Comparative analysis of geotypic variations in the proteome of Nostoc commune.

Cyanobacterium Nostoc commune is a filamentous terrestrial prokaryotic organism widely distributed, which suggest its high adaptive potential to environmental or abiotic stress. Physiological parameters and proteomic analysis were performed in two accession of N. commune with the aim to elucidate the differences of physiological trails between distant geotypes, namely Antarctic (AN) and central European (CE). The result obtained clearly showed that the AN geotype demonstrates elevated levels of total phenols, flavonoids, carotenoids, and phycobiliproteins, indicative of its adaptation to environmental stress as referred by comparison to CE sample. Additionally, we employed LC-MS analysis to investigate the proteomes of N. commune from AN and CE geotypes. In total, 1147 proteins were identified, among which 646 proteins expressed significant (up-regulation) changes in both accessions. In the AN geotype, 83 exclusive proteins were identified compared to 25 in the CE geotype. Functional classification of the significant proteins showed a large fraction involved in photosynthesis, amino acid metabolism, carbohydrate metabolism and protein biosynthesis. Further analysis revealed some defense-related proteins such as, superoxide dismutase (SOD) and glutathione reductase, which are rather explicitly expressed in the AN N. commune. The last two proteins suggest a more stressful condition in AN N. commune. In summary, our findings highlight biochemical processes that safeguard the AN geotype of N. commune from extreme environmental challenges, not recorded in CE accession, probably due to less stressful environment in Europe. This study brings the first ever proteomic analysis of N. commune, emphasizing the need for additional investigations into the climate adaptation of this species with rather plastic genome.

Integrated multi-omics analysis reveals drought stress response mechanism in chickpea (Cicer arietinum L.).

Drought is one of the major constraints limiting chickpea productivity. To unravel complex mechanisms regulating drought response in chickpea, we generated transcriptomics, proteomics, and metabolomics datasets from root tissues of four contrasting drought-responsive chickpea genotypes: ICC 4958, JG 11, and JG 11+ (drought-tolerant), and ICC 1882 (drought-sensitive) under control and drought stress conditions. Integration of transcriptomics and proteomics data identified enriched hub proteins encoding isoflavone 4'-O-methyltransferase, UDP-d-glucose/UDP-d-galactose 4-epimerase, and delta-1-pyrroline-5-carboxylate synthetase. These proteins highlighted the involvement of pathways such as antibiotic biosynthesis, galactose metabolism, and isoflavonoid biosynthesis in activating drought stress response mechanisms. Subsequently, the integration of metabolomics data identified six metabolites (fructose, galactose, glucose, myoinositol, galactinol, and raffinose) that showed a significant correlation with galactose metabolism. Integration of root-omics data also revealed some key candidate genes underlying the drought-responsive "QTL-hotspot" region. These results provided key insights into complex molecular mechanisms underlying drought stress response in chickpea.

Cell-type proteomic and metabolomic resolution of early and late grain filling stages of wheat endosperm.

The nutritional value of wheat grains, particularly their protein and metabolite composition, is a result of the grain-filling process, especially in the endosperm. Here, we employ laser microdissection (LMD) combined with shotgun proteomics and metabolomics to generate a cell type-specific proteome and metabolome inventory of developing wheat endosperm at the early (15 DAA) and late (26 DAA) grain-filling stages. We identified 1803 proteins and 41 metabolites from four different cell types (aleurone (AL), sub-aleurone (SA), starchy endosperm (SE) and endosperm transfer cells (ETCs). Differentially expressed proteins were detected, 67 in the AL, 31 in the SA, 27 in the SE and 50 in the ETCs between these two-time points. Cell-type accumulation of specific SUT and GLUT transporters, sucrose converting and starch biosynthesis enzymes correlate well with the respective sugar metabolites, suggesting sugar upload and starch accumulation via nucellar projection and ETC at 15 DAA in contrast to the later stage at 26 DAA. Changes in various protein levels between AL, SA and ETC support this metabolic switch from 15 to 26 DAA. The distinct spatial and temporal abundances of proteins and metabolites revealed a contrasting activity of nitrogen assimilation pathways, e.g. for GOGAT, GDH and glutamic acid, in the different cell types from 15 to 26 DAA, which can be correlated with specific protein accumulation in the endosperm. The integration of cell-type specific proteome and metabolome data revealed a complex metabolic interplay of the different cell types and a functional switch during grain development and grain-filling processes.

Low dose ribosomal DNA P-loop mutation affects development and enforces autophagy in Arabidopsis.

