Analysis of widely targeted metabolites of quinoa sprouts (Chenopodium quinoa Willd.) under saline-alkali stress provides new insights into nutritional value.

Quinoa sprouts are a green vegetable rich in bioactive chemicals, which have multiple health benefits. However, there is limited information on the overall metabolic profiles of quinoa sprouts and the metabolite changes caused by saline-alkali stress. Here, a UHPLC-MS/MS-based widely targeted metabolomics technique was performed to comprehensively evaluate the metabolic profiles of quinoa sprouts and characterize its metabolic response to saline-alkali stress. A total of 930 metabolites were identified of which 232 showed significant response to saline-alkali stress. The contents of lipids and amino acids were significantly increased, while the contents of flavonoids and phenolic acids were significantly reduced under saline-alkali stress. Moreover, the antioxidant activities of quinoa sprouts were significantly affected by saline-alkali stress. The enrichment analysis of the differentially accumulated metabolites revealed that flavonoid, amino acid and carbohydrate biosynthesis/metabolism pathways responded to saline-alkali stress. This study provided an important theoretical basis for evaluating the nutritional value of quinoa sprouts and the changes in metabolites in response to saline-alkali stress.

The Glycine soja cytochrome P450 gene GsCYP82C4 confers alkaline tolerance by promoting reactive oxygen species scavenging.

Recent studies have demonstrated the crucial role of Cytochrome P450 enzymes (CYPs) in the production of secondary metabolites, phytohormones and antioxidants in plants. However, their functional characterization specifically under alkaline stress remains elusive. CYP82C4 was the key gene screened from a family of wild soybean CYPs in our previous studies. The aim of this present study was to clone the Glycine soja GsCYP82C4 gene and characterize its functions in Arabidopsis and Glycine max. The results showed that the GsCYP82C4 gene displayed a high expression in different plant tissues at mature stages compared to young stages. Further, higher temporal expression of the GsCYP82C4 gene was noted at 6, 12 and 24 h time points after alkali treatment in leaves compared to roots. In addition, overexpression of GsCYP82C4 improved alkaline stress tolerance in Arabidopsis via increased root lengths and fresh biomass and strengthened the antioxidant defense system via a reduction in superoxide radicals in transgenic lines compared to wild type (WT) and atcyp82c4 mutants. Further, the expression levels of stress-related marker genes were up-regulated in GsCYP82C4 OX lines under alkali stress. The functional analysis of GsCYP82C4 overexpression in soybean displayed better hairy root growth, increased fresh weight, higher antioxidant enzyme activities and reduced lipid peroxidation rates in OX lines compared to the soybean WT (K599) line. In total, our study displayed positive roles of GsCYP82C4 overexpression in both Arabidopsis and Glycine max to alleviate alkaline stress via altering expression abundance of stress responsive genes, stronger roots, higher antioxidant enzyme activities as well as reduced rates of lipid peroxidation and superoxide radicals.

A DUF966 gene family member OsDSR3 positively regulates alkali stress tolerance in rice.

Rice growth and production are severely constrained by alkali stress. However, the mechanism underlying the rice tolerance to alkali stress is unclear. OsDSR3, a novel gene from the domains of unknown function 966 (DUF966) family, was identified and characterized for its function in the response of rice to alkali stress. The result of this study clearly showed that alkali stress significantly induced OsDSR3 expression level. Moreover, the expression of OsDSR3 was up-regulated by drought, salt, cold, H2O2 and abscisic acid (ABA), and down-regulated by gibberellic acid (GA3), and 2,4-Dichlorophenoxyacetic acid (2,4-D) treatments. Subcellular localization exhibited that OsDSR3 was detected in the nucleus and membrane. OsDSR3-overexpressing (OsDSR3-OE) plants showed higher tolerance to alkali stress than the wild-type (WT). In contrast, OsDSR3 knockout (OsDSR3-KO) mutants were more vulnerable to alkali stress. The differentially expressed genes (DEGs) among OsDSR3-OE and WT seedlings were mainly enriched in porphyrin and chlorophyll, starch and sucrose, and carotenoid metabolic pathways. Among these DEGs, 26 were identified as potential alkali stress-responsive genes, including several up-regulated genes like OsHAK5, OsGRX23 and OsNIR2. Consistent with the expression profiles of metabolic pathways-related genes, most of the metabolite contents and metabolite synthases activities were improved in OsDSR3-OE lines and decreased in OsDSR3-KO lines compared to WT. This may explain the higher tolerance of OE lines and lower tolerance of KO lines to alkali stress. These findings suggested that OsDSR3 positively regulates rice tolerance to alkali stress, which will help to elucidate the molecular mechanism underlying rice alkali tolerance.

