Drought resistance strategies in minor millets: a review.

MAIN CONCLUSION: The review discusses growth and drought-response mechanisms in minor millets under three themes: drought escape, drought avoidance and drought tolerance. Drought is one of the most prominent abiotic stresses impacting plant growth, performance, and productivity. In the context of climate change, the prevalence and severity of drought is expected to increase in many agricultural regions worldwide. Millets (coarse grains) are a group of small-seeded grasses cultivated in arid and semi-arid regions throughout the world and are an important source of food and feed for humans and livestock. Although minor millets, i.e., foxtail millet, finger millet, proso millet, barnyard millet, kodo millet and little millet are generally hardier and more drought-resistant than cereals and major millets (sorghum and pearl millet), understanding their responses, processes and strategies in response to drought is more limited. Here, we review drought resistance strategies in minor millets under three themes: drought escape (e.g., short crop cycle, short vegetative period, developmental plasticity and remobilization of assimilates), drought avoidance (e.g., root traits for better water absorption and leaf traits to control water loss), and drought tolerance (e.g., osmotic adjustment, maintenance of photosynthetic ability and antioxidant potential). Data from 'omics' studies are summarized to provide an overview of the molecular mechanisms important in drought tolerance. In addition, the final section highlights knowledge gaps and challenges to improving minor millets. This review is intended to enhance major cereals and millet per se in light of climate-related increases in aridity.

Impact of Heavy Metals on Cold Acclimation of Salix viminalis Roots.

In nature, plants are exposed to a range of climatic conditions. Those negatively impacting plant growth and survival are called abiotic stresses. Although abiotic stresses have been extensively studied separately, little is known about their interactions. Here, we investigate the impact of long-term mild metal exposure on the cold acclimation of Salix viminalis roots using physiological, transcriptomic, and proteomic approaches. We found that, while metal exposure significantly affected plant morphology and physiology, it did not impede cold acclimation. Cold acclimation alone increased glutathione content and glutathione reductase activity. It also resulted in the increase in transcripts and proteins belonging to the heat-shock proteins and related to the energy metabolism. Exposure to metals decreased antioxidant capacity but increased catalase and superoxide dismutase activity. It also resulted in the overexpression of transcripts and proteins related to metal homeostasis, protein folding, and the antioxidant machinery. The simultaneous exposure to both stressors resulted in effects that were not the simple addition of the effects of both stressors taken separately. At the antioxidant level, the response to both stressors was like the response to metals alone. While this should have led to a reduction of frost tolerance, this was not observed. The impact of the simultaneous exposure to metals and cold acclimation on the transcriptome was unique, while at the proteomic level the cold acclimation component seemed to be dominant. Some genes and proteins displayed positive interaction patterns. These genes and proteins were related to the mitigation and reparation of oxidative damage, sugar catabolism, and the production of lignans, trehalose, and raffinose. Interestingly, none of these genes and proteins belonged to the traditional ROS homeostasis system. These results highlight the importance of the under-studied role of lignans and the ROS damage repair and removal system in plants simultaneously exposed to multiple stressors.

Exploration of the Diversity of Vicine and Convicine Derivatives in Faba Bean (Vicia faba L.) Cultivars: Insights from LC-MS/MS Spectra.

While numerous Fabaceae seeds are a good nutritional source of high-quality protein, the use of some species is hampered by toxic effects caused by exposure to metabolites that accumulate in the seeds. One such species is the faba or broad bean (Vicia faba L.), which accumulates vicine and convicine. These two glycoalkaloids cause favism, the breakdown of red blood cells in persons with a glucose-6-phosphate dehydrogenase deficiency. Because this is the most common enzyme deficiency worldwide, faba bean breeding efforts have focused on developing cultivars with low levels of these alkaloids. Consequently, quantification methods have been developed; however, they quantify vicine and convicine only and not the derivatives of these compounds that potentially generate the same bio-active molecules. Based on the recognition of previously unknown (con)vicine-containing compounds, we screened the fragmentation spectra of LC-MS/MS data from five faba bean cultivars using the characteristic fragments generated by (con)vicine. This resulted in the recognition of more than a hundred derivatives, of which 89 were tentatively identified. (Con)vicine was mainly derivatized through the addition of sugars, hydroxycinnamic acids, and dicarboxylic acids, with a group of compounds composed of two (con)vicine residues linked by dicarboxyl fatty acids. In general, the abundance profiles of the different derivatives in the five cultivars mimicked that of vicine and convicine, but some showed a derivative-specific profile. The description of the (con)vicine diversity will impact the interpretation of future studies on the biosynthesis of (con)vicine, and the content in potentially bio-active alkaloids in faba beans may be higher than that represented by the quantification of vicine and convicine alone.

