Harnessing enzyme cofactors and plant metabolism: an essential partnership.

Cofactors are fundamental to the catalytic activity of enzymes. Additionally, because plants are a critical source of several cofactors (i.e., including their vitamin precursors) within the context of human nutrition, there have been several studies aiming to understand the metabolism of coenzymes and vitamins in plants in detail. For example, compelling evidence has been brought forth regarding the role of cofactors in plants; specifically, it is becoming increasingly clear that an adequate supply of cofactors in plants directly affects their development, metabolism, and stress responses. Here, we review the state-of-the-art knowledge on the significance of coenzymes and their precursors with regard to general plant physiology and discuss the emerging functions attributed to them. Furthermore, we discuss how our understanding of the complex relationship between cofactors and plant metabolism can be used for crop improvement.

Clinical and immunological outcomes of SARS-CoV-2-infected vaccine responders, vaccine non-responders, and unvaccinated patients evaluated for neutralizing monoclonal antibody treatment at a single German tertiary care center: a retrospective cohort study with prospective follow-up.

PURPOSE: This study assessed the clinical and immunological outcomes of SARS-CoV-2-infected patients with risk factors for severe disease depending on their immunological status. METHODS: In this retrospective study with single follow-up visit, clinical outcome and humoral immunity was monitored in SARS-CoV-2 infected patients at risk. The results were compared based on the patients' initial immunological status: unvaccinated (UV), patients who did not develop neutralizing antibodies after vaccination (vaccine non-responders, VNR), and patients who expressed neutralizing antibodies after vaccination (vaccine responders, VR). Patients who lacked neutralizing antibodies (VNR and UV) were treated with nMABs. RESULTS: In total, 113 patients at risk of severe COVID-19 consented to participate in the study. VR and UV were not admitted to the hospital. During the observation period, UVs had the highest rate of SARS-CoV-2 re-infections. Three of 41 VNRs (7.3%) were hospitalized due to severe COVID-19, with two of them having undergone iatrogenic B-cell depletion. The humoral immune response after infection was significantly lower in the VNR group than in the VR group in terms of anti-N, anti-receptor-binding domain (RBD), anti-S antibody titers, and anti-S antibody avidity. In a sub-analysis of VNR, B cell-deficient non-responders had significantly lower levels of anti-N antibodies and anti-S avidity after infection than other VNRs. CONCLUSION: VNR, particularly B-cell-depleted VNR, remained at risk of hospitalization due to COVID-19. In the VR group, however, no clinical complications or severe disease were observed, despite not receiving nMAbs. Tailoring the administration of nMABs according to patient vaccination and immunological status may be advisable.

Mitochondrial and peroxisomal NAD+ uptake are important for improved photosynthesis and seed yield under elevated CO2 concentrations.

As sessile organisms, plants must adapt their physiology and developmental processes to cope with challenging environmental circumstances, such as the ongoing elevation in atmospheric carbon dioxide (CO2 ) levels. Nicotinamide adenine dinucleotide (NAD+ ) is a cornerstone of plant metabolism and plays an essential role in redox homeostasis. Given that plants impaired in NAD metabolism and transport often display growth defects, low seed production and disturbed stomatal development/movement, we hypothesized that subcellular NAD distribution could be a candidate for plants to exploit the effects of CO2 fertilization. We report that an efficient subcellular NAD+ distribution is required for the fecundity-promoting effects of elevated CO2 levels. Plants with reduced expression of either mitochondrial (NDT1 or NDT2) or peroxisomal (PXN) NAD+ transporter genes grown under elevated CO2 exhibited reduced total leaf area compared with the wild-type while PXN mutants also displayed reduced leaf number. NDT2 and PXN lines grown under elevated CO2 conditions displayed reduced rosette dry weight and lower photosynthetic rates coupled with reduced stomatal conductance. Interestingly, high CO2 doubled seed production and seed weight in the wild-type, whereas the mutants were less responsive to increases in CO2 levels during reproduction, producing far fewer seeds than the wild-type under both CO2 conditions. These data highlight the importance of mitochondrial and peroxisomal NAD+ uptake mediated by distinct NAD transporter proteins to modulate photosynthesis and seed production under high CO2 levels.