Oxalic acid secretion alleviates saline-alkali stress in alfalfa by improving photosynthetic characteristics and antioxidant activity.

Saline-alkali stress significantly affects the growth and yield of alfalfa (Medicago sativa L.). Organic acid secretion is crucial in alleviating abiotic stress-induced damage in plants. In this study, we evaluated the contents of the major organic acids secreted by the roots of tolerant (ZD) and sensitive (LYL) varieties of alfalfa under saline-alkali stress and investigated the effects of these organic acids on the growth, and physiological functions of alfalfa. Our results indicated that the oxalic acid (OA) content was the highest among the organic acids secreted from alfalfa roots under saline-alkali stress, and oxalic acid content was the most significantly different between the two varieties, ZD and LYL, compared to the contents of the other organic acids. Oxalic acid alleviated the inhibition of alfalfa growth caused by saline-alkali stress, improved photosynthetic characteristics, reduced the accumulation of reactive oxygen species, and increased the activity of antioxidant enzymes and content of osmoregulatory substances. Furthermore, oxalic acid resulted in significantly increased expression of genes involved in photosynthesis and antioxidant system in alfalfa under saline-alkali stress. This study revealed the effects of oxalic acid secreted by the root system on stress-related physiological processes, providing valuable insights into the functions of root secretions in plant saline-alkali resistance.

Integrated Metabolomics and Transcriptomics Analyses Highlight the Flavonoid Compounds Response to Alkaline Salt Stress in Glycyrrhiza uralensis Leaves.

Glycyrrhiza uralensis is a saline-alkali-tolerant plant whose aerial parts are rich in flavonoids; however, the role of these flavonoids in saline-alkali tolerance remains unclear. Herein, we performed physiological, metabolomics, and transcriptomics analyses in G. uralensis leaves under alkaline salt stress for different durations. Alkaline salt stress stimulated excessive accumulation of reactive oxygen species and consequently destroyed the cell membrane, causing cell death, and G. uralensis initiated osmotic regulation and the antioxidant system to respond to stress. In total, 803 metabolites, including 244 flavonoids, were detected via metabolomics analysis. Differentially altered metabolites and differentially expressed genes were coenriched in flavonoid-related pathways. Genes such as novel.4890, Glyur001511s00039602, and Glyur000775s00025737 were highly expressed, and flavonoid metabolites such as 2'-hydroxygenistein, apigenin, and 3-O-methylquercetin were upregulated. Thus, flavonoids as nonenzymatic antioxidants play an important role in stress tolerance. These findings provide novel insights into the response of G. uralensis to alkaline salt stress.

MWCNTs Alleviated saline-alkali stress by optimizing photosynthesis and sucrose metabolism in rice seedling.