Molecular insights into plant desiccation tolerance: transcriptomics, proteomics and targeted metabolite profiling in Craterostigma plantagineum.

The resurrection plant Craterostigma plantagineum possesses an extraordinary capacity to survive long-term desiccation. To enhance our understanding of this phenomenon, complementary transcriptome, soluble proteome and targeted metabolite profiling was carried out on leaves collected from different stages during a dehydration and rehydration cycle. A total of 7348 contigs, 611 proteins and 39 metabolites were differentially abundant across the different sampling points. Dynamic changes in transcript, protein and metabolite levels revealed a unique signature characterizing each stage. An overall low correlation between transcript and protein abundance suggests a prominent role for post-transcriptional modification in metabolic reprogramming to prepare plants for desiccation and recovery. The integrative analysis of all three data sets was performed with an emphasis on photosynthesis, photorespiration, energy metabolism and amino acid metabolism. The results revealed a set of precise changes that modulate primary metabolism to confer plasticity to metabolic pathways, thus optimizing plant performance under stress. The maintenance of cyclic electron flow and photorespiration, and the switch from C3 to crassulacean acid metabolism photosynthesis, may contribute to partially sustain photosynthesis and minimize oxidative damage during dehydration. Transcripts with a delayed translation, ATP-independent bypasses, alternative respiratory pathway and 4-aminobutyric acid shunt may all play a role in energy management, together conferring bioenergetic advantages to meet energy demands upon rehydration. This study provides a high-resolution map of the changes occurring in primary metabolism during dehydration and rehydration and enriches our understanding of the molecular mechanisms underpinning plant desiccation tolerance. The data sets provided here will ultimately inspire biotechnological strategies for drought tolerance improvement in crops.

The Roots of Plant Frost Hardiness and Tolerance.

Frost stress severely affects agriculture and agroforestry worldwide. Although many studies about frost hardening and resistance have been published, most of them focused on the aboveground organs and only a minority specifically targets the roots. However, roots and aboveground tissues have different physiologies and stress response mechanisms. Climate models predict an increase in the magnitude and frequency of late-frost events, which, together with an observed loss of soil insulation, will greatly decrease plant primary production due to damage at the root level. Molecular and metabolic responses inducing root cold hardiness are complex. They involve a variety of processes related to modifications in cell wall composition, maintenance of the cellular homeostasis and the synthesis of primary and secondary metabolites. After a summary of the current climatic models, this review details the specificity of freezing stress at the root level and explores the strategies roots developed to cope with freezing stress. We then describe the level to which roots can be frost hardy, depending on their age, size category and species. After that, we compare the environmental signals inducing cold acclimation and frost hardening in the roots and aboveground organs. Subsequently, we discuss how roots sense cold at a cellular level and briefly describe the following signal transduction pathway, which leads to molecular and metabolic responses associated with frost hardening. Finally, the current options available to increase root frost tolerance are explored and promising lines of future research are discussed.

Repair of sub-lethal freezing damage in leaves of Arabidopsis thaliana.

