Topoisomerase VI participates in an insulator-like function that prevents H3K9me2 spreading.

The organization of the genome into transcriptionally active and inactive chromatin domains requires well-delineated chromatin boundaries and insulator functions in order to maintain the identity of adjacent genomic loci with antagonistic chromatin marks and functionality. In plants that lack known chromatin insulators, the mechanisms that prevent heterochromatin spreading into euchromatin remain to be identified. Here, we show that DNA Topoisomerase VI participates in a chromatin boundary function that safeguards the expression of genes in euchromatin islands within silenced heterochromatin regions. While some transposable elements are reactivated in mutants of the Topoisomerase VI complex, genes insulated in euchromatin islands within heterochromatic regions of the Arabidopsis thaliana genome are specifically down-regulated. H3K9me2 levels consistently increase at euchromatin island loci and decrease at some transposable element loci. We further show that Topoisomerase VI physically interacts with S-adenosylmethionine synthase methionine adenosyl transferase 3 (MAT3), which is required for H3K9me2. A Topoisomerase VI defect affects MAT3 occupancy on heterochromatic elements and its exclusion from euchromatic islands, thereby providing a possible mechanistic explanation to the essential role of Topoisomerase VI in the delimitation of chromatin domains.

Genome-wide transcriptional responses to water deficit during seed development in Pisum sativum, focusing on sugar transport and metabolism.

Agriculture is particularly impacted by global changes, drought being a main limiting factor of crop production. Here, we focus on pea (Pisum sativum), a model legume cultivated for its seed nutritional value. A water deficit (WD) was applied during its early reproductive phase, harvesting plant organs at two key developmental stages, either at the embryonic or the seed-filling stages. We combined phenotypic, physiological and transcriptome analyses to better understand the adaptive response to drought. First, we showed that apical growth arrest is a major phenotypic indicator of water stress. Sugar content was also greatly impacted, especially leaf fructose and starch contents. Our RNA-seq analysis identified 2001 genes regulated by WD in leaf, 3684 genes in root and 2273 genes in embryonic seed, while only 80 genes were regulated during seed-filling. Hence, a large transcriptional reprogramming occurred in response to WD in seeds during early embryonic stage, but no longer during the later stage of nutritional filling. Biological processes involved in transcriptional regulation, carbon transport and metabolism were greatly regulated by WD in both source and sink organs, as illustrated by the expression of genes encoding transcription factors, sugar transporters and enzymes of the starch synthesis pathway. We then looked at the transcriptomic changes during seed development, highlighting a transition from monosaccharide utilization at the embryonic stage to sucrose transport feeding the starch synthesis pathway at the seed-filling stage. Altogether, our study presents an integrative picture of sugar transport and metabolism in response to drought and during seed development at a genome-wide level.

Carbon fluxes and environmental interactions during legume development, with a specific focus on Pisum sativum.

Grain legumes are major food crops cultivated worldwide for their seeds with high nutritional content. To answer the growing concern about food safety and protein autonomy, legume cultivation must increase in the coming years. In parallel, current agricultural practices are facing environmental challenges, including global temperature increase and more frequent and severe episodes of drought stress. Crop yield directly relies on carbon allocation and is particularly affected by these global changes. We review the current knowledge on source-sink relationships and carbon resource allocation at all developmental stages, from germination to vegetative growth and seed production in grain legumes, focusing on pea (Pisum sativum). We also discuss how these source-sink relationships and carbon fluxes are influenced by biotic and abiotic factors. Major agronomic traits, including seed yield and quality, are particularly impacted by drought, temperatures, salinity, waterlogging, or pathogens and can be improved through the promotion of beneficial soil microorganisms or through optimized plant carbon resource allocation. Altogether, our review highlights the need for a better understanding of the cellular and molecular mechanisms regulating carbon fluxes from source leaves to sink organs, roots, and seeds. These advancements will further improve our understanding of yield stability and stress tolerance and contribute to the selection of climate-resilient crops.

Genome-wide identification of invertases in Fabaceae, focusing on transcriptional regulation of Pisum sativum invertases in seed subjected to drought.

