-dark and changes in metabolites at five time points spanning a single 24-h period. R-dark differed among the treatments and with time of day. However, there was no significant interaction between time and growth temperature, indicating that the diel cycle does not alter thermal acclimation of R-dark. Amino acids were highly responsive to the diel cycle and growth temperature, and many were negatively correlated with carbohydrates and with organic acids of the tricarboxylic acid (TCA) cycle. Organic TCA intermediates were significantly altered by the diel cycle irrespective of growth temperature, which we attributed to light-dependent regulatory control of TCA enzyme activities. Collectively, our study shows that environmental disruption of the balance between respiratory substrate supply and demand is corrected for by shifts in TCA-dependent metabolites.

Mitochondria have critical functions in the acclimation to abiotic and biotic stresses. Adverse environmental conditions lead to increased demands in energy supply and metabolic intermediates, which are provided by mitochondrial ATP production and the tricarboxylic acid (TCA) cycle. Mitochondria also play a role as stress sensors to adjust nuclear gene expression via retrograde signalling with the transcription factor (TF) ANAC017 and the kinase CDKE1 key components to integrate various signals into this pathway. To determine the importance of mitochondria as sensors of stress and their contribution in the tolerance to adverse growth conditions, a comparative phenotypical, physiological and transcriptomic characterisation of Arabidopsis mitochondrial signalling mutants (cdke1/rao1 and anac017/rao2) and a set of contrasting accessions was performed after applying the complex compound stress of submergence. Our results showed that impaired mitochondrial retrograde signalling leads to increased sensitivity to the stress treatments. The multi-factorial approach identified a network of 702 co-expressed genes, including several WRKY TFs, overlapping in the transcriptional responses in the mitochondrial signalling mutants and stress-sensitive accessions. Functional characterisation of two WRKY TFs (WRKY40 and WRKY45), using both knockout and overexpressing lines, confirmed their role in conferring tolerance to submergence. Together, the results revealed that acclimation to submergence is dependent on mitochondrial retrograde signalling, and underlying transcriptional re-programming is used as an adaptation mechanism.

International audience; In angiosperms, double fertilization triggers the concomitant development of two closely juxtaposed tissues, the embryo and the endosperm. Successful seed development and germination require constant interactions between these tissues, which occur across their common interface. The embryo–endosperm interface is a complex and poorly understood compound apoplast comprising components derived from both tissues, across which nutrients transit to fuel embryo development. Interface properties, which affect molecular diffusion and thus communication, are themselves dynamically regulated by molecular and physical dialogues between the embryo and endosperm. We review the current understanding of embryo–endosperm interactions, with a focus on the structure, properties, and function of their shared interface. Concentrating on Arabidopsis, but with reference to other species, we aim to situate recent findings within the broader context of seed physiology, developmental biology, and genetic factors such as parental conflicts over resource allocation.

Reactive oxygen species (ROS) are key signalling molecules that enable cells to rapidly respond to different stimuli. In plants, ROS play a crucial role in abiotic and biotic stress sensing, integration of different environmental signals and activation of stress-response networks, thus contributing to the establishment of defence mechanisms and plant resilience. Recent advances in the study of ROS signalling in plants include the identification of ROS receptors and key regulatory hubs that connect ROS signalling with other important stress-response signal transduction pathways and hormones, as well as new roles for ROS in organelle-to-organelle and cell-to-cell signalling. Our understanding of how ROS are regulated in cells by balancing production, scavenging and transport has also increased. In this Review, we discuss these promising developments and how they might be used to increase plant resilience to environmental stress.

The central metabolic regulator SnRK1 controls plant growth and survival upon activation by energy depletion, but detailed molecular insight into its regulation and downstream targets is limited. Here we used phosphoproteomics to infer the sucrose-dependent processes targeted upon starvation by kinases as SnRK1, corroborating the relation of SnRK1 with metabolic enzymes and transcriptional regulators, while also pointing to SnRK1 control of intracellular trafficking. Next, we integrated affinity purification, proximity labelling and crosslinking mass spectrometry to map the protein interaction landscape, composition and structure of the SnRK1 heterotrimer, providing insight in its plant-specific regulation. At the intersection of this multi-dimensional interactome, we discovered a strong association of SnRK1 with class II T6P synthase (TPS)-like proteins. Biochemical and cellular assays show that TPS-like proteins function as negative regulators of SnRK1. Next to stable interactions with the TPS-like proteins, similar intricate connections were found with known regulators, suggesting that plants utilize an extended kinase complex to fine-tune SnRK1 activity for optimal responses to metabolic stress.

