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Transcriptional regulation is important for plants to respond to toxic effects of aluminium (Al). However, our current knowledge to these events is confined to a few transcription factors. Here, we functionally characterized a rice bean (Vigna umbellata) NAC-type transcription factor, VuNAR1, in terms of Al stress response. We demonstrated that rice bean VuNAR1 is a nuclear-localized transcriptional activator, whose expression was specifically upregulated by Al in roots but not in shoot. VuNAR1 overexpressing Arabidopsis plants exhibit improved Al resistance via Al exclusion. However, VuNAR1-mediated Al exclusion is independent of the function of known Al-resistant genes. Comparative transcriptomic analysis revealed that VuNAR1 specifically regulates the expression of genes associated with protein phosphorylation and cell wall modification in Arabidopsis. Transient expression assay demonstrated the direct transcriptional activation of cell wall-associated receptor kinase 1 (WAK1) by VuNAR1. Moreover, yeast one-hybrid assays and MEME motif searches identified a new VuNAR1-specific binding motif in the promoter of WAK1. Compared with wild-type Arabidopsis plants, VuNAR1 overexpressing plants have higher WAK1 expression and less pectin content. Taken together, our results suggest that VuNAR1 regulates Al resistance by regulating cell wall pectin metabolism via directly binding to the promoter of WAK1 and induce its expression.

AIM A modified repair technique for traumatic cloaca caused by obstetric anal sphincter injury was evaluated, and its feasibility and functional outcome were investigated. METHODS A retrospective review of 23 consecutively enrolled patients diagnosed with traumatic cloaca who underwent the modified repair technique between September 2010 and August 2018 was performed. Demographic, clinical feature, operative and follow-up data were recorded. RESULTS The patients diagnosed with traumatic cloaca who underwent surgical repair after obstetric anal sphincter injury had a median time from obstetric injury of 24 (12-35) years. The median preoperative Wexner faecal incontinence score was 16 (14-17). The postoperative hospital stay was 6 (6-7) days. The median postoperative Wexner faecal incontinence score decreased to 2 (2-3). The anal resting pressure increased from 9.00 (5.25-11.50) mmHg to 56.00 (55.00-65.75) mmHg (P

Buckwheat (Fagopyrum esculentum) exhibits high Al tolerance, but only a few genes have been functionally characterized. We previously characterized a half-type ABC transporter, Fagopyrum esculentum sensitive to Al rhizotoxicity1 (FeSTAR1), in buckwheat Al tolerance. This study aims to investigate whether and how another half-type ABC transporter (FeSTAR2) plays role in Al tolerance in buckwheat. The expression of FeSTAR2 and complementation test in Arabidopsis als3 mutant was examined. The interaction between FeSTAR1 and FeSTAR2 and subcellular location were analyzed by bimolecular fluorescence complementation (BiFC) and yeast two-hybrid (Y2H) assays. Al rapidly and specifically induced FeSTAR2 expression. Having transmembrane domains, FeSTAR2 localizes to membrane. BiFC and Y2H assays showed that FeSTAR2 could interact with FeSTAR1 which contains only nucleotide binding domain. Intriguingly, interaction between FeSTAR1 and FeSTAR2 altered their locations. Both heterologous expression of FeSTAR2 in als3 and exogenous UDP-glucose rescued its Al hypersensitivity of als3 mutant, suggesting that involvement of FeSTAR2 in Al tolerance requires UDP-glucose. Furthermore, Al-induced inhibition of xyloglucan endotransglucosylase (XET) activity in both atstar1 and als3 mutants could be restored by UDP-glucose. Our results indicate that FeSTAR2 interacts with FeSTAR1 to form an ABC transporter to regulate Al tolerance by vesicular transport of UDP-glucose which affects hemicellulose metabolism by regulating XET activity.