Saline and alkali stress affects the growth and development, survival rate, and final yield of rice, while new nano materials can have a positive effect on rice growth. In order to investing the effects of carboxymethyl multi walled carbon nanotubes (MWCNTs) on the growth and development of rice seedlings under salt alkali stress, rice seedlings were cultured using rice variety "Songjing 3" using nutrient solution water culture method. The effects of MWCNTs on water absorption capacity, leaf photosynthesis, and sucrose metabolism of rice seedlings under 50 mmol/L saline-alkali stress (1NaCl: 9Na2SO4: 9NaHCO3: 1Na2CO3) conditions were investigated. The results showed that MWCNTs can improve the water use ability of roots and leaves, especially the water absorption ability of roots, which provides a guarantee for the improvement of rice biomass and the enhancement of leaf photosynthetic capacity under adverse conditions. After treatment with MWCNTs, the photosynthetic rate (Pn), stomatal conductance (gs), and transpiration rate (Tr) of leaves increased significantly, and the photochemical quenching value (qP), photochemical quantum efficiency value (Fv/Fm), and electron transfer rate value (ETR) of chlorophyll fluorescence parameters increased significantly, which is beneficial to the improvement of the PSII photosynthetic system. MWCNTs treatment promoted the increase of photosynthetic pigment content in leaves under salt and alkali stress, improved the ratio of Chla and Chlb parameters, increased the activities of key photosynthetic enzymes (RUBPCase and PEPCase) in leaves, increased the value of total lutein cycle pool (VAZ), and significantly enhanced the deepoxidation effect of lutein cycle (DEPS), which can effectively alleviate the stomatal and non stomatal constraints on leaf photosynthesis caused by salt and alkali stress. MWCNTs treatment significantly enhanced the activities of sucrose phosphate synthase (SPS) and sucrose synthase (SS) under salt and alkali stress, and decreased the activities of soluble acid invertase (SAInv) and alkaline/neutral invertase (A/N-Inv), indicating that MWCNTs promoted sucrose synthesis while inhibiting sucrose decomposition, thereby promoting sucrose accumulation in rice leaves. This study can provide theoretical and experimental basis for the application of MWCNTs to the production of rice under salt and alkali stress, and can find a new way for rice production in saline and alkaline lands.

The Transcription Factor ZmNAC89 Gene Is Involved in Salt Tolerance in Maize (Zea mays L.).

The NAC gene family has transcription factors specific to plants, which are involved in development and stress response and adaptation. In this study, ZmNAC89, an NAC gene in maize that plays a role in saline-alkaline tolerance, was isolated and characterized. ZmNAC89 was localized in the nucleus and had transcriptional activation activity during in vitro experiments. The expression of ZmNAC89 was strongly upregulated under saline-alkaline, drought and ABA treatments. Overexpression of the ZmNAC89 gene in transgenic Arabidopsis and maize enhanced salt tolerance at the seedling stage. Differentially expressed genes (DEGs) were then confirmed via RNA-sequencing analysis with the transgenic maize line. GO analyses showed that oxidation-reduction process-regulated genes were involved in ZmNAC89-mediated salt-alkaline stress. ZmNAC89 may regulate maize saline-alkali tolerance through the REDOX pathway and ABA signal transduction pathway. From 140 inbred maize lines, 20 haplotypes and 16 SNPs were found in the coding region of the ZmNAC89 gene, including the excellent haplotype HAP20. These results contribute to a better understanding of the response mechanism of maize to salt-alkali stress and marker-assisted selection during maize breeding.

A Soybean Sucrose Non-Fermenting Protein Kinase 1 Gene, GmSNF1, Positively Regulates Plant Response to Salt and Salt-Alkali Stress in Transgenic Plants.

Soybean is one of the most widely grown oilseed crops worldwide. Several unfavorable factors, including salt and salt-alkali stress caused by soil salinization, affect soybean yield and quality. Therefore, exploring the molecular basis of salt tolerance in plants and developing genetic resources for genetic breeding is important. Sucrose non-fermentable protein kinase 1 (SnRK1) belongs to a class of Ser/Thr protein kinases that are evolutionarily highly conserved direct homologs of yeast SNF1 and animal AMPKs and are involved in various abiotic stresses in plants. The GmPKS4 gene was experimentally shown to be involved with salinity tolerance. First, using the yeast two-hybrid technique and bimolecular fluorescence complementation (BiFC) technique, the GmSNF1 protein was shown to interact with the GmPKS4 protein. Second, the GmSNF1 gene responded positively to salt and salt-alkali stress according to qRT-PCR analysis, and the GmSNF1 protein was localized in the nucleus and cytoplasm using subcellular localization assay. The GmSNF1 gene was then heterologously expressed in yeast, and the GmSNF1 gene was tentatively identified as having salt and salt-alkali tolerance function. Finally, the salt-alkali tolerance function of the GmSNF1 gene was demonstrated by transgenic Arabidopsis thaliana, soybean hairy root complex plants overexpressing GmSNF1 and GmSNF1 gene-silenced soybean using VIGS. These results indicated that GmSNF1 might be useful in genetic engineering to improve plant salt and salt-alkali tolerance.

