Dynamics and genetic regulation of leaf nutrient concentration in barley based on hyperspectral imaging and machine learning.

Biofortification, the enrichment of nutrients in crop plants, is of increasing importance to improve human health. The wild barley nested association mapping (NAM) population HEB-25 was developed to improve agronomic traits including nutrient concentration. Here, we evaluated the potential of high-throughput hyperspectral imaging in HEB-25 to predict leaf concentration of 15 mineral nutrients, sampled from two field experiments and four developmental stages. Particularly accurate predictions were obtained by partial least squares regression (PLS) modeling of leaf concentrations for N, P and K reaching coefficients of determination of 0.90, 0.75 and 0.89, respectively. We recognized nutrient-specific patterns of variation of leaf nutrient concentration between developmental stages. A number of quantitative trait loci (QTL) associated with the simultaneous expression of leaf nutrients were detected, indicating their potential co-regulation in barley. For example, the wild barley allele of QTL-4H-1 simultaneously increased leaf concentration of N, P, K and Cu. Similar effects of the same QTL were previously reported for nutrient concentrations in grains, supporting a potential parallel regulation of N, P, K and Cu in leaves and grains of HEB-25. Our study provides a new approach for nutrient assessment in large-scale field experiments to ultimately select genes and genotypes supporting plant biofortification.

Untargeted metabotyping to study phenylpropanoid diversity in crop plants.

Plant genebanks constitute a key resource for breeding to ensure crop yield under changing environmental conditions. Because of their roles in a range of stress responses, phenylpropanoids are promising targets. Phenylpropanoids comprise a wide array of metabolites; however, studies regarding their diversity and the underlying genes are still limited for cereals. The assessment of barley diversity via genotyping-by-sequencing is in rapid progress. Exploring these resources by integrating genetic association studies to in-depth metabolomic profiling provides a valuable opportunity to study barley phenylpropanoid metabolism; but poses a challenge by demanding large-scale approaches. Here, we report an LC-PDA-MS workflow for barley high-throughput metabotyping. Without prior construction of a species-specific library, this method produced phenylpropanoid-enriched metabotypes with which the abundance of putative metabolic features was assessed across hundreds of samples in a single-processed data matrix. The robustness of the analytical performance was tested using a standard mix and extracts from two selected cultivars: Scarlett and Barke. The large-scale analysis of barley extracts showed (1) that barley flag leaf profiles were dominated by glycosylation derivatives of isovitexin, isoorientin, and isoscoparin; (2) proved the workflow's capability to discriminate within genotypes; (3) highlighted the role of glycosylation in barley phenylpropanoid diversity. Using the barley S42IL mapping population, the workflow proved useful for metabolic quantitative trait loci purposes. The protocol can be readily applied not only to explore the barley phenylpropanoid diversity represented in genebanks but also to study species whose profiles differ from those of cereals: the crop Helianthus annuus (sunflower) and the model plant Arabidopsis thaliana.

Function and Regulation of Chloroplast Peroxiredoxin IIE.

Peroxiredoxins (PRX) are thiol peroxidases that are highly conserved throughout all biological kingdoms. Increasing evidence suggests that their high reactivity toward peroxides has a function not only in antioxidant defense but in particular in redox regulation of the cell. Peroxiredoxin IIE (PRX-IIE) is one of three PRX types found in plastids and has previously been linked to pathogen defense and protection from protein nitration. However, its posttranslational regulation and its function in the chloroplast protein network remained to be explored. Using recombinant protein, it was shown that the peroxidatic Cys121 is subjected to multiple posttranslational modifications, namely disulfide formation, S-nitrosation, S-glutathionylation, and hyperoxidation. Slightly oxidized glutathione fostered S-glutathionylation and inhibited activity in vitro. Immobilized recombinant PRX-IIE allowed trapping and subsequent identification of interaction partners by mass spectrometry. Interaction with the 14-3-3 υ protein was confirmed in vitro and was shown to be stimulated under oxidizing conditions. Interactions did not depend on phosphorylation as revealed by testing phospho-mimicry variants of PRX-IIE. Based on these data it is proposed that 14-3-3υ guides PRX­IIE to certain target proteins, possibly for redox regulation. These findings together with the other identified potential interaction partners of type II PRXs localized to plastids, mitochondria, and cytosol provide a new perspective on the redox regulatory network of the cell.