The Arabidopsis electron-transfer flavoprotein:ubiquinone oxidoreductase is required during normal seed development and germination.

The importance of the alternative donation of electrons to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex has been demonstrated. However, the functional significance of this pathway during seed development and germination remains to be elucidated. To assess the function of this pathway, we performed a detailed metabolic and transcriptomic analysis of Arabidopsis mutants to test the molecular consequences of a dysfunctional ETF/ETFQO pathway. We demonstrate that the disruption of this pathway compromises seed germination in the absence of an external carbon source and also impacts seed size and yield. Total protein and storage protein content is reduced in dry seeds, whilst sucrose levels remain invariant. Seeds of ETFQO and related mutants were also characterized by an altered fatty acid composition. During seed development, lower levels of fatty acids and proteins accumulated in the etfqo-1 mutant as well as in mutants in the alternative electron donors isovaleryl-CoA dehydrogenase (ivdh-1) and d-2-hydroxyglutarate dehydrogenase (d2hgdh1-2). Furthermore, the content of several amino acids was increased in etfqo-1 mutants during seed development, indicating that these mutants are not using such amino acids as alternative energy source for respiration. Transcriptome analysis revealed alterations in the expression levels of several genes involved in energy and hormonal metabolism. Our findings demonstrated that the alternative pathway of respiration mediated by the ETF/ETFQO complex affects seed germination and development by directly adjusting carbon storage during seed filling. These results indicate a role for the pathway in the normal plant life cycle to complement its previously defined roles in the response to abiotic stress.

The interplay between autophagy and chloroplast vesiculation pathways under dark-induced senescence.

In cellular circumstances where carbohydrates are scarce, plants can use alternative substrates for cellular energetic maintenance. In plants, the main protein reserve is present in the chloroplast, which contains most of the total leaf proteins and represents a rich source of nitrogen and amino acids. Autophagy plays a key role in chloroplast breakdown, a well-recognised symptom of both natural and stress-induced plant senescence. Remarkably, an autophagic-independent route of chloroplast degradation associated with chloroplast vesiculation (CV) gene was previously demonstrated. During extended darkness, CV is highly induced in the absence of autophagy, contributing to the early senescence phenotype of atg mutants. To further investigate the role of CV under dark-induced senescence conditions, mutants with low expression of CV (amircv) and double mutants amircv1xatg5 were characterised. Following darkness treatment, no aberrant phenotypes were observed in amircv single mutants; however, amircv1xatg5 double mutants displayed early senescence and altered dismantling of chloroplast and membrane structures under these conditions. Metabolic characterisation revealed that the functional lack of both CV and autophagy leads to higher impairment of amino acid release and differential organic acid accumulation during starvation conditions. The data obtained are discussed in the context of the role of CV and autophagy, both in terms of cellular metabolism and the regulation of chloroplast degradation.

Bradykinin promotes murine melanoma cell migration and invasion through endogenous production of superoxide and nitric oxide.

Spatial confinement and temporal regulation of signaling by nitric oxide (NO) and reactive oxygen species (ROS) occurs in cancer cells. Signaling mediated by NO and ROS was investigated in two sub clones of the murine melanoma B16F10-Nex2 cell line, Nex10C and Nex8H treated or not with bradykinin (BK). The sub clone Nex10C, similar to primary site cells, has a low capacity for colonizing the lungs, whereas the sub clone Nex8H, similar to metastatic cells, corresponds to a highly invasive melanoma. BK-treated Nex10C cells exhibited a transient increase in NO and an inhibition in basal O2- levels. Inhibition of endogenous NO production by l-NAME resulted in detectable levels of O2-. l-NAME promoted Rac1 activation and enhanced Rac1-PI3K association. l-NAME in the absence of BK resulted in Nex10C cell migration and invasion, suggesting that NO is a negative regulator of O2- mediated cell migration and cell invasion. BK-treated Nex8H cells sustained endogenous NO production through the activation of NOS3. NO activated Rac1 and promoted Rac1-PI3K association. NO stimulated cell migration and cell invasion through a signaling axis involving Ras, Rac1 and PI3K. In conclusion, a role for O2- and NO as positive regulators of Rac1-PI3K signaling associated with cell migration and cell invasion is proposed respectively for Nex10C and Nex8H murine melanoma cells.