Aboveground plant-arthropod interactions are typically complex, involving herbivores, predators, pollinators, and various other guilds that can strongly affect plant fitness, directly or indirectly, and individually, synergistically, or antagonistically. However, little is known about how ongoing natural selection by these interacting guilds shapes the evolution of plants, i.e., how they affect the differential survival and reproduction of genotypes due to differences in phenotypes in an environment. Recent technological advances, including next-generation sequencing, metabolomics, and gene-editing technologies along with traditional experimental approaches (e.g., quantitative genetics experiments), have enabled far more comprehensive exploration of the genes and traits involved in complex ecological interactions. Connecting different levels of biological organization (genes to communities) will enhance the understanding of evolutionary interactions in complex communities, but this requires a multidisciplinary approach. Here, we review traditional and modern methods and concepts, then highlight future avenues for studying the evolution of plant-arthropod interactions (e.g., plant-herbivore-pollinator interactions). Besides promoting a fundamental understanding of plant-associated arthropod communities’ genetic background and evolution, such knowledge can also help address many current global environmental challenges.

Cross-species network analysis enables identification and validation of growth regulators in Arabidopsis. With the need to increase plant productivity, one of the challenges plant scientists are facing is to identify genes that play a role in beneficial plant traits. Moreover, even when such genes are found, it is generally not trivial to transfer this knowledge about gene function across species to identify functional orthologs. Here, we focused on the leaf to study plant growth. First, we built leaf growth transcriptional networks in Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and aspen (Populus tremula). Next, known growth regulators, here defined as genes that when mutated or ectopically expressed alter plant growth, together with cross-species conserved networks, were used as guides to predict novel Arabidopsis growth regulators. Using an in-depth literature screening, 34 out of 100 top predicted growth regulators were confirmed to affect leaf phenotype when mutated or overexpressed and thus represent novel potential growth regulators. Globally, these growth regulators were involved in cell cycle, plant defense responses, gibberellin, auxin, and brassinosteroid signaling. Phenotypic characterization of loss-of-function lines confirmed two predicted growth regulators to be involved in leaf growth (NPF6.4 and LATE MERISTEM IDENTITY2). In conclusion, the presented network approach offers an integrative cross-species strategy to identify genes involved in plant growth and development.

Key message A dehydration-inducible Arabidopsis CIN-like TCP gene, TCP13, acts as a key regulator of plant growth in leaves and roots under dehydration stress conditions. Abstract Plants modulate their shape and growth in response to environmental stress. However, regulatory mechanisms underlying the changes in shape and growth under environmental stress remain elusive. The CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family of transcription factors (TFs) are key regulators for limiting the growth of leaves through negative effect of auxin response. Here, we report that stress-inducible CIN-like TCP13 plays a key role in inducing morphological changes in leaves and growth regulation in leaves and roots that confer dehydration stress tolerance in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing TCP13 (35Spro::TCP13OX) exhibited leaf rolling, and reduced leaf growth under osmotic stress. The 35Spro::TCP13OX transgenic leaves showed decreased water loss from leaves, and enhanced dehydration tolerance compared with their control counterparts. Plants overexpressing a chimeric repressor domain SRDX-fused TCP13 (TCP13pro::TCP13SRDX) showed severely serrated leaves and enhanced root growth. Transcriptome analysis of TCP13pro::TCP13SRDX transgenic plants revealed that TCP13 affects the expression of dehydration- and abscisic acid (ABA)-regulated genes. TCP13 is also required for the expression of dehydration-inducible auxin-regulated genes, INDOLE-3-ACETIC ACID5 (IAA5) and LATERAL ORGAN BOUNDARIES (LOB) DOMAIN 1 (LBD1). Furthermore, tcp13 knockout mutant plants showed ABA-insensitive root growth and reduced dehydration-inducible gene expression. Our findings provide new insight into the molecular mechanism of CIN-like TCP that is involved in both auxin and ABA response under dehydration stress.

