Alternative oxidase 1a and 1d enable metabolic flexibility during Ala catabolism in Arabidopsis.

Ala is a central metabolite in leaf cells whose abundance is related to pyruvate (Pyr) metabolism and nocturnal respiration rates. Exposure of Arabidopsis (Arabidopsis thaliana) leaf disks to certain exogenous amino acids including Ala led to substantial increases in nighttime respiration rates as well as increases in alternative oxidase (AOX) 1d transcript and protein levels. During Ala treatment, AOX1d accumulation, but not AOX1a accumulation, was dependent upon the catabolism of Ala. Complete loss of AOX expression in aox1a aox1d leaf disks did not significantly affect oxygen consumption rates (OCR) under Ala treatment, indicating that AOX capacity per se was not essential for respiratory stimulation by Ala. Rather, Ala treatments caused induction of select antioxidant mechanisms in leaf disks, including a large increase of the ascorbate pool, which was substantially more oxidized in aox1a aox1d leaf disks. Furthermore, we observed differences in the accumulation of a sequence of TCA cycle intermediates from Pyr to 2-oxoglutarate (2-OG) in wild type (WT) upon Ala treatment that did not occur in aox1a aox1d leaf disks. The results indicate that AOX induction during enhanced Ala catabolism in leaves mediates mitochondrial redox status, allowing greater metabolic flexibility in mitochondrial organic acid metabolism.

High-Throughput Oxygen Consumption Measurements in Leaf Tissue Using Oxygen Sensitive Fluorophores.

Respiratory rate measurements are crucial assays to understand mitochondrial biochemistry as well as metabolic regulation within tissues. Several technologies currently exist that can measure plant respiratory oxygen consumption or carbon dioxide evolution rates over short durations by either isolated mitochondria or plant tissues. Here we describe recently developed alternative methods for measuring tissue oxygen consumption rates (OCRs) using systems reliant on oxygen sensitive fluorophores. The methods described have distinct experimental advantages: they can allow high-throughput and long-duration measurements; and they are particularly suited to investigating the metabolic regulation of respiration by comparing OCRs among treatments or genotypes.

Alternative oxidase (AOX) 1a and 1d limit proline-induced oxidative stress and aid salinity recovery in Arabidopsis.

Proline (Pro) catabolism and reactive oxygen species production have been linked in mammals and Caenorhabditis elegans, while increases in leaf respiration rate follow Pro exposure in plants. Here, we investigated how alternative oxidases (AOXs) of the mitochondrial electron transport chain accommodate the large, atypical flux resulting from Pro catabolism and limit oxidative stress during Pro breakdown in mature Arabidopsis (Arabidopsis thaliana) leaves. Following Pro treatment, AOX1a and AOX1d accumulate at transcript and protein levels, with AOX1d approaching the level of the typically dominant AOX1a isoform. We therefore sought to determine the function of both AOX isoforms under Pro respiring conditions. Oxygen consumption rate measurements in aox1a and aox1d leaves suggested these AOXs can functionally compensate for each other to establish enhanced AOX catalytic capacity in response to Pro. Generation of aox1a.aox1d lines showed complete loss of AOX proteins and activity upon Pro treatment, yet full respiratory induction in response to Pro remained possible via the cytochrome pathway. However, aox1a.aox1d leaves displayed symptoms of elevated oxidative stress and suffered increased oxidative damage during Pro metabolism compared to the wild-type (WT) or the single mutants. During recovery from salt stress, when relatively high rates of Pro catabolism occur naturally, photosynthetic rates in aox1a.aox1d recovered slower than in the WT or the single aox lines, showing that both AOX1a and AOX1d are beneficial for cellular metabolism during Pro drawdown following osmotic stress. This work provides physiological evidence of a beneficial role for AOX1a but also the less studied AOX1d isoform in allowing safe catabolism of alternative respiratory substrates like Pro.

Metabolite Regulatory Interactions Control Plant Respiratory Metabolism via Target of Rapamycin (TOR) Kinase Activation.

Respiration rate measurements provide an important readout of energy expenditure and mitochondrial activity in plant cells during the night. As plants inhabit a changing environment, regulatory mechanisms must ensure that respiratory metabolism rapidly and effectively adjusts to the metabolic and environmental conditions of the cell. Using a high-throughput approach, we have directly identified specific metabolites that exert transcriptional, translational, and posttranslational control over the nighttime O2 consumption rate (RN) in mature leaves of Arabidopsis (Arabidopsis thaliana). Multi-hour RN measurements following leaf disc exposure to a wide array of primary carbon metabolites (carbohydrates, amino acids, and organic acids) identified phosphoenolpyruvate (PEP), Pro, and Ala as the most potent stimulators of plant leaf RN Using metabolite combinations, we discovered metabolite-metabolite regulatory interactions controlling RN Many amino acids, as well as Glc analogs, were found to potently inhibit the RN stimulation by Pro and Ala but not PEP. The inhibitory effects of amino acids on Pro- and Ala-stimulated RN were mitigated by inhibition of the Target of Rapamycin (TOR) kinase signaling pathway. Supporting the involvement of TOR, these inhibitory amino acids were also shown to be activators of TOR kinase. This work provides direct evidence that the TOR signaling pathway in plants responds to amino acid levels by eliciting regulatory effects on respiratory energy metabolism at night, uniting a hallmark mechanism of TOR regulation across eukaryotes.

Blau Syndrome Associated with Nucleotide-binding Oligomerization Domain Containing 2 Mutation in a Baby from Malaysia.

Blau syndrome (BS) is a very rare autosomal dominant juvenile inflammatory disorder caused by mutation in nucleotide-binding oligomerization domain containing 2 (NOD2). Usually, dermatitis is the first symptom that appears in the 1st year of life. About 220 BS cases with confirmed NOD2 mutation have been reported. However, the rarity and lack of awareness of the disease, especially in the regions where genetic tests are very limited, often result in late diagnosis and misdiagnosis. Here, we report a de novo BS case from Malaysia, which may be the first report from southeast Asia. PCR and DNA sequencing of peripheral blood mononuclear cells were performed to screen the entire coding region of NOD2 gene. A heterozygous c.1000C>T transition in exon 4, p. R334W, of the NOD2 gene was identified in the patient. This report further reaffirms the ubiquitousness of the disease and recurrency of p. R334W mutation.