Metabolic adaptations leading to an enhanced lignification in wheat roots under salinity stress.

Analysis of salinity tolerance processes in wheat has focused on salt exclusion from shoots while root phenotypes have received limited attention. Here, we consider the varying phenotypic response of four bread wheat varieties that differ in their type and degree of salt tolerance and assess their molecular responses to salinity and changes in root cell wall lignification. These varieties were Westonia introgressed with Nax1 and Nax2 root sodium transporters (HKT1;4-A and HKT1;5-A) that reduce Na+ accumulation in leaves, as well as the 'tissue tolerant' Portuguese landrace Mocho de Espiga Branca that has a mutation in the homologous gene HKT1;5-D and has high Na+ concentration in leaves. These three varieties were compared with the relatively more salt-sensitive cultivar Gladius. Through the use of root histochemical analysis, ion concentrations, as well as differential proteomics and targeted metabolomics, we provide an integrated view of the wheat root response to salinity. We show different metabolic re-arrangements in energy conversion, primary metabolic machinery and phenylpropanoid pathway leading to monolignol production in a genotype and genotype by treatment-dependent manner that alters the extent and localisation of root lignification which correlated with an improved capacity of wheat roots to cope better under salinity stress.

Variation in leaf dark respiration among C3 and C4 grasses is associated with use of different substrates.

Measurements of respiratory properties have often been made at a single time point either during daytime using dark-adapted leaves or during nighttime. The influence of the day-night cycle on respiratory metabolism has received less attention but is crucial to understand photosynthesis and photorespiration. Here, we examined how CO2- and O2-based rates of leaf dark respiration (Rdark) differed between midday (after 30-min dark adaptation) and midnight in 8 C3 and C4 grasses. We used these data to calculate the respiratory quotient (RQ; ratio of CO2 release to O2 uptake), and assessed relationships between Rdark and leaf metabolome. Rdark was higher at midday than midnight, especially in C4 species. The day-night difference in Rdark was more evident when expressed on a CO2 than O2 basis, with the RQ being higher at midday than midnight in all species, except in rice (Oryza sativa). Metabolomic analyses showed little correlation of Rdark or RQ with leaf carbohydrates (sucrose, glucose, fructose, or starch) but strong multivariate relationships with other metabolites. The results suggest that rates of Rdark and differences in RQ were determined by several concurrent CO2-producing and O2-consuming metabolic pathways, not only the tricarboxylic acid cycle (organic acids utilization) but also the pentose phosphate pathway, galactose metabolism, and secondary metabolism. As such, Rdark was time-, type- (C3/C4) and species-dependent, due to the use of different substrates.

Photosynthesis in newly developed leaves of heat-tolerant wheat acclimates to long-term nocturnal warming.

We examined photosynthetic traits of pre-existing and newly developed flag leaves of four wheat genotypes grown in controlled-environment experiments. In newly developed leaves, acclimation of the maximum rate of net CO2 assimilation (An) to warm nights (i.e. increased An) was associated with increased capacity of Rubisco carboxylation and photosynthetic electron transport, with Rubisco activation state probably contributing to increased Rubisco activity. Metabolite profiling linked acclimation of An to greater accumulation of monosaccharides and saturated fatty acids in leaves; these changes suggest roles for osmotic adjustment of leaf turgor pressure and maintenance of cell membrane integrity. By contrast, where An decreased under warm nights, the decline was related to lower stomatal conductance and rates of photosynthetic electron transport. Decreases in An occurred despite higher basal PSII thermal stability in all genotypes exposed to warm nights: Tcrit of 45-46.5 °C in non-acclimated versus 43.8-45 °C in acclimated leaves. Pre-existing leaves showed no change in An-temperature response curves, except for an elite heat-tolerant genotype. These findings illustrate the impact of night-time warming on the ability of wheat plants to photosynthesize during the day, thereby contributing to explain the impact of global warming on crop productivity.