Pollen development is dependent on the tapetum, a sporophytic anther cell layer surrounding the microspores that functions in pollen wall formation but is also essential for meiosis-associated development. There is clear evidence of crosstalk and co-regulation between the tapetum and microspores, but how this is achieved is currently not characterized. ABORTED MICROSPORES (AMS), a tapetum transcription factor, is important for pollen wall formation, but also has an undefined role in early pollen development. We conducted a detailed investigation of chromosome behaviour, cytokinesis, radial microtubule array (RMA) organization, and callose formation in the ams mutant. Early meiosis initiates normally in ams, shows delayed progression after the pachytene stage, and then fails during late meiosis, with disorganized RMA, defective cytokinesis, abnormal callose formation, and microspore degeneration, alongside abnormal tapetum development. Here, we show that selected meiosis-associated genes are directly repressed by AMS, and that AMS is essential for late meiosis progression. Our findings indicate that AMS has a dual function in tapetum-meiocyte crosstalk by playing an important regulatory role during late meiosis, in addition to its previously characterized role in pollen wall formation. AMS is critical for RMA organization, callose deposition, and therefore cytokinesis, and is involved in the crosstalk between the gametophyte and sporophytic tissues, which enables synchronous development of tapetum and microspores. The tapetum transcription factor ABORTED MICROSPORES is key to tapetum-meiocyte crosstalk by enabling late meiosis progression, cytokinesis, radial microtubule array organization, and callose deposition.

Insufficient dietary intake of micronutrients contributes to the onset of deficiencies termed hidden hunger—a global health problem affecting approximately 2 billion people. Vitamin B1 (thiamine) and vitamin B6 (pyridoxine) are essential micronutrients because of their roles as enzymatic cofactors in all organisms. Metabolic engineering attempts to biofortify rice endosperm—a poor source of several micronutrients leading to deficiencies when consumed monotonously—have led to only minimal improvements in vitamin B1 and B6 contents. To determine if rice germplasm could be exploited for biofortification of rice endosperm, we screened 59 genetically diverse accessions under greenhouse conditions for variation in vitamin B1 and vitamin B6 contents across three tissue types (leaves, unpolished and polished grain). Accessions from low, intermediate and high vitamin categories that had similar vitamin levels in two greenhouse experiments were chosen for in-depth vitamer profiling and selected biosynthesis gene expression analyses. Vitamin B1 and B6 contents in polished seeds varied almost 4-fold. Genes encoding select vitamin B1 and B6 biosynthesis de novo enzymes (THIC for vitamin B1, PDX1.3a–c and PDX2 for vitamin B6) were differentially expressed in leaves across accessions contrasting in their respective vitamin contents. These expression levels did not correlate with leaf and unpolished seed vitamin contents, except for THIC expression in leaves that was positively correlated with total vitamin B1 contents in polished seeds. This study expands our knowledge of diversity in micronutrient traits in rice germplasm and provides insights into the expression of genes for vitamin B1 and B6 biosynthesis in rice., Frontiers in Plant Science, 13, ISSN:1664-462X

Mitochondrial ACONITASE3 is important for the acclimation to submergence stress by integrating carbon and nitrogen metabolism and impacting stress signaling pathways. Flooding causes severe crop losses in many parts of the world. Genetic variation in flooding tolerance exists in many species; however, there are few examples for the identification of tolerance genes and their underlying function. We conducted a genome-wide association study (GWAS) in 387 Arabidopsis (Arabidopsis thaliana) accessions. Plants were subjected to prolonged submergence followed by desubmergence, and seven traits (score, water content, Fv/Fm, and concentrations of nitrate, chlorophyll, protein, and starch) were quantified to characterize their acclimation responses. These traits showed substantial variation across the range of accessions. A total of 35 highly significant single-nucleotide polymorphisms (SNPs) were identified across the 20 GWA datasets, pointing to 22 candidate genes, with functions in TCA cycle, DNA modification, and cell division. Detailed functional characterization of one candidate gene, ACONITASE3 (ACO3), was performed. Chromatin immunoprecipitation followed by sequencing showed that a single nucleotide polymorphism in the ACO3 promoter co-located with the binding site of the master regulator of retrograde signaling ANAC017, while subcellular localization of an ACO3-YFP fusion protein confirmed a mitochondrial localization during submergence. Analysis of mutant and overexpression lines determined changes in trait parameters that correlated with altered submergence tolerance and were consistent with the GWAS results. Subsequent RNA-seq experiments suggested that impairing ACO3 function increases the sensitivity to submergence by altering ethylene signaling, whereas ACO3 overexpression leads to tolerance by metabolic priming. These results indicate that ACO3 impacts submergence tolerance through integration of carbon and nitrogen metabolism via the mitochondrial TCA cycle and impacts stress signaling during acclimation to stress.