Under conditions of aluminum (Al) toxicity, which severely inhibits root growth in acidic soils, plants rapidly alter their gene expression to optimize physiological fitness for survival. Abscisic acid (ABA) has been suggested as a mediator between Al stress and gene expression, but the underlying mechanisms remain largely unknown. Here, we investigated ABA-mediated Al-stress responses, using integrated physiological and molecular biology approaches. We demonstrate that Al stress caused ABA accumulation in the root apex of rice bean (Vigna umbellata [Thunb.] Ohwi & Ohashi), which positively regulated Al tolerance. However, this was not associated with known Al-tolerance mechanisms. Transcriptomic analysis revealed that nearly one-third of the responsive genes were shared between the Al-stress and ABA treatments. We further identified a transcription factor, ABI5, as being positively involved in Al tolerance. Arabidopsis abi5 mutants displayed increased sensitivity to Al, which was not related to the regulation of AtALMT1 and AtMATE expression. Functional categorization of ABI5-mediated genes revealed the importance of cell wall modification and osmoregulation in Al tolerance, a finding supported by osmotic stress treatment on Al tolerance. Our results suggest that ABA signal transduction pathways provide an additional layer of regulatory control over Al tolerance in plants.

Buckwheat (Fagopyrum esculentum) is highly tolerant to Al stress, but the molecular mechanisms remain largely unknown. This study aims to investigate a half-type ABC transporter gene (FeSTAR1) with respect to Al tolerance. The expression of FeSTAR1 was examined and complementation test in atstar1 mutant was conducted. Furthermore, Al distribution and cell wall polysaccharides content were analyzed. FeSTAR1 is an ABC transporter protein with nucleotide binding domain, but lack of transmembrane domain. Consistently, FeSTAR1 is a soluble protein, localizing to both cytoplasm and nucleus. Al rapidly and specifically induced FeSTAR1 expression. Heterologous expression of FeSTAR1 in atstar1 rescued its Al tolerance, and exogenous applied UDP-glucose could alleviate Al sensitivity of atstar1 mutant, suggesting the connection between FeSTAR1 and UDP-glucose in terms of Al tolerance. Furthermore, FeSTAR1 complemented lines accumulated less Al in root cell wall than atstar1 mutant. Further cell wall fraction analysis showed that Al was largely confined to cell wall hemicellulose1, at which Al content was significantly lower in complemented lines. Consistent with Al distribution in different cell wall polysaccharides, complemented lines had lower hemicellulose1 content. Our results indicate that FeSTAR1 is involved in Al resistance via possibly cell wall matrix polysaccharides metabolism in buckwheat.

Aluminum (Al)-induced organic acid secretion from the root apex is an important Al resistance mechanism. However, it remains unclear how plants fine-tune root organic acid secretion which can contribute significantly to the loss of fixed carbon from the plant. Here, we demonstrate that Al-induced citrate secretion from the rice bean root apex is biphasic, consisting of an early phase with low secretion and a later phase of large citrate secretion. We isolated and characterized VuMATE2 as a possible second citrate transporter in rice bean functioning in tandem with VuMATE1, which we previously identified. The time-dependent kinetics of VuMATE2 expression correlates well with the kinetics of early phase root citrate secretion. Ectopic expression of VuMATE2 in Arabidopsis resulted in increased root citrate secretion and Al resistance. Electrophysiological analysis of Xenopus oocytes expressing VuMATE2 indicated VuMATE2 mediates anion efflux. However, the expression regulation of VuMATE2 differs from VuMATE1. While a protein translation inhibitor suppressed Al-induced VuMATE1 expression, it releases VuMATE2 expression. Yeast one-hybrid assays demonstrated that a previously identified transcription factor, VuSTOP1, interacts with the VuMATE2 promoter at a GGGAGG cis-acting motif. Thus, we demonstrate that plants adapt to Al toxicity by fine-tuning root citrate secretion with two separate root citrate transport systems.