Influence of radiologic pattern and the presence of diffuse parenchymal lung disease on outcome in ventilated patients with COVID-19 pneumonia: impact on prognosis.

BACKGROUND: Suspected organising pneumonia (OP) is a common finding in patients with severe coronavirus disease 2019 (COVID-19), but the impact on outcomes of the radiological patterns of diffuse parenchymal lung disease on outcome of these patients is still uncertain. AIMS: Investigate the presence of radiological images compatible with OP and its association with clinical outcomes in patients with COVID-19 submitted to invasive mechanical ventilation (IMV). METHODS: Retrospective, unicentric cohort study composed of patients who required IMV and underwent chest computerized tomography to investigate secondary complications of COVID-19. We compared patients with radiological findings characteristic of suspected OP with those without this condition. The main outcome was hospital mortality. RESULTS: Two hundred and ten patients were included, and 65 had signals compatible with OP. All patients with suspected OP were treated with corticosteroids. There was no difference in IVM-free days until day 28 between the groups (median, 0 days; interquartile range [IQR], 0-14.8) in the group with suspected OP vs 0 days (IQR, 0-11) in the group without suspected OP (P = 0.14). In univariate analysis, the presence of suspected OP was associated with lower hospital mortality; however, after correction for potential confounding variables, it was not associated with the outcome, even after matching by propensity score in patients without this condition. CONCLUSION: OP radiologic pattern in patients with severe COVID-19 is not associated with worse outcomes.

Acute phase reaction to lipopolysaccharide-induced mastitis in early lactation dairy cows fed nitrogenic, glucogenic, or lipogenic diets.

The availability of certain macronutrients is likely to influence the capacity of the immune system. Therefore, we investigated the acute phase response to intramammary (i.mam.) lipopolysaccharide (LPS) in dairy cows fed a nitrogenic diet (n = 10) high in crude protein, a glucogenic diet (n = 11) high in carbohydrates and glucogenic precursors, or a lipogenic diet (n = 11) high in lipids. Thirty-two dairy cows were fed one of the dietary concentrates directly after calving until the end of trial at 27 ± 3 days in milk (mean ± standard deviation). In wk 3 of lactation, 20 µg of LPS was i.mam. injected in one quarter, and sterile NaCl (0.9%) in the contralateral quarter. Milk samples of the LPS-challenged and control quarter were taken hourly from before (0 h) until 9 h after LPS challenge and analyzed for milk amyloid A (MAA), haptoglobin (HP), and IL-8. In addition, blood samples were taken in the morning, and composite milk samples at morning and evening milkings, from 1 d before until 3 d after LPS challenge, and again on d 9, to determine serum amyloid A (SAA) and HP in blood, and MAA and HP in milk. The mRNA abundance of various immunological and metabolic factors in blood leukocytes was quantified by quantitative reverse-transcription PCR from samples taken at -18, -1, 6, 9, and 23 h relative to LPS application. The dietary concentrates did not affect any of the parameters in blood, milk, and leukocytes. The IL-8 was increased from 2 h, HP from 2 to 3 h, and MAA from 6 h relative to the LPS administration in the milk of the challenged quarter and remained elevated until 9 h. The MAA and HP were also increased at 9 h after LPS challenge in whole-udder composite milk, whereas HP and SAA in blood were increased only after 23 h. All 4 parameters were decreased again on d 9. Similar for all groups, the mRNA abundance of HP and the heat shock protein family A increased after the LPS challenge, whereas the mRNA expression of the tumor necrosis factor α and the leukocyte integrin ß 2 subunit (CD18) were decreased at 6 h after LPS challenge. The glucose transporter (GLUT)1 mRNA abundance decreased after LPS, whereas that of the GLUT3 increased, and that of the GLUT4 was not detectable. The mRNA abundance of GAPDH was increased at 9 h after LPS and remained elevated. The acute phase protein response was detected earlier in milk compared with blood indicating mammary production. However, immunological responses to LPS were not affected by the availability of specific macronutrients provided by the different diets.