International audience; In cereals, the root system is mainly composed of post-embryonic shoot-borne roots, named crown roots. The CROWN ROOTLESS1 (CRL1) transcription factor, belonging to the ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES DOMAIN (ASL/LBD) family, is a key regulator of crown root initiation in rice (Oryza sativa). Here, we show that CRL1 can bind, both in vitro and in vivo, not only the LBD-box, a DNA sequence recognized by several ASL/LBD transcription factors, but also another not previously identified DNA motif that was named CRL1-box. Using rice protoplast transient transactivation assays and a set of previously identified CRL1-regulated genes, we confirm that CRL1 transactivates these genes if they possess at least a CRL1-box or an LBD-box in their promoters. In planta, ChIP-qPCR experiments targeting two of these genes that include both a CRL1- and an LBD-box in their promoter show that CRL1 binds preferentially to the LBD-box in these promoter contexts. CRISPR/Cas9-targeted mutation of these two CRL1-regulated genes, which encode a plant Rho GTPase (OsROP) and a basic helix-loop-helix transcription factor (OsbHLH044), show that both promote crown root development. Finally, we show that OsbHLH044 represses a regulatory module, uncovering how CRL1 regulates specific processes during crown root formation.

27 páginas.- 7 figuras.- 2 tablas.- 157 referencias.- Supplementary data The following supplementary data are available at JXB online., Phenolic metabolism is rewired during internode development and in response to cinnamyl alcohol dehydrogenase perturbation in sorghum, with different sets of genes and accumulating compounds. The molecular mechanisms associated with secondary cell wall (SCW) deposition in sorghum remain largely uncharacterized. Here, we employed untargeted metabolomics and large-scale transcriptomics to correlate changes in SCW deposition with variation in global gene expression profiles and metabolite abundance along an elongating internode of sorghum, with a major focus on lignin and phenolic metabolism. To gain deeper insight into the metabolic and transcriptional changes associated with pathway perturbations, a bmr6 mutant [with reduced cinnamyl alcohol dehydrogenase (CAD) activity] was analyzed. In the wild type, internode development was accompanied by an increase in the content of oligolignols, p-hydroxybenzaldehyde, hydroxycinnamate esters, and flavonoid glucosides, including tricin derivatives. We further identified modules of genes whose expression pattern correlated with SCW deposition and the accumulation of these target metabolites. Reduced CAD activity resulted in the accumulation of hexosylated forms of hydroxycinnamates (and their derivatives), hydroxycinnamaldehydes, and benzenoids. The expression of genes belonging to one specific module in our co-expression analysis correlated with the differential accumulation of these compounds and contributed to explaining this metabolic phenotype. Metabolomics and transcriptomics data further suggested that CAD perturbation activates distinct detoxification routes in sorghum internodes. Our systems biology approach provides a landscape of the metabolic and transcriptional changes associated with internode development and with reduced CAD activity in sorghum., This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) via the BIOEN Young Investigators Awards research grant (FAPESP no. 2015/02527-1). This study was financed in part by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior-Brasil (CAPES) -Finance Code 001, by FEDER/Agencia Estatal de Investigacion with the Spanish Projects AGL2017-83036-R and PID2020-118968RB-I00, and by Junta de Andalucia (Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades, project P20_00017). IC is indebted to Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) for the research fellowship 302927/2018-2. SSF was funded for a post-doctoral fellowship (FAPESP n0. 2016/06917-1). MSS was funded for a master's fellowship (FAPESP no. 2015/18361-5). AVAP was funded for a scientific initiation fellowship (FAPESP no. 2016/06714-3). LGAL was funded for a scientific initiation fellowship (FAPESP no. 2019/14604-1).