BACKGROUND: The detrimental effects of global climate change direct more attention to the survival and productivity of plants during periods of highly fluctuating temperatures. In particular in temperate climates in spring, temperatures can vary between above-zero and freezing temperatures, even during a single day. Freeze-thaw cycles cause cell membrane lesions that can lead to tissue damage and plant death. Whereas the processes of cold acclimation and freeze-thaw injury are well documented, not much is known about the recovery of plants after a freezing event. We therefore addressed the following questions: i. how does the severity of freezing damage influence repair; ii. how are respiration and content of selected metabolites influenced during the repair process; and iii. how do transcript levels of selected genes respond during repair? RESULTS: We have investigated the recovery from freezing to sub-lethal temperatures in leaves of non-acclimated and cold acclimated Arabidopsis thaliana plants over a period of 6 days. Fast membrane repair and recovery of photosynthesis were observed 1 day after recovery (1D-REC) and continued until 6D-REC. A substantial increase in respiration accompanied the repair process. In parallel, concentrations of sugars and proline, acting as compatible solutes during freezing, remained unchanged or declined, implicating these compounds as carbon and nitrogen sources during recovery. Similarly, cold-responsive genes were mainly down regulated during recovery of cold acclimated leaves. In contrast, genes involved in cell wall remodeling and ROS scavenging were induced during recovery. Interestingly, also the expression of genes encoding regulatory proteins, such as 14-3-3 proteins, was increased suggesting their role as regulators of repair processes. CONCLUSIONS: Recovery from sub-lethal freezing comprised membrane repair, restored photosynthesis and increased respiration rates. The process was accompanied by transcriptional changes including genes encoding regulatory proteins redirecting the previous cold response to repair processes, e.g. to cell wall remodeling, maintenance of the cellular proteome and to ROS scavenging. Understanding of processes involved in repair of freeze-thaw injury increases our knowledge on plant survival in changing climates with highly fluctuating temperatures.

Does long-term cadmium exposure influence the composition of pectic polysaccharides in the cell wall of Medicago sativa stems?

BACKGROUND: The heavy metal cadmium (Cd) accumulates in the environment due to anthropogenic influences. It is unessential and harmful to all life forms. The plant cell wall forms a physical barrier against environmental stress and changes in the cell wall structure have been observed upon Cd exposure. In the current study, changes in the cell wall composition and structure of Medicago sativa stems were investigated after long-term exposure to Cd. Liquid chromatography coupled to mass spectrometry (LC-MS) for quantitative protein analysis was complemented with targeted gene expression analysis and combined with analyses of the cell wall composition. RESULTS: Several proteins determining for the cell wall structure changed in abundance. Structural changes mainly appeared in the composition of pectic polysaccharides and data indicate an increased presence of xylogalacturonan in response to Cd. Although a higher abundance and enzymatic activity of pectin methylesterase was detected, the total pectin methylation was not affected. CONCLUSIONS: An increased abundance of xylogalacturonan might hinder Cd binding in the cell wall due to the methylation of its galacturonic acid backbone. Probably, the exclusion of Cd from the cell wall and apoplast limits the entry of the heavy metal into the symplast and is an important factor during tolerance acquisition.

Selection of Appropriate Reference Genes for Gene Expression Analysis under Abiotic Stresses in Salix viminalis.

Salix viminalis is a fast growing willow species with potential as a plant used for biomass feedstock or for phytoremediation. However, few reference genes (RGs) for quantitative real-time polymerase chain reaction (qPCR) are available in S. viminalis, thereby limiting gene expression studies. Here, we investigated the expression stability of 14 candidate reference genes (RGs) across various organs exposed to five abiotic stresses (cold, heat, drought, salt, and poly-metals). Four RGs ranking algorithms, namely geNormPLUS, BestKeeper, NormFinder, and GrayNorm were applied to analyze the qPCR data and the outputs were merged into consensus lists with RankAggreg, a rank aggregation algorithm. In addition, the optimal RG combinations were determined with geNormPLUS and GrayNorm. The genes that were the most stable in the roots were TIP41 and CDC2. In the leaves, TIP41 was the most stable, followed by EF1b and ARI8, depending on the condition tested. Conversely, GAPDH and ß-TUB, two genes commonly used for qPCR data normalization were the least stable across all organs. Nevertheless, both geNormPLUS and GrayNorm recommended the use of a combination of genes rather than a single one. These results are valuable for research of transcriptomic responses in different S. viminalis organs.

Insights into the molecular regulation of monolignol-derived product biosynthesis in the growing hemp hypocotyl.