Autophagy is required for lipid homeostasis during dark-induced senescence.

Autophagy is an evolutionarily conserved mechanism that mediates the degradation of cytoplasmic components in eukaryotic cells. In plants, autophagy has been extensively associated with the recycling of proteins during carbon-starvation conditions. Even though lipids constitute a significant energy reserve, our understanding of the function of autophagy in the management of cell lipid reserves and components remains fragmented. To further investigate the significance of autophagy in lipid metabolism, we performed an extensive lipidomic characterization of Arabidopsis (Arabidopsis thaliana) autophagy mutants (atg) subjected to dark-induced senescence conditions. Our results revealed an altered lipid profile in atg mutants, suggesting that autophagy affects the homeostasis of multiple lipid components under dark-induced senescence. The acute degradation of chloroplast lipids coupled with the differential accumulation of triacylglycerols (TAGs) and plastoglobuli indicates an alternative metabolic reprogramming toward lipid storage in atg mutants. The imbalance of lipid metabolism compromises the production of cytosolic lipid droplets and the regulation of peroxisomal lipid oxidation pathways in atg mutants.

The significance of WRKY45 transcription factor in metabolic adjustments during dark-induced leaf senescence.

Plants are constantly exposed to environmental changes that affect their performance. Metabolic adjustments are crucial to controlling energy homoeostasis and plant survival, particularly during stress. Under carbon starvation, coordinated reprogramming is initiated to adjust metabolic processes, which culminate in premature senescence. Notwithstanding, the regulatory networks that modulate transcriptional control during low energy remain poorly understood. Here, we show that the WRKY45 transcription factor is highly induced during both developmental and dark-induced senescence. The overexpression of Arabidopsis WRKY45 resulted in an early senescence phenotype characterized by a reduction of maximum photochemical efficiency of photosystem II and chlorophyll levels in the later stages of darkness. The detailed metabolic characterization showed significant changes in amino acids coupled with the accumulation of organic acids in WRKY45 overexpression lines during dark-induced senescence. Furthermore, the markedly upregulation of alternative oxidase (AOX1a, AOX1d) and electron transfer flavoprotein/ubiquinone oxidoreductase (ETFQO) genes suggested that WRKY45 is associated with a dysregulation of mitochondrial signalling and the activation of alternative respiration rather than amino acids catabolism regulation. Collectively our results provided evidence that WRKY45 is involved in the plant metabolic reprogramming following carbon starvation and highlight the potential role of WRKY45 in the modulation of mitochondrial signalling pathways.

Reduced auxin signalling through the cyclophilin gene DIAGEOTROPICA impacts tomato fruit development and metabolism during ripening.

Auxin is an important hormone playing crucial roles during fruit growth and ripening; however, the metabolic impact of changes in auxin signalling during tomato (Solanum lycopersicum L.) ripening remains unclear. Here, we investigated the significance of changes in auxin signalling during different stages of fruit development by analysing changes in tomato fruit quality and primary metabolism using mutants with either lower or higher auxin sensitivity [diageotropica (dgt) and entire mutants, respectively]. Altered auxin sensitivity modifies metabolism, through direct impacts on fruit respiration and fruit growth. We verified that the dgt mutant plants exhibit reductions in fruit set, total fruit dry weight, fruit size, number of seeds per fruit, and fresh weight loss during post-harvest. Sugar accumulation was associated with delayed fruit ripening in dgt, probably connected with reduced ethylene levels and respiration, coupled with a lower rate of starch degradation. In contrast, despite exhibiting parthenocarpy, increased auxin perception (entire) did not alter fruit ripening, leading to only minor changes in primary metabolism. By performing a comprehensive analysis, our results connect auxin signalling and metabolic changes during tomato fruit development, indicating that reduced auxin signalling led to extensive changes in sugar concentration and starch metabolism during tomato fruit ripening.

