STOP1-regulated SMALL AUXIN UP RNA55 (SAUR55) is involved in proton/malate co-secretion for Al tolerance in Arabidopsis.

Proton (H+) release is linked to aluminum (Al)-enhanced organic acids (OAs) excretion from the roots under Al rhizotoxicity in plants. It is well-reported that the Al-enhanced organic acid excretion mechanism is regulated by SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1), a zinc-finger TF that regulates major Al tolerance genes. However, the mechanism of H+ release linked to OAs excretion under Al stress has not been fully elucidated. Recent physiological and molecular-genetic studies have implicated the involvement of SMALL AUXIN UP RNAs (SAURs) in the activation of plasma membrane H+-ATPases for stress responses in plants. We hypothesized that STOP1 is involved in the regulation of Al-responsive SAURs, which may contribute to the co-secretion of protons and malate under Al stress conditions. In our transcriptome analysis of the roots of the stop1 (sensitive to proton rhizotoxicity1) mutant, we found that STOP1 regulates the transcription of one of the SAURs, namely SAUR55. Furthermore, we observed that the expression of SAUR55 was induced by Al and repressed in the STOP1 T-DNA insertion knockout (KO) mutant (STOP1-KO). Through in silico analysis, we identified a functional STOP1-binding site in the promoter of SAUR55. Subsequent in vitro and in vivo studies confirmed that STOP1 directly binds to the promoter of SAUR55. This suggests that STOP1 directly regulates the expression of SAUR55 under Al stress. We next examined proton release in the rhizosphere and malate excretion in the T-DNA insertion KO mutant of SAUR55 (saur55), in conjunction with STOP1-KO. Both saur55 and STOP1-KO suppressed rhizosphere acidification and malate release under Al stress. Additionally, the root growth of saur55 was sensitive to Al-containing media. In contrast, the overexpressed line of SAUR55 enhanced rhizosphere acidification and malate release, leading to increased Al tolerance. These associations with Al tolerance were also observed in natural variations of Arabidopsis. These findings demonstrate that transcriptional regulation of SAUR55 by STOP1 positively regulates H+ excretion via PM H+-ATPase 2 which enhances Al tolerance by malate secretion from the roots of Arabidopsis. The activation of PM H+-ATPase 2 by SAUR55 was suggested to be due to PP2C.D2/D5 inhibition by interaction on the plasma membrane with its phosphatase. Furthermore, RNAi-suppression of NtSTOP1 in tobacco shows suppression of rhizosphere acidification under Al stress, which was associated with the suppression of SAUR55 orthologs, which are inducible by Al in tobacco. It suggests that transcriptional regulation of Al-inducible SAURs by STOP1 plays a critical role in OAs excretion in several plant species as an Al tolerance mechanism.

Role of PPARα in inflammatory response of C2C12 myotubes.

Recent studies have shown a role of inflammation in muscle atrophy and sarcopenia. However, no anti-inflammatory pharmacotherapy has been established for the treatment of sarcopenia. Here, we investigate the potential role of PPARα and its ligands on inflammatory response and PGC-1α gene expression in LPS-treated C2C12 myotubes. Knockdown of PPARα, whose expression was upregulated upon differentiation, augmented IL-6 or TNFα gene expression. Conversely, PPARα overexpression or its activation by ligands suppressed 2-h LPS-induced cytokine expression, with pemafibrate attenuating NF-κB or STAT3 phosphorylation. Of note, reduction of PGC-1α gene expression by LPS treatment for 24 hours was partially reversed by fenofibrate. Our data demonstrate a critical inhibitory role of PPARα in inflammatory response of C2C12 myotubes and suggest a future possibility of PPARα ligands as a candidate for anti-inflammatory therapy against sarcopenia.

The transcription factors, STOP1 and TCP20, are required for root system architecture alterations in response to nitrate deficiency.

