Anaerobic Response Research Articles

Acquisition of hypoxia inducibility by oxygen sensing N-terminal cysteine oxidase in spermatophytes.

N-terminal cysteine oxidases (NCOs) use molecular oxygen to oxidise the amino-terminal cysteine of specific proteins, thereby initiating the proteolytic N-degron pathway. To expand the characterisation of the plant family of NCOs (plant cysteine oxidases [PCOs]), we performed a phylogenetic analysis across different taxa in terms of sequence similarity and transcriptional regulation. Based on this survey, we propose a distinction of PCOs into two main groups. A-type PCOs are conserved across all plant species and are generally unaffected at the messenger RNA level by oxygen availability. Instead, B-type PCOs appeared in spermatophytes to acquire transcriptional regulation in response to hypoxia. The inactivation of two A-type PCOs in Arabidopsis thaliana, PCO4 and PCO5, is sufficient to activate the anaerobic response in young seedlings, whereas the additional removal of B-type PCOs leads to a stronger induction of anaerobic genes and impairs plant growth and development. Our results show that both PCO types are required to regulate the anaerobic response in angiosperms. Therefore, while it is possible to distinguish two clades within the PCO family, we conclude that they all contribute to restrain the anaerobic transcriptional programme in normoxic conditions and together generate a molecular switch to toggle the hypoxic response.

28 Blood Lactate Response of Yearling Horses on a Progressive Workload

Abstract Blood lactate measurements following exercise provide valuable insight into the metabolic response to different workloads, but minimal research has been published involving yearling horses. Therefore, the objective of the current study was to determine blood lactate response to increasing exercise intensity in yearling horses, hypothesizing that blood lactate levels would peak immediately following exercise, and gradually decrease over a 3 h post-exercise period. To test this, 32 Quarter horses of similar background (480-520 d of age; 16 geldings and 16 fillies) were randomly assigned to one of four groups (n = 8/group) and exercised using an 8-panel free-stall walker. Exercise bouts were 30 min each, divided equally in clockwise and counterclockwise directions. Test 1 consisted of walking at 1.75m/s, and test 2 consisted of horses alternating between the walk and trot at 1.75 and 4.0m/s, respectively. Blood samples were obtained at the onset (PRE) and immediately (H0), half (H0.5), one (H1), and three (H3) h post-exercise. Mean blood lactate was determined using two portable lactate analyzers previously validated for use in horses. Data were analyzed using PROC MIXED in SAS with PRE values used as a covariate. There was a group × test × time interaction (P < 0.01), where group 3 reached peak lactate concentration at H0 during test 2 (1.37 ± 0.08 mmol/L), while all other groups increased blood lactate concentrations over time to H3 post. Mean lactate post-exercise indicates that horses were able to clear lactate without a sufficient increase in systemic distribution. Values remained below the anaerobic threshold, similar to mature horses, which suggests that sub-maximal exercise in young horses does not elicit an anaerobic response and lactate accumulation.

Genome-wide identification of alcohol dehydrogenase (ADH) gene family under waterlogging stress in wheat (Triticum aestivum).

BackgroundAlcohol dehydrogenase (ADH) plays an important role in plant survival under anaerobic conditions. Although some research about ADH in many plants have been carried out, the bioinformatics analysis of the ADH gene family from Triticum aestivum and their response to abiotic stress is unclear.MethodsA total of 22 ADH genes were identified from the wheat genome, and these genes could be divided into two subfamilies (subfamily I and subfamily II). All TaADH genes belonged to the Medium-chain ADH subfamily. Sequence alignment analysis showed that all TaADH proteins contained a conservative GroES-like domain and Zinc-binding domain. A total of 64 duplicated gene pairs were found, and the Ka/Ks value of these gene pairs was less than 1, which indicated that these genes were relatively conservative and did not change greatly in the process of duplication.ResultsThe organizational analysis showed that nine TaADH genes were highly expressed in all organs, and the rest of TaADH genes had tissue specificity. Cis-acting element analysis showed that almost all of the TaADH genes contained an anaerobic response element. The expression levels of ADH gene in waterlogging tolerant and waterlogging sensitive wheat seeds were analyzed by quantitative real-time PCR (qRT-PCR). This showed that some key ADH genes were significantly responsive to waterlogging stress at the seed germination stage, and the response of waterlogging tolerant and waterlogging sensitive wheat seeds to waterlogging stress was regulated by different ADH genes. The results may be helpful to further study the function of TaADH genes and to determine the candidate gene for wheat stress resistance breeding.

