Brassica rapa CURLY LEAF is a major H3K27 methyltransferase regulating flowering time.

MAIN CONCLUSION: In Brassica rapa, the epigenetic modifier BraA.CLF orchestrates flowering by modulating H3K27me3 levels at the floral integrator genes FT, SOC1, and SEP3, thereby influencing their expression. CURLY LEAF (CLF) is the catalytic subunit of the plant Polycomb Repressive Complex 2 that mediates the trimethylation of histone H3 lysine 27 (H3K27me3), an epigenetic modification that leads to gene silencing. While the function of CURLY LEAF (CLF) has been extensively studied in Arabidopsis thaliana, its role in Brassica crops is barely known. In this study, we focused on the Brassica rapa homolog of CLF and found that the loss-of-function mutant braA.clf-1 exhibits an accelerated flowering together with pleiotropic phenotypic alterations compared to wild-type plants. In addition, we carried out transcriptomic and H3K27me3 genome-wide analyses to identify the genes regulated by BraA.CLF. Interestingly, we observed that several floral regulatory genes, including the B. rapa homologs of FT, SOC1 and SEP3, show reduced H3K27me3 levels and increased transcript levels compared to wild-type plants, suggesting that they are direct targets of BraA.CLF and key players in regulating flowering time in this crop. In addition, the results obtained will enhance our understanding of the epigenetic mechanisms regulating key developmental traits and will aid to increase crop yield by engineering new Brassica varieties with different flowering time requirements.

Conserved and distinct roles of H3K27me3 demethylases regulating flowering time in Brassica rapa.

Epigenetic regulation is necessary for optimal organism development and preservation of gene expression profiles in the cell. In plants, the trimethylation of histone H3 lysine 27 (H3K27me3) is a silencing epigenetic mark relevant for developmental transitions like flowering. The floral transition is a key agronomic trait; however, the epigenetic mechanisms of flowering time regulation in crops remain poorly understood. Here we study the Jumonji H3K27me3 demethylases BraA.REF6 and BraA.ELF6 in Brassica rapa. Phenotypic characterization of novel mutant lines and genome-wide H3K27me3 chromatin immunoprecipitation and transcriptomic analyses indicated that BraA.REF6 plays a greater role than BraA.ELF6 in fine-tuning H3K27me3 levels. In addition, we found that braA.elf6 mutants were early flowering due to high H3K27me3 levels at B. rapa homologs of the floral repressor FLC. Unlike mutations in Arabidopsis thaliana, braA.ref6 mutants were late flowering without altering the expression of B. rapa FLC genes. Remarkably, we found that BraA.REF6 regulated a number of gibberellic acid (GA) biosynthetic genes, including a homolog of GA1, and that GA-treatment complemented the late flowering mutant phenotype. This study increases our understanding of the epigenetic regulation of flowering time in B. rapa, highlighting conserved and distinct regulatory mechanisms between model and crop species.

High ambient temperature leads to reduced FT expression and delayed flowering in Brassica rapa via a mechanism associated with H2A.Z dynamics.

Flowering time is a relevant agronomic trait because is crucial for the optimal formation of seeds and fruits. The genetic pathways controlling this developmental phase transition have been studied extensively in Arabidopsis thaliana. These pathways converge in a small number of genes including FT, the so-called florigen, which integrates environmental cues like ambient temperature. Nevertheless, detailed and functional studies about flowering time in Brassica crops are scarce. Here we study the role of the FT Brassica rapa homologues and the effect of high ambient temperature on flowering time in this crop. Phenotypic characterization and gene-expression analyses suggest that BraA.FT.a (BraA02g016700.3C) is decisive for initiating floral transition; consequently, braA.ft.a loss-of-function and hypomorphic mutations result in late flowering phenotypes. We also show that high ambient temperature delays B. rapa floral transition by reducing BraA.FT.a expression. Strikingly, these expression changes are associated with increased histone H2A.Z levels and less accessible chromatin configuration of the BraA.FT.a locus at high ambient temperature. Interestingly, increased H2A.Z levels at high ambient temperature were also observed for other B. rapa temperature-responsive genes. Previous reports delimited that Arabidopsis flowers earlier at high ambient temperature due to reduced H2A.Z incorporation in the FT locus. Our data reveal a conserved chromatin-mediated mechanism in B. rapa and Arabidopsis in which the incorporation of H2A.Z at FT chromatin in response to warm ambient temperature results in different flowering time responses. This work will help to develop improved Brassica crop varieties with flowering time requirements to cope with global warming. OPEN RESEARCH BADGES: This article has earned an Open Materials Badge for making publicly available the components of the research methodology needed to reproduce the reported procedure and analysis. Methods are available at protocols.iodx.doi.org/10.17504/protocols.io.zmff43n.