Evidence of a Near-Threshold Resonance in

A narrow near-threshold proton-emitting resonance (E_{x}=11.4 MeV, J^{π}=1/2^{+}, and Γ_{p}=4.4 keV) was directly observed in ^{11}B via proton resonance scattering. This resonance was previously inferred in the ß-delayed proton emission of the neutron halo nucleus ^{11}Be. The good agreement between both experimental results serves as a ground to confirm the existence of such exotic decay and the particular behavior of weakly bound nuclei coupled to the continuum. R-matrix analysis shows a sizable partial decay width for both, proton and α (Γ_{α}=11 keV) emission channels.

Sex-specific differences in reproductive life-history traits of the moss Weissia jamaicensis.

PREMISE: We investigated sex-specific differences in the life-history traits of a metapopulation of the dioicous moss Weissia jamaicensis. Field observations revealed high rates of fertilization, which is uncommon for most dioicous bryophytes. We raised four hypotheses associated with the way the reproductive traits are related to the fertilization rate in this metapopulation. METHODS: We sampled 10 patches of the metapopulation and quantified sexual expression, sex ratio, reproductive success, and reproductive allocation. The ramets were classified as male, non-sporophytic female, sporophytic female, or non-sex-expressing. Thirty ramets from each of the categories expressing sex were placed for regeneration to test the effect of reproductive allocation on this trait. RESULTS: We found greater expression of the female function in all patches, implying a female bias in the metapopulation. The number of male ramets was variable in each patch and did not affect reproductive success. At the prezygotic level, the allocation of resources to the male function was higher. However, the large allocation of resources to sporophyte development in sporophytic females, which exceeded allocations at prezygotic levels, was related to the higher mortality rate of these ramets, suggesting reproductive cost. CONCLUSIONS: The prezygotic ramets that allocated the greatest amount of resources to reproduction expressed sex less frequently, biasing the sex ratio toward the sex that allocated the least amount of resources to reproduction. Overall, the ramets that allocated the greatest amount of resources to reproduction had the lowest regeneration rate, suggesting reproductive cost.

Knockdown of the inducible nitric oxide synthase (NOS2) splicing variant S3 promotes autophagic cell death from nitrosative stress in SW480 human colon cancer cells.

Low levels of nitric oxide (NO) produced by constitutively expressed inducible NO synthase (NOS2) in tumor cells may be an important factor in their development. NOS2 expression is associated with high mortality rates for various cancers. Alternative splicing of NOS2 down-regulates its enzymatic activity, resulting in decreased intracellular NO concentrations. Specific probes to detect alternative splicing of NOS2 were used in two isogenic human colon cancer cell lines derived either from the primary tumor (SW480) or from a lymph node metastasis (SW620). Splicing variant of NOS2 S3, lacking exons 9, 10, and 11, was overexpressed in SW480 cells. NOS2 S3 was silenced in SW480 cells. Flow-cytometry analysis was used to estimate the intracellular NO levels and to analyze the cell cycle of the studied cell lines. Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine apoptosis and autophagy markers. SW480 and SW620 cells expressed NOS2 S3. Overexpression of the NOS2 S3 in SW480 cells downregulated intracellular NO levels. SW480 cells with knocked down NOS2 S3 (referred to as S3C9 cells) had higher intracellular levels of NO compared to the wild-type SW480 cells under serum restriction. Higher NO levels resulted in the loss of viability of S3C9 cells, which was associated with autophagy. Induction of autophagy by elevated intracellular NO levels in S3C9 cells under serum restriction, suggests that autophagy operates as a cytotoxic response to nitrosative stress. The expression of NOS2 S3 plays an important role in regulating intracellular NO production and maintaining viability in SW480 cells under serum restriction. These findings may prove significant in the design of NOS2/NO-based therapies for colon cancer.