Functional identification of ANR genes in apple (Malus halliana) that reduce saline-alkali stress tolerance.

As one of the major abiotic stresses restricting the development of global agriculture, saline-alkali stress causes osmotic stress, ion poisoning, ROS damage and high pH damage, which seriously restrict sustainable development of fruit industry. Therefore, it is essential to develop and cultivate saline-alkali-resistant apple rootstocks to improve the yield and quality of apples in China. Based on transcriptome data, MhANR (LOC114827797), which is significantly induced by saline-alkali stress, was cloned from Malus halliana. The physicochemical properties, evolutionary relationships and cis-acting elements were analysed. Subsequently, the tolerance of MhANR overexpression in Arabidopsis thaliana, tobacco, and apple calli to saline-alkali stress was verified through genetic transformation. Transgenic plants contained less Chl a, Chl b and proline, SOD, POD and CAT activity, and higher relative electrical conductivity (REC) compared to WT plants under saline-alkali stress. In addition, expression of saline-alkali stress-related genes in overexpressed apple calli were also lower than in WT calli, including the antioxidant genes (MhSOD and MhCAT^), the Na+ transporter genes (MhCAX5, MhCAX5, MhSOS1, MhALT1), and the H+ -ATPase genes (MhAHA2 and MhAHA8), while expression of the K+ transporter genes (MhSKOR and MhNHX4) were higher. Expression of MhANR reduced tolerance of A. thaliana, tobacco, and apple calli to saline-alkali stress by regulating osmoregulatory substances, chlorophyll content, antioxidant enzyme activity, and expression of saline-alkali stress-related genes. This research provides a theoretical basis for cultivating apple rootstocks with effective saline-alkali stress tolerance.

Isolation and Identification of a Phosphate-Solubilizing Pantoea dispersa with a Saline-Alkali Tolerance and Analysis of Its Growth-Promoting Effects on Silage Maize Under Saline-Alkali Field Conditions.

Phosphate-solubilizing bacteria (PSB) are microorganisms that can dissolve insoluble phosphorus (P) to accessible forms. This study aimed to screen saline-alkali-tolerant PSB and analyze its growth promoting properties, and evaluate its effects on the growth, quality, soil nutrient balance, and enzyme activities of silage maize in the field. We isolated six phosphate-solubilizing strains from rhizosphere soil of silage maize planted in saline-alkali land, and FC-1 with the best P-solubilizing effect was used for further study. The morphological, physiological and biochemical analysis, and 16S rDNA and housekeeping gene atpD sequencing were performed for identification. FC-1 was identified as Pantoea dispersa and had high P solubility. The phosphate solubility of FC-1 using four P sources ranged from 160.79 to 270.22 mg l-1. FC-1 produced indole-3-acetic acid (IAA) and decreased the pH of the growth media by secreting organic acids, including citric acid, malic acid, succinic acid, and acetic acid. The results of a field experiment indicated that FC-1 treatment increased the height, stem diameter, fresh weight, dry weight, starch content, crude protein content, and total P content of silage maize by 9.8, 9.2, 12.6, 11.7, 12.6, 18.3, and 17.4%, respectively. The nitrogen, potassium, phosphorus, and organic matter contents in the rhizosphere soil of silage maize increased by 29.8, 17.1, 17.9, and 25.3%, respectively; urease, catalase, sucrase, and alkaline phosphatase levels also increased by 24.7, 26.7, 24.0, and 19.5%, respectively. FC-1 promoted the growth of silage maize by improving nutrient metabolism and enzyme activities in saline-alkali soil and may be an effective alternative to fertilizers.

Collagen membrane loaded with doxycycline through hydroxypropyl chitosan microspheres for the early reconstruction of alkali-burned cornea.