BACKGROUND: Lignin and lignans are both derived from the monolignol pathway. Despite the similarity of their building blocks, they fulfil different functions in planta. Lignin strengthens the tissues of the plant, while lignans are involved in plant defence and growth regulation. Their biosyntheses are tuned both spatially and temporally to suit the development of the plant (water conduction, reaction to stresses). We propose to study the general molecular events related to monolignol-derived product biosynthesis, especially lignin. It was previously shown that the growing hemp hypocotyl (between 6 and 20 days after sowing) is a valid system to study secondary growth and the molecular events accompanying lignification. The present work confirms the validity of this system, by using it to study the regulation of lignin and lignan biosynthesis. Microscopic observations, lignin analysis, proteomics, together with in situ laccase and peroxidase activity assays were carried out to understand the dynamics of lignin synthesis during the development of the hemp hypocotyl. RESULTS: Based on phylogenetic analysis and targeted gene expression, we suggest a role for the hemp dirigent and dirigent-like proteins in lignan biosynthesis. The transdisciplinary approach adopted resulted in the gene- and protein-level quantification of the main enzymes involved in the biosynthesis of monolignols and their oxidative coupling (laccases and class III peroxidases), in lignin deposition (dirigent-like proteins) and in the determination of the stereoconformation of lignans (dirigent proteins). CONCLUSIONS: Our work sheds light on how, in the growing hemp hypocotyl, the provision of the precursors needed to synthesize the aromatic biomolecules lignin and lignans is regulated at the transcriptional and proteomic level.

Changes in the Proteome of Medicago sativa Leaves in Response to Long-Term Cadmium Exposure Using a Cell-Wall Targeted Approach.

Accumulation of cadmium (Cd) shows a serious problem for the environment and poses a threat to plants. Plants employing various cellular and molecular mechanisms to limit Cd toxicity and alterations of the cell wall structure were observed upon Cd exposure. This study focuses on changes in the cell wall protein-enriched subproteome of alfalfa (Medicago sativa) leaves during long-term Cd exposure. Plants grew on Cd-contaminated soil (10 mg/kg dry weight (DW)) for an entire season. A targeted approach was used to sequentially extract cell wall protein-enriched fractions from the leaves and quantitative analyses were conducted with two-dimensional difference gel electrophoresis (2D DIGE) followed by protein identification with matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time of flight (TOF/TOF) mass spectrometry. In 212 spots that showed a significant change in intensity upon Cd exposure a single protein was identified. Of these, 163 proteins are predicted to be secreted and involved in various physiological processes. Proteins of other subcellular localization were mainly chloroplastic and decreased in response to Cd, which confirms the Cd-induced disturbance of the photosynthesis. The observed changes indicate an active defence response against a Cd-induced oxidative burst and a restructuring of the cell wall, which is, however, different to what is observed in M. sativa stems and will be discussed.

Novel Insights from Comparative In Silico Analysis of Green Microalgal Cellulases.

The assumption that cellulose degradation and assimilation can only be carried out by heterotrophic organisms was shattered in 2012 when it was discovered that the unicellular green alga, Chlamydomonas reinhardtii (Cr), can utilize cellulose for growth under CO2-limiting conditions. Publications of genomes/transcriptomes of the colonial microalgae, Gonium pectorale (Gp) and Volvox carteri (Vc), between 2010⁻2016 prompted us to look for cellulase genes in these algae and to compare them to cellulases from bacteria, fungi, lower/higher plants, and invertebrate metazoans. Interestingly, algal catalytic domains (CDs), belonging to the family GH9, clustered separately and showed the highest (33⁻42%) and lowest (17⁻36%) sequence identity with respect to cellulases from invertebrate metazoans and bacteria, respectively, whereas the identity with cellulases from plants was only 27⁻33%. Based on comparative multiple alignments and homology models, the domain arrangement and active-site architecture of algal cellulases are described in detail. It was found that all algal cellulases are modular, consisting of putative novel cysteine-rich carbohydrate-binding modules (CBMs) and proline/serine-(PS) rich linkers. Two genes were found to encode a protein with a putative Ig-like domain and a cellulase with an unknown domain, respectively. A feature observed in one cellulase homolog from Gp and shared by a spinach cellulase is the existence of two CDs separated by linkers and with a C-terminal CBM. Dockerin and Fn-3-like domains, typically found in bacterial cellulases, are absent in algal enzymes. The targeted gene expression analysis shows that two Gp cellulases consisting, respectively, of a single and two CDs were upregulated upon filter paper addition to the medium.

Combining -Omics to Unravel the Impact of Copper Nutrition on Alfalfa (Medicago sativa) Stem Metabolism.