Enhancing crop yields through improvements in the efficiency of photosynthesis and respiration.

The rate with which crop yields per hectare increase each year is plateauing at the same time that human population growth and other factors increase food demand. Increasing yield potential ( Y p ) of crops is vital to address these challenges. In this review, we explore a component of Y p that has yet to be optimised - that being improvements in the efficiency with which light energy is converted into biomass ( ε c ) via modifications to CO2 fixed per unit quantum of light (α), efficiency of respiratory ATP production ( ε prod ) and efficiency of ATP use ( ε use ). For α, targets include changes in photoprotective machinery, ribulose bisphosphate carboxylase/oxygenase kinetics and photorespiratory pathways. There is also potential for ε prod to be increased via targeted changes to the expression of the alternative oxidase and mitochondrial uncoupling pathways. Similarly, there are possibilities to improve ε use via changes to the ATP costs of phloem loading, nutrient uptake, futile cycles and/or protein/membrane turnover. Recently developed high-throughput measurements of respiration can serve as a proxy for the cumulative energy cost of these processes. There are thus exciting opportunities to use our growing knowledge of factors influencing the efficiency of photosynthesis and respiration to create a step-change in yield potential of globally important crops.

Wheat mitochondrial respiration shifts from the tricarboxylic acid cycle to the GABA shunt under salt stress.

Mitochondrial respiration and tricarboxylic acid (TCA) cycle activity are required during salt stress in plants to provide ATP and reductants for adaptive processes such as ion exclusion, compatible solute synthesis and reactive oxygen species (ROS) detoxification. However, there is a poor mechanistic understanding of how salinity affects mitochondrial metabolism, particularly respiratory substrate source. To determine the mechanism of respiratory changes under salt stress in wheat leaves, we conducted an integrated analysis of metabolite content, respiratory rate and targeted protein abundance measurements. Also, we investigated the direct effect of salt on mitochondrial enzyme activities. Salt-treated wheat leaves exhibit higher respiration rate and extensive metabolite changes. The activity of the TCA cycle enzymes pyruvate dehydrogenase complex and the 2-oxoglutarate dehydrogenase complex were shown to be directly salt-sensitive. Multiple lines of evidence showed that the γ-aminobutyric acid (GABA) shunt was activated under salt treatment. During salt exposure, key metabolic enzymes required for the cyclic operation of the TCA cycle are physiochemically inhibited by salt. This inhibition is overcome by increased GABA shunt activity, which provides an alternative carbon source for mitochondria that bypasses salt-sensitive enzymes, to facilitate the increased respiration of wheat leaves.

Diel- and temperature-driven variation of leaf dark respiration rates and metabolite levels in rice.

Leaf respiration in the dark (Rdark ) is often measured at a single time during the day, with hot-acclimation lowering Rdark at a common measuring temperature. However, it is unclear whether the diel cycle influences the extent of thermal acclimation of Rdark , or how temperature and time of day interact to influence respiratory metabolites. To examine these issues, we grew rice under 25°C : 20°C, 30°C : 25°C and 40°C : 35°C day : night cycles, measuring Rdark and changes in metabolites at five time points spanning a single 24-h period. Rdark differed among the treatments and with time of day. However, there was no significant interaction between time and growth temperature, indicating that the diel cycle does not alter thermal acclimation of Rdark . Amino acids were highly responsive to the diel cycle and growth temperature, and many were negatively correlated with carbohydrates and with organic acids of the tricarboxylic acid (TCA) cycle. Organic TCA intermediates were significantly altered by the diel cycle irrespective of growth temperature, which we attributed to light-dependent regulatory control of TCA enzyme activities. Collectively, our study shows that environmental disruption of the balance between respiratory substrate supply and demand is corrected for by shifts in TCA-dependent metabolites.

Energy costs of salt tolerance in crop plants.