Serendipita indica is a plant growth-promoting fungus. It is a natural soil dweller that can colonize the roots of a wide range of plants, including cultivated crops. S. indica has been reported to improve plant nutrient uptake and increase stress tolerance when inoculated into the soil. The present study was undertaken to study the effect of volatile organic compounds (VOCs) of S. indica on salt-stressed Ocimum basilicum ‘Fin vert’ in vitro, either in a culture vessel with a semi-solid medium or via a modified temporary immersion bioreactor system (SETIS). For all salt concentrations, VOCs of S. indica significantly improved plant growth in both semi-solid medium and SETIS bioreactors. This resulted in heavier and taller plants, more shoots per plant, and longer roots. This was even observed for the control without salt. At 9 g/L NaCl, plants with Serendipita were able to give longer roots than those without (1.2 cm vs. 0.0 and 1.7 cm vs. 1.7 cm) in the semi-solid medium and SETIS, respectively. Nevertheless, the VOCs were not able to make the plant salt tolerant to this high concentration. The increase in total phenolic and flavonoid content and radical scavenging suggest that the antioxidant defense system is triggered by the S. indica VOCs. In the semi-solid system, without VOCs, 1 g/L NaCl led to an increase in total chlorophyll content (TCC) and a significant decrease in TCC was further measured only at 6 g/L NaCl or more. However, when VOCs were added, the bleaching effect of the salt was partially restored, even at 6 and 9 g/L NaCl. A significant decrease in TCC was also measured in the SETIS system at 6 g/L NaCl or more and treatment with VOC did not make any difference. An exception was 9 g/L, where the VOC-treated plants produced more than three times more chlorophyll than the non-treated plants. These findings will encourage the application of Serendipita indica for stress reduction. In addition, the proposed original adaptation of a temporary immersion system will be instrumental to investigate stress reduction associated with volatile compounds and better understand their mechanism of action.

Description, Plants regulate the distribution of electrons to activate chloroplast metabolism at the onset of light., Photosynthetically produced electrons provide energy for various metabolic pathways, including carbon reduction. Four Calvin-Benson cycle enzymes and several other plastid proteins are activated in the light by reduction of specific cysteines via thioredoxins, a family of electron transporters operating in redox regulation networks. How does this network link the photosynthetic chain with cellular metabolism? Using a time-resolved redox proteomic method, we have investigated the redox network in vivo during the dark–to–low light transition. We show that redox states of some thioredoxins follow the photosynthetic linear electron transport rate. While some redox targets have kinetics compatible with an equilibrium with one thioredoxin (TRXf), reduction of other proteins shows specific kinetic limitations, allowing fine-tuning of each redox-regulated step of chloroplast metabolism. We identified five new redox-regulated proteins, including proteins involved in Mg2+ transport and 1O2 signaling. Our results provide a system-level functional view of the photosynthetic redox regulation network.

Various kinds of primary metabolisms in plants are modulated through sulfate metabolism, and sulfotransferases (SOTs), which are engaged in sulfur metabolism, catalyze sulfonation reactions. In this study, a genome-wide approach was utilized for the recognition and characterization of SOT family genes in the significant nutritional crop potato (Solanum tuberosum L.). Twenty-nine putative StSOT genes were identified in the potato genome and were mapped onto the nine S. tuberosum chromosomes. The protein motifs structure revealed two highly conserved 5 '-phosphosulfate-binding (5 ' PSB) regions and a 3 '-phosphate-binding (3 ' PB) motif that are essential for sulfotransferase activities. The protein-protein interaction networks also revealed an interesting interaction between SOTs and other proteins, such as PRTase, APS-kinase, protein phosphatase, and APRs, involved in sulfur compound biosynthesis and the regulation of flavonoid and brassinosteroid metabolic processes. This suggests the importance of sulfotransferases for proper potato growth and development and stress responses. Notably, homology modeling of StSOT proteins and docking analysis of their ligand-binding sites revealed the presence of proline, glycine, serine, and lysine in their active sites. An expression essay of StSOT genes via potato RNA-Seq data suggested engagement of these gene family members in plants' growth and extension and responses to various hormones and biotic or abiotic stimuli. Our predictions may be informative for the functional characterization of the SOT genes in potato and other nutritional crops.