Identification of two fnr genes and characterisation of their role in the anaerobic switch in Sphingopyxis granuli strain TFA

Sphingopyxis granuli strain TFA is able to grow on the organic solvent tetralin as the only carbon and energy source. The aerobic catabolic pathway for tetralin, the genes involved and their regulation have been fully characterised. Unlike most of the bacteria belonging to the sphingomonads group, this strain is able to grow in anoxic conditions by respiring nitrate, though not nitrite, as the alternative electron acceptor. In this work, two fnr-like genes, fnrN and fixK, have been identified in strain TFA. Both genes are functional in E. coli and Sphingopyxis granuli although fixK, whose expression is apparently activated by FnrN, seems to be much less effective than fnrN in supporting anaerobic growth. Global transcriptomic analysis of a ΔfnrN ΔfixK double mutant and identification of Fnr boxes have defined a minimal Fnr regulon in this bacterium. However, expression of a substantial number of anaerobically regulated genes was not affected in the double mutant. Additional regulators such regBA, whose expression is also activated by Fnr, might also be involved in the anaerobic response. Anaerobically induced stress response genes were not regulated by Fnr but apparently induced by stress conditions inherent to anaerobic growth, probably due to accumulation of nitrite and nitric oxide.

Genome-wide association studies using 50K rice genic SNP chip unveil genetic architecture for anaerobic germination of deep-water rice population of Assam, India.

North Eastern part of India such as Assam is inundated by flood every year where the farmers are forced to grow the traditional tall deep-water rice. Genetic improvement of this type of rice is slow because of insufficient knowledge about their genetic architecture and population structure. In the present investigation, the genetic diversity architecture of 94 deep-water rice genotypes of Assam and association mapping strategy was, for the first time, applied to determine the significant SNPs and genes for deep-water rice. These genotypes are known for their unique elongation ability under deep-water condition. The anaerobic germination (AG) related trait-associated genes identified here can provide affluent resources for rice breeding especially in flood-prone areas. We investigated the genome-wide association studies (GWAS) using 50K rice genic SNP chip across 94 deep-water rice genotypes collected from different flood-prone districts/villages of Assam. Population structure and diversity analysis revealed that these genotypes were stratified into four sub-populations. Using GWAS approach, 20 significant genes were identified and found to be associated with AG-related traits. Of them, two most relevant genes (OsXDH1and SSXT) have been identified which explain phenotypic variability (R2 > 20%) in the population. These genes were located in Chr 3 (LOC_Os03g31550) which encodes for enzyme xanthine dehydrogenase 1(OsXDH1) and in Chr 12 (LOC_Os12g31350) which encodes for SSXT family protein. Both of these genes were found to be associated with anaerobic response index (increase in the coleoptile length under water in anaerobic condition with respect to control), respectively. Interestingly, OsXDH1is involved in purine catabolism pathway and acts as a scavenger of reactive oxygen species in plants, whereas SSXT is GRF1-interacting factor 3. These two candidate genes associated with AG of deep-water rice have been found to be reported for the first time. Thus, this study provides a greater resource for breeders not only for improvement of deep-water rice, but also for AG tolerant variety useful for direct-seeded rice in flood-affected areas.

Effects of ccpA gene deficiency in Lactobacillus delbrueckii subsp. bulgaricus under aerobic conditions as assessed by proteomic analysis

