The phosphorylated pathway of serine biosynthesis links plant growth with nitrogen metabolism.

Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot be compensated by photorespiratory serine biosynthesis. Using isotope labeling, we show that PPSB-deficiency impairs the synthesis of proteins and purine nucleotides in plants. Furthermore, deficiency in PPSB-mediated serine biosynthesis leads to a strong accumulation of metabolites related to nitrogen metabolism. This result corroborates 15N-isotope labeling in which we observed an increased enrichment in labeled amino acids in PPSB-deficient plants. Expression studies indicate that elevated ammonium uptake and higher glutamine synthetase/glutamine oxoglutarate aminotransferase (GS/GOGAT) activity causes this phenotype. Metabolic analyses further show that elevated nitrogen assimilation and reduced amino acid turnover into proteins and nucleotides are the most likely driving forces for changes in respiratory metabolism and amino acid catabolism in PPSB-deficient plants. Accordingly, we conclude that even though photorespiration generates high amounts of serine in plants, PPSB-derived serine is more important for plant growth and its deficiency triggers the induction of nitrogen assimilation, most likely as an amino acid starvation response.

Pinpointing secondary metabolites that shape the composition and function of the plant microbiome.

One of the major questions in contemporary plant science involves determining the functional mechanisms that plants use to shape their microbiome. Plants produce a plethora of chemically diverse secondary metabolites, many of which exert bioactive effects on microorganisms. Several recent publications have unequivocally shown that plant secondary metabolites affect microbiome composition and function. These studies have pinpointed that the microbiome can be influenced by a diverse set of molecules, including: coumarins, glucosinolates, benzoxazinoids, camalexin, and triterpenes. In this review, we summarize the role of secondary metabolites in shaping the plant microbiome, highlighting recent literature. A body of knowledge is now emerging that links specific plant metabolites with distinct microbial responses, mediated via defined biochemical mechanisms. There is significant potential to boost agricultural sustainability via the targeted enhancement of beneficial microbial traits, and here we argue that the newly discovered links between root chemistry and microbiome composition could provide a new set of tools for rationally manipulating the plant microbiome.

Nitrogen Substrate Utilization in Three Rhizosphere Bacterial Strains Investigated Using Proteomics.

Nitrogen metabolism in the rhizosphere microbiome plays an important role in mediating plant nutrition, particularly under low inputs of mineral fertilizers. However, there is relatively little mechanistic information about which genes and metabolic pathways are induced by rhizosphere bacterial strains to utilize diverse nitrogen substrates. Here we investigate nitrogen substrate utilization in three taxonomically diverse bacterial strains previously isolated from Arabidopsis roots. The three strains represent taxa that are consistently detected as core members of the plant microbiome: Pseudomonas, Streptomyces, and Rhizobium. We use phenotype microarrays to determine the nitrogen substrate preferences of these strains, and compare the experimental results vs. computational simulations of genome-scale metabolic network models obtained with EnsembleFBA. Results show that all three strains exhibit generalistic nitrogen substrate preferences, with substrate utilization being well predicted by EnsembleFBA. Using label-free quantitative proteomics, we document hundreds of proteins in each strain that exhibit differential abundance values following cultivation on five different nitrogen sources: ammonium, glutamate, lysine, serine, and urea. The proteomic response to these nitrogen sources was strongly strain-dependent, with lysine nutrition eliciting widespread protein-level changes in Pseudomonas sp. Root9, whereas Rhizobium sp. Root491 showed relatively stable proteome composition across different nitrogen sources. Our results give new protein-level information about the specific transporters and enzymes induced by diverse rhizosphere bacterial strains to utilize organic nitrogen substrates.

Recent advances in the role of plant metabolites in shaping the root microbiome.

The last decade brought great progress in describing the repertoire of microbes associated with plants and identifying principles of their interactions. Metabolites exuded by plant roots have been considered candidates for the mechanisms by which plants shape their root microbiome. Here, we review the evidence for several plant metabolites affecting plant interaction with microbes belowground. We also discuss the development of new approaches to study the mechanisms of such interaction that will help to elucidate the metabolic networks in the rhizosphere.

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.

Root-specific camalexin biosynthesis controls the plant growth-promoting effects of multiple bacterial strains.