// Bin Yu 1, 2, 3, * , Jia-Meng Xu 1, 3, * , Shan Li 1, 3, * , Cheng Chen 1, 3 , Rui-Xin Chen 1, 3 , Lei Wang 4 , Yan Zhang 3, 5 and Ming-Hui Wang 1, 3 1 College of Mathematics and Physics, Qingdao University of Science and Technology, Qingdao 266061, China 2 CAS Key Laboratory of Geospace Environment, Department of Geophysics and Planetary Science, University of Science and Technology of China, Hefei 230026, China 3 Bioinformatics and Systems Biology Research Center, Qingdao University of Science and Technology, Qingdao 266061, China 4 Key Laboratory of Eco-chemical Engineering, Ministry of Education, Laboratory of Inorganic Synthesis and Applied Chemistry, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China 5 College of Electromechanical Engineering, Qingdao University of Science and Technology, Qingdao 266061, China * These authors contributed equally to this work and are joint First Authors Correspondence to: Bin Yu, email: yubin@qust.edu.cn Keywords: gene regulatory networks, multiple time-delayed, dynamic Bayesian network, comprehensive score model, network structure profiles Received: July 20, 2017 Accepted: August 27, 2017 Published: September 23, 2017 ABSTRACT Gene regulatory networks (GRNs) research reveals complex life phenomena from the perspective of gene interaction, which is an important research field in systems biology. Traditional Bayesian networks have a high computational complexity, and the network structure scoring model has a single feature. Information-based approaches cannot identify the direction of regulation. In order to make up for the shortcomings of the above methods, this paper presents a novel hybrid learning method (DBNCS) based on dynamic Bayesian network (DBN) to construct the multiple time-delayed GRNs for the first time, combining the comprehensive score (CS) with the DBN model. DBNCS algorithm first uses CMI2NI (conditional mutual inclusive information-based network inference) algorithm for network structure profiles learning, namely the construction of search space. Then the redundant regulations are removed by using the recursive optimization algorithm (RO), thereby reduce the false positive rate. Secondly, the network structure profiles are decomposed into a set of cliques without loss, which can significantly reduce the computational complexity. Finally, DBN model is used to identify the direction of gene regulation within the cliques and search for the optimal network structure. The performance of DBNCS algorithm is evaluated by the benchmark GRN datasets from DREAM challenge as well as the SOS DNA repair network in Escherichia coli , and compared with other state-of-the-art methods. The experimental results show the rationality of the algorithm design and the outstanding performance of the GRNs.

Information on the subcellular localization of Gram-negative bacterial proteins is of great significance to study the pathogenesis, drug design and discovery of certain diseases. Protein subcellular localization is an important part of proteomics, while providing new opportunities and challenges for chemometrics. Since the prediction of protein subcellular localization can help to understand their function and the role played by their metabolic processes, a number of protein subcellular localization prediction methods have been developed in recent years. In this paper, we propose a novel method by combining wavelet denoising with support vector machine to predict the subcellular localization of proteins for the first time. Firstly, the features of the protein sequence are extracted by Chou's pseudo amino acid composition (PseAAC), then the feature information of the extracted is denoised by two-dimensional (2-D) wavelet. Finally, the optimal feature vectors are input to the SVM classifier to predict subcellular location of the Gram-negative bacterial proteins. Quite promising predictions are obtained using the jackknife test and compared with other predictive methods. The results indicate that the method proposed in this paper can remarkably improve the prediction accuracy of protein subcellular localization, and it can be used to predict the other attributes of proteins.

The mechanisms involved in the regulation of gene expression in response to phosphate (Pi) deficiency have been extensively studied, but their chromatin-level regulation remains poorly understood. We examined the role of histone acetylation in response to Pi deficiency by using the histone deacetylase complex1 (hdc1) mutant. Genes involved in root system architecture (RSA) remodeling were analyzed by quantitative real-time polymerase chain reaction (qPCR) and chromatin immunoprecipitation qPCR. We demonstrate that histone H3 acetylation increased under Pi deficiency, and the hdc1 mutant was hypersensitive to Pi deficiency, with primary root growth inhibition and increases in root hair number. Concomitantly, Pi deficiency repressed HDC1 protein abundances. Under Pi deficiency, hdc1 accumulated higher concentrations of Fe3+ in the root tips and had higher expression of genes involved in RSA remodeling, such as ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (ALMT1), LOW PHOSPHATE ROOT1 (LPR1), and LPR2 compared with wild-type plants. Furthermore, Pi deficiency enriched the histone H3 acetylation of ALMT1 and LPR1. Finally, genetic evidence showed that LPR1/2 was epistatic to HDC1 in regulating RSA remodeling. Our results suggest a chromatin-level control of Pi starvation responses in which HDC1-mediated histone H3 deacetylation represses the transcriptional activation of genes involved in RSA remodeling in Arabidopsis.