BackgroundAerobic growth provides benefits in biomass yield and stress tolerance of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). Catabolite control protein A (CcpA) is a master regulator involved in the aerobic and anaerobic growth, metabolic production and stress response in L. bulgaricus, but its potential molecular mechanisms remains unclear. The aim of this study is to elucidate the role of CcpA in L. bulgaricus in aerobic growth at the proteomic perspective.ResultsThe differential proteomic analysis was performed on the L. bulgaricus ATCC11842 and its ccpA inactivated mutant strain using iTRAQ technology. A total of 132 differentially expressed proteins were obtained, among which 58 were up-regulated and 74 were down-regulated. These proteins were mainly involved in the cellular stress response, carbohydrate and energy metabolism, amino acid transport and protein synthesis, genetic information processing. Moreover, inactivation of ccpA negatively affected the expression of key enzymes involved in glycolysis pathway, while it enhanced the expression of proteins related to the pyruvate pathway, supporting the conclusion that CcpA mediated the shift from homolactic fermentation to mixed acid fermentation in L. bulgaricus.ConclusionsOverall, these results showed that the role of CcpA in L. bulgaricus as a pleiotropic regulator in aerobic metabolism and stress response. This proteomic analysis also provide new insights into the CcpA-mediated regulatory network of L. bulgaricus and potential strategies to improve the production of starter and probiotic cultures based on the metabolic engineering of global regulators.

Genetic variability, character association and path analysis for anaerobic germination traits in rice (Oryza sativa L.)

Study on genetic parameters, character associations and path coefficients of 107 rice genotypes for anaerobic germination traits revealed high variability, heritability and genetic advance as per cent of mean for seedling vigour index. High heritability and genetic advance as per cent of mean was also recorded for shoot length, root length, seedling dry weight and anaerobic response index. Further, all anaerobic germination traits studied had recorded high positive association with anaerobic response index. Among these, shoot length had recorded high positive direct and indirect effects and is identified as effective selection criterion for improvement of anaerobic response index towards tolerance to anaerobic conditions during germination for use under direct seeding.

Principal component analysis for anaerobic germination traits in rice (<em>Oryza sativa </em>L.)

Principal component analysis was carried out to assess the diversity among 107 rice genotypes with regards to anaerobic germination tolerance traits. Results revealed three principal components with eigen value > 1. These components contributed for a total variability of 75.780 per cent. Component 1 (PC 1) had contributed maximum 27.288 per cent while PC 2 had contributed to 26.060 per cent and PC 3 had contributed to 22.331 per cent towards the total variability. The characters, namely, anaerobic response index and germination per cent explained maximum variance in PC 1. The results of 2D scatter diagram revealed maximum diversity for MTU 1140. Hybridization of MTU 1140 with MTU 1010, identified as widely diverse genotypes in the study is expected to result in high yielding transgressive segregants with high yield and tolerance for germination under anaerobic conditions.

Anaerobic Transcription by OxyR: A Novel Paradigm for Nitrosative Stress.

Significance: S-nitrosylation, the post-translational modification by nitric oxide (NO) to form S-nitrosothiols (SNOs), regulates diverse aspects of cellular function, and aberrant S-nitrosylation (nitrosative stress) is implicated in disease, from neurodegeneration to cancer. Essential roles for S-nitrosylation have been demonstrated in microbes, plants, and animals; notably, bacteria have often served as model systems for elucidation of general principles. Recent Advances: Recent conceptual advances include the idea of a molecular code through which proteins sense and differentiate S-nitrosothiol (SNO) from alternative oxidative modifications, providing the basis for specificity in SNO signaling. In Escherichia coli, S-nitrosylation relies on an enzymatic cascade that regulates, and is regulated by, the transcription factor OxyR under anaerobic conditions. S-nitrosylated OxyR activates an anaerobic regulon of >100 genes that encode for enzymes that both mediate S-nitrosylation and protect against nitrosative stress. Critical Issues: Mitochondria originated from endosymbiotic bacteria and generate NO under hypoxic conditions, analogous to conditions in E. coli. Nitrosative stress in mitochondria has been implicated in Alzheimer's and Parkinson's disease, among others. Many proteins that are S-nitrosylated in mitochondria are also S-nitrosylated in E. coli. Insights into enzymatic regulation of S-nitrosylation in E. coli may inform the identification of disease-relevant regulatory machinery in mammalian systems. Future Directions: Using E. coli as a model system, in-depth analysis of the anaerobic response controlled by OxyR may lead to the identification of enzymatic mechanisms regulating S-nitrosylation in particular, and hypoxic signaling more generally, providing novel insights into analogous mechanisms in mammalian cells and within dysfunctional mitochondria that characterize neurodegenerative diseases.