Plants in their natural ecosystems interact with numerous microorganisms, but how they influence their microbiota is still elusive. We observed that sulfatase activity in soil, which can be used as a measure of rhizosphere microbial activity, is differently affected by Arabidopsis accessions. Following a genome-wide association analysis of the variation in sulfatase activity we identified a candidate gene encoding an uncharacterized cytochrome P450, CYP71A27 Loss of this gene resulted in 2 different and independent microbiota-specific phenotypes: A lower sulfatase activity in the rhizosphere and a loss of plant growth-promoting effect by Pseudomonas sp. CH267. On the other hand, tolerance to leaf pathogens was not affected, which agreed with prevalent expression of CYP71A27 in the root vasculature. The phenotypes of cyp71A27 mutant were similar to those of cyp71A12 and cyp71A13, known mutants in synthesis of camalexin, a sulfur-containing indolic defense compound. Indeed, the cyp71A27 mutant accumulated less camalexin in the roots upon elicitation with silver nitrate or flagellin. Importantly, addition of camalexin complemented both the sulfatase activity and the loss of plant growth promotion by Pseudomonas sp. CH267. Two alleles of CYP71A27 were identified among Arabidopsis accessions, differing by a substitution of Glu373 by Gln, which correlated with the ability to induce camalexin synthesis and to gain fresh weight in response to Pseudomonas sp. CH267. Thus, CYP71A27 is an additional component in the camalexin synthesis pathway, contributing specifically to the control of plant microbe interactions in the root.

Metabolic niches in the rhizosphere microbiome: new tools and approaches to analyse metabolic mechanisms of plant-microbe nutrient exchange.

Plants nourish rhizospheric microbes via provision of carbon substrates, and the composition of the microbiome is strongly influenced by metabolic phenomena such as niche differentiation, competitive exclusion, and cross-feeding. Despite intensive investigations of the taxonomic structure in root microbiomes, there is relatively little biochemical knowledge of the metabolic niches occupied by microbial strains in the rhizosphere. Here, we review new tools and approaches that are boosting our knowledge of the metabolic mechanisms that shape the composition of the root microbiome. New studies have elucidated biochemical pathways that mediate root colonisation and pathogen suppression, and synthetic communities are emerging as a powerful tool to understand microbe-microbe interactions. Knowledge of root exudate composition is being advanced by new metabolomics methodologies, which have highlighted that specific exudate components can inhibit pathogen growth, and that certain metabolites can recruit mutualistic strains according to substrate uptake preferences. Microbial genomics is rapidly advancing, with large collections of isolated rhizosphere strains and mutant libraries giving new insights into the metabolic mechanisms of root colonisation. Exometabolomics is emerging as a powerful methodology for directly observing microbial uptake of root metabolites, and also for profiling microbial cross-feeding. Integrative studies using these resources should enable rapid advances, particularly when applied to standardised experimental set-ups and model synthetic communities.

Exometabolomic Profiling of Bacterial Strains as Cultivated Using Arabidopsis Root Extract as the Sole Carbon Source.

The ability of microorganisms to use root-derived metabolites as growth substrates is a key trait for success in the rhizospheric niche. However, few studies describe which specific metabolites are consumed or to what degree microbial strains differ in their substrate consumption patterns. Here, we present a liquid chromatography-mass spectrometry (MS) exometabolomic study of three bacterial strains cultivated using either glucose or Arabidopsis thaliana root extract as the sole carbon source. Two of the strains were previously isolated from field-grown Arabidopsis roots, the other is Escherichia coli, included as a comparison. When cultivated on root extract, a set of 62 MS features were commonly taken up by all three strains, with m/z values matching components of central metabolism (including amino acids and purine or pyrimidine derivatives). Escherichia coli took up very few MS features outside this commonly consumed set, whereas the root-inhabiting strains took up a much larger number of MS features, many with m/z values matching plant-specific metabolites. These measurements define the metabolic niche that each strain potentially occupies in the rhizosphere. Furthermore, we document many MS features released by these strains that could play roles in cross-feeding, antibiosis, or signaling. We present our methodological approach as a foundation for future studies of rhizosphere exometabolomics.

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.

Opportunities for wheat proteomics to discover the biomarkers for respiration-dependent biomass production, stress tolerance and cytoplasmic male sterility.