VANTED: A Tool for Integrative Visualization and Analysis of -Omics Data.

The investigation of biological systems from different perspectives leads, due to novel -omics technologies, to large-scale, heterogeneous, and complex datasets. To elucidate molecular programs that control biological systems growth and development the integration and analysis of these -omics data remains challenging. Network-integrated visualizations based on graphical standards support intuitive exploration and interpretation of -omics data within the functional context. This integrated vision of the biological system to be studied tries to extract all hidden information for deepening our understanding and reveals new biological insights.The method described here gives detailed instructions on the generation of such an integrative visualization of -omics data in the context of networks presented in the Systems Biology Graphical Notation (SBGN) using VANTED; a software tool for systems biology applications. An example illustrates the application of the method for metabolomics and proteomics data integration and analysis using a primary metabolic pathway, for the model crop potato.

Nitrogen Deficiency Induced Alterations in the Root Proteome of a Pair of Potato (Solanum tuberosum L.) Varieties Contrasting for their Response to Low N.

Improving crop nitrogen use efficiency is important both from the economic and the environmental viewpoint. Here, the aim is to highlight differences between the proteomic response of the roots of two potato cultivars contrasting in their response to nitrogen (N) deficiency, in an effort to understand which proteins and metabolic pathways contribute to the tolerance of N deprivation. The two cultivars ''Topas'' (tolerant) and ''Lambada'' (sensitive) are grown under both an N sufficient and an N deficient regime, using an in vitro-based cultivation system. Responsive proteins are identified and quantified using label-free quantitative shotgun proteomics. The contrasting cultivars differed with respect to components of the glutamine synthetase/glutamine oxoglutarate aminotransferase pathway, tricarboxylic acid cycle, the glycolysis/gluconeogenesis pathway as well as protein and amino acid synthesis machinery. Additional differences are associated with protein catabolism and defense mechanisms.

Comparative shoot proteome analysis of two potato (Solanum tuberosum L.) genotypes contrasting in nitrogen deficiency responses in vitro.

Aiming at a better understanding of the physiological and biochemical background of nitrogen use efficiency, alterations in the shoot proteome under N-deficiency were investigated in two contrasting potato genotypes grown in vitro with 60 and 7.5mM N, respectively. A gel based proteomic approach was applied to identify candidate proteins associated with genotype specific responses to N-deficiency. 21% of the detected proteins differed in abundance between the two genotypes. Between control and N-deficiency conditions 19.5% were differentially accumulated in the sensitive and 15% in the tolerant genotype. 93% of the highly N-deficiency responsive proteins were identified by MALDI TOF/TOF mass spectrometry. The major part was associated with photosynthesis, carbohydrate metabolism, stress response and regulation. Differential accumulation of enzymes involved in the Calvin cycle and glycolysis suggest activation of alternative carbohydrate pathways. In the tolerant genotype, increased abundance under N-deficiency was also found for enzymes involved in chlorophyll synthesis and stability of enzymes, which increase photosynthetic carbon fixation efficiency. Out of a total of 106 differentially abundant proteins, only eight were detected in both genotypes. Our findings suggest that mutually responsive proteins reflect universal stress responses while adaptation to N-deficiency in metabolic pathways is more genotype specific. SIGNIFICANCE: Nitrogen losses from arable farm land considerably contribute to environmental pollution. In potato, this is a special problem due cultivation on light soils, irrigation and the shallow root system. Therefore, breeding of cultivars with improved nitrogen use efficiency and stable yields under reduced N fertilization is an important issue. Knowledge of genotype dependent adaptation to N-deficiency at the proteome level can help to understand regulation of N efficiency and development of N-efficient cultivars.