Rewired cellular signaling coordinates sugar and hypoxic responses for anaerobic xylose fermentation in yeast.

Microbes can be metabolically engineered to produce biofuels and biochemicals, but rerouting metabolic flux toward products is a major hurdle without a systems-level understanding of how cellular flux is controlled. To understand flux rerouting, we investigated a panel of Saccharomyces cerevisiae strains with progressive improvements in anaerobic fermentation of xylose, a sugar abundant in sustainable plant biomass used for biofuel production. We combined comparative transcriptomics, proteomics, and phosphoproteomics with network analysis to understand the physiology of improved anaerobic xylose fermentation. Our results show that upstream regulatory changes produce a suite of physiological effects that collectively impact the phenotype. Evolved strains show an unusual co-activation of Protein Kinase A (PKA) and Snf1, thus combining responses seen during feast on glucose and famine on non-preferred sugars. Surprisingly, these regulatory changes were required to mount the hypoxic response when cells were grown on xylose, revealing a previously unknown connection between sugar source and anaerobic response. Network analysis identified several downstream transcription factors that play a significant, but on their own minor, role in anaerobic xylose fermentation, consistent with the combinatorial effects of small-impact changes. We also discovered that different routes of PKA activation produce distinct phenotypes: deletion of the RAS/PKA inhibitor IRA2 promotes xylose growth and metabolism, whereas deletion of PKA inhibitor BCY1 decouples growth from metabolism to enable robust fermentation without division. Comparing phosphoproteomic changes across ira2Δ and bcy1Δ strains implicated regulatory changes linked to xylose-dependent growth versus metabolism. Together, our results present a picture of the metabolic logic behind anaerobic xylose flux and suggest that widespread cellular remodeling, rather than individual metabolic changes, is an important goal for metabolic engineering.

Functional analysis of the role of CcpA in Lactobacillus plantarum grown on fructooligosaccharides or glucose: a transcriptomic perspective

BackgroundThe catabolite control protein A (CcpA) is a master regulator of many important cellular processes in Gram-positive bacteria. In Lactobacillus plantarum, CcpA directly or indirectly controls the transcription of a large number of genes that are involved in carbohydrate metabolism, aerobic and anaerobic growth, stress response and metabolite production, but its role in response to different carbon sources remains unclear.ResultsHere a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth phase of wild-type and ccpA mutant strains of L. plantarum ST-III using fructooligosaccharides (FOS) or glucose as the sole carbon source. The inactivation of ccpA significantly affected the growth and production of metabolites under both carbon sources. About 15% of the total genes were significantly altered between wild-type and ccpA strains grown on glucose and the value is deceased to 12% when these two strains were compared on FOS, while only 7% were obviously changed due to the loss of CcpA when comparing strains grown on glucose and FOS. Although most of the differentially expressed genes mediated by CcpA are glucose dependent, FOS can also induce carbon catabolite repression (CCR) through the CcpA pathway. Moreover, the inactivation of ccpA led to a transformation from homolactic fermentation to mixed fermentation under aerobic conditions. CcpA can control genes directly by binding in the regulatory region of the target genes (mixed fermentation), indirectly through local regulators (fatty acid biosynthesis), or have a double effect via direct and indirect regulation (FOS metabolism).ConclusionOverall, our results show that CcpA plays a central role in response to carbon source and availability of L. plantarum and provide new insights into the complex and extended regulatory network of lactic acid bacteria.

Age-dependent regulation of ERF-VII transcription factor activity in Arabidopsis thaliana.

The Group VII Ethylene Responsive Factors (ERFs-VII) RAP2.2 and RAP2.12 have been mainly characterized with regard to their contribution as activators of fermentation in plants. However, transcriptional changes measured in conditions that stabilize these transcription factors exceed the mere activation of this biochemical pathway, implying additional roles performed by the ERF-VIIs in other processes. We evaluated gene expression in transgenic Arabidopsis lines expressing a stabilized form of RAP2.12, or hampered in ERF-VII activity, and identified genes affected by this transcriptional regulator and its homologs, including some involved in oxidative stress response, which are not universally induced under anaerobic conditions. The contribution of the ERF-VIIs in regulating this set of genes in response to chemically induced or submergence-stimulated mitochondria malfunctioning was found to depend on the plant developmental stage. A similar age-dependent mechanism also restrained ERF-VII activity upon the core-hypoxic genes, independently of the N-end rule pathway, which is accounted for the control of the anaerobic response. To conclude, this study shed new light on a dual role of ERF-VII proteins under submergence: as positive regulators of the hypoxic response and as repressors of oxidative-stress related genes, depending on the developmental stage at which plants are challenged by stress conditions.