Wheat has served as a key species for characterising fundamental aspects of mitochondrial biochemistry and respiratory physiology. Respiratory traits are linked to many important agronomic properties, so identifying the proteins that carry out these molecular processes would define a new set of targets for wheat breeding. To date, systematic proteomic investigations into wheat mitochondria have lagged behind other species, due to the size and complexity of the wheat genome. However this situation is changing with new sequence data increasing the power of proteomics applied to wheat. In this review, we argue that the impact of wheat mitochondrial proteomics on wheat respiratory traits can be improved through integrating data from current proteomics approaches with knowledge from the wheat respiration literature. We present a historical overview of biochemical and physiological studies of mitochondrial respiration in wheat, highlighting respiratory properties linked to agronomically important traits, such as biomass production, stress tolerance and cytoplasmic male sterility. Also, we summarise the current status of the wheat mitochondrial proteome and present a predicted set of 2000 probable mitochondrial proteins from Triticum urartu. Finally, we present a set of strategies outlining how future proteomics experiments can be applied to wheat mitochondria, by targeting studies to build on pre-existing knowledge.

Finding the Subcellular Location of Barley, Wheat, Rice and Maize Proteins: The Compendium of Crop Proteins with Annotated Locations (cropPAL).

Barley, wheat, rice and maize provide the bulk of human nutrition and have extensive industrial use as agricultural products. The genomes of these crops each contains >40,000 genes encoding proteins; however, the major genome databases for these species lack annotation information of protein subcellular location for >80% of these gene products. We address this gap, by constructing the compendium of crop protein subcellular locations called crop Proteins with Annotated Locations (cropPAL). Subcellular location is most commonly determined by fluorescent protein tagging of live cells or mass spectrometry detection in subcellular purifications, but can also be predicted from amino acid sequence or protein expression patterns. The cropPAL database collates 556 published studies, from >300 research institutes in >30 countries that have been previously published, as well as compiling eight pre-computed subcellular predictions for all Hordeum vulgare, Triticum aestivum, Oryza sativa and Zea mays protein sequences. The data collection including metadata for proteins and published studies can be accessed through a search portal http://crop-PAL.org. The subcellular localization information housed in cropPAL helps to depict plant cells as compartmentalized protein networks that can be investigated for improving crop yield and quality, and developing new biotechnological solutions to agricultural challenges.

Assessment of respiration in isolated plant mitochondria using Clark-type electrodes.

Mitochondrial respiration involves two key gas exchanges, the consumption of oxygen and the release of carbon dioxide. The ability to measure the consumption of oxygen via Clark-type electrodes has been one of the key techniques for advancing our knowledge of mitochondrial function in whole organisms, tissue samples, cells, and isolated subcellular fractions. In plants, oxygen electrode analyses provided the first evidence for some of the unique respiratory properties of plant mitochondria. This chapter briefs the principles of respiration and oxidative phosphorylation, how oxygen consumption measurements can be used to assess the quality of isolated mitochondrial preparations, and how these measurements can answer important questions in plant biochemistry and physiology. Finally, it presents instructions on assembling the oxygen electrode apparatus and how to conduct various assays.

Exposure of barley plants to low Pi leads to rapid changes in root respiration that correlate with specific alterations in amino acid substrates.

The majority of inorganic phosphate (Pi ) stress studies in plants have focused on the response after growth has been retarded. Evidence from transcript analysis, however, shows that a Pi -stress specific response is initiated within minutes of transfer to low Pi and in crop plants precedes the expression of Pi transporters and depletion of vacuolar Pi reserves by days. In order to investigate the physiological and metabolic events during early exposure to low Pi in grain crops, we monitored the response of whole barley plants during the first hours following Pi withdrawal. Lowering the concentration of Pi led to rapid changes in root respiration and leaf gas exchange throughout the early phase of the light course. Combining amino and organic acid analysis with (15) N labelling we show a root-specific effect on nitrogen metabolism linked to specific substrates of respiration as soon as 1 h following Pi withdrawal; this explains the respiratory responses observed and was confirmed by stimulation of respiration by exogenous addition of these respiratory substrates to roots. The rapid adjustment of substrates for respiration in roots during short-term Pi -stress is highlighted and this could help guide roots towards Pi -rich soil patches without compromising biomass accumulation of the plant.

Proteins with high turnover rate in barley leaves estimated by proteome analysis combined with in planta isotope labeling.