Proteomic analysis of two divergently responding potato genotypes (Solanum tuberosum L.) following osmotic stress treatment in vitro.

UNLABELLED: Starch potatoes (Solanum tuberosum L.) are of interest for production of starch, ethanol, and biopolymers. Due to the predicted increase in drought periods, the breeding of starch potatoes for drought tolerance is essential. This study aims to elucidate the physiological mechanisms that give rise to drought tolerance. Two genotypes contrasting in drought tolerance were compared. We applied osmotic stress which is a known component of drought stress under in vitro conditions. Shoot tips were harvested after 11days of culture on control medium and medium supplied with 0.2M sorbitol. Their proteomes were analyzed using two-dimensional isoelectric focussing sodium dodecyl sulphate polyacrylamide gel electrophoresis (2D-IEF/SDS-PAGE). Of a total of 679 distinct protein spots, 118 and 20 spots with differential abundance were found in the sensitive and the tolerant genotype, respectively, after the application of stress. Using mass spectrometry, the proteins in 100 differentially abundant spots were identified; a majority of these proteins were from the chloroplast. For the sensitive genotype, an increase in the abundance of proteinase inhibitors and their precursors, changes in stress responsive proteins and an altered RNA/DNA-binding response were observed. The differentially abundant spots of the tolerant genotype comprised one chaperone and one hydrogen peroxide detoxifying protein. BIOLOGICAL SIGNIFICANCE: Our findings reveal that the two genotypes have different responses to osmotic stress in terms of protein degradation and reactive oxygen species (ROS) scavenging and production. Our data suggest that the tolerant genotype might adjust to the applied stress more quickly. A comparative temporal analysis might provide further insights into these rapid changes and assist in the development of biomarkers.

Label-free proteome profiling reveals developmental-dependent patterns in young barley grains.

UNLABELLED: Due to its importance as a cereal crop worldwide, high interest in the determination of factors influencing barley grain quality exists. This study focusses on the elucidation of protein networks affecting early grain developmental processes. NanoLC-based separation coupled to label-free MS detection was applied to gain insights into biochemical processes during five different grain developmental phases (pre-storage until storage phase, 3days to 16days after flowering). Multivariate statistics revealed two distinct developmental patterns during the analysed grain developmental phases: proteins showed either highest abundance in the middle phase of development - in the transition phase - or at later developmental stages - within the storage phase. Verification of developmental patterns observed by proteomic analysis was done by applying hypothesis-driven approaches, namely Western Blot analysis and enzyme assays. High general metabolic activity of the grain with regard to protein synthesis, cell cycle regulation, defence against oxidative stress, and energy production via photosynthesis was observed in the transition phase. Proteins upregulated in the storage phase are related towards storage protein accumulation, and interestingly to the defence of storage reserves against pathogens. A mixed regulatory pattern for most enzymes detected in our study points to regulatory mechanisms at the level of protein isoforms. BIOLOGICAL SIGNIFICANCE: In-depth understanding of early grain developmental processes of cereal caryopses is of high importance as they influence final grain weight and quality. Our knowledge about these processes is still limited, especially on proteome level. To identify key mechanisms in early barley grain development, a label-free data-independent proteomics acquisition approach has been applied. Our data clearly show, that proteins either exhibit highest expression during cellularization and the switch to the storage phase (transition phase, 5-7 DAF), or during storage product accumulation (10-16 DAF). The results highlight versatile cellular metabolic activity in the transition phase and strong convergence towards storage product accumulation in the storage phase. Notably, both phases are characterized by particular protective mechanism, such as scavenging of oxidative stress and defence against pathogens, during the transition and the storage phase, respectively.