Nitrate distribution in soils is often heterogeneous. Plants have adapted to this by modifying their root system architecture (RSA). Previous studies showed that NITRATE-TRANSPORTER1.1 (NRT1.1), which also transports auxin, helps inhibit lateral root primordia (LRP) emergence in nitrate-poor patches, by preferentially transporting auxin away from the LRP. In this study, we identified the regulatory system for this response involving the transcription factor (TF), SENSITIVE-TO-PROTON-RHIZOTOXICITY1 (STOP1), which is accumulated in the nuclei of LRP cells under nitrate deficiency and directly regulates Arabidopsis NRT1.1 expression. Mutations in STOP1 mimic the root phenotype of the loss-of-function NRT1.1 mutant under nitrate deficiency, compared to wild-type plants, including increased LR growth and higher DR5promoter activity (i.e., higher LRP auxin signaling/activity). Nitrate deficiency-induced LR growth inhibition was almost completely reversed when STOP1 and the TF, TEOSINTE-BRANCHED1,-CYCLOIDEA,-PCF-DOMAIN-FAMILY-PROTEIN20 (TCP20), a known activator of NRT1.1 expression, were both mutated. Thus, the STOP1-TCP20 system is required for activation of NRT1.1 expression under nitrate deficiency, leading to reduced LR growth in nitrate-poor regions. We found this STOP1-mediated system is more active as growth media becomes more acidic, which correlates with reductions in soil nitrate as the soil pH becomes more acidic. STOP1 has been shown to be involved in RSA modifications in response to phosphate deficiency and increased potassium uptake, hence, our findings indicate that root growth regulation in response to low availability of the major fertilizer nutrients, nitrogen, phosphorus and potassium, all involve STOP1, which may allow plants to maintain appropriate root growth under the complex and varying soil distribution of nutrients.

Cowpea NAC1/NAC2 transcription factors improve growth and tolerance to drought and heat in transgenic cowpea through combined activation of photosynthetic and antioxidant mechanisms.

NAC (NAM/ATAF1/2/CUC2) transcription factors are central switches of growth and stress responses in plants. However, unpredictable interspecies conservation of function and regulatory targets makes the well-studied NAC orthologs inapt for pulse engineering. The knowledge of suitable NAC candidates in hardy pulses like cowpea (Vigna unguiculata (L.) Walp.) is still in infancy, hence warrants immediate biotechnological intervention. Here, we showed that overexpression of two native NAC genes (VuNAC1 and VuNAC2) promoted germinative, vegetative, and reproductive growth and conferred multiple abiotic stress tolerance in a commercial cowpea variety. The transgenic lines displayed increased leaf area, thicker stem, nodule-rich denser root system, early flowering, higher pod production (∼3.2-fold and ∼2.1-fold), and greater seed weight (10.3% and 6.0%). In contrast, transient suppression of VuNAC1/2 caused severe growth retardation and flower inhibition. The overexpressor lines showed remarkable tolerance to major yield-declining terminal stresses, such as drought, salinity, heat, and cold, and recovered growth and seed production by boosting photosynthetic activity, water use efficiency, membrane integrity, Na+ /K+ homeostasis, and antioxidant activity. The comparative transcriptome study indicated consolidated activation of genes involved in chloroplast development, photosynthetic complexes, cell division and expansion, cell wall biogenesis, nutrient uptake and metabolism, stress response, abscisic acid, and auxin signaling. Unlike their orthologs, VuNAC1/2 direct synergistic transcriptional tuning of stress and developmental signaling to avoid unwanted trade-offs. Their overexpression governs the favorable interplay of photosynthesis and reactive oxygen species regulation to improve stress recovery, nutritional sufficiency, biomass, and production. This unconventional balance of strong stress tolerance and agronomic quality is useful for translational crop research and molecular breeding of pulses.

Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity.