Arabidopsis contains hundreds of ribosomal DNA copies organized within the nucleolar organizing regions (NORs) in chromosomes 2 and 4. There are four major types of variants of rDNA, VAR1-4, based on the polymorphisms of 3' external transcribed sequences. The variants are known to be differentially expressed during plant development. We created a mutant by the CRISPR-Cas9-mediated excision of ~ 25 nt from predominantly NOR4 ribosomal DNA copies, obtaining mosaic mutational events on ~ 5% of all rDNA copies. The excised region consists of P-loop and Helix-82 segments of 25S rRNA. The mutation led to allelic, dosage-dependent defects marked by lateral root inhibition, reduced size, and pointy leaves, all previously observed for defective ribosomal function. The mutation in NOR4 led to dosage compensation from the NOR2 copies by elevated expression of VAR1 in mutants and further associated single-nucleotide variants, thus, resulting in altered rRNA sub-population. Furthermore, the mutants exhibited rRNA maturation defects specifically in the minor pathway typified by 32S pre-rRNA accumulation. Density-gradient fractionation and subsequent RT-PCR of rRNA analyses revealed that mutated copies were not incorporated into the translating ribosomes. The mutants in addition displayed an elevated autophagic flux as shown by the autophagic marker GFP-ATG8e, likely related to ribophagy.

Quantifying the impact of dynamic plant-environment interactions on metabolic regulation.

A plant's genome encodes enzymes, transporters and many other proteins which constitute metabolism. Interactions of plants with their environment shape their growth, development and resilience towards adverse conditions. Although genome sequencing technologies and applications have experienced triumphantly rapid development during the last decades, enabling nowadays a fast and cheap sequencing of full genomes, prediction of metabolic phenotypes from genotype × environment interactions remains, at best, very incomplete. The main reasons are a lack of understanding of how different levels of molecular organisation depend on each other, and how they are constituted and expressed within a setup of growth conditions. Phenotypic plasticity, e.g., of the genetic model plant Arabidopsis thaliana, has provided important insights into plant-environment interactions and the resulting genotype x phenotype relationships. Here, we summarize previous and current findings about plant development in a changing environment and how this might be shaped and reflected in metabolism and its regulation. We identify current challenges in the study of plant development and metabolic regulation and provide an outlook of how methodological workflows might support the application of findings made in model systems to crops and their cultivation.

Ferroptosis is the key cellular process mediating Bisphenol A responses in Chlamydomonas and a promising target for enhancing microalgae-based bioremediation.

Microplastics are one of the major pollutants in aquatic environments. Among their components, Bisphenol A (BPA) is one of the most abundant and dangerous, leading to endocrine disorders deriving even in different types of cancer in mammals. However, despite this evidence, the xenobiotic effects of BPA over plantae and microalgae still need to be better understood at the molecular level. To fill this gap, we characterized the physiological and proteomic response of Chlamydomonas reinhardtii during long-term BPA exposure by analyzing physiological and biochemical parameters combined with proteomics. BPA imbalanced iron and redox homeostasis, disrupting cell function and triggering ferroptosis. Intriguingly, this microalgae defense against this pollutant is recovering at both molecular and physiological levels while starch accumulation at 72 h of BPA exposure. In this work, we addressed the molecular mechanisms involved in BPA exposure, demonstrating for the first time the induction of ferroptosis in a eukaryotic alga and how ROS detoxification mechanisms and other specific proteomic rearrangements reverted this situation. These results are of great significance not only for understanding the BPA toxicology or exploring the molecular mechanisms of ferroptosis in microalgae but also for defining novel target genes for microplastic bioremediation efficient strain development.

Proteomics for abiotic stresses in legumes: present status and future directions.

Legumes are the most important crop plants in agriculture, contributing 27% of the world's primary food production. However, productivity and production of Legumes is reduced due to increasing environmental stress. Hence, there is a pressing need to understand the molecular mechanism involved in stress response and legumes adaptation. Proteomics provides an important molecular approach to investigate proteins involved in stress response. Both the gel-based and gel-free-based techniques have significantly contributed to understanding the proteome regulatory network in leguminous plants. In the present review, we have discussed the role of different proteomic approaches (2-DE, 2 D-DIGE, ICAT, iTRAQ, etc.) in the identification of various stress-responsive proteins in important leguminous crops, including soybean, chickpea, cowpea, pigeon pea, groundnut, and common bean under variable abiotic stresses including heat, drought, salinity, waterlogging, frost, chilling and metal toxicity. The proteomic analysis has revealed that most of the identified differentially expressed proteins in legumes are involved in photosynthesis, carbohydrate metabolism, signal transduction, protein metabolism, defense, and stress adaptation. The proteomic approaches provide insights in understanding the molecular mechanism of stress tolerance in legumes and have resulted in the identification of candidate genes used for the genetic improvement of plants against various environmental stresses. Identifying novel proteins and determining their expression under different stress conditions provide the basis for effective engineering strategies to improve stress tolerance in crop plants through marker-assisted breeding.