Invertases are key enzymes for carbon metabolism, cleaving sucrose into energy-rich and signaling metabolites, glucose and fructose. Invertases play pivotal roles in development and stress response, determining yield and quality of seed production. In this context, the repertoire of invertase gene families is critically scarce in legumes. Here, we performed a systematic search for invertase families in 16 Fabaceae genomes. For instance, we identified 19 invertase genes in the model plant Medicago and 17 accessions in the agronomic crop Pisum sativum. Our comprehensive phylogenetic analysis sets a milestone for the scientific community as we propose a new nomenclature to correctly name plant invertases. Thus, neutral invertases were classified into four clades of cytosolic invertase (CINV). Acid invertases were classified into two cell wall invertase clades (CWINV) and two vacuolar invertase clades (VINV). Then, we explored transcriptional regulation of the pea invertase family, focusing on seed development and water stress. Invertase expression decreased sharply from embryogenesis to seed-filling stages, consistent with higher sucrose and lower monosaccharide contents. The vacuolar invertase PsVINV1.1 clearly marked the transition between both developmental stages. We hypothesize that the predominantly expressed cell wall invertase, PsCWINV1.2, may drive sucrose unloading towards developing seeds. The same candidates, PsVINV1.1 and PsCWINV1.2, were also regulated by water deficit during embryonic stage. We suggest that PsVINV1.1 along with vacuolar sugar transporters maintain cellular osmotic pressure and PsCWINV1.2 control hexose provision, thereby ensuring embryo survival in drought conditions. Altogether, our findings provide novel insights into the regulation of plant carbon metabolism in a challenging environment.

Beneficial rhizobacteria Pseudomonas simiae WCS417 induce major transcriptional changes in plant sugar transport.

Plants live in close relationships with complex populations of microorganisms, including rhizobacterial species commonly referred to as plant growth-promoting rhizobacteria (PGPR). PGPR are able to improve plant productivity, but the molecular mechanisms involved in this process remain largely unknown. Using an in vitro experimental system, the model plant Arabidopsis thaliana, and the well-characterized PGPR strain Pseudomonas simiae WCS417r (PsWCS417r), we carried out a comprehensive set of phenotypic and gene expression analyses. Our results show that PsWCS417r induces major transcriptional changes in sugar transport and in other key biological processes linked to plant growth, development, and defense. Notably, we identified a set of 13 genes of the SWEET and ERD6-like sugar transporter gene families whose expression is up- or down-regulated in response to seedling root inoculation with the PGPR or exposure to their volatile compounds. Using a reverse genetic approach, we demonstrate that SWEET11 and SWEET12 are functionally involved in the interaction and its plant growth-promoting effects, possibly by controlling the amount of sugar transported from the shoot to the root and to the PGPR. Altogether, our study reveals that PGPR-induced beneficial effects on plant growth and development are associated with changes in plant sugar transport.

Sugars en route to the roots. Transport, metabolism and storage within plant roots and towards microorganisms of the rhizosphere.

In plants, the root is a typical sink organ that relies exclusively on the import of sugar from the aerial parts. Sucrose is delivered by the phloem to the most distant root tips and, en route to the tip, is used by the different root tissues for metabolism and storage. Besides, a certain portion of this carbon is exuded in the rhizosphere, supplied to beneficial microorganisms and diverted by parasitic microbes. The transport of sugars toward these numerous sinks either occurs symplastically through cell connections (plasmodesmata) or is apoplastically mediated through membrane transporters (MST, mononsaccharide tranporters, SUT/SUC, H+/sucrose transporters and SWEET, Sugar will eventually be exported transporters) that control monosaccharide and sucrose fluxes. Here, we review recent progresses on carbon partitioning within and outside roots, discussing membrane transporters involved in plant responses to biotic and abiotic factors.

Stress-induced chromatin changes in plants: of memories, metabolites and crop improvement.

Exposure of plants to adverse environmental conditions leads to extensive transcriptional changes. Genome-wide approaches and gene function studies have revealed the importance of chromatin-level control in the regulation of stress-responsive gene expression. Advances in understanding chromatin modifications implicated in plant stress response and identifying proteins involved in chromatin-mediated transcriptional responses to stress are briefly presented in this review. We then highlight how chromatin-mediated gene expression changes can be coupled to the metabolic status of the cell, since many of the chromatin-modifying proteins involved in transcriptional regulation depend on cofactors and metabolites that are shared with enzymes in basic metabolism. Lastly, we discuss the stability and heritability of stress-induced chromatin changes and the potential of chromatin-based strategies for increasing stress tolerance of crops.