Obesity is an important health problem, which can be prevented through appetite control. Ghrelin is an appetite-stimulating hormone considered to promote obesity. Thus, we examined whether gastric stretching affects ghrelin secretion. We investigated the role of transient receptor potential vanilloid 4 (TRPV4) in gastric glands in the regulation of ghrelin secretion. TRPV4 immunostaining was performed in tissue samples from 57 patients who underwent gastrectomy. TRPV4 expression was compared between patients with (body mass index (BMI) ≥ 30) and without (BMI <30) obesity. For in vitro experiments, we used MGN3-1 cells, a ghrelin-producing cell line derived from mice. To investigate the bioactivity of TRPV4, MGN3-1 cells were treated with TRPV4 agonists and antagonists, and changes in intracellular Ca2+ concentration were confirmed. The concentration of ghrelin in the cell supernatant was measured using the ELISA with and without 120% stretch stimulation. TRPV4 expression was significantly higher in patients with obesity than in those without at all sites, except the fornix. Immunostaining confirmed the expression of TRPV4 in MGN3-1 cells. TRPV4 agonist administration increased intracellular Ca2+ concentration and ghrelin secretion in MGN3-1 cells, whereas the administration of the agonist combined with the antagonist decreased intracellular Ca2+ concentration and ghrelin secretion. Ghrelin secretion significantly increased in response to a 120% stretch in MGN3-1 cells. However, secretion was not increased by stretch when cells were treated with a TRPV4 antagonist. TRPV4 regulates ghrelin secretion in response to stretch in the stomach, which may affect body weight.

Enhanced osmotic adjustment, antioxidant defense, and photosynthesis efficiency under drought and heat stress of transgenic cowpea overexpressing an engineered DREB transcription factor.

Cowpea is sensitive to drought and heat stress, particularly at the reproductive stages of development. Both stresses limit growth and yield, and their effect is more devastating when occurring concurrently. Dehydration-responsive element-binding protein 2A (DREB2A) is an important signaling hub integrating information about two different abiotic stresses, drought and heat. We identified VuDREB2A as a canonical DREB ortholog in cowpea, activating downstream stress-responsive genes by binding to DREs in their promoter. Post-translational modification of a negative regulatory domain (NRD) within the VuDREB2A protein prevents its degradation. Targeted deletion of the NRD produces a stable and constitutively active form VuDREB2A-CA. However, there is very little evidence of its practical utility under field conditions. This study overexpressed the VuDREB2A-CA in a popular cowpea variety and conducted drought- and heat-tolerance experiments across various stress regimes. Transgenic cowpea exhibited significant tolerance with consistently higher yield when exposed to over 30-d drought stress and 3-d exposure to high temperature (28 °C-52 °C) without any pleiotropic alterations. The transgenic lines showed higher photosynthetic efficiency, osmotic adjustment, antioxidant defense, thermotolerance, and significantly higher survival and increased biomass than the wild type. Late embryogenesis abundant 5, heat shock protein 70, dehydrin, mitogen-activated protein kinase 2/4, isoflavonoid reductase, and myoinositol phosphate synthase were upregulated in transgenic lines under drought and heat stress. Through transcriptome analysis of the transgenic lines, we found significant up-regulation of various stress-responsive cowpea genes, having DRE in their promoter. Our results suggest that overexpression of VuDREB2A could improve cowpea production under drought and high temperatures.

Progressive loss of the thyroid tissue integrity visualised by serial CT scans.

A man in his 70s developed thyrotoxicosis due to painless thyroiditis after starting nivolumab, which was subsequently followed by severe hypothyroidism. We diagnosed him as chronic thyroiditis, initiated levothyroxine supplementation and treated appropriately. Retrospective CT images of the thyroid gland during the clinical course revealed that the CT attenuation value was high at first but gradually decreased. The high-density signal of the normal thyroid tissues reflects its function of concentrating inorganic iodine, and the progressive decrease of the CT density in the present case can be viewed as a reflection of the thyroid destruction and progressive loss of iodine during the clinical course of the development of chronic thyroiditis. Considering the high incidence rate of functional thyroid disease in patients treated with immune checkpoint inhibitors, CT density of the thyroid gland needs to be paid attention to as the first sign of thyroiditis in this patient population.

Quantitative Analysis of Redox Pool (NAD + , NADH Content) in Plant Samples Under Aluminum Stress.