Correction: Ramakrishnan et al. The Dynamism of Transposon Methylation for Plant Development and Stress Adaptation. Int. J. Mol. Sci. 2021, 22, 11387 https://doi.org/10.3390/ijms232214107 Plant development processes are regulated by epigenetic alterations that shape nuclear structure, gene expression, and phenotypic plasticity; these alterations can provide the plant with protection from environmental stresses. During plant growth and development, these processes play a significant role in regulating gene expression to remodel chromatin structure. These epigenetic alterations are mainly regulated by transposable elements (TEs) whose abundance in plant genomes results in their interaction with genomes. Thus, TEs are the main source of epigenetic changes and form a substantial part of the plant genome. Furthermore, TEs can be activated under stress conditions, and activated elements cause mutagenic effects and substantial genetic variability. This introduces novel gene functions and structural variation in the insertion sites and primarily contributes to epigenetic modifications. Altogether, these modifications indirectly or directly provide the ability to withstand environmental stresses. In recent years, many studies have shown that TE methylation plays a major role in the evolution of the plant genome through epigenetic process that regulate gene imprinting, thereby upholding genome stability. The induced genetic rearrangements and insertions of mobile genetic elements in regions of active euchromatin contribute to genome alteration, leading to genomic stress. These TE-mediated epigenetic modifications lead to phenotypic diversity, genetic variation, and environmental stress tolerance. Thus, TE methylation is essential for plant evolution and stress adaptation, and TEs hold a relevant military position in the plant genome. High-throughput techniques have greatly advanced the understanding of TE-mediated gene expression and its associations with genome methylation and suggest that controlled mobilization of TEs could be used for crop breeding. However, development application in this area has been limited, and an integrated view of TE function and subsequent processes is lacking. In this review, we explore the enormous diversity and likely functions of the TE repertoire in adaptive evolution and discuss some recent examples of how TEs impact gene expression in plant development and stress adaptation.

Originally conceived as harmful metabolic byproducts, reactive oxygen species (ROS) are now recognized as an integral part of numerous cellular programs. Thanks to their diverse physicochemical properties, compartmentalized production, and tight control exerted by the antioxidant machinery they activate signaling pathways that govern plant growth, development, and defense. Excessive ROS levels are often driven by adverse changes in environmental conditions, ultimately causing oxidative stress. The associated negative impact on cellular constituents have been a major focus of decade-long research efforts to improve the oxidative stress resilience by boosting the antioxidant machinery in model and crop species. We highlight the role of enzymatic and non-enzymatic antioxidants as integral factors of multiple signaling cascades beyond their mere function to prevent oxidative damage under adverse abiotic stress conditions.

Reactive oxygen species (ROS) generated in response to infections often activate immune responses in eukaryotes including plants. In plants, ROS are primarily produced by plasma membrane-bound NADPH oxidases called respiratory burst oxidase homologue (Rboh). Surprisingly, Rbohs can also promote the infection of plants by certain pathogens, including plant parasitic cyst nematodes. The Arabidopsis genome contains 10 Rboh genes (RbohA-RbohJ). Previously, we showed that cyst nematode infection causes a localised ROS burst in roots, mediated primarily by RbohD and RbohF. We also found that plants deficient in RbohD and RbohF (rbohD/F) exhibit strongly decreased susceptibility to cyst nematodes, suggesting that Rboh-mediated ROS plays a role in promoting infection. However, little information is known of the mechanism by which Rbohs promote cyst nematode infection. Here, using detailed genetic and biochemical analyses, we identified WALLS ARE THIN1 (WAT1), an auxin transporter, as a downstream target of Rboh-mediated ROS during parasitic infections. We found that WAT1 is required to modulate the host's indole metabolism, including indole-3-acetic acid levels, in infected cells and that this reprogramming is necessary for successful establishment of the parasite. In conclusion, this work clarifies a unique mechanism that enables cyst nematodes to use the host's ROS for their own benefit.

Metacaspases are essential cysteine proteases present in plants, fungi, and protists that are regulated by calcium binding and proteolytic maturation through mechanisms not yet understood. Here, we developed and validated activity-based probes for the three main metacaspase types, and used them to study calcium-mediated activation of metacaspases from their precursors in vitro. By combining substrate-inspired tetrapeptide probes containing an acyloxymethylketone (AOMK) reactive group, with purified representatives of type-I, type-II, and type-III metacaspases, we were able to demonstrate that labeling of mature metacaspases is strictly dependent on calcium. The probe with the highest affinity for all metacaspases also labels higher molecular weight proteoforms of all three metacaspases only in the presence of calcium, displaying the active, unprocessed metacaspase intermediates. Our data suggest that metacaspase activation proceeds through previously unknown active intermediates that are formed upon calcium binding, before precursor processing.