Shotgun proteomics profiling of chia seeds (Salvia hispanica L.) reveals genotypic differential responses to viability loss.

Introduction: Exposure to elevated temperatures and relative humidity expedites the seed aging process, finally leading to seed viability loss. In this context, certain proteins play a pivotal role in safeguarding the longevity of seeds. However, the seedproteomic response to loss viability in Salvia hispanica L., commonly known as chia, remains incompletely understood. Methods: This work explores the application of proteomics as a potent tool for uncovering molecular responses to viability loss caused by artificial aging in two chia genotypes, WN and MN. Results: By using a quantitative label-free proteomics analysis (LC-MS/MS), 1787 proteins wereidentified in chia seeds at a 95% confidence level, including storage proteins, heat shock proteins (HSPs), late embryogenesis abundant proteins (LEA),oleosins, reactive oxygen species (ROS)-related enzymes, and ribosomal proteins. A relatively low percentage of exclusive proteins were identified in viable and non-viable seeds. However, proteins exhibiting differential abundancebetween samples indicated variations in the genotype and physiological status. Specifically, the WN genotype showed 130 proteins with differential abundancecomparing viable and non-viable seeds, while MN displayed changes in the abundance of 174 proteins. While both showed a significant decrease in keyproteins responsible for maintaining seed functionality, longevity, and vigor withhigh-temperature and humidity conditions, such as LEA proteins or HSPs, ROS, and oleosins, distinct responses between genotypes were noted, particularly in ribosomal proteins that were accumulated in MN and diminished in WN seeds. Discussion: Overall, the results emphasize the importance of evaluating changes in proteins of viable and non-viable seeds as they offer valuable insights into the underlying biological mechanisms responsible for the maintenance of chia seed integrity throughout high-temperature and humidity exposure.

Shotgun proteomics of quinoa seeds reveals chitinases enrichment under rainfed conditions.

Quinoa is an Andean crop whose cultivation has been extended to many different parts of the world in the last decade. It shows a great capacity for adaptation to diverse climate conditions, including environmental stressors, and, moreover, the seeds are very nutritious in part due to their high protein content, which is rich in essential amino acids. They are gluten-free seeds and contain good amounts of other nutrients such as unsaturated fatty acids, vitamins, or minerals. Also, the use of quinoa hydrolysates and peptides has been linked to numerous health benefits. Altogether, these aspects have situated quinoa as a crop able to contribute to food security worldwide. Aiming to deepen our understanding of the protein quality and function of quinoa seeds and how they can vary when this crop is subjected to water-limiting conditions, a shotgun proteomics analysis was performed to obtain the proteomes of quinoa seeds harvested from two different water regimes in the field: rainfed and irrigated conditions. Differentially increased levels of proteins determined in seeds from each field condition were analysed, and the enrichment of chitinase-related proteins in seeds harvested from rainfed conditions was found. These proteins are described as pathogen-related proteins and can be accumulated under abiotic stress. Thus, our findings suggest that chitinase-like proteins in quinoa seeds can be potential biomarkers of drought. Also, this study points to the need for further research to unveil their role in conferring tolerance when coping with water-deficient conditions.

Unveiling changes in rhizosphere-associated bacteria linked to the genotype and water stress in quinoa.

Drought is among the main abiotic factors causing agronomical losses worldwide. To minimize its impact, several strategies have been proposed, including the use of plant growth-promoting bacteria (PGPBs), as they have demonstrated roles in counteracting abiotic stress. This aspect has been little explored in emergent crops such as quinoa, which has the potential to contribute to reducing food insecurity. Thus, here we hypothesize that the genotype, water environment and the type of inoculant are determining factors in shaping quinoa rhizosphere bacterial communities, affecting plant performance. To address this, two different quinoa cultivars (with contrasting water stress tolerance), two water conditions (optimal and limiting water conditions) and different soil infusions were used to define the relevance of these factors. Different bacterial families that vary among genotypes and water conditions were identified. Certain families were enriched under water stress conditions, such as the Nocardioidaceae, highly present in the water-sensitive cultivar F15, or the Pseudomonadaceae, Burkholderiaceae and Sphingomonadaceae, more abundant in the tolerant cultivar F16, which also showed larger total polyphenol content. These changes demonstrate that the genotype and environment highly contribute to shaping the root-inhabiting bacteria in quinoa, and they suggest that this plant species is a great source of PGPBs for utilization under water-liming conditions.

