Non-Isocyanate Synthesis of Aliphatic Polyurethane by BiCl3-Catalyzed Transurethanization Polycondensation.

Non-isocyanate polyurethane synthesis by non-Sn catalysis is an essential challenge toward green polyurethane synthesis. Bismuth compounds are attractive candidates due to their low cost, low toxicity, and availability to urethane chemistry. This work applied various Bi catalysts to the self-polycondensation of a bishydroxyurethane monomer and found BiCl3 to be an excellent catalyst through optimization. The catalytic activity and price of BiCl3 are comparable to those of Bu2SnO, while its toxicity is significantly low. BiCl3 is, therefore, a promising alternative to Sn-based catalysts in non-isocyanate polyurethane synthesis.

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.

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.

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.

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.

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.

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 GWAS of PGIP1 Identifies STOP1-Dependent and STOP1-Independent Regulation of PGIP1 in Aluminum Stress Signaling in Arabidopsis.

To elucidate the unknown regulatory mechanisms involved in aluminum (Al)-induced expression of POLYGALACTURONASE-INHIBITING PROTEIN 1 (PGIP1), which is one of the downstream genes of SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) regulating Al-tolerance genes, we conducted a genome-wide association analysis of gene expression levels (eGWAS) of PGIP1 in the shoots under Al stress using 83 Arabidopsis thaliana accessions. The eGWAS, conducted through a mixed linear model, revealed 17 suggestive SNPs across the genome having the association with the expression level variation in PGIP1. The GWAS-detected SNPs were directly located inside transcription factors and other genes involved in stress signaling, which were expressed in response to Al. These candidate genes carried different expression level and amino acid polymorphisms. Among them, three genes encoding NAC domain-containing protein 27 (NAC027), TRX superfamily protein, and R-R-type MYB protein were associated with the suppression of PGIP1 expression in their mutants, and accordingly, the system affected Al tolerance. We also found the involvement of Al-induced endogenous nitric oxide (NO) signaling, which induces NAC027 and R-R-type MYB genes to regulate PGIP1 expression. In this study, we provide genetic evidence that STOP1-independent NO signaling pathway and STOP1-dependent regulation in phosphoinositide (PI) signaling pathway are involved in the regulation of PGIP1 expression under Al stress.

Comparative RNA-Seq analysis of the root revealed transcriptional regulation system for aluminum tolerance in contrasting indica rice of North East India.

Expression pattern of aluminum (Al) tolerance genes is one of the major determinants of Al avoidance/tolerance within plant cultivars. We have performed transcriptome analysis of two contrasting (Al-tolerant, Disang; Al-sensitive, Joymati) cultivars of India's North Eastern region, an indica rice diversity hotspot, on exposure to excess Al3+ treatment in acidic condition. Co-expression analysis and SNPs enrichment analysis proposed the role of both trans-acting and cis-acting polymorphisms in Al signaling in the Al-tolerant cultivar. We proposed ten major genes, including arginine decarboxylase, phytase, and beta-glucosidase aggregating factor as candidates responsible for Al tolerance based on transcriptome analysis. Al3+ stress led to changes in the alternative splicing profile of the Al-tolerant cultivar. These studies demonstrated the transcriptional variations affiliated to Al avoidance/tolerance in contrasting indica rice of North East India and provided us with several candidate genes responsible for Al tolerance.

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.

A single-population GWAS identified AtMATE expression level polymorphism caused by promoter variants is associated with variation in aluminum tolerance in a local Arabidopsis population.

Organic acids (OA) are released from roots in response to aluminum (Al), conferring an Al tolerance to plants that is regulated by OA transporters such as ALMT (Al-activated malate transporter) and multi-drug and toxic compound extrusion (MATE). We have previously reported that the expression level polymorphism (ELP) of AtALMT1 is strongly associated with variation in Al tolerance among natural accessions of Arabidopsis. However, although AtMATE is also expressed following Al exposure and contributes to Al tolerance, whether AtMATE contributes to the variation of Al tolerance and the molecular mechanisms of ELP remains unclear. Here, we dissected the natural variation in AtMATE expression level in response to Al at the root using diverse natural accessions of Arabidopsis. Phylogenetic analysis revealed that more than half of accessions belonging to the Central Asia (CA) population show markedly low AtMATE expression levels, while the majority of European populations show high expression levels. The accessions of the CA population with low AtMATE expression also show significantly weakened Al tolerance. A single-population genome-wide association study (GWAS) of AtMATE expression in the CA population identified a retrotransposon insertion in the AtMATE promoter region associated with low gene expression levels. This may affect the transcriptional regulation of AtMATE by disrupting the effect of a cis-regulatory element located upstream of the insertion site, which includes AtSTOP1 (sensitive to proton rhizotoxicity 1) transcription factor-binding sites revealed by chromatin immunoprecipitation-qPCR analysis. Furthermore, the GWAS performed without the accessions expressing low levels of AtMATE, excluding the effect of AtMATE promoter polymorphism, identified several candidate genes potentially associated with AtMATE expression.