During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication and are hypersensitive to replication stress. Here, we report the identification of the polα-2 mutant, mutated in the catalytic subunit of DNA polymerase α, as a suppressor mutant of wee1. The mutated protein appears to be less stable, causing a loss of interaction with its subunits and resulting in a prolonged S-phase.

Nitrogen (N) is fundamental to plant growth, development and yield. Genes underlying N utilization and assimilation are well-characterized, but mechanisms underpinning plasticity of different phenotypes in response to N remain elusive. Here, using Arabidopsis thaliana accessions, we dissected the genetic architecture of plasticity in early and late rosette diameter, flowering time and yield, in response to three levels of N in the soil. Furthermore, we found that the plasticity in levels of primary metabolites were related with the plasticities of the studied traits. Genome-wide association analysis identified three significant associations for phenotypic plasticity, one for early rosette diameter and two for flowering time. We confirmed that the gene At1g19880, hereafter named as PLASTICITY OF ROSETTE TO NITROGEN 1 (PROTON1), encoding for a regulator of chromatin condensation 1 (RCC1) family protein, conferred plasticity of rosette diameter in response to N. Treatment of PROTON1 T-DNA line with salt implied that the reduced plasticity of early rosette diameter was not a general growth response to stress. We further showed that plasticities of growth and flowering-related traits differed between environmental cues, indicating decoupled genetic programs regulating these traits. Our findings provide a prospective to identify genes that stabilize performance under fluctuating environments.

Departamento de Biotecnología (INIA), The success in the response of a plant to a pest depends on the regulatory networks that connect plant perception and plant response. Meta-analyses of transcriptomic responses are valuable tools to discover novel mechanisms in the plant/herbivore interplay. Considering the quantity and quality of available transcriptomic analyses, Arabidopsis thaliana was selected to test the ability of comprehensive meta-analyses to disentangle plant responses. The analysis of the transcriptomic data showed a general induction of biological processes commonly associated with the response to herbivory, like jasmonate signaling or glucosinolate biosynthesis. However, an uneven induction of many genes belonging to these biological categories was found, which was likely associated with the particularities of each specific Arabidopsis-herbivore interaction. A thorough analysis of the responses to the lepidopteran Pieris rapae and the spider mite Tetranychus urticae highlighted specificities in the perception and signaling pathways associated with the expression of receptors and transcription factors. This information was translated to a variable alteration of secondary metabolic pathways. In conclusion, transcriptomic meta-analysis has been revealed as a potent way to sort out relevant physiological processes in the plant response to herbivores. Translation of these transcriptomic-based analyses to crop species will permit a more appropriate design of biotechnological programs., Research was supported by the Ministerio de Economía, Industria y Competitividad (MEIC, Grants BIO2017-83472-R, RED2018-102407-T, and RyC17MESFB) and by the Convenio Plurianual between Comunidad de Madrid (CM) and Universidad Politécnica de Madrid (UPM) through the Programa de Apoyo a la Realización de Proyectos de I+D para Jóvenes Investigadores (APOYO-jovenes-SUR6Q9-22-Y). RyC grant (RyC2017-21814) from the MEIC and Plan Propio from the UPM financed MES. Programa Propio UPM financed AG., 15 Pág.

Soil microbial communities hold great potential for sustainable and ecologically compatible agriculture. Although numerous plant-beneficial bacterial strains from a wide range of taxonomic groups have been reported, very little evidence is available on the plant-beneficial role of bacteria from the genus Caulobacter. Here, the mode of action of a Caulobacter strain, designated RHG1, which had originally been identified through a microbial screen for plant growth-promoting (PGP) bacteria in maize (Zea mays), is investigated in Arabidopsis thaliana. RHG1 colonized both roots and shoots of Arabidopsis, promoted lateral root formation in the root, and increased leaf number and leaf size in the shoot. The genome of RHG1 was sequenced and was utilized to look for PGP factors. Our data revealed that the bacterial production of nitric oxide, auxins, cytokinins, or 1-aminocyclopropane-1-carboxylate deaminase as PGP factors could be excluded. However, the analysis of brassinosteroid mutants suggests that an unknown PGP mechanism is involved that impinges directly or indirectly on the pathway of this growth hormone.