Corneal alkali burn is one of the most devastating ophthalmic emergencies correlated with remarkable morbidity resulting in severe visual impairment. Appropriate intervention in the acute phase determines the eventual outcome for later corneal restoration treatment. Since the epithelium plays an essential role in inhibiting inflammation and promoting tissue repair, sustained anti-matrix metalloproteinases (MMPs) and pro-epithelialization are the prior remedies during the first week. In this study, a drug-loaded collagen membrane (Dox-HCM/Col) that could be sutured to overlay the burned cornea was developed to accelerate the early reconstruction. Doxycycline (Dox), a specific inhibitor of MMPs, was encapsulated in collagen membrane (Col) through hydroxypropyl chitosan microspheres (HCM) to develop Dox-HCM/Col, affording a preferable pro-epithelialization microenvironment and an in-situ controlled release. Results showed that loading HCM into Col prolonged the release time to 7 days, and Dox-HCM/Col could significantly suppress the expression of MMP-9 and -13 in vitro and in vivo. Furthermore, the membrane accelerated the corneal complete re-epithelialization and promoted early reconstruction within the first week. Overall, Dox-HCM/Col was a promising biomaterial membrane for treating alkali-burned cornea in the early stage, and our attempt may provide a clinically feasible method for the ocular surface reconstruction.

Transitory alkali exposure on meibomian gland orifices induces meibomian gland dysfunction.

PURPOSE: To determine pathological changes of meibomian glands (MGs) after transient exposure of the rat eyelid margin to alkali solution. METHODS: Filter paper infiltrated with 1 N sodium hydroxide solution was applied to the eyelid margin of Sprague-Dawley rats for 30 s under general anesthesia, without touching the conjunctiva, after which the ocular surface and eyelid margin were examined by slit-lamp microscopy. In vivo confocal microscopy and stereomicroscopy were subsequently applied to observe MG morphology on day 5, day 10 and day 30 post alkali injury. Eyelid cross-sections were processed for H&E staining, Oil red O staining and immunofluorescent staining. RESULTS: After alkali injury, there was marked plugging of MG orifices, telangiectasia and hypertrophy of the eyelid margin, while corneal epithelium was intact at post-injury days 5 and 10. However, 30 days after alkali injury, mild corneal epithelial damage was observed. Degeneration of MG acini was observed at days 5 and became aggravated at days 10 and 30, along with MG duct dilation and acini loss. Oil red O staining showed lipid accumulation in the dilated duct. Inflammatory cell infiltration and the presence of apoptotic cells was seen in the MG loci 5 days post injury, but diminished at days 10 and 30. Cytokeratin 10 expression was increased in dilated duct, while cytokeratin 14, PPAR-γ, Ki67 and LRIG1 expression were decreased in the acini of injured loci. CONCLUSIONS: Transitory alkali exposure of the rat eyelid margin obstructs the MG orifice and induces pathological changes of MG dysfunction.

Integrated metabolomic and transcriptomic strategies to reveal alkali-resistance mechanisms in wild soybean during post-germination growth stage.

MAIN CONCLUSION: The keys to alkali-stress resistance of barren-tolerant wild soybean lay in enhanced reutilization of reserves in cotyledons as well as improved antioxidant protection and organic acid accumulation in young roots. Soil alkalization of farmlands is increasingly serious, adversely restricting crop growth and endangering food security. Here, based on integrated analysis of transcriptomics and metabolomics, we systematically investigated changes in cotyledon weight and young root growth in response to alkali stress in two ecotypes of wild soybean after germination to reveal alkali-resistance mechanisms in barren-tolerant wild soybean. Compared with barren-tolerant wild soybean, the dry weight of common wild soybean cotyledons under alkali stress decreased slowly and the length of young roots shortened. In barren-tolerant wild soybean, nitrogen-transport amino acids asparagine and glutamate decreased in cotyledons but increased in young roots, and nitrogen-compound transporter genes and genes involved in asparagine metabolism were significantly up-regulated in both cotyledons and young roots. Moreover, isocitric, succinic, and L-malic acids involved in the glyoxylate cycle significantly accumulated and the malate synthetase gene was up-regulated in barren-tolerant wild soybean cotyledons. In barren-tolerant wild soybean young roots, glutamate and glycine related to glutathione metabolism increased significantly and the glutathione reductase gene was up-regulated. Pyruvic acid and citric acid involved in pyruvate-citrate metabolism increased distinctly and genes encoding pyruvate decarboxylase and citrate synthetase were up-regulated. Integrated analysis showed that the keys to alkali-stress resistance of barren-tolerant wild soybean lay in enhanced protein decomposition, amino acid transport, and lipolysis in cotyledons as well as improved antioxidant protection and organic acid accumulation in young roots. This study provides new ideas for the exploitation and utilization of wild soybean resources.