Integration of a Relationship-focused Counseling Intervention with Delivery of the Dapivirine Ring for HIV Prevention to Women in Johannesburg: Results of the CHARISMA Pilot Study.

Biomedical, female-initiated HIV prevention methods can help reduce disproportionately high HIV rates among women in sub-Saharan Africa, but male partner resistance and intimate partner violence (IPV) may impact ability to ensure effective use. To support consistent use of the dapivirine vaginal ring (VR), we pilot-tested the impact of the CHARISMA relationship counseling intervention ("CHARISMA") with women enrolled in the multi-site open-label Microbicide Trials Network (MTN) 025/HOPE trial at the Wits Reproductive Health and HIV Research Institute (Wits RHI) site in Johannesburg, South Africa. Lay counselors used a 42-item tool with five subscales to assess relationships and IPV and provide tailored counseling at enrolment, followed by a booster counselling session at Month 1 and follow-up checks at Months 3 and 6. We evaluated potential impact by examining self-reported ring disclosure to partners, partner clinic attendance, self-reported incident social harms (SH) and IPV, and biomarkers of ring adherence at Wits RHI. We subsequently compared these outcomes at three comparator HOPE study sites using multivariable regression models. Comparator study sites were purposively selected as those most similar to Wits RHI for baseline characteristics identified a priori. At Wits RHI, 95 of 96 (99%) HOPE participants enrolled into the CHARISMA pilot study. Mean age was 30, 36.8% lived with a partner, and 85.3% received their partner's financial support. During the six months of pilot study follow-up, participants reported: ring use disclosure to partners at 72.7% visits; 4.3% partners attending the research clinic; one partner-related SH; and 9.5% experienced incident IPV. The mean level of dapivirine released from returned used rings was 3.4 mg (SD 1.56), suggesting moderate adherence. Participants in the CHARISMA pilot had high background prevalence and incidence of IPV but were nevertheless able to adhere to ring use, and some male partners came to the research clinic. In adjusted regression models, compared to Wits RHI, partner clinic attendance was lower at all comparator sites; and significantly so at Site A (aRR 0.12, 95% CI 0.00-0.98). Sites B and C had lower levels of dapivirine released (suggesting lower adherence), but this difference was not significant. Site B women were more likely to report ring disclosure to partners at FU visits (aRR 1.12, 95% CI 1.00-1.25). IPV reported during follow-up was significantly lower at Site B (aRR 0.20, 95% CI 0.04-0.98, p = 0.047). CHARISMA taught women skills to decide on levels of ring-use disclosure to partners or others; therefore it is difficult to interpret differences in ring disclosure to partners with other sites. Similarly, CHARISMA heightened participants' awareness of abuse, possibly increasing IPV reports. Testing CHARISMA under fully-powered controlled conditions will improve understanding of its impact on women's relationships and ability to use female-initiated HIV prevention methods.

Serious complications and recurrences after pelvic organ prolapse surgery for 2309 women in the VIGI-MESH registry.