Genome-Wide Association Study and Genomic Prediction Elucidate the Distinct Genetic Architecture of Aluminum and Proton Tolerance in Arabidopsis thaliana.

Under acid soil conditions, Al stress and proton stress can occur, reducing root growth and function. However, these stressors are distinct, and tolerance to each is governed by multiple physiological processes. To better understand the genes that underlie these coincidental but experimentally separable stresses, a genome-wide association study (GWAS) and genomic prediction (GP) models were created for approximately 200 diverse Arabidopsis thaliana accessions. GWAS and genomic prediction identified 140/160 SNPs associated with Al and proton tolerance, respectively, which explained approximately 70% of the variance observed. Reverse genetics of the genes in loci identified novel Al and proton tolerance genes, including TON1-RECRUITING MOTIF 28 (AtTRM28) and THIOREDOXIN H-TYPE 1 (AtTRX1), as well as genes known to be associated with tolerance, such as the Al-activated malate transporter, AtALMT1. Additionally, variation in Al tolerance was partially explained by expression level polymorphisms of AtALMT1 and AtTRX1 caused by cis-regulatory allelic variation. These results suggest that we successfully identified the loci that regulate Al and proton tolerance. Furthermore, very small numbers of loci were shared by Al and proton tolerance as determined by the GWAS. There were substantial differences between the phenotype predicted by genomic prediction and the observed phenotype for Al tolerance. This suggested that the GWAS-undetectable genetic factors (e.g., rare-allele mutations) contributing to the variation of tolerance were more important for Al tolerance than for proton tolerance. This study provides important new insights into the genetic architecture that produces variation in the tolerance of acid soil.

Sensitive to Proton Rhizotoxicity1 Regulates Salt and Drought Tolerance of Arabidopsis thaliana through Transcriptional Regulation of CIPK23.

The transcription factor sensitive to proton rhizotoxicity 1 (STOP1) regulates multiple stress tolerances. In this study, we confirmed its involvement in NaCl and drought tolerance. The root growth of the T-DNA insertion mutant of STOP1 (stop1) was sensitive to NaCl-containing solidified MS media. Transcriptome analysis of stop1 under NaCl stress revealed that STOP1 regulates several genes related to salt tolerance, including CIPK23. Among all available homozygous T-DNA insertion mutants of the genes suppressed in stop1, only cipk23 showed a NaCl-sensitive root growth phenotype comparable to stop1. The CIPK23 promoter had a functional STOP1-binding site, suggesting a strong CIPK23 suppression led to NaCl sensitivity of stop1. This possibility was supported by in planta complementation of CIPK23 in the stop1 background, which rescued the short root phenotype under NaCl. Both stop1 and cipk23 exhibited a drought tolerant phenotype and increased abscisic acid-regulated stomatal closure, while the complementation of CIPK23 in stop1 reversed these traits. Our findings uncover additional pleiotropic roles of STOP1 mediated by CIPK23, which regulates various ion transporters including those regulating K+-homeostasis, which may induce a trade-off between drought tolerance and other traits.

Involvement of phosphatidylinositol metabolism in aluminum-induced malate secretion in Arabidopsis.

To identify the upstream signaling of aluminum-induced malate secretion through aluminum-activated malate transporter 1 (AtALMT1), a pharmacological assay using inhibitors of human signal transduction pathways was performed. Early aluminum-induced transcription of AtALMT1 and other aluminum-responsive genes was significantly suppressed by phosphatidylinositol 4-kinase (PI4K) and phospholipase C (PLC) inhibitors, indicating that the PI4K-PLC metabolic pathway activates early aluminum signaling. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and PI4K reduced aluminum-activated malate transport by AtALMT1, suggesting that both the PI3K and PI4K metabolic pathways regulate this process. These results were validated using T-DNA insertion mutants of PI4K and PI3K-RNAi lines. A human protein kinase inhibitor, putatively inhibiting homologous calcineurin B-like protein-interacting protein kinase and/or Ca-dependent protein kinase in Arabidopsis, suppressed late-phase aluminum-induced expression of AtALMT1, which was concomitant with the induction of an AtALMT1 repressor, WRKY46, and suppression of an AtALMT1 activator, Calmodulin-binding transcription activator 2 (CAMTA2). In addition, a human deubiquitinase inhibitor suppressed aluminum-activated malate transport, suggesting that deubiquitinases can regulate this process. We also found a reduction of aluminum-induced citrate secretion in tobacco by applying inhibitors of PI3K and PI4K. Taken together, our results indicated that phosphatidylinositol metabolism regulates organic acid secretion in plants under aluminum stress.