PANOMICS at the interface of root-soil microbiome and BNI.

Nitrification and denitrification are soil biological processes responsible for large nitrogen losses from agricultural soils and generation of the greenhouse gas (GHG) N2O. Increased use of nitrogen fertilizer and the resulting decline in nitrogen use efficiency (NUE) are a major concern in agroecosystems. This nitrogen cycle in the rhizosphere is influenced by an intimate soil microbiome-root exudate interaction and biological nitrification inhibition (BNI). A PANOMICS approach can dissect these processes. We review breakthroughs in this area, including identification and characterization of root exudates by metabolomics and proteomics, which facilitate better understanding of belowground chemical communications and help identify new biological nitrification inhibitors (BNIs). We also address challenges for advancing the understanding of the role root exudates play in biotic and abiotic stresses.

Root exudation of contrasting drought-stressed pearl millet genotypes conveys varying biological nitrification inhibition (BNI) activity.

Roots secrete a vast array of low molecular weight compounds into the soil broadly referred to as root exudates. It is a key mechanism by which plants and soil microbes interact in the rhizosphere. The effect of drought stress on the exudation process and composition is rarely studied, especially in cereal crops. This study focuses on comparative metabolic profiling of the exudates from sensitive and tolerant genotypes of pearl millet after a period of drought stress. We employed a combined platform of gas and liquid chromatography coupled to mass spectrometry to cover both primary and secondary metabolites. The results obtained demonstrate that both genotype and drought stress have a significant impact on the concentration and composition of root exudates. The complexity and function of these differential root exudates are discussed. To reveal the potential effect of root exudates on the soil microbial community after a period of drought stress, we also tested for biological nitrification inhibition (BNI) activity. The analysis revealed a genotype-dependent enhancement of BNI activity after a defined period of drought stress. In parallel, we observed a genotype-specific relation of elongated root growth and root exudation under drought stress. These data suggest that the drought stress-dependent change in root exudation can manipulate the microbial soil communities to adapt and survive under harsh conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s00374-021-01578-w.

Heat-induced proteomic changes in anthers of contrasting rice genotypes under variable stress regimes.

Heat stress drastically affects anther tissues resulting in poor plant fertility, necessitating an urgent need to determine the key proteome regulation associated with mature anther in response to heat stress. We identified several genotype - specific protein alterations in rice anthers of Moroberekan (Japonica, heat sensitive), IR64 (Indica, moderately heat tolerant), and Nagina22 (Aus, heat tolerant) in the short-term (ST_HS; one cycle of 42°C, 4 hours before anthesis) and long-term (LT_HS; 6 cycles of 38°C, 6 hours before anthesis) heat stress. The proteins upregulated in long-term heat stress in Nagina22 were enriched in biological processes related to unfolded protein binding and carboxylic acid metabolism, including amino acid metabolism. In short-term heat stress, Nagina22 anthers were enriched in proteins associated with vitamin E biosynthesis and GTPase activator activity. In contrast, downregulated proteins were related to ribosomal proteins. The expression of different Hsp20 and DnaJ was genotype specific. Overall, the heat response in Nagina22 was associated with its capacity for adequate metabolic control and cellular homeostasis, which may be critical for its higher reproductive thermotolerance. This study improves our understanding of thermotolerance mechanisms in rice anthers during anthesis and lays a foundation for breeding thermotolerant varieties via molecular breeding.

Spatial distribution of proteins and metabolites in developing wheat grain and their differential regulatory response during the grain filling process.