Boosting crop yields with plant steroids.

Plant sterols and steroid hormones, the brassinosteroids (BRs), are compounds that exert a wide range of biological activities. They are essential for plant growth, reproduction, and responses to various abiotic and biotic stresses. Given the importance of sterols and BRs in these processes, engineering their biosynthetic and signaling pathways offers exciting potentials for enhancing crop yield. In this review, we focus on how alterations in components of sterol and BR metabolism and signaling or application of exogenous steroids and steroid inhibitors affect traits of agronomic importance. We also discuss areas for future research and identify the fine-tuning modulation of endogenous BR content as a promising strategy for crop improvement.

Integration of stress-related and reactive oxygen species-mediated signals by Topoisomerase VI in Arabidopsis thaliana.

Environmental stress often leads to an increased production of reactive oxygen species that are involved in plastid-to-nucleus retrograde signaling. Soon after the release of singlet oxygen ((1)O(2)) in chloroplasts of the flu mutant of Arabidopsis, reprogramming of nuclear gene expression reveals a rapid transfer of signals from the plastid to the nucleus. We have identified extraplastidic signaling constituents involved in (1)O(2)-initiated plastid-to-nucleus signaling and nuclear gene activation after mutagenizing a flu line expressing the luciferase reporter gene under the control of the promoter of a (1)O(2)-responsive AAA-ATPase gene (At3g28580) and isolating second-site mutations that lead to a constitutive up-regulation of the reporter gene or abrogate its (1)O(2)-dependent up-regulation. One of these mutants, caa39, turned out to be a weak mutant allele of the Topoisomerase VI (Topo VI) A-subunit gene with a single amino acid substitution. Transcript profile analysis of flu and flu caa39 mutants revealed that Topo VI is necessary for the full activation of AAA-ATPase and a set of (1)O(2)-responsive transcripts in response to (1)O(2). Topo VI binds to the promoter of the AAA-ATPase and other (1)O(2)-responsive genes, and hence could directly regulate their expression. Under photoinhibitory stress conditions, which enhance the production of (1)O(2) and H(2)O(2), Topo VI regulates (1)O(2)-responsive and H(2)O(2)-responsive genes in a distinct manner. These results suggest that Topo VI acts as an integrator of multiple signals generated by reactive oxygen species formed in plants under adverse environmental conditions.

A suite of Lotus japonicus starch mutants reveals both conserved and novel features of starch metabolism.

The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (Arabidopsis thaliana) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth.

Unearthing the Plant Growth-Promoting Traits of Bacillus megaterium RmBm31, an Endophytic Bacterium Isolated From Root Nodules of Retama monosperma.

Plants live in association with complex populations of microorganisms, including Plant Growth-Promoting Rhizobacteria (PGPR) that confer to plants an improved growth and enhanced stress tolerance. This large and diverse group includes endophytic bacteria that are able to colonize the internal tissues of plants. In the present study, we have isolated a nonrhizobial species from surface sterilized root nodules of Retama monosperma, a perennial leguminous species growing in poor and high salinity soils. Sequencing of its genome reveals this endophytic bacterium is a Bacillus megaterium strain (RmBm31) that possesses a wide range of genomic features linked to plant growth promotion. Furthermore, we show that RmBm31 is able to increase the biomass and positively modify the root architecture of seedlings of the model plant species Arabidopsis thaliana both in physical contact with its roots and via the production of volatile organic compounds. Lastly, we investigated the molecular mechanisms implicated in RmBm31 plant beneficial effects by carrying out a transcriptional analysis on a comprehensive set of phytohormone-responsive marker genes. Altogether, our results demonstrate that RmBm31 displays plant growth-promoting traits of potential interest for agricultural applications.

Comparison of the Molecular Responses of Tolerant, Susceptible and Highly Susceptible Grapevine Cultivars During Interaction With the Pathogenic Fungus Eutypa lata.