Copper can be found in the environment at concentrations ranging from a shortage up to the threshold of toxicity for plants, with optimal growth conditions situated in between. The plant stem plays a central role in transferring and distributing minerals, water and other solutes throughout the plant. In this study, alfalfa is exposed to different levels of copper availability, from deficiency to slight excess, and the impact on the metabolism of the stem is assessed by a non-targeted proteomics study and by the expression analysis of key genes controlling plant stem development. Under copper deficiency, the plant stem accumulates specific copper chaperones, the expression of genes involved in stem development is decreased and the concentrations of zinc and molybdenum are increased in comparison with the optimum copper level. At the optimal copper level, the expression of cell wall-related genes increases and proteins playing a role in cell wall deposition and in methionine metabolism accumulate, whereas copper excess imposes a reduction in the concentration of iron in the stem and a reduced abundance of ferritins. Secondary ion mass spectrometry (SIMS) analysis suggests a role for the apoplasm as a copper storage site in the case of copper toxicity.

Unravelling the effect of sucrose and cold pretreatment on cryopreservation of potato through sugar analysis and proteomics.

Apical shoot tips were dissected from donor plants (cultured in several conditions) and cryopreserved using the droplet-vitrification technique. The effect of two preculture treatments (sucrose pretreatment medium or cold-culturing during two weeks) on donor plants of four potato species (Solanum commersonii, S. juzepcukii, S. ajanhuiri, and Solanum tuberosum) was studied. Post-cryopreservation meristem growth and plant recovery were influenced by the treatments, but the effect on the regeneration was strongly genotype-dependent. The highest post-rewarming plant recovery percentage was obtained using meristems dissected from donor plants of S. commersonii cultured on sucrose pretreatment medium or cold-cultured. Both preculture conditions also enhanced plant recovery in S. juzepcukii compared to control cultures. Cold preculture, however, proved to be undesirable for S. tuberosum whereas sucrose pretreatment had a positive impact on the plant regeneration of this species. The determination of changes in the concentration of soluble sugars revealed sugar accumulation, especially of sucrose and the raffinose family of oligosaccharides (RFOs), which can be linked to tolerance towards the cryopreservation. Additionally, a study of the proteome of the donor plantlets after the pretreatments by 2D-fluorescence difference gel electrophoresis (DIGE) was carried out to identify differentially abundant proteins. Carbon metabolism-related proteins, together with stress-response and oxidative-homeostasis related proteins were the main class of proteins that changed in abundance after the pretreatments. Our results suggest that oxidative homeostasis-related proteins and sugars may be associated with the improved tolerance to cryopreservation and the ability to cold acclimate by S. commersonii in contrast to the other genotypes. The increased accumulation of sucrose and RFOs play a fundamental role in the response to stress in potato and may help to acquire tolerance to cryopreservation.

The impact of dietary Laminaria digitata and alginate lyase supplementation on the weaned piglet liver: A comprehensive proteomics and metabolomics approach.

The brown seaweed Laminaria digitata, a novel feedstuff for weaned piglets, has potentially beneficial prebiotic properties. However, its recalcitrant cell wall challenges digestion in monogastrics. Alginate lyase is a promising supplement to mitigate this issue. This study's aim was to investigate the impact of incorporating 10% dietary Laminaria digitata, supplemented with alginate lyase, on the hepatic proteome and metabolome of weaned piglets. These diets introduced minor variations to the metabolome and caused significant shifts in the proteome. Dietary seaweed provided a rich source of n-3 PUFAs that could signal hepatic fatty acid oxidation (FABP, ACADSB and ALDH1B1). This may have affected the oxidative stability of the tissue, requiring an elevated abundance of GST for regulation. The presence of reactive oxygen species likely inflicted protein damage, triggering increased proteolytic activity (LAPTM4B and PSMD4). Alginate lyase supplementation augmented the number of differentially abundant proteins, which included GBE1 and LDHC, contributing to maintain circulating glucose levels by mobilizing glycogen stores and branched-chain amino acids. The enzymatic supplementation with alginate lyase amplified the effects of the seaweed-only diet. An additional filter was employed to test the effect of missing values on the proteomics analysis, which is discussed from a technical perspective. SIGNIFICANCE: Brown seaweeds such as Laminaria digitata have prebiotic and immune-modulatory components, such as laminarin, that can improve weaned piglet health. However, they have recalcitrant cell wall polysaccharides, such as alginate, that can elicit antinutritional effects on the monogastric digestive system. The aim of this study was to evaluate the effect of a high level of dietary L. digitata and alginate lyase supplementation on the hepatic metabolism of weaned piglets, using high throughput Omics approaches.