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor of numerous enzymatic reactions found in all living cells. Pyridine nucleotides (NAD + and NADH) are also key players in signaling through reactive oxygen species (ROS), being crucial in the regulation of both ROS-producing and ROS-consuming systems in plants. NAD content is a powerful modulator of metabolic integration, protein de-acetylation, and DNA repair. The balance between NAD oxidized and reduced forms, i.e ., the NADH/NAD + ratio, indicates the redox state of a cell, and it is a measurement that reflects the metabolic health of cells. Here we present an easy method to estimate the NAD + and NADH content enzymatically, using alcohol dehydrogenase (ADH), an oxido-reductase enzyme, and with MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) as the substrate and 1-methoxy PMS (1-Methoxy-5-methylphenazinium methyl sulfate) as the electron carrier. MTT is reduced to a purple formazan, which is then detected. We used Arabidopsis leaf samples exposed to aluminum toxicity and under untreated control conditions. NADH/NAD + connects many aspects of metabolism and plays vital roles in plant developmental processes and stress responses. Therefore, it is fundamental to determine the status of NADH/NAD + under stress.

How do plants remember drought?

MAIN CONCLUSION: Plants develop both short-term and transgenerational memory of drought stress through epigenetic regulation of transcription for a better response to subsequent exposure. Recurrent spells of droughts are more common than a single drought, with intermittent moist recovery intervals. While the detrimental effects of the first drought on plant structure and physiology are unavoidable, if survived, plants can memorize the first drought to present a more robust response to the following droughts. This includes a partial stomatal opening in the watered recovery interval, higher levels of osmoprotectants and ABA, and attenuation of photosynthesis in the subsequent exposure. Short-term drought memory is regulated by ABA and other phytohormone signaling with transcriptional memory behavior in various genes. High levels of methylated histones are deposited at the drought-tolerance genes. During the recovery interval, the RNA polymerase is stalled to be activated by a pause-breaking factor in the subsequent drought. Drought leads to DNA demethylation near drought-response genes, with genetic control of the process. Progenies of the drought-exposed plants can better adapt to drought owing to the inheritance of particular methylation patterns. However, a prolonged watered recovery interval leads to loss of drought memory, mediated by certain demethylases and chromatin accessibility factors. Small RNAs act as critical regulators of drought memory by altering transcript levels of drought-responsive target genes. Further studies in the future will throw more light on the genetic control of drought memory and the interplay of genetic and epigenetic factors in its inheritance. Plants from extreme environments can give queues to understanding robust memory responses at the ecosystem level.

Overexpression of cowpea NAC transcription factors promoted growth and stress tolerance by boosting photosynthetic activity in Arabidopsis.

ATAF-like NAC transcription factors are bonafide regulators of stress-signaling. However, their overexpression often exerts growth-retardation by activating ABA-hypersensitivity, chloroplast-degradation, or carbon-starvation. To improve tolerance to multiple stress complying with growth sustainability, we examined two ATAF orthologs, VuNAC1 and VuNAC2, isolated from a drought-hardy cowpea genotype, for a harmonized regulation of stress and growth signaling. The genes were induced by dehydration, NaCl, polyethylene glycol, heat, cold, ABA, and light. Analysis of the promoter-elements and regulatory network corroborated the integration of circadian, hormonal, stress, developmental, and nutrition signals, being VuNAC1/2 the central transcriptional-switch interfacing growth and stress responses. The constitutive gene overexpression in Arabidopsis resulted in an improved embryonic, rosette, and inflorescence growth, under optimum as well as limiting nutrition, in association with increased photosynthetic activity and stomatal-density. The transgenic seedlings manifested tolerance to dehydration, salinity, aluminum, cadmium, and H2O2 toxicity, in addition to ABA-mediated seed dormancy and hypersensitivity. The soil-grown plants survived severe drought and hypersalinity by maintaining the water-status and membrane integrity through the accumulation of stress protectants, such as proline, glutathione, and ascorbate. Unlike their orthologs from other species, VuNAC1/2 conferred tolerance to multiple abiotic stresses in line with improved growth attributes via regulation of photosynthetic controls and nutritional balance, suggesting growth being a crucial component of stress-tolerance and recovery. Such unique stress-responsive transcription factors, which also confer photosynthetic gain, could be sustainable biotechnological tools for developing stress-tolerant crops and translating the improved growth into yield without unintended trade-offs.