Assessment of the changes in seed yield and nutritional quality of quinoa grown under rainfed Mediterranean environments.

Climate change is considered a serious threat to agriculture and food security. It is linked to rising temperatures and water shortages, conditions that are expected to worsen in the coming decades. Consequently, the introduction of more drought-tolerant crops is required. Quinoa (Chenopodium quinoa Willd.) has received great attention worldwide due to the nutritional properties of its seeds and its tolerance to abiotic stress. In this work, the agronomic performance and seed nutritional quality of three quinoa varieties were studied during two consecutive years (2019-2020) under three water environmental conditions of Southwestern Europe (irrigated conditions, fresh rainfed, and hard rainfed) with the goal of determining the impact of rainfed conditions on this crop performance. High precipitations were recorded during the 2020 growing season resulting in similar grain yield under irrigation and fresh rainfed conditions. However, in 2019, significant yield differences with penalties under water-limiting conditions were found among the evaluated environmental conditions. Furthermore, nutritional and metabolomic differences were observed among seeds harvested from different water environments including the progressive accumulation of glycine betaine accompanied by an increase in saponin and a decrease in iron with water limitation. Generally, water-limiting environments were associated with increased protein contents and decreased yields preserving a high nutritional quality despite particular changes. Overall, this work contributes to gaining further knowledge about how water availability affects quinoa field performance, as it might impact both seed yield and quality. It also can help reevaluate rainfed agriculture, as water deficit can positively impact the nutritional quality of seeds.

Genome-wide analysis of the H3K27me3 epigenome and transcriptome in Brassica rapa.

BACKGROUND: Genome-wide maps of histone modifications have been obtained for several plant species. However, most studies focus on model systems and do not enforce FAIR data management principles. Here we study the H3K27me3 epigenome and associated transcriptome of Brassica rapa, an important vegetable cultivated worldwide. FINDINGS: We performed H3K27me3 chromatin immunoprecipitation followed by high-throughput sequencing and transcriptomic analysis by 3'-end RNA sequencing from B. rapa leaves and inflorescences. To analyze these data we developed a Reproducible Epigenomic Analysis pipeline using Galaxy and Jupyter, packaged into Docker images to facilitate transparency and reuse. We found that H3K27me3 covers roughly one-third of all B. rapa protein-coding genes and its presence correlates with low transcript levels. The comparative analysis between leaves and inflorescences suggested that the expression of various floral regulatory genes during development depends on H3K27me3. To demonstrate the importance of H3K27me3 for B. rapa development, we characterized a mutant line deficient in the H3K27 methyltransferase activity. We found that braA.clf mutant plants presented pleiotropic alterations, e.g., curly leaves due to increased expression and reduced H3K27me3 levels at AGAMOUS-like loci. CONCLUSIONS: We characterized the epigenetic mark H3K27me3 at genome-wide levels and provide genetic evidence for its relevance in B. rapa development. Our work reveals the epigenomic landscape of H3K27me3 in B. rapa and provides novel genomics datasets and bioinformatics analytical resources. We anticipate that this work will lead the way to further epigenomic studies in the complex genome of Brassica crops.

Boron deficiency inhibits root growth by controlling meristem activity under cytokinin regulation.

Significant advances have been made in the last years trying to identify regulatory pathways that control plant responses to boron (B) deficiency. Still, there is a lack of a deep understanding of how they act regulating growth and development under B limiting conditions. Here, we analyzed the impact of B deficit on cell division leading to root apical meristem (RAM) disorganization. Our results reveal that inhibition of cell proliferation under the regulatory control of cytokinins (CKs) is an early event contributing to root growth arrest under B deficiency. An early recovery of QC46:GUS expression after transferring B-deficient seedlings to control conditions revealed a role of B in the maintenance of QC identity whose loss under deficiency occurred at later stages of the stress. Additionally, the D-type cyclin CYCD3 overexpressor and triple mutant cycd3;1-3 were used to evaluate the effect on mitosis inhibition at the G1-S boundary. Overall, this study supports the hypothesis that meristem activity is inhibited by B deficiency at early stages of the stress as it does cell elongation. Likewise, distinct regulatory mechanisms seem to take place depending on the severity of the stress. The results presented here are key to better understand early signaling responses under B deficiency.