Physiological and proteome study of sunflowers exposed to a polymetallic constraint.

The new energy requirements of the growing world population together with the actual ecological trend of phytoremediation have made challenging the cultivation of energetic crops on nonagricultural lands, such as those contaminated with trace elements. In this study, phenotypical characterization and biochemical analyses were combined to emphasize the global response of young sunflowers (Helianthus annuus L.) grown in hydroponic media contaminated with different Cd, Ni, and Zn concentrations. Leaves and roots of sunflowers reaching the stage "2-extended leaves" and exposed to different trace metal concentrations were harvested and analyzed by 2D-DIGE in order to study in depth the molecular responses of the young plants upon the polymetallic exposure. Proteomics confirmed the observed global reduction in growth and development. If photosynthetic light reactions and carbon metabolism were the most affected in leaves, in roots significant disruptions were observed in proteins involved in respiration, oxidative balance, protein and gene expression, and in the induction of programmed cell death. Elemental analyses of the plantlets indicated a profound impact of the treatment resulting in misbalance in essential micronutrients. Altogether, this study highlights the sensitivity of the sunflower to a polymetallic pollution and indicates that its use as a remediative tool of trace element polluted soils is limited.

A physiological and proteomic study of poplar leaves during ozone exposure combined with mild drought.

The occurrence of high-ozone concentrations during drought episodes is common considering that they are partially caused by the same meteorological phenomena. It was suggested that mild drought could protect plants against ozone-induced damage by causing the closure of stomata and preventing the entry of ozone into the leaves. The present experiment attempts to create an overview of the changes in cellular processes in response to ozone, mild drought and a combined treatment based on the use of 2D-DiGE to compare the involved proteins, and a number of supporting analyses. Morphological symptoms were worst in the combined treatment, indicating a severe stress, but fewer proteins were differentially abundant in the combined treatment than for ozone alone. Stomatal conductance was slightly lowered in the combined treatment. Shifts in carbon metabolism indicated that the metabolism changed to accommodate for protective measures and changes in the abundance of proteins involved in redox protection indicated the presence of an oxidative stress. This study allowed identifying a set of proteins that changed similarly during ozone and drought stress, indicative of crosstalk in the molecular response of plants exposed to these stresses. The abundance of other key proteins changed only when the plants are exposed to specific conditions. Together this indicates the coexistence of generalized and specialized responses to different conditions.

Two traditional maize inbred lines of contrasting technological abilities are discriminated by the seed flour proteome.

The seed proteome of two traditional maize inbred lines (pb269 and pb369) contrasting in grain hardness and in preferable use for bread-making was evaluated. The pb269 seeds, of flint type (i.e., hard endosperm), are preferably used by manufacturers, while pb369 (dent, soft endosperm) is rejected. The hypothesis that the content and relative amounts of specific proteins in the maize flour are relevant for such discrimination of the inbred lines was tested. The flour proteins were sequentially extracted following the Osborne fractionation (selective solubilization), and the four Osborne fractions were submitted to two-dimensional electrophoresis (2DE). The total amount of protein extracted from the seeds was not significantly different, but pb369 flour exhibited significantly higher proportions of salt-extracted proteins (globulins) and ethanol-extracted proteins (alcohol-soluble prolamins). The proteome analysis allowed discrimination between the two inbred lines, with pb269 demonstrating higher heterogeneity than pb369. From the 967 spots (358 common to both lines, 208 specific to pb269, and 401 specific to pb369), 588 were submitted to mass spectrometry (MS). Through the combined use of trypsin and chymotrypsin it was possible to identify proteins in 436 spots. The functional categorization in combination with multivariate analysis highlighted the most discriminant biological processes (carbohydrate metabolic process, response to stress, chitin catabolic process, oxidation-reduction process) and molecular function (nutrient reservoir activity). The inbred lines exhibited quantitative and qualitative differences in these categories. Differences were also revealed in the amounts, proportions, and distribution of several groups of storage proteins, which can have an impact on the organization of the protein body and endosperm hardness. For some proteins (granule-bound starch synthase-1, cyclophilin, zeamatin), a change in the protein solubility rather than in the total amount extracted was observed, which reveals distinct in vivo associations and/or changes in binding strength between the inbred lines. Our approach produced information that relates protein content, relative protein content, and specific protein types to endosperm hardness and to the preferable use for "broa" bread-making.

