EARLY NODULIN93 acts via cytochrome c oxidase to alter respiratory ATP production and root growth in plants.

EARLY NODULIN 93 (ENOD93) has been genetically associated with biological nitrogen fixation in legumes and nitrogen use efficiency in cereals, but its precise function is unknown. We show that hidden Markov models define ENOD93 as a homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 regulates cytochrome oxidase (CIV), influencing the generation of a mitochondrial proton motive force in yeast (Saccharomyces cerevisiae). Knockout of ENOD93 in Arabidopsis (Arabidopsis thaliana) causes a short root phenotype and early flowering. ENOD93 is associated with a protein complex the size of CIV in mitochondria, but neither CIV abundance nor its activity changed in ruptured organelles of enod93. However, a progressive loss of ADP-dependent respiration rate was observed in intact enod93 mitochondria, which could be recovered in complemented lines. Mitochondrial membrane potential was higher in enod93 in a CIV-dependent manner, but ATP synthesis and ADP depletion rates progressively decreased. The respiration rate of whole enod93 seedlings was elevated, and root ADP content was nearly double that in wild type without a change in ATP content. We propose that ENOD93 and HYPOXIA-INDUCED GENE DOMAIN 2 (HIGD2) are the functional equivalent of yeast RCF2 but have remained undiscovered in many eukaryotic lineages because they are encoded by two distinct genes.

The diversity of substrates for plant respiration and how to optimize their use.

Plant respiration is a foundational biological process with the potential to be optimized to improve crop yield. To understand and manipulate the outputs of respiration, the inputs of respiration-respiratory substrates-need to be probed in detail. Mitochondria house substrate catabolic pathways and respiratory machinery, so transport into and out of these organelles plays an important role in committing substrates to respiration. The large number of mitochondrial carriers and catabolic pathways that remain unidentified hinder this process and lead to confusion about the identity of direct and indirect respiratory substrates in plants. The sources and usage of respiratory substrates vary and are increasing found to be highly regulated based on cellular processes and environmental factors. This review covers the use of direct respiratory substrates following transport through mitochondrial carriers and catabolism under normal and stressed conditions. We suggest the introduction of enzymes not currently found in plant mitochondria to enable serine and acetate to be direct respiratory substrates in plants. We also compare respiratory substrates by assessing energetic yields, availability in cells, and their full or partial oxidation during cell catabolism. This information can assist in decisions to use synthetic biology approaches to alter the range of respiratory substrates in plants. As a result, respiration could be optimized by introducing, improving, or controlling specific mitochondrial transporters and mitochondrial catabolic pathways.

The mitochondrial pyruvate carrier (MPC) complex mediates one of three pyruvate-supplying pathways that sustain Arabidopsis respiratory metabolism.

Malate oxidation by plant mitochondria enables the generation of both oxaloacetate and pyruvate for tricarboxylic acid (TCA) cycle function, potentially eliminating the need for pyruvate transport into mitochondria in plants. Here, we show that the absence of the mitochondrial pyruvate carrier 1 (MPC1) causes the co-commitment loss of its putative orthologs, MPC3/MPC4, and eliminates pyruvate transport into Arabidopsis thaliana mitochondria, proving it is essential for MPC complex function. While the loss of either MPC or mitochondrial pyruvate-generating NAD-malic enzyme (NAD-ME) did not cause vegetative phenotypes, the lack of both reduced plant growth and caused an increase in cellular pyruvate levels, indicating a block in respiratory metabolism, and elevated the levels of branched-chain amino acids at night, a sign of alterative substrate provision for respiration. 13C-pyruvate feeding of leaves lacking MPC showed metabolic homeostasis was largely maintained except for alanine and glutamate, indicating that transamination contributes to the restoration of the metabolic network to an operating equilibrium by delivering pyruvate independently of MPC into the matrix. Inhibition of alanine aminotransferases when MPC1 is absent resulted in extremely retarded phenotypes in Arabidopsis, suggesting all pyruvate-supplying enzymes work synergistically to support the TCA cycle for sustained plant growth.

Metabolic evidence for distinct pyruvate pools inside plant mitochondria.

The majority of the pyruvate inside plant mitochondria is either transported into the matrix from the cytosol via the mitochondria pyruvate carrier (MPC) or synthesized in the matrix by alanine aminotransferase (AlaAT) or NAD-malic enzyme (NAD-ME). Pyruvate from these origins could mix into a single pool in the matrix and contribute indistinguishably to respiration via the pyruvate dehydrogenase complex (PDC), or these molecules could maintain a degree of independence in metabolic regulation. Here we demonstrate that feeding isolated mitochondria with uniformly labelled 13C-pyruvate and unlabelled malate enables the assessment of pyruvate contribution from different sources to intermediate production in the tricarboxylic acid cycle. Imported pyruvate was the preferred source for citrate production even when the synthesis of NAD-ME-derived pyruvate was optimized. Genetic or pharmacological elimination of MPC activity removed this preference and allowed an equivalent amount of citrate to be generated from the pyruvate produced by NAD-ME. Increasing the mitochondrial pyruvate pool size by exogenous addition affected only metabolites from pyruvate transported by MPC, whereas depleting the pyruvate pool size by transamination to alanine affected only metabolic products derived from NAD-ME. PDC was more membrane-associated than AlaAT and NAD-ME, suggesting that the physical organization of metabolic machinery may influence metabolic rates. Together, these data reveal that the respiratory substrate supply in plants involves distinct pyruvate pools inside the matrix that can be flexibly mixed on the basis of the rate of pyruvate transport from the cytosol. These pools are independently regulated and contribute differentially to organic acid export from plant mitochondria.

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics.

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100-1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.