Grain filling and grain development are essential biological processes in the plant's life cycle, eventually contributing to the final seed yield and quality in all cereal crops. Studies of how the different wheat (Triticum aestivum L.) grain components contribute to the overall development of the seed are very scarce. We performed a proteomics and metabolomics analysis in four different developing components of the wheat grain (seed coat, embryo, endosperm, and cavity fluid) to characterize molecular processes during early and late grain development. In-gel shotgun proteomics analysis at 12, 15, 20, and 26 days after anthesis (DAA) revealed 15 484 identified and quantified proteins, out of which 410 differentially expressed proteins were identified in the seed coat, 815 in the embryo, 372 in the endosperm, and 492 in the cavity fluid. The abundance of selected protein candidates revealed spatially and temporally resolved protein functions associated with development and grain filling. Multiple wheat protein isoforms involved in starch synthesis such as sucrose synthases, starch phosphorylase, granule-bound and soluble starch synthase, pyruvate phosphate dikinase, 14-3-3 proteins as well as sugar precursors undergo a major tissue-dependent change in abundance during wheat grain development suggesting an intimate interplay of starch biosynthesis control. Different isoforms of the protein disulfide isomerase family as well as glutamine levels, both involved in the glutenin macropolymer pattern, showed distinct spatial and temporal abundance, revealing their specific role as indicators of wheat gluten quality. Proteins binned into the functional category of cell growth/division and protein synthesis/degradation were more abundant in the early stages (12 and 15 DAA). At the metabolome level all tissues and especially the cavity fluid showed highly distinct metabolite profiles. The tissue-specific data are integrated with biochemical networks to generate a comprehensive map of molecular processes during grain filling and developmental processes.

Proteomics Data Analysis for the Identification of Proteins and Derived Proteotypic Peptides of Potential Use as Putative Drought Tolerance Markers for Quercus ilex.

Drought is one of the main causes of mortality in holm oak (Quercus ilex) seedlings used in reforestation programs. Although this species shows high adaptability to the extreme climate conditions prevailing in Southern Spain, its intrinsic genetic variability may play a role in the differential response of some populations and individuals. The aim of this work was to identify proteins and derived proteotypic peptides potentially useful as putative markers for drought tolerance in holm oak by using a targeted post-acquisition proteomics approach. For this purpose, we used a set of proteins identified by shotgun (LC-MSMS) analysis in a drought experiment on Q. ilex seedlings from four different provenances (viz. the Andalusian provinces Granada, Huelva, Cadiz and Seville). A double strategy involving the quantification of proteins and target peptides by shotgun analysis and post-acquisition data analysis based on proteotypic peptides was used. To this end, an initial list of proteotypic peptides from proteins highly represented under drought conditions was compiled that was used in combination with the raw files from the shotgun experiment to quantify the relative abundance of the fragment's ion peaks with the software Skyline. The most abundant peptides under drought conditions in at least two populations were selected as putative markers of drought tolerance. A total of 30 proteins and 46 derived peptides belonging to the redox, stress-related, synthesis,-folding and degradation, and primary and secondary metabolism functional groups were thus identified. Two proteins (viz., subtilisin and chaperone GrpE protein) were found at increased levels in three populations, which make them especially interesting for validation drought tolerance markers in subsequent experiments.

Heat stress response mechanisms in pollen development.

Being rooted in place, plants are faced with the challenge of responding to unfavourable local conditions. One such condition, heat stress, contributes massively to crop losses globally. Heatwaves are predicted to increase, and it is of vital importance to generate crops that are tolerant to not only heat stress but also to several other abiotic stresses (e.g. drought stress, salinity stress) to ensure that global food security is protected. A better understanding of the molecular mechanisms that underlie the temperature stress response in pollen will be a significant step towards developing effective breeding strategies for high and stable production in crop plants. While most studies have focused on the vegetative phase of plant growth to understand heat stress tolerance, it is the reproductive phase that requires more attention as it is more sensitive to elevated temperatures. Every phase of reproductive development is affected by environmental challenges, including pollen and ovule development, pollen tube growth, male-female cross-talk, fertilization, and embryo development. In this review we summarize how pollen is affected by heat stress and the molecular mechanisms employed during the stress period, as revealed by classical and -omics experiments.

Magnetic Field Induced Changes in the Shoot and Root Proteome of Barley (Hordeum vulgare L.).

The geomagnetic field (GMF) has been present since the beginning of plant evolution. Recently, some researchers have focused their efforts on employing magnetic fields (MFs) higher than GMF to improve the seed germination, growth, and harvest of agriculturally important crop plants, as the use of MFs is an inexpensive and environment-friendly technique. In this study, we have employed different treatments of MF at 7 mT (milliTesla) at different time points of exposure, including 1, 3, and 6 h. The extended exposure was followed by five consecutive days at 6 h per day in barley seeds. The results showed a positive impact of MF on growth characteristics for 5-day-old seedlings, including seed germination rate, root and shoot length, and biomass weight. Furthermore, ~5 days of delay of flowering in pre-treated plants was also observed. We used a shotgun proteomics approach to identify changes in the protein signatures of root and shoot tissues under MF effects. In total, we have identified 2,896 proteins. Thirty-eight proteins in the shoot and 15 proteins in the root showed significant changes under the MF effect. Proteins involved in primary metabolic pathways were increased in contrast to proteins with a metal ion binding function, proteins that contain iron ions in their structure, and proteins involved in electron transfer chain, which were all decreased significantly in the treated tissues. The upregulated proteins' overall biological processes included carbohydrate metabolic process, oxidation-reduction process, and cell redox homeostasis, while down-regulated processes included translation and protein refolding. In general, shoot response was more affected by MF effect than root tissue, leading to the identification of 41 shoot specific proteins. This study provides an initial insight into the proteome regulation response to MF during barley's seedling stage.