OBJECTIVE: To assess the incidence of serious complications and reoperations for recurrence after surgery for pelvic organ prolapse (POP) and compare the three most common types of repair. DESIGN: Prospective cohort study using a registry. SETTING: Nineteen French surgical centres. POPULATION: A total of 2309 women participated between 2017 and 2019. METHODS: A multivariate analysis including an inverse probability of treatment weighting approach was used to obtain three comparable groups. MAIN OUTCOME MEASURES: Serious complications and subsequent reoperations for POP recurrence. RESULTS: The median follow-up time was 17.6 months. Surgeries were native tissue vaginal repairs (n = 504), transvaginal mesh placements (n = 692) and laparoscopic sacropexies with mesh (n = 1113). Serious complications occurred among 52 women (2.3%), and reoperation for POP recurrence was required for 32 women (1.4%). At 1 year the cumulative weighted incidence of serious complications was 1.8% for native tissue vaginal repair, 3.9% for transvaginal mesh and 2.2% for sacropexy, and the rates for reoperation for recurrence of POP were 1.5, 0.7 and 1.1%, respectively. Compared with native tissue vaginal repair, the risk of serious complications was higher in the transvaginal mesh group (weighted hazard ratio, wHR 3.84, 95% CI 2.43-6.08) and the sacropexy group (wHR 2.48, 95% CI 1.45-4.23), whereas the risk of reoperation for prolapse recurrence was lower in both the transvaginal mesh (wHR 0.22, 95% CI 0.13-0.39) and sacropexy (wHR 0.29, 95% CI 0.18-0.47) groups. CONCLUSIONS: Our results suggest that native tissue vaginal repairs have the lowest risk of serious complications but the highest risk of reoperation for recurrence. These results are useful for informing women and for shared decision making. TWEETABLE ABSTRACT: Laparoscopic sacropexy had fewer serious complications than transvaginal mesh and fewer reoperations for recurrence than vaginal repair.

The role of the electron-transfer flavoprotein: ubiquinone oxidoreductase following carbohydrate starvation in Arabidopsis cell cultures.

KEY MESSAGE: The functional absence of the electron-transfer flavoprotein: ubiquinone oxidoreductase (ETFQO) directly impacts electrons donation to the mitochondrial electron transport chain under carbohydrate-limiting conditions without major impacts on the respiration of cell cultures. Alternative substrates (e.g., amino acids) can directly feed electrons into the mitochondrial electron transport chain (mETC) via the electron transfer flavoprotein/electron-transfer flavoprotein: ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports plant respiration during stress situations. By using a cell culture system, here we investigated the responses of Arabidopsis thaliana mutants deficient in the expression of ETFQO (etfqo-1) following carbon limitation and supplied with amino acids. Our results demonstrate that isovaleryl-CoA dehydrogenase (IVDH) activity was induced during carbon limitation only in wild-type and that these changes occurred concomit with enhanced protein content. By contrast, neither the activity nor the total amount of IVDH was altered in etfqo-1 mutants. We also demonstrate that the activities of mitochondrial complexes in etfqo-1 mutants, display a similar pattern as in wild-type cells. Our findings suggest that the defect of ETFQO protein culminates with an impaired functioning of the IVDH, since no induction of IVDH activity was observed. However, the functional absence of the ETFQO seems not to cause major impacts on plant respiration under carbon limiting conditions, most likely due to other alternative electron entry pathways.

Elevated carbon assimilation and metabolic reprogramming in tomato high pigment mutants support the increased production of pigments.

KEY MESSAGE: High pigment mutants in tomato (Solanum lycopersicum L.), a loss of function in the control of photomorphogenesis, with greater pigment production, show altered growth, greater photosynthesis, and a metabolic reprogramming. High pigment mutations cause plants to be extremely responsive to light and produce excessive pigmentation as well as fruits with high levels of health-beneficial nutrients. However, the association of these traits with changes in the physiology and metabolism of leaves remains poorly understood. Here, we performed a detailed morphophysiological and metabolic characterization of high pigment 1 (hp1) and high pigment 2 (hp2) mutants in tomato (Solanum lycopersicum L. 'Micro-Tom') plants under different sunlight conditions (natural light, 50% shading, and 80% shading). These mutants occur in the DDB1 (hp1) and DET1 (hp2) genes, which are related to the regulation of photomorphogenesis and chloroplast development. Our results demonstrate that these mutations delay plant growth and height, by affecting physiological and metabolic parameters at all stages of plant development. Although the mutants were characterized by higher net CO2 assimilation, lower stomatal limitation, and higher carboxylation rates, with anatomical changes that favour photosynthesis, we found that carbohydrate levels did not increase, indicating a change in the energy flow. Shading minimized the differences between mutants and the wild type or fully reversed them in the phenotype at the metabolic level. Our results indicate that the high levels of pigments in hp1 and hp2 mutants represent an additional energy cost for these plants and that extensive physiological and metabolic reprogramming occurs to support increased pigment biosynthesis.