Agriculture is expanding into regions that are affected by salinity. This review considers the energetic costs of salinity tolerance in crop plants and provides a framework for a quantitative assessment of costs. Different sources of energy, and modifications of root system architecture that would maximize water vs ion uptake are addressed. Energy requirements for transport of salt (NaCl) to leaf vacuoles for osmotic adjustment could be small if there are no substantial leaks back across plasma membrane and tonoplast in root and leaf. The coupling ratio of the H+ -ATPase also is a critical component. One proposed leak, that of Na+ influx across the plasma membrane through certain aquaporin channels, might be coupled to water flow, thus conserving energy. For the tonoplast, control of two types of cation channels is required for energy efficiency. Transporters controlling the Na+ and Cl- concentrations in mitochondria and chloroplasts are largely unknown and could be a major energy cost. The complexity of the system will require a sophisticated modelling approach to identify critical transporters, apoplastic barriers and root structures. This modelling approach will inform experimentation and allow a quantitative assessment of the energy costs of NaCl tolerance to guide breeding and engineering of molecular components.

Cold sensitivity of mitochondrial ATP synthase restricts oxidative phosphorylation in Arabidopsis thaliana.

The combined action of the electron transport chain (ETC) and ATP synthase is essential in determining energy efficiency in plants, and so is important for cellular biosynthesis, growth and development. Owing to the sessile nature of plants, mitochondria must operate over a wide temperature range in the environment, necessitating a broad temperature tolerance of their biochemical reactions. We investigated the temperature response of mitochondrial respiratory processes in isolated mitochondria and intact plants of Arabidopsis thaliana and considered the effect of instantaneous responses to temperature and acclimation responses to low temperatures. We show that at 4°C the plant mitochondrial ATP synthase is differentially inhibited compared with other elements of the respiratory pathway, leading to decreased ADP : oxygen ratios and a limitation to the rate of ATP synthesis. This effect persists in vivo and cannot be overcome by cold-temperature acclimation of plants. This mechanism adds a new element to the respiratory acclimation model and provides a direct means of temperature perception by plant mitochondria. This also provides an alternative explanation for non-phosphorylating ETC bypass mechanisms, like the alternative oxidase to maintain respiratory rates, albeit at lower ATP synthesis efficiency, in response to the sensitivity of ATP synthase to the prevailing temperature.

Resource: Mapping the Triticum aestivum proteome.

Yield and quality improvement of bread wheat (Triticum aestivum) is a focus in efforts to meet new demands from population growth and changing human diets. As the complexity of the wheat genome is unravelled, determining how it is used to build the protein machinery of wheat plants is a key next step in explaining detailed aspects of wheat growth and development. The specific functions of wheat organs during vegetative development and the role of metabolism, protein degradation and remobilisation in driving grain production are the foundations of crop performance and have recently become accessible through studies of the wheat proteome. We present a large scale, publicly accessible proteome mapping of wheat consisting of 24 organ and developmental samples. Tissue specific sub-proteomes and ubiquitously expressed markers of the wheat proteome are identified, alongside hierarchical assessment of protein functional classes, their presence in different tissues and correlations between the abundance of functional classes of proteins. Gene-specific identifications and protein family relationships are accounted for in the organisation of the data and 202 new protein-coding transcripts identified by proteogenomic mapping. The interactive database will serve as a vehicle to build, refine and deposit confirmed targeted proteomic assays for wheat proteins and protein families to assess function (www.wheatproteome.org).

Stepwise Evolution of a Buried Inhibitor Peptide over 45 My.