Under conditions of aluminum (Al) toxicity, which severely inhibits root growth in acidic soils, plants rapidly alter their gene expression to optimize physiological fitness for survival. Abscisic acid (ABA) has been suggested as a mediator between Al stress and gene expression, but the underlying mechanisms remain largely unknown. Here, we investigated ABA-mediated Al-stress responses, using integrated physiological and molecular biology approaches. We demonstrate that Al stress caused ABA accumulation in the root apex of rice bean (Vigna umbellata [Thunb.] OhwiOhashi), which positively regulated Al tolerance. However, this was not associated with known Al-tolerance mechanisms. Transcriptomic analysis revealed that nearly one-third of the responsive genes were shared between the Al-stress and ABA treatments. We further identified a transcription factor, ABI5, as being positively involved in Al tolerance. Arabidopsis abi5 mutants displayed increased sensitivity to Al, which was not related to the regulation of AtALMT1 and AtMATE expression. Functional categorization of ABI5-mediated genes revealed the importance of cell wall modification and osmoregulation in Al tolerance, a finding supported by osmotic stress treatment on Al tolerance. Our results suggest that ABA signal transduction pathways provide an additional layer of regulatory control over Al tolerance in plants.

Being an Al-accumulating crop, buckwheat detoxifies and tolerates Al not only in roots but also in leaves. While much progress has recently been made toward Al toxicity and resistance mechanisms in roots, little is known about the molecular basis responsible for detoxification and tolerance processes in leaves. Here, we carried out transcriptome analysis of buckwheat leaves in response to Al stress (20 µM, 24 h). We obtained 33,931 unigenes with 26,300 unigenes annotated in the NCBI database, and identified 1063 upregulated and 944 downregulated genes under Al stress. Functional category analysis revealed that genes related to protein translation, processing, degradation and metabolism comprised the biological processes most affected by Al, suggesting that buckwheat leaves maintain flexibility under Al stress by rapidly reprogramming their physiology and metabolism. Analysis of genes related to transcription regulation revealed that a large proportion of chromatin-regulation genes are specifically downregulated by Al stress, whereas transcription factor genes are overwhelmingly upregulated. Furthermore, we identified 78 upregulated and 22 downregulated genes that encode transporters. Intriguingly, only a few genes were overlapped with root Al-regulated transporter genes, which include homologs of AtMATE, ALS1, STAR1, ALS3 and a divalent ion symporter. In addition, we identified a subset of genes involved in development, in which genes associated with flowering regulation were important. Based on these data, it is proposed that buckwheat leaves develop conserved and distinct mechanisms to cope with Al toxicity.

Aluminum (Al)-induced organic acid secretion from the root apex is an important Al resistance mechanism. However, it remains unclear how plants fine-tune root organic acid secretion which can contribute significantly to the loss of fixed carbon from the plant. Here, we demonstrate that Al-induced citrate secretion from the rice bean root apex is biphasic, consisting of an early phase with low secretion and a later phase of large citrate secretion. We isolated and characterized VuMATE2 as a possible second citrate transporter in rice bean functioning in tandem with VuMATE1, which we previously identified. The time-dependent kinetics of VuMATE2 expression correlates well with the kinetics of early phase root citrate secretion. Ectopic expression of VuMATE2 in Arabidopsis resulted in increased root citrate secretion and Al resistance. Electrophysiological analysis of Xenopus oocytes expressing VuMATE2 indicated VuMATE2 mediates anion efflux. However, the expression regulation of VuMATE2 differs from VuMATE1. While a protein translation inhibitor suppressed Al-induced VuMATE1 expression, it releases VuMATE2 expression. Yeast one-hybrid assays demonstrated that a previously identified transcription factor, VuSTOP1, interacts with the VuMATE2 promoter at a GGGAGG cis-acting motif. Thus, we demonstrate that plants adapt to Al toxicity by fine-tuning root citrate secretion with two separate root citrate transport systems.