Responses of Rice and Related Cadmium Transporter Genes to the Passivating Microbial Agent MBLHAP.

Alkali-producing microorganisms and hydroxyapatite (HAP), a chemical passivation agent, have a certain remediation effect on cadmium (Cd) -contaminated soil. They can decrease the available Cd content in the soil to a certain extent and reduce the overall Cd content of rice planted in the soil. The Cd-contaminated soil was treated with the passivating bacterial agent that had been developed. Changes in the Cd concentration of rice leaves and soil were observed. Real-time PCR was used to analyse the expression levels of Cd transport protein genes in rice. Then, we determined the activities of super-oxide dismutase (SOD), catalase (CAT) and peroxidase (POD) at different stages of rice growth. The results showed that after HAP, alkali-producing microorganisms and passivating microbial agents were applied to the Cd treated soil. The total Cd content in rice leaves was reduced by 66.80%, 80.32% and 81.35%. The expression differences of genes related to Cd transporter proteins were measured, and the results showed that the changes in gene regulation were consistent with the changes in Cd content of rice leaves. The changes in SOD activity, CAT activity and POD activity further indicated that the three enzymes could alleviate the adverse effects of Cd stress by regulating the related enzyme activities in rice. In conclusion, alkali-producing microorganisms, HAP and passivating bacterial agents can effectively reduce the toxicity of Cd to rice, and reduce the absorption and accumulation of Cd in rice leaves.

Insights into the genomic evolution and the alkali tolerance mechanisms of Agaricus sinodeliciosus by comparative genomic and transcriptomic analyses.

Agaricus sinodeliciosus is a rare wild edible mushroom from northwest China, and grows naturally in mild saline-alkali soil, which is also unusual in mushrooms. A. sinodeliciosus represents a potential model organism for explaining saline-alkali tolerance mechanisms and revealing related physiological processes in mushrooms. Here, we provide a high-quality genome of A. sinodeliciosus. Comparative genomic analyses reveal A. sinodeliciosus has numerous changes to its genome organization after a solitary evolutionary history under saline-alkali environments, such as gene family contraction, retrotransposon expansion and rapid evolution of adaptative genes. Our saline and alkali tolerance tests show that mycelium growth and fruit body formation of this species are effected by mild alkalinity. Transcriptomic analyses reveal that genes involved in carbon and nitrogen utilization, cell stability and fruit body formation of A. sinodeliciosus could be activated under mildly alkaline conditions. In particular, the 'starch and sucrose metabolism', 'biosynthesis of amino acids' and 'phenylpropanoid biosynthesis' pathways are important for mildly alkaline tolerance of A. sinodeliciosus. Like plants and arbuscular mycorrhizal fungi, in the rot fungus A. sinodeliciosus, the biosynthesis of intracellular small molecules could be enhanced to counter osmotic and oxidative stresses caused by mild alkalinity, and the biosynthesis of monolignol could be suppressed to increase cell wall infiltrates under mildly alkaline conditions. This research provides an understanding of the genomic evolution and mechanisms of A. sinodeliciosus in tolerance to saline-alkali environments. The A. sinodeliciosus genome constitutes a valuable resource for evolutionary and ecological studies of Agaricus.

PS II Subunit P in Lilium pumilum (LpPsbP) Confers Saline-Alkali Resistance to the Plant by Scavenging ROS.