The de novo evolution of genes and the novel proteins they encode has stimulated much interest in the contribution such innovations make to the diversity of life. Most research on this de novo evolution focuses on transcripts, so studies on the biochemical steps that can enable completely new proteins to evolve and the time required to do so have been lacking. Sunflower Preproalbumin with SFTI-1 (PawS1) is an unusual albumin precursor because in addition to producing albumin it also yields a potent, bicyclic protease-inhibitor called SunFlower Trypsin Inhibitor-1 (SFTI-1). Here, we show how this inhibitor peptide evolved stepwise over tens of millions of years. To trace the origin of the inhibitor peptide SFTI-1, we assembled seed transcriptomes for 110 sunflower relatives whose evolution could be resolved by a chronogram, which allowed dates to be estimated for the various stages of molecular evolution. A genetic insertion event in an albumin precursor gene ∼45 Ma introduced two additional cleavage sites for protein maturation and conferred duality upon PawS1-Like genes such that they also encode a small buried macrocycle. Expansion of this region, including two Cys residues, enlarged the peptide ∼34 Ma and made the buried peptides bicyclic. Functional specialization into a protease inhibitor occurred ∼23 Ma. These findings document the evolution of a novel peptide inside a benign region of a pre-existing protein. We illustrate how a novel peptide can evolve without de novo gene evolution and, critically, without affecting the function of what becomes the protein host.

Editorial for Special Issue "Plant Mitochondria".

The primary function of mitochondria is respiration, where catabolism of substrates is coupled to adenosine triphosphate (ATP) synthesis via oxidative phosphorylation (OxPhos). [...].

We Are Not Alone: The iMOP Initiative and Its Roles in a Biology- and Disease-Driven Human Proteome Project.

Mapping of the human proteome has advanced significantly in recent years and will provide a knowledge base to accelerate our understanding of how proteins and protein networks can affect human health and disease. However, providing solutions to human health challenges will likely fail if insights are exclusively based on studies of human samples and human proteomes. In recent years, it has become evident that human health depends on an integrated understanding of the many species that make human life possible. These include the commensal microorganisms that are essential to human life, pathogens, and food species as well as the classic model organisms that enable studies of biological mechanisms. The Human Proteome Organization (HUPO) initiative on multiorganism proteomes (iMOP) works to support proteome research undertaken on nonhuman species that remain widely under-studied compared with the progress in human proteome research. This perspective argues the need for further research on multiple species that impact human life. We also present an update on recent progress in model organisms, microbiota, and food species, address the emerging problem of antibiotics resistance, and outline how iMOP activities could lead to a more inclusive approach for the human proteome project (HPP) to better support proteome research aimed at improving human health and furthering knowledge on human biology.

Connecting salt stress signalling pathways with salinity-induced changes in mitochondrial metabolic processes in C3 plants.

Salinity exerts a severe detrimental effect on crop yields globally. Growth of plants in saline soils results in physiological stress, which disrupts the essential biochemical processes of respiration, photosynthesis, and transpiration. Understanding the molecular responses of plants exposed to salinity stress can inform future strategies to reduce agricultural losses due to salinity; however, it is imperative that signalling and functional response processes are connected to tailor these strategies. Previous research has revealed the important role that plant mitochondria play in the salinity response of plants. Review of this literature shows that 2 biochemical processes required for respiratory function are affected under salinity stress: the tricarboxylic acid cycle and the transport of metabolites across the inner mitochondrial membrane. However, the mechanisms by which components of these processes are affected or react to salinity stress are still far from understood. Here, we examine recent findings on the signal transduction pathways that lead to adaptive responses of plants to salinity and discuss how they can be involved in and be affected by modulation of the machinery of energy metabolism with attention to the role of the tricarboxylic acid cycle enzymes and mitochondrial membrane transporters in this process.

Proteomic profiling of mature leaves from oil palm (Elaeis guineensis Jacq.).