Eutypa lata is the causal agent of eutypa dieback, one of the most destructive grapevine trunk disease that causes severe economic losses in vineyards worldwide. This fungus causes brown sectorial necrosis in wood which affect the vegetative growth. Despite intense research efforts made in the past years, no cure currently exists for this disease. Host responses to eutypa dieback are difficult to address because E. lata is a wood pathogen that causes foliar symptoms several years after infection. With the aim to classify the level of susceptibility of grapevine cultivars to the foliar symptoms caused by E. lata, artificial inoculations of Merlot, Cabernet Sauvignon, and Ugni Blanc were conducted over 3 years. Merlot was the most tolerant cultivar, whereas Ugni Blanc and Cabernet Sauvignon exhibited higher and differential levels of susceptibility. We took advantage of their contrasting phenotypes to explore their defense responses, including the activation of pathogenesis-related (PR) genes, oxylipin and phenylpropanoid pathways and the accumulation of stilbenes. These analyses were carried out using the millicell system that enables the molecular dialogue between E. lata mycelium and grapevine leaves to take place without physical contact. Merlot responded to E. lata by inducing the expression of a large number of defense-related genes. On the contrary, Ugni Blanc failed to activate such defense responses despite being able to perceive the fungus. To gain insight into the role of carbon partitioning in E. lata infected grapevine, we monitored the expression of plant genes involved in sugar transport and cleavage, and measured invertase activities. Our results evidence a coordinated up-regulation of VvHT5 and VvcwINV genes, and a stimulation of the cell wall invertase activity in leaves of Merlot elicited by E. lata, but not in Ugni Blanc. Altogether, this study indicates that the degree of cultivar susceptibility is associated with the activation of host defense responses, including extracellular sucrolytic machinery and hexose uptake during the grapevine/E. lata interaction. Given the role of these activities in governing carbon allocation through the plant, we postulate that the availability of sugar resources for either the host or the fungus is crucial for the outcome of the interaction.

Evolutionary trails of plant steroid genes.

Plant steroids - brassinosteroids (BRs) and their precursors, phytosterols - play a major role in plant growth, development, stress tolerance, and have high potential for agricultural applications. Currently, this prospect is limited by a lack of information about their evolution and expression dynamics (spatial and temporal) across plant species. The increasing number of sequenced genomes offers an opportunity for evolutionary studies that might help to prioritize functional analyses with the aim to improve crop yield and stress tolerance. In this review we provide a glimpse of the origin, evolution, and functional conservation of phytosterol and BR genes in the green plant lineage using comparative sequence and expression analyses of publicly available datasets.

Root starch reserves are necessary for vigorous re-growth following cutting back in Lotus japonicus.

Perenniality and vegetative re-growth vigour represent key agronomic traits in forage legume (Fabaceae) species. The known determinants of perenniality include the conservation of the vegetative meristem during and after the flowering phase, and the separation of flowering from senescence. The ability of the plants to store nutrient resources in perennial organs and remobilize them may also play an important role in the perennial growth habit, and in determining the capacity of the plant to re-grow following grazing or from one season to the next. To examine the importance of stored starch, we examined the vegetative re-growth vigour following cutting back of a unique collection of Lotus japonicus mutants impaired in their ability to synthesize or degrade starch. Our results establish that starch stored in the roots is important for re-growth vigour in Lotus japonicus. We extended this analysis to a collection of Lotus (trefoil) species and two ecotypes of Lotus japonicus displaying a large variation in their carbohydrate resource allocation. There was a positive correlation between root starch content and re-growth vigour in these natural variants, and a good general correlation between high re-growth vigour and the perennial life-form. We discuss the relationship between perenniality and the availability of root carbohydrates for re-growth.

From squalene to brassinolide: the steroid metabolic and signaling pathways across the plant kingdom.

The plant steroid hormones, brassinosteroids (BRs), and their precursors, phytosterols, play major roles in plant growth, development, and stress tolerance. Here, we review the impressive progress made during recent years in elucidating the components of the sterol and BR metabolic and signaling pathways, and in understanding their mechanism of action in both model plants and crops, such as Arabidopsis and rice. We also discuss emerging insights into the regulations of these pathways, their interactions with other hormonal pathways and multiple environmental signals, and the putative nature of sterols as signaling molecules.