Protein turnover is a key component in cellular homeostasis; however, there is little quantitative information on degradation kinetics for individual plant proteins. We have used (15)N labeling of barley (Hordeum vulgare) plants and gas chromatography-mass spectrometry analysis of free amino acids and liquid chromatography-mass spectrometry analysis of proteins to track the enrichment of (15)N into the amino acid pools in barley leaves and then into tryptic peptides derived from newly synthesized proteins. Using information on the rate of growth of barley leaves combined with the rate of degradation of (14)N-labeled proteins, we calculate the turnover rates of 508 different proteins in barley and show that they vary by more than 100-fold. There was approximately a 9-h lag from label application until (15)N incorporation could be reliably quantified in extracted peptides. Using this information and assuming constant translation rates for proteins during the time course, we were able to quantify degradation rates for several proteins that exhibit half-lives on the order of hours. Our workflow, involving a stringent series of mass spectrometry filtering steps, demonstrates that (15)N labeling can be used for large-scale liquid chromatography-mass spectrometry studies of protein turnover in plants. We identify a series of abundant proteins in photosynthesis, photorespiration, and specific subunits of chlorophyll biosynthesis that turn over significantly more rapidly than the average protein involved in these processes. We also highlight a series of proteins that turn over as rapidly as the well-known D1 subunit of photosystem II. While these proteins need further verification for rapid degradation in vivo, they cluster in chlorophyll and thiamine biosynthesis.

Plant mitochondrial proteomics.

Mitochondria are responsible for a number of major biochemical processes in plant cells including oxidative phosphorylation and photorespiration. Traditionally their primary role has been viewed as the oxidation of organic acids via the tricarboxylic acid cycle and the synthesis of ATP coupled to the transfer of electrons to O2. More recently its role in the synthesis of many metabolites such as amino acids, lipids, and vitamins has been revealed. They also contain large number of transporters including members of the mitochondrial carrier substrate family (MCSF) that allow the exchange of metabolites with the cytosol. Mitochondria also contain their own genome and actively transcribe and translate a set of proteins that are coordinated with proteins encoded by the nuclear genome to produce large multisubunit enzymes. To reveal the full diversity of metabolism carried out by mitochondria significant efforts have sought to uncover the protein profile of mitochondria from both crops and model plants. Successful proteomic analysis depends on the preparation of high-quality isolated mitochondria, coupled to high-resolution proteomic techniques for identification, quantitation, and assessment of the degree of contamination by other organelles and cellular compartments. Here we outline a mitochondrial isolation protocol that can be applied to a range of plant tissues, and detail methods of assessing the quality and purity of the resultant sample, including calculations of respiratory control ratio, marker enzyme assays, differential in-gel electrophoresis, and quantitative gel-free mass spectrometry.

Investigating the role of respiration in plant salinity tolerance by analyzing mitochondrial proteomes from wheat and a salinity-tolerant Amphiploid (wheat × Lophopyrum elongatum).

The effect of salinity on mitochondrial properties was investigated by comparing the reference wheat variety Chinese Spring (CS) to a salt-tolerant amphiploid (AMP). The octoploid AMP genotype was previously generated by combining hexaploid bread wheat (CS) with the diploid wild wheatgrass adapted to salt marshes, Lophopyrum elongatum. Here we used a combination of physiological, biochemical, and proteomic analyses to explore the mitochondrial and respiratory response to salinity in these two genotypes. The AMP showed greater growth tolerance to salinity treatments and altered respiration rate in both roots and shoots. A proteomic workflow of 2D-DIGE and MALDI TOF/TOF mass spectrometry was used to compare the protein composition of isolated mitochondrial samples from roots and shoots of both genotypes, following control or salt treatment. A large set of mitochondrial proteins were identified as responsive to salinity in both genotypes, notably enzymes involved in detoxification of reactive oxygen species. Genotypic differences in mitochondrial composition were also identified, with AMP exhibiting a higher abundance of manganese superoxide dismutase, serine hydroxymethyltransferase, aconitase, malate dehydrogenase, and ß-cyanoalanine synthase compared to CS. We present peptide fragmentation spectra derived from some of these AMP-specific protein spots, which could serve as biomarkers to track superior protein variants.

Degradation rate of mitochondrial proteins in Arabidopsis thaliana cells.