Oil palm is one of the most productive oil bearing crops grown in Southeast Asia. Due to the dwindling availability of agricultural land and increasing demand for high yielding oil palm seedlings, clonal propagation is vital to the oil palm industry. Most commonly, leaf explants are used for in vitro micropropagation of oil palm and to optimize this process it is important to unravel the physiological and molecular mechanisms underlying somatic embryo production from leaves. In this study, a proteomic approach was used to determine protein abundance of mature oil palm leaves. To do this, leaf proteins were extracted using TCA/acetone precipitation protocol and separated by 2DE. A total of 191 protein spots were observed on the 2D gels and 67 of the most abundant protein spots that were consistently observed were selected for further analysis with 35 successfully identified using MALDI TOF/TOF MS. The majority of proteins were classified as being involved in photosynthesis, metabolism, cellular biogenesis, stress response, and transport. This study provides the first proteomic assessment of oil palm leaves in this important oil crop and demonstrates the successful identification of selected proteins spots using the Malaysian Palm Oil Board (MPOB) Elaeis guineensis EST and NCBI-protein databases. The MS data have been deposited in the ProteomeXchange Consortium database with the data set identifier PXD001307.

The Protein Corona of PEGylated PGMA-Based Nanoparticles is Preferentially Enriched with Specific Serum Proteins of Varied Biological Function.

The composition of the protein corona formed on poly(ethylene glycol)-functionalized (PEGylated) poly(glycidyl methacrylate) (PGMA) nanoparticles (NPs) was qualitatively and quantitatively compared to the protein corona on non-PEGylated PGMA NPs. Despite the reputation of PEGylated NPs for stealth functionality, we demonstrate the preferential enrichment of specific serum proteins of varied biological function in the protein corona on PEGylated NPs when compared to non-PEGylated NPs. Additionally, we suggest that the base material of polymeric NPs plays a role in the preferential enrichment of select serum proteins to the hard corona.

INTERMEDIATE CLEAVAGE PEPTIDASE55 Modifies Enzyme Amino Termini and Alters Protein Stability in Arabidopsis Mitochondria.

Precursor proteins containing mitochondrial peptide signals are cleaved after import by a mitochondrial processing peptidase. In yeast (Saccharomyces cerevisiae) and human (Homo sapiens), intermediate cleavage peptidase55 (ICP55) plays a role in stabilizing mitochondrial proteins by the removal of single amino acids from mitochondrial processing peptidase-processed proteins. We have investigated the role of a metallopeptidase (At1g09300) from Arabidopsis (Arabidopsis thaliana) that has sequence similarity to yeast ICP55. We identified this protein in mitochondria by mass spectrometry and have studied its function in a transfer DNA insertion line (icp55). Monitoring of amino-terminal peptides showed that Arabidopsis ICP55 was responsible for the removal of single amino acids, and its action explained the -3 arginine processing motif of a number of mitochondrial proteins. ICP55 also removed single amino acids from mitochondrial proteins known to be cleaved at nonconserved arginine sites, a subset of mitochondrial proteins specific to plants. Faster mitochondrial protein degradation rates not only for ICP55 cleaved protein but also for some non-ICP55 cleaved proteins were observed in Arabidopsis mitochondrial samples isolated from icp55 than from the wild type, indicating that a complicated protease degradation network has been affected. The lower protein stability of isolated mitochondria and the lack of processing of target proteins in icp55 were complemented by transformation with the full-length ICP55. Analysis of in vitro degradation rates and protein turnover rates in vivo of specific proteins indicated that serine hydroxymethyltransferase was affected in icp55. The maturation of serine hydroxymethyltransferase by ICP55 is unusual, as it involves breaking an amino-terminal diserine that is not known as an ICP55 substrate in other organisms and that is typically considered a sequence that stabilizes rather than destabilizes a protein.

Diverse cyclic seed peptides in the Mexican zinnia (Zinnia haageana).