Visual and histological evaluation of the effects of trafermin in a dog oronasal fistula model.

The standard procedure to treat oronasal fistula in dogs requires tooth extraction to close the fistula; hence, the subject would lose its tooth. In this study, trafermin was applied to four dog models with oronasal fistula to investigate the periodontal tissue regenerative effects of trafermin in the treatment without tooth extraction. A fistula was created along the palatal side of each upper canine tooth. One of the fistulae was filled with trafermin, whereas that on the contralateral side was left unfilled as a control. The results showed a significant decrease in the non-calcified periodontal tissue volume on the trafermin side after the fourth week. In addition, oronasal fistula closure was visually and histologically confirmed at the eighth week on the trafermin side of all four models.

Maturity-onset diabetes of the young type 5, presenting as diabetic ketoacidosis with alkalemia: A report of a case.

A 34-year-old man visited our Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan, because of dry mouth and weight loss. His plasma glucose level was 32.8 mmol/L and serum levels of ketone bodies were increased, but with metabolic alkalemia. He was also suffering from renal tubular hypomagnesemia and hypokalemia. Abdominal computed tomography showed bilateral renal cysts. These findings were suggestive of maturity-onset diabetes of the young type 5. Genetic testing showed heterozygous hepatocyte nuclear factor 1 beta gene deletion. In the present case, it seemed reasonable to view hepatocyte nuclear factor 1 beta gene deletion as the common cause of maturity-onset diabetes of the young type 5-associated diabetic ketoacidosis and tubular malfunction-induced hypokalemic alkalosis. This case exemplifies the importance of hepatocyte nuclear factor 1 beta gene abnormality as a potential cause of diabetic ketoacidosis with alkalemia.

A mouse model of weight gain after nicotine withdrawal.

Smoking cessation increases body weight. The underlying mechanisms, however, have not been fully understood. We here report an establishment of a mouse model that exhibits an augmented body weight gain after nicotine withdrawal. High fat diet-fed mice were infused with nicotine for two weeks, and then with vehicle for another two weeks using osmotic minipumps. Body weight increased immediately after nicotine cessation and was significantly higher than that of mice continued on nicotine. Mice switched to vehicle consumed more food than nicotine-continued mice during the first week of cessation, while oxygen consumption was comparable. Elevated expression of orexigenic agouti-related peptide was observed in the hypothalamic appetite center. Pair-feeding experiment revealed that the accelerated weight gain after nicotine withdrawal is explained by enhanced energy intake. As a showcase of an efficacy of pharmacologic intervention, exendin-4 was administered and showed a potent suppression of energy intake and weight gain in mice withdrawn from nicotine. Our current model provides a unique platform for the investigation of the changes of energy regulation after smoking cessation.

Synergistic and antagonistic pleiotropy of STOP1 in stress tolerance.

SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) is a master transcription factor (TF) that regulates genes encoding proteins critical for cellular pH homeostasis. STOP1 also causes pleiotropic effects in both roots and shoots associated with various stress tolerances. STOP1-regulated genes in roots synergistically confer tolerance to coexisting stress factors in acid soil, and root-architecture remodeling for superior phosphorus acquisition. Additionally, STOP1 confers salt tolerance to roots under low-potassium conditions. By contrast, STOP1 antagonistically functions in shoots to promote hypoxia tolerance but to suppress drought tolerance. In this review, we discuss how these synergetic- and antagonistic-pleiotropic effects indicate that STOP1 is a central hub of stress regulation and that the harmonization of STOP1-regulated traits is essential for plant adaptation to various environments.