(1) Background: The growth of plants is impacted by salinity and alkali, Lilium pumilum (L. pumilum) is an ornamental plant with strong resistance to salinity and alkali, while the LpPsbP gene is helpful to fully understand the Saline-Alkali tolerance of L. pumilum. (2) Methods: Gene cloning, bioinformatics analysis, expression of fusion protein, determination of physiological indices of plant after Saline-Alkali stress, yeast two-hybrid screening, luciferase complementation assay, chromosome walking to obtain the promoter sequence, and then analyzed by PlantCARE. (3) Results: The LpPsbP gene was cloned and the fusion protein was purified. The transgenic plants had higher Saline-Alkali resistance than the wild type. A total of eighteen proteins interacting with LpPsbP were screened, and nine sites in the promoter sequence were analyzed. (4) Conclusion: Under Saline-Alkali or oxidative stress, L. pumilum will promote the expression of LpPsbP, which will then directly scavenge reactive oxygen species (ROS) in order to protect its photosystem II, reduce its damage, and thus improve the Saline-Alkali resistance of the plant. Moreover, according to some of the literature and the following experiments, two additional speculations are developed on the mechanisms of how two newly found objects, namely jasmonic acid (JA) and FoxO protein, could be involved in ROS scavenging processes were made.

Upadacitinib inhibits corneal inflammation and neovascularization by suppressing M1 macrophage infiltration in the corneal alkali burn model.

Alkali burn-induced corneal inflammation and subsequent corneal neovascularization (CNV) are major causes of corneal opacity and vision loss. M1 macrophages play a central role in inflammation and CNV. Therefore, modulation of M1 macrophage polarization is a promising strategy for corneal alkali burns. Here, we illustrate the effect and underlying mechanisms of upadacitinib on corneal inflammation and CNV induced by alkali burns in mice. The corneas of BALB/c mice were administered with 1 M NaOH for 30 s and randomly assigned to the vehicle group and the upadacitinib-treated group. Corneal opacity and corneal epithelial defects were assessed clinically. Quantitative real-time PCR (qRT-PCR), immunohistochemistry, and western blot analysis were performed to detect M1 macrophage polarization and CD31+ corneal blood vessels. The results showed that upadacitinib notably decreased corneal opacity, and promoted corneal wound healing. On day 7 and 14 after alkali burns, upadacitinib significantly suppressed CNV. Corneal alkali injury caused M1 macrophage recruitment in the cornea. In contrast to the vehicle, upadacitinib suppressed M1 macrophage infiltration and decreased the mRNA expression levels of inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1ß, and vascular endothelial growth factor A (VEGF-A) in alkali-injured corneas. Moreover, upadacitinib dose-dependently inhibited M1 macrophage polarization by suppressing interferon (IFN)-γ-/lipopolysaccharide-stimulated STAT1 activation in vitro. Our findings reveal that upadacitinib can efficiently alleviate alkali-induced corneal inflammation and neovascularization by inhibiting M1 macrophage infiltration. These data demonstrate that upadacitinib is an effective drug for the treatment of corneal alkali burns.

Genome-Wide Characterization and Expression Profiling of the GRAS Gene Family in Salt and Alkali Stresses in Miscanthus sinensis.

The GRAS family genes encode plant-specific transcription factors that play important roles in a diverse range of developmental processes and abiotic stress responses. However, the information of GRAS gene family in the bioenergy crop Miscanthus has not been available. Here, we report the genome-wide identification of GRAS gene family in Micanthus sinensis. A total of 123 MsGRAS genes were identified, which were divided into ten subfamilies based on the phylogenetic analysis. The co-linearity analysis revealed that 59 MsGRAS genes experienced segmental duplication, forming 35 paralogous pairs. The expression of six MsGRAS genes in responding to salt, alkali, and mixed salt-alkali stresses was analyzed by transcriptome and real-time quantitative PCR (RT-qPCR) assays. Furthermore, the role of MsGRAS60 in salt and alkali stress response was characterized in transgenic Arabidopsis. The MsGRAS60 overexpression lines exhibited hyposensitivity to abscisic acid (ABA) treatment and resulted in compromised tolerance to salt and alkali stresses, suggesting that MsGRAS60 is a negative