From tolerance to acute metabolic deregulation: contribution of proteomics to dig into the molecular response of alder species under a polymetallic exposure.

Alnus spp. are actinorhizal trees commonly found in wet habitats and able to grow effectively in soil slightly contaminated with metal trace- elements. Two clones belonging to two Alnus species, namely, A. incana and A. glutinosa, were grown in hydroponics and exposed for 9 weeks to a Cd + Ni + Zn polymetallic constraint. Although responding by a similar decrease in total biomass production, the proteomic analysis associated with the study of various biochemical parameters including carbohydrate and mineral analyses revealed that the two clones have a distinct stress-responsive behavior. All parameters indicated that the roots, the organ in direct contact with the media, are more affected than the leaves. In fact, in A. glutinosa the response was almost completely confined to the roots, whereas many proteins change significantly in the roots and in the leaves of the treated A. incana. In both clones, the changes affected a broad range of metabolic processes such as redox regulation and energy metabolism and induced the production of pathogenesis-related proteins. In particular, changes in the accumulation of bacterial proteins that were not identified as coming from the known symbionts of Alnus were reported. Further investigation should be performed to identify their origin and exact role in the plant response to the polymetallic exposure tested here.

Proteomic changes associated with freeze-thaw injury and post-thaw recovery in onion (Allium cepa L.) scales.

The ability of plants to recover from freeze-thaw injury is a critical component of freeze-thaw stress tolerance. To investigate the molecular basis of freeze-thaw recovery, here we compared the proteomes of onion scales from unfrozen control (UFC), freeze-thaw injured (INJ), and post-thaw recovered (REC) treatments. Injury-related proteins (IRPs) and recovery-related proteins (RRPs) were differentiated according to their accumulation patterns. Many IRPs decreased right after thaw without any significant re-accumulation during post-thaw recovery, while others were exclusively induced in INJ tissues. Most IRPs are antioxidants, stress proteins, molecular chaperones, those induced by physical injury or proteins involved in energy metabolism. Taken together, these observations suggest that while freeze-thaw compromises the constitutive stress protection and energy supply in onion scales, it might also recruit 'first-responders' (IRPs that were induced) to mitigate such injury. RRPs, on the other hand, are involved in the injury-repair program during post-thaw environment conducive for recovery. Some RRPs were restored in REC tissues after their first reduction right after thaw, while others exhibit higher abundance than their 'constitutive' levels. RRPs might facilitate new cellular homeostasis, potentially by re-establishing ion homeostasis and proteostasis, cell-wall remodelling, reactive oxygen species (ROS) scavenging, defence against possible post-thaw infection, and regulating the energy budget to sustain these processes.

Integrated -omics: a powerful approach to understanding the heterogeneous lignification of fibre crops.

Lignin and cellulose represent the two main components of plant secondary walls and the most abundant polymers on Earth. Quantitatively one of the principal products of the phenylpropanoid pathway, lignin confers high mechanical strength and hydrophobicity to plant walls, thus enabling erect growth and high-pressure water transport in the vessels. Lignin is characterized by a high natural heterogeneity in its composition and abundance in plant secondary cell walls, even in the different tissues of the same plant. A typical example is the stem of fibre crops, which shows a lignified core enveloped by a cellulosic, lignin-poor cortex. Despite the great value of fibre crops for humanity, however, still little is known on the mechanisms controlling their cell wall biogenesis, and particularly, what regulates their spatially-defined lignification pattern. Given the chemical complexity and the heterogeneous composition of fibre crops' secondary walls, only the use of multidisciplinary approaches can convey an integrated picture and provide exhaustive information covering different levels of biological complexity. The present review highlights the importance of combining high throughput -omics approaches to get a complete understanding of the factors regulating the lignification heterogeneity typical of fibre crops.