The biotechnological importance of the plant-specific NAC transcription factor family in crop improvement.

Climate change, malnutrition, and food insecurity are the inevitable challenges being faced by the agriculture sector today. Plants are susceptible to extreme temperatures during the crucial phases of flowering and seed development, and elevated carbon levels also lead to yield losses. Productivity is also affected by floods and droughts. Therefore, increasing plant yield and stress tolerance are the priorities to be met through novel biotechnological interventions. The contributions of NAC genes towards enhancing plant survivability under stress is well known. Here we focus on the potential of NAC genes in the regulation of abiotic stress tolerance, secondary cell wall synthesis, lateral root development, yield potential, seed size and biomass, ROS signaling, leaf senescence, and programmed cell death. Once naturally tolerant candidate NAC genes have been identified, and the nature of their association with growth and fitness against multi-environmental stresses has been determined, they can be exploited for building inherent tolerance in future crops via transgenic technologies. An update on the latest developments is provided in this review, which summarizes the current understanding of the roles of NAC in the establishment of various stress-adaptive mechanisms in model and food crop plants.

High affinity promoter binding of STOP1 is essential for early expression of novel aluminum-induced resistance genes GDH1 and GDH2 in Arabidopsis.

Malate efflux from roots, which is regulated by the transcription factor STOP1 (SENSITIVE-TO-PROTON-RHIZOTOXICITY1) and mediates aluminum-induced expression of ALUMINUM-ACTIVATED-MALATE-TRANSPORTER1 (AtALMT1), is critical for aluminum resistance in Arabidopsis thaliana. Several studies showed that AtALMT1 expression in roots is rapidly observed in response to aluminum; this early induction is an important mechanism to immediately protect roots from aluminum toxicity. Identifying the molecular mechanisms that underlie rapid aluminum resistance responses should lead to a better understanding of plant aluminum sensing and signal transduction mechanisms. In this study, we observed that GFP-tagged STOP1 proteins accumulated in the nucleus soon after aluminum treatment. The rapid aluminum-induced STOP1-nuclear localization and AtALMT1 induction were detected in the presence of a protein synthesis inhibitor, suggesting that post-translational regulation is involved in these events. STOP1 also regulated rapid aluminum-induced expression for other genes that carry a functional/high-affinity STOP1-binding site in their promoter, including STOP2, GLUTAMATE-DEHYDROGENASE1 and 2 (GDH1 and 2). However STOP1 did not regulate Al resistance genes which have no functional STOP1-binding site such as ALUMINUM-SENSITIVE3, suggesting that the binding of STOP1 in the promoter is essential for early induction. Finally, we report that GDH1 and 2 which are targets of STOP1, are novel aluminum-resistance genes in Arabidopsis.

Expression genome-wide association study identifies that phosphatidylinositol-derived signalling regulates ALUMINIUM SENSITIVE3 expression under aluminium stress in the shoots of Arabidopsis thaliana.

To identify unknown regulatory mechanisms leading to aluminium (Al)-induction of the Al tolerance gene ALS3, we conducted an expression genome-wide association study (eGWAS) for ALS3 in the shoots of 95 Arabidopsis thaliana accessions in the presence of Al. The eGWAS was conducted using a mixed linear model with 145,940 genome-wide single nucleotide polymorphisms (SNPs) and the association results were validated using reverse genetics. We found that many SNPs from the eGWAS were associated with genes related to phosphatidylinositol metabolism as well as stress signal transduction, including Ca2+signals, inter-connected in a co-expression network. Of these, PLC9, CDPK32, ANAC071, DIR1, and a hypothetical protein (AT4G10470) possessed amino acid sequence/ gene expression level polymorphisms that were significantly associated with ALS3 expression level variation. Furthermore, T-DNA insertion mutants of PLC9, CDPK32, and ANAC071 suppressed shoot ALS3 expression in the presence of Al. This study clarified the regulatory mechanisms of ALS3 expression in the shoot and provided genetic evidence of the involvement of phosphatidylinositol-derived signal transduction under Al stress.