The turnover of the proteomes of organelles in plant cells are known to be governed by both whole cell and organelle-specific processes. However, the rate and specificity of this protein turnover has not been explored in depth to understand how it affects different organellar processes. Here we have used progressive ¹5N labeling of Arabidopsis cells, and focused on the turnover rate of proteins in mitochondria. We provide estimates of degradation rate (K(d)) for 224 mitochondrial proteins, showing a range of over 50-fold in K(d). Protein complexes, most notably the respiratory chain complexes, had K(d) values that were generally coordinated and we have interpreted these measurements to outline how protein K(d) differs within protein complexes and between functional categories. The fastest turnover rates were reported for DNA/RNA metabolism enzymes, chaperones, and proteases.

Application of selected reaction monitoring mass spectrometry to field-grown crop plants to allow dissection of the molecular mechanisms of abiotic stress tolerance.

One major constraint upon the application of molecular crop breeding approaches is the small number of genes linked to agronomically desirable traits through defined biochemical mechanisms. Proteomic investigations of crop plants under abiotic stress treatments have identified many proteins that differ in control versus stress comparisons, however, this broad profiling of cell physiology is poorly suited to ranking the effects and identifying the specific proteins that are causative in agronomically relevant traits. Here we will reason that insights into a protein's function, its biochemical process and links to stress tolerance are more likely to arise through approaches that evaluate these differential abundances of proteins and include varietal comparisons, precise discrimination of protein isoforms, enrichment of functionally related proteins, and integration of proteomic datasets with physiological measurements of both lab and field-grown plants. We will briefly explain how applying the emerging proteomic technology of multiplexed selective reaction monitoring mass spectrometry with its accuracy and throughput can facilitate and enhance these approaches and provide a clear means to rank the growing cohort of stress responsive proteins. We will also highlight the benefit of integrating proteomic analyses with cultivar-specific genetic databases and physiological assessments of cultivar performance in relevant field environments for revealing deeper insights into molecular crop improvement.

Differential induction of mitochondrial machinery by light intensity correlates with changes in respiratory metabolism and photorespiration in rice leaves.

The light responsiveness of mitochondrial function was investigated through changes in mitochondrial composition and metabolism in rice (Oryza sativa) shoots. The mitochondrial proteome and metabolite abundances under low light, (LL, 100 µmol m(-2) s(-1) ), and high light (HL, 700 µmol m(-2) s(-1) ) were measured along with information on shoot photosynthetic, respiratory and photorespiratory activity. Specific steps in mitochondrial tricarboxylic acid (TCA) cycle metabolism were decreased under HL, correlating with lower respiration rate under HL. The abundance of mitochondrial enzymes in branch chain metabolism was reduced under HL/LL, and correlated with a decrease in the abundance of a range of amino acids in the HL/LL. Mitochondrial nucleoside diphosphate kinase was increased under LL/HL treatments. Significant accumulation of glycine decarboxylase P, T subunits and serine hydroxymethyltransferase occurred in response to light. The abundance of the glycine decarboxylase (GDC) H subunit proteins was not changed by HL/LL treatments, and the abundance of GDC L subunit protein was halved under HL, indicating a change in the stoichiometry of GDC subunits, while photorespiration was fourfold higher in LL- than in HL-treated plants. Insights into these light-dependent phenomena and their importance for understanding the initiation of photorespiration in rice and adaptation of mitochondria to function in photosynthetic cells are discussed.

Mitochondrial composition, function and stress response in plants.

The primary function of mitochondria is respiration, where catabolism of substrates is coupled to ATP synthesis via oxidative phosphorylation. In plants, mitochondrial composition is relatively complex and flexible and has specific pathways to support photosynthetic processes in illuminated leaves. This review begins with outlining current models of mitochondrial composition in plant cells, with an emphasis upon the assembly of the complexes of the classical electron transport chain (ETC). Next, we focus upon the comparative analysis of mitochondrial function from different tissue types. A prominent theme in the plant mitochondrial literature involves linking mitochondrial composition to environmental stress responses, and this review then gives a detailed outline of how oxidative stress impacts upon the plant mitochondrial proteome with particular attention to the role of transition metals. This is followed by an analysis of the signaling capacity of mitochondrial reactive oxygen species, which studies the transcriptional changes of stress responsive genes as a framework to define specific signals emanating from the mitochondrion. Finally, specific mitochondrial roles during exposure to harsh environments are outlined, with attention paid to mitochondrial delivery of energy and intermediates, mitochondrial support for photosynthesis, and mitochondrial processes operating within root cells that mediate tolerance to anoxia and unfavorable soil chemistries. [Formula: see text] [ A. Harvey Millar (Corresponding author)].