STOP1 regulates the expression of HsfA2 and GDHs that are critical for low-oxygen tolerance in Arabidopsis.

The SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) transcription factor regulates gene expression associated with multiple stress tolerances in plant roots. In this study, we investigated the mechanism responsible for the sensitivity of the stop1 mutant to low-oxygen stress in Arabidopsis. Transcriptomic analyses revealed that two genes involved in low-oxygen tolerance, namely GLUTAMATE DEHYDROGENASE 1 (GDH1) and GDH2, showed lower expression levels in the stop1 mutant than in the wild-type. Sensitivity of the gdh1gdh2 double-mutant to low-oxygen conditions was partly attributable to the low-oxygen sensitivity of the stop1 mutant. Two transcription factors, STOP2 and HEAT SHOCK FACTOR A2 (HsfA2), were expressed at lower levels in the stop1 mutant. An in planta complementation assay indicated that CaMV35S::STOP2 or CaMV35S::HsfA2 partially rescued the low-oxygen tolerance of the stop1 mutant, which was concomitant with recovered expression of genes regulating low-pH tolerance and genes encoding molecular chaperones. Prediction of cis-elements and in planta promoter assays revealed that STOP1 directly activated the expression of HsfA2. Similar STOP1-dependent low-oxygen sensitivity was detected in tobacco. Suppression of NtSTOP1 induced low-oxygen sensitivity, which was associated with lower expression levels of NtHsfA2 and NtGDHs compared with the wild-type. Our results indicated that STOP1 pleiotropically regulates low-oxygen tolerance by transcriptional regulation.

Agrobacterium rhizogenes-mediated hairy roots transformation as a tool for exploring aluminum-responsive genes function.

AIM: To develop a useful alternative approach to evaluate the gene function in hairy roots. METHODS: Arabidopsis and tobacco (wild-type or mutant) were a host for Agrobacterium rhizogenes transformation. RESULTS: The hairy roots formation efficiency ranged from 53 to 98% in tobacco and 53 to 66% in Arabidopsis. Hairy and intact roots showed similar gene expression pattern in response to salt and aluminum stress. Genomic polymerase chain reaction and fluorescent images showed high rate (>80%) of co-integration of T-DNAs and uniform cell transformation without use of any antibiotic selection. Whole processes of hairy roots were completed within 1 month after the infection of Agrobacterium. CONCLUSION: Aluminum-responsive orthologous gene function could be evaluated by NtSTOP1-KD and Atstop1 as a host for hairy roots transformation.

Comparative genome-wide analysis of WRKY transcription factors in two Asian legume crops: Adzuki bean and Mung bean.

The seminal participation of WRKY transcription factors in plant development, metabolism and in the governance of defense mechanism implicated their gaining importance for genomic and functional studies. The recent release of draft genome sequences of two legume crops, Adzuki bean (Vigna angularis) and Mung bean (Vigna radiata) has paved the way for characterization of WRKY gene family in these crops. We found 84 WRKY genes in Adzuki bean (VaWRKY) and 85 WRKY genes in Mung bean (VrWRKY). Based on the phylogenetic analysis, VaWRKY genes were classified into three groups with 15 members in Group I, 56 members in Group II, and 13 members in Group III, which was comparable to VrWRKY distribution in Mung bean, 16, 56 and 13 members in Group I, II and III, respectively. The few tandem and segmental duplication events suggested that recent duplication plays no prominent role in the expansion VaWRKY and VrWRKY genes. The illustration of gene-structure and their encoded protein-domains further revealed the nature of WRKY proteins. Moreover, the identification of abiotic or biotic stress-responsive cis-regulatory elements in the promoter regions of some WRKY genes provides fundamental insights for their further implementation in stress-tolerance and genetic improvement of agronomic traits.