Multiomics approach unravels fertility transition in a pigeonpea line for a two-line hybrid system.

Pigeonpea [Cajanus cajan (L.) Millsp.] is a pulse crop cultivated in the semi-arid regions of Asia and Africa. It is a rich source of protein and capable of alleviating malnutrition, improving soil health and the livelihoods of small-holder farmers. Hybrid breeding has provided remarkable improvements for pigeonpea productivity, but owing to a tedious and costly seed production system, an alternative two-line hybrid technology is being explored. In this regard, an environment-sensitive male sterile line has been characterized as a thermosensitive male sterile line in pigeonpea precisely responding to day temperature. The male sterile and fertile anthers from five developmental stages were studied by integrating transcriptomics, proteomics and metabolomics supported by precise phenotyping and scanning electron microscopic study. Spatio-temporal analysis of anther transcriptome and proteome revealed 17 repressed DEGs/DEPs in sterile anthers that play a critical role in normal cell wall morphogenesis and tapetal cell development. The male fertility to sterility transition was mainly due to a perturbation in auxin homeostasis, leading to impaired cell wall modification and sugar transport. Limited nutrient utilization thus leads to microspore starvation in response to moderately elevated day temperature which could be restored with auxin-treatment in the male sterile line. Our findings outline a molecular mechanism that underpins fertility transition responses thereby providing a process-oriented two-line hybrid breeding framework for pigeonpea.

Genomic footprints of repeated evolution of CAM photosynthesis in a Neotropical species radiation.

The adaptive radiation of Bromeliaceae (pineapple family) is one of the most diverse among Neotropical flowering plants. Diversification in this group was facilitated by shifts in several adaptive traits or "key innovations" including the transition from C3 to CAM photosynthesis associated with xeric (heat/drought) adaptation. We used phylogenomic approaches, complemented by differential gene expression (RNA-seq) and targeted metabolite profiling, to address the mechanisms of C3 /CAM evolution in the extremely species-rich bromeliad genus, Tillandsia, and related taxa. Evolutionary analyses of whole-genome sequencing and RNA-seq data suggest that evolution of CAM is associated with coincident changes to different pathways mediating xeric adaptation in this group. At the molecular level, C3 /CAM shifts were accompanied by gene expansion of XAP5 CIRCADIAN TIMEKEEPER homologs, a regulator involved in sugar- and light-dependent regulation of growth and development. Our analyses also support the re-programming of abscisic acid-related gene expression via differential expression of ABF2/ABF3 transcription factor homologs, and adaptive sequence evolution of an ENO2/LOS2 enolase homolog, effectively tying carbohydrate flux to abscisic acid-mediated abiotic stress response. By pinpointing different regulators of overlapping molecular responses, our results suggest plausible mechanistic explanations for the repeated evolution of correlated adaptive traits seen in a textbook example of an adaptive radiation.

Structural and Functional Heat Stress Responses of Chloroplasts of Arabidopsis thaliana.

Temperature elevations constitute a major threat to plant performance. In recent years, much was learned about the general molecular mode of heat stress reaction of plants. The current research focuses on the integration of the knowledge into more global networks, including the reactions of cellular compartments. For instance, chloroplast function is central for plant growth and survival, and the performance of chloroplasts is tightly linked to the general status of the cell and vice versa. We examined the changes in photosynthesis, chloroplast morphology and proteomic composition posed in Arabidopsisthaliana chloroplasts after a single or repetitive heat stress treatment over a period of two weeks. We observed that the acclimation is potent in the case of repetitive application of heat stress, while a single stress results in lasting alterations. Moreover, the physiological capacity and its adjustment are dependent on the efficiency of the protein translocation process as judged from the analysis of mutants of the two receptor units of the chloroplast translocon, TOC64, and TOC33. In response to repetitive heat stress, plants without TOC33 accumulate Hsp70 proteins and plants without TOC64 have a higher content of proteins involved in thylakoid structure determination when compared to wild-type plants.