Hypoxia-responsive ERFs involved in postdeastringency softening of persimmon fruit.

SummaryRemoval of astringency by endogenously formed acetaldehyde, achieved by postharvest anaerobic treatment, is of critical importance for many types of persimmon fruit. Although an anaerobic environment accelerates de‐astringency, it also has the deleterious effect of promoting excessive softening, reducing shelf life and marketability. Some hypoxia‐responsive ethylene response factors (ERFs) participate in anaerobic de‐astringency, but their role in accelerated softening was unclear. Undesirable rapid softening induced by high CO 2 (95%) was ameliorated by adding the ethylene inhibitor 1‐MCP (1 μL/L), resulting in reduced astringency while maintaining firmness, suggesting that CO 2‐induced softening involves ethylene signalling. Among the hypoxia‐responsive genes, expression of eight involved in fruit cell wall metabolism (Dkβ‐gal1/4, DkEGase1, DkPE1/2, DkPG1, DkXTH9/10) and three ethylene response factor genes (DkERF8/16/19) showed significant correlations with postdeastringency fruit softening. Dual‐luciferase assay indicated that DkERF8/16/19 could trans‐activate the DkXTH9 promoter and this interaction was abolished by a mutation introduced into the C‐repeat/dehydration‐responsive element of the DkXTH9 promoter, supporting the conclusion that these DkERFs bind directly to the DkXTH9 promoter and regulate this gene, which encodes an important cell wall metabolism enzyme. Some hypoxia‐responsive ERF genes are involved in deastringency and softening, and this linkage was uncoupled by 1‐MCP. Fruit of the Japanese cultivar ‘Tonewase’ provide a model for altered anaerobic response, as they lost astringency yet maintained firmness after CO 2 treatment without 1‐MCP and changes in cell wall enzymes and ERFs did not occur.

In Silico Characterization and Functional Validation of Cell Wall Modification Genes Imparting Waterlogging Tolerance in Maize.

Cell wall modification (CWM) promotes the formation of aerenchyma in roots under waterlogging conditions as an adaptive mechanism. Lysigenous aerenchyma formation in roots improves oxygen transfer in plants, which highlights the importance of CWM as a focal point in waterlogging stress tolerance. We investigated the structural and functional compositions of CWM genes and their expression patterns under waterlogging conditions in maize. Cell wall modification genes were identified for 3 known waterlogging-responsive cis-acting regulatory elements, namely, GC motif, anaerobic response elements, and G-box, and 2 unnamed elements. Structural motifs mapped in CWM genes were represented in genes regulating waterlogging stress-tolerant pathways, including fermentation, glycolysis, programmed cell death, and reactive oxygen species signaling. The highly aligned regions of characterized and uncharacterized CWM proteins revealed common structural domains amongst them. Membrane spanning regions present in the protein structures revealed transmembrane activity of CWM proteins in the plant cell wall. Cell wall modification proteins had interacted with ethylene-responsive pathway regulating genes (E3 ubiquitin ligases RNG finger and F-box) in a maize protein-protein interaction network. Cell wall modification genes had also coexpressed with energy metabolism, programmed cell death, and reactive oxygen species signaling, regulating genes in a single coexpression cluster. These configurations of CWM genes can be used to modify the protein expression in maize under waterlogging stress condition. Our study established the importance of CWM genes in waterlogging tolerance, and these genes can be used as candidates in introgression breeding and genome editing experiments to impart tolerance in maize hybrids.