Acyl Activating Enzyme3 (AAE3) was identified to be involved in the catabolism of oxalate, which is critical for seed development and defense against fungal pathogens. However, the role of AAE3 protein in abiotic stress responses is unknown. Here, we investigated the role of rice bean (Vigna umbellata) VuAAE3 in Al tolerance. Recombinant VuAAE3 protein has specific activity against oxalate, with Km = 121 ± 8.2 µm and Vmax of 7.7 ± 0.88 µmol min−1 mg−1 protein, indicating it functions as an oxalyl-CoA synthetase. VuAAE3-GFP localization suggested that this enzyme is a soluble protein with no specific subcellular localization. Quantitative reverse transcription-PCR and VuAAE3 promoter-GUS reporter analysis showed that the expression induction of VuAAE3 is mainly confined to rice bean root tips. Accumulation of oxalate was induced rapidly by Al stress in rice bean root tips, and exogenous application of oxalate resulted in the inhibition of root elongation and VuAAE3 expression induction, suggesting that oxalate accumulation is involved in Al-induced root growth inhibition. Furthermore, overexpression of VuAAE3 in tobacco (Nicotiana tabacum) resulted in the increase of Al tolerance, which was associated with the decrease of oxalate accumulation. In addition, NtMATE and NtALS3 expression showed no difference between transgenic lines and wild-type plants. Taken together, our results suggest that VuAAE3-dependent turnover of oxalate plays a critical role in Al tolerance mechanisms.

Aluminium (Al)-activated citrate secretion plays an important role in Al resistance in a number of plant species, such as rice bean (Vigna umbellata). This study further characterized the regulation of VuMATE1, an aluminium-activated citrate transporter. Al stress induced VuMATE1 expression, followed by the secretion of citrate. Citrate secretion was specific to Al stress, whereas VuMATE1 expression was not, which could be explained by a combined regulation of VuMATE1 expression and Al-specific activation of VuMATE1 protein. Pre-treatment with a protein translation inhibitor suppressed VuMATE1 expression, indicating that de novo biosynthesis of proteins is required for gene expression. Furthermore, post-treatment with a protein translation inhibitor inhibited citrate secretion, indicating that post-transcriptional regulation of VuMATE1 is critical for citrate secretion. Protein kinase and phosphatase inhibitor studies showed that reversible phosphorylation was important not only for transcriptional regulation of VuMATE1 expression but also for post-translational regulation of VuMATE1 protein activity. These results suggest that citrate secretion is dependent on both transcriptional and post-transcriptional regulation of VuMATE1. Additionally, VuMATE1 promoter–β-glucuronidase fusion lines revealed that VuMATE1 expression was restricted to the root apex and was entirely Al induced, indicating the presence of cis-acting elements regulating root tip-specific and Al-inducible gene expression, which will be an important resource for genetic improvement of plant Al resistance.

Grain amaranth (Amaranthus hypochondriacus L.) is abundant in oxalate and can secrete oxalate under aluminium (Al) stress. However, the features of Al-induced secretion of organic acid anions (OA) and potential genes responsible for OA secretion are poorly understood. Here, Al-induced OA secretion in grain amaranth roots was characterized by ion charomatography and enzymology methods, and suppression subtractive hybridization (SSH) together with quantitative real-time PCR (qRT-PCR) was used to identify up-regulated genes that are potentially involved in OA secretion. The results showed that grain amaranth roots secrete both oxalate and citrate in response to Al stress. The secretion pattern, however, differs between oxalate and citrate. Neither lanthanum chloride (La) nor cadmium chloride (Cd) induced OA secretion. A total of 84 genes were identified as up-regulated by Al, in which six genes were considered as being potentially involved in OA secretion. The expression pattern of a gene belonging to multidrug and toxic compound extrusion (MATE) family, AhMATE1, was in close agreement with that of citrate secretion. The expression of a gene encoding tonoplast dicarboxylate transporter and four genes encoding ATP-binding cassette transporters was differentially regulated by Al stress, but the expression pattern was not correlated well with that of oxalate secretion. Our results not only reveal the secretion pattern of oxalate and citrate from grain amaranth roots under Al stress, but also provide some genetic information that will be useful for further characterization of genes involved in Al toxicity and tolerance mechanisms.