Effect of different dietary regimens at dry-off on performance, metabolism, and immune system in dairy cows.

Concentrate withdrawal and feed restriction are commonly used to reduce milk production and to facilitate dry-off, but may impair immune function in dairy cows. We investigated the effect of feed rations providing different amounts of nutrients in combination with feed restriction on performance, endocrine, and metabolic responses, as well as on leukocyte function before and after abrupt dry-off. Forty-three cows were studied from d 12 before until d 6 after dry-off (56 d before scheduled calving). Cows were fed experimental concentrates rich in crude protein (nitrogenic, n = 14), glucogenic precursors (glucogenic, n = 14), or lipids (lipogenic, n = 15). On d 3 before dry-off, total feed allowance was restricted to 50% in half of the animals of each dietary group, whereas feed allowance remained unchanged in the other animals. Performance parameters (milk yield, milk composition, and dry matter intake) were recorded, and daily blood and milk samples were taken and analyzed for various metabolic and endocrine parameters. Additionally, activity and mRNA abundance of several genes in leukocytes were measured at selected time points before and after feed restriction and dry-off, respectively. Feed restriction immediately resulted in a negative energy balance and decreased milk production. Concomitantly, concentrations of nonesterified fatty acids increased, whereas insulin, insulin-like growth factor-1, and glucagon decreased. After dry-off, energy balance turned positive and plasma nonesterified fatty acids decreased. Plasma glucose, insulin, and insulin-like growth factor-1 concentrations increased in all groups after dry-off. Glucose, insulin, and glucagon concentrations in plasma were higher in nonrestricted compared with restricted animals after dry-off. The experimental concentrate types marginally affected the investigated metabolic and endocrine factors, with the exception of elevated milk and plasma urea concentrations in cows fed the nitrogenic concentrate. Chemotactic and phagocytic activity of leukocytes were not affected by diets, feed restriction, or dry-off. Likewise, blood leukocyte mRNA abundance encoding for tumor necrosis factor α (TNF), heat shock protein family A (HSP70), and the glucose transporters (GLUT) 1 and 3 remained unchanged throughout the study period. Overall, the short-term negative energy balance induced by feed restriction was temporarily accompanied by metabolic adaptations, but did not alter the studied factors related to the immune system. Metabolic and endocrine adaptations supporting milk synthesis were continued during the first days after dry-off despite cessation of milking. Thus, the abrupt dry-off resulted in a short-term increase of glucose and triglyceride concentrations, with a delayed endocrine response to re-establish nutrient homeostasis in blood.

Germination and in vitro development of mature zygotic embryos and protein profile of seedlings of wild and cultivated Hevea brasiliensis.

The main factors governing Hevea brasiliensis germination and seedling establishment remains unclear. We examined the effect of growth regulators Indole 3-Acetic Acid (IAA) and 6-Benzylaminopurine (BAP), and their interactions on germination and the development of mature zygotic embryos (MZE) and protein profile of Hevea brasiliensis seedlings from wild and cultivated (clone PB 250) genotypes. Embryonic axes excised from seeds (wild and clone PB 250) were inoculated in Murashige and Skoog medium (control) and supplemented with IAA (3 µM) and BAP (6 µM) individually and their combination (3 µM IAA + 6 µM BAP). For both genotypes, the mature embryos displayed a high percentage of germination and establishment, and the seedlings were characterized by protein bands ranging from 7 to 30 kDa. Notably, the wild genotype showed proteins in the 14 kDa range, which may be associated with one of the major rubber elongation factors (REF). The wild and clone genotypes presented different behavior and strategies in relation to the protein profile in the presence of different growth regulators. Although the latex biosynthetic pathway and its mechanisms of regulation still remain largely unknown, our results aid in our understanding of the dynamics of proteins in different rubber tree clones in vitro.