A Potassium-Dependent Oxygen Sensing Pathway Regulates Plant Root Hydraulics

Aerobic organisms survive low oxygen (O2) through activation of diverse molecular, metabolic, and physiological responses. In most plants, root water permeability (in other words, hydraulic conductivity, Lpr) is downregulated under O2 deficiency. Here, we used a quantitative genetics approach in Arabidopsis to clone Hydraulic Conductivity of Root 1 (HCR1), a Raf-like MAPKKK that negatively controls Lpr. HCR1 accumulates and is functional under combined O2 limitation and potassium (K(+)) sufficiency. HCR1 regulates Lpr and hypoxia responsive genes, through the control of RAP2.12, a key transcriptional regulator of the core anaerobic response. A substantial variation of HCR1 in regulating Lpr is observed at the Arabidopsis species level. Thus, by combinatorially integrating two soil signals, K(+) and O2 availability, HCR1 modulates the resilience of plants to multiple flooding scenarios.

Involvement of DkTGA1 Transcription Factor in Anaerobic Response Leading to Persimmon Fruit Postharvest De-Astringency.

Persimmon fruit are unique in accumulating proanthocyanidins (tannins) during development, which cause astringency in mature fruit. In ‘Mopanshi’ persimmon, astringency can be removed by treatment with 95% CO2, which increases the concentrations of ethanol and acetaldehyde by glycolysis, and precipitates the soluble tannin. A TGA transcription factor, DkTGA1, belonging to the bZIP super family, was isolated from an RNA-seq database and real-time quantitative PCR indicated that DkTGA1 was up-regulated by CO2 treatment, in concert with the removal of astringency from persimmon fruit. Dual-luciferase assay revealed that DkTGA1 had a small (less than 2-fold), but significant effect on the promoters of de-astringency-related genes DkADH1, DkPDC2 and DkPDC3, which encode enzymes catalyzing formation of acetaldehyde and ethanol. A combination of DkTGA1 and a second transcription factor, DkERF9, shown previously to be related to de-astringency, showed additive effects on the activation of the DkPDC2 promoter. Yeast one-hybrid assay showed that DkERF9, but not DkTGA1, could bind to the DkPDC2 promoter. Thus, although DkTGA1 expression is positively associated with persimmon fruit de-astringency, trans-activation analyses with DkPDC2 indicates it is likely to act by binding indirectly DkPDC2 promoter, might with helps of DkERF9.

Effects of training at a walk on conventional and underwater treadmills on fiber properties and metabolic responses of superficial digital flexor and gluteal muscles to high-speed exercise in horses.

To compare effects of training on conventional and underwater treadmills on fiber properties and metabolic responses of the superficial digital flexor (SDF) and gluteal muscles to high-speed exercise in horses. 6 unconditioned Quarter Horse-type horses. 6 horses were walked on underwater and conventional treadmills for 5 d/wk (maximum, 40 min/d) for 8 weeks in a randomized crossover design (60-day detraining period). Horses underwent a standardized exercise test (SET) at high speed before and after training. Analyte concentrations and fiber characteristics were measured in muscle biopsy specimens obtained from horses before and after each SET. Lactate concentration increased 2- to 3-fold in SDF and gluteal muscle after SETs. No training effect was identified on muscle fiber type composition, type II fiber diameter, muscle analyte concentrations, blood lactate concentration, or heart rate responses. Maximum diameters of type I fibers decreased significantly in gluteal muscle with conventional treadmill training and decreased in SDF muscle with both types of training, with maximum diameters greater for horses after underwater versus conventional treadmill training. No change was identified in minimum fiber diameters. SETs involving near-maximal exertion resulted in an anaerobic response in SDF and gluteal muscles of horses. Eight weeks of conventional or underwater treadmill training resulted in minor changes in type I muscle fiber sizes, with no effect on muscle metabolic or heart rate responses to SETs. After rehabilitation involving underwater treadmills, training at progressing speeds is recommended for horses to develop the required fitness for speed work.

Constitutively expressed ERF-VII transcription factors redundantly activate the core anaerobic response in Arabidopsis thaliana

Plant adaptation to hypoxic conditions is mediated by the transcriptional activation of genes involved in the metabolic reprogramming of plant cells to cope with reduced oxygen availability. Recent studies indicated that members of the group VII of the Ethylene Responsive Transcription Factor (ERFs) family act as positive regulators of this molecular response. In the current study, the five ERF-VII transcription factors of Arabidopsis thaliana were compared to infer a hierarchy in their role with respect to the anaerobic response. When the activity of each transcription factor was tested on a set of hypoxia-responsive promoters, RAP2.2, RAP2.3 and RAP2.12 appeared to be the most powerful activators. RAP2.12 was further dissected in transactivation assays in Arabidopsis protoplasts to identify responsible regions for transcriptional activation. An ultimate C-terminal motif was identified as sufficient to drive gene transcription. Finally, using realtime RT-PCR in single and double mutants for the corresponding genes, we confirmed that RAP2.2 and RAP2.12 exert major control upon the anaerobic response.