A new family of small plant peptides was recently described and found to be widespread throughout the Millereae and Heliantheae tribes of the sunflower family Asteraceae. These peptides originate from the post-translational processing of unusual seed-storage albumin genes, and have been termed PawS-derived peptides (PDPs). The prototypic family member is a 14-residue cyclic peptide with potent trypsin inhibitory activity named SunFlower Trypsin Inhibitor (SFTI-1). In this study we present the features of three new PDPs discovered in the seeds of the sunflower species Zinnia haageana by a combination of de novo transcriptomics and liquid chromatography-mass spectrometry. Two-dimensional solution NMR spectroscopy was used to elucidate their structural characteristics. All three Z. haageana peptides have well-defined folds with a head-to-tail cyclized peptide backbone and a single disulfide bond. Although two possess an anti-parallel ß-sheet structure, like SFTI-1, the Z. haageana peptide PDP-21 has a more irregular backbone structure. Despite structural similarities with SFTI-1, PDP-20 was not able to inhibit trypsin, thus the functional roles of these peptides is yet to be discovered. Defining the structural features of the small cyclic peptides found in the sunflower family will be useful for guiding the exploitation of these peptides as scaffolds for grafting and protein engineering applications.

Selected reaction monitoring to determine protein abundance in Arabidopsis using the Arabidopsis proteotypic predictor.

In reverse genetic knockout (KO) studies that aim to assign function to specific genes, confirming the reduction in abundance of the encoded protein will often aid the link between genotype and phenotype. However, measuring specific protein abundance is particularly difficult in plant research, where only a limited number of antibodies are available. This problem is enhanced when studying gene families or different proteins derived from the same gene (isoforms), as many antibodies cross react with more than one protein. We show that utilizing selected reaction monitoring (SRM) mass spectrometry allows researchers to confirm protein abundance in mutant lines, even when discrimination between very similar proteins is needed. Selecting the best peptides for SRM analysis to ensure that protein- or gene-specific information can be obtained requires a series of steps, aids, and interpretation. To enable this process in Arabidopsis (Arabidopsis thaliana), we have built a Web-based tool, the Arabidopsis Proteotypic Predictor, to select candidate SRM transitions when no previous mass spectrometry evidence exists. We also provide an in-depth analysis of the theoretical Arabidopsis proteome and its use in selecting candidate SRM peptides to establish assays for use in determining protein abundance. To test the effectiveness of SRM mass spectrometry in determining protein abundance in mutant lines, we selected two enzymes with multiple isoforms, aconitase and malate dehydrogenase. Selected peptides were quantified to estimate the abundance of each of the two mitochondrial isoforms in wild-type, KO, double KO, and complemented plant lines. We show that SRM protein analysis is a sensitive and rapid approach to quantify protein abundance differences in Arabidopsis for specific and highly related enzyme isoforms.

Succinate dehydrogenase assembly factor 2 is needed for assembly and activity of mitochondrial complex II and for normal root elongation in Arabidopsis.

Mitochondria complex II (succinate dehydrogenase, SDH) plays a central role in respiratory metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. We report the identification of an SDH assembly factor by analysis of T-DNA insertions in At5g51040, a protein with unknown function that was identified by mass spectrometry analysis as a low abundance mitochondrial protein. This gene is co-expressed with a number of genes encoding mitochondrial proteins, including SDH1-1, and has low partial sequence similarity to human SDHAF2, a protein required for flavin-adenine dinucleotide (FAD) insertion into SDH. In contrast to observations of other SDH deficient lines in Arabidopsis, the sdhaf2 line did not affect photosynthetic rate or stomatal conductance, but instead showed inhibition of primary root elongation with early lateral root emergence, presumably due to the low SDH activity caused by the reduced abundance of SDHAF2. Both roots and leaves showed succinate accumulation but different responses in the abundance of other organic acids and amino acids assayed. Isolated mitochondria showed lowered SDH1 protein abundance, lowered maximal SDH activity and less protein-bound flavin-adenine dinucleotide (FAD) at the molecular mass of SDH1 in the gel separation. The short root phenotype and SDH function of sdhaf2 was fully complemented by transformation with SDHAF2. Application of the SDH inhibitor, malonate, phenocopied the sdhaf2 root architecture in WT. Whole root respiratory assays showed no difference between WT and sdhaf2, but micro-respirometry of the tips of roots clearly showed low oxygen consumption in sdhaf2 which could explain a metabolic deficit responsible for root tip growth.