A Trihelix DNA Binding Protein Counterbalances Hypoxia-Responsive Transcriptional Activation in Arabidopsis

Author SummaryRespiratory metabolism in land plants requires oxygen availability to be able to generate ATP, which is essential for biosynthetic processes. Cellular hypoxia can be triggered as a consequence of environmental events (mainly floods), anatomical constraints (low tissue permeability to gases), or elevated cellular respiration, and it is unfavorable to growth due to the resultant decline in ATP. The adaptation of plants to fluctuating oxygen levels inside tissues requires the dynamic regulation of mechanisms that ensure cell viability and ultimately organism survival, but only a few molecular components of this homeostatic network are known. Direct hypoxia-sensing entails the posttranslational stabilization of a subgroup of plant ethylene-responsive factor (ERF) transcription factors, which coordinate the expression of hypoxia-inducible genes. Turnover of these ERFs is determined by an oxygen-dependent pathway of proteasomal degradation. Here, we demonstrate that the hypoxia-inducible transcription factor gene HRA1 is transcriptionally activated upon ERF-VII RAP2.12 stabilization and encodes a trihelix DNA binding protein that functionally interacts with RAP2.12 to curtail its activity. In addition to its negative regulation of RAP2.12, HRA1 negatively regulates activation of its own promoter. This RAP2.12-HRA1 control unit allows plants to modulate the extent of the response to hypoxia, including anaerobic enzyme production, to levels that improve endurance of the stress. Our results emphasize the importance of a strategy that can counterbalance energy-inefficient survival responses.

The Effect of Land and Underwater Treadmill Training on Metabolic Responses to Exercise and Properties of Superficial Digital Flexor and Gluteal Muscle

IntroductionResistance training provided by underwater (UWT) treadmills may enhance strength and fatigue resistance of superficial digital flexor muscle thereby reducing tendon injury. This study examined effects of UWT and land (LT) treadmill training on SDF and gluteal muscle.MethodsSix unfit horses (3 × 2 crossover design, 60d detraining period) walked on UWT and LT for 5d/week for 8 weeks (up to 40 min/day). A standardized exercise test (SET) was performed 2 weeks before, and after L or UW training, and V180 bpm and La10.5 m/s calculated. Resting and post‐SET gluteal and SDF muscle biopsies were obtained. Muscle [glycogen], [lactate] and [ATP], fiber type composition and min/max fiber diameters were analysed. Comparisons were made using GLM or ANOVA, mean ± SD and significance of P<0.05.ResultsPost‐exercise SDF muscle lactate increased 3–4 fold to 30 ± 12 (UWT) and 41 ± 15 (LT) mmol/kg dw post‐exercise. Post‐exercise gluteal muscle lactate increased 2 fold to 55 ± 18 (UWT) and 62 ± 18 (LT) mmol/kg. No significant differences were found between UW and L post‐exercise muscle [glycogen], [lactate] and [ATP], V180 or LA10.5 or fiber type composition. Gluteal fiber diameters decreased significantly with UW and L training. Fiber diameters in SDF muscle decreased with training to a significantly lesser extent after UW vs. L training.ConclusionsAt maximal speed, both SDF and gluteal muscle have a significant anaerobic response which was not impacted by UWT or LT training at a walk. UWT may be of benefit by attenuating the training induced decrease in fiber sizes in SDF muscle thereby enhancing SDF muscle strength.Ethical Animal ResearchAll procedures were approved by the Institutional Animal Care and Use Committee of the University of Minnesota. Sources of funding: Supported by University of Minnesota Equine Center with funds provided by the Minnesota Racing Commission, University of Minnesota Agricultural Experiment Station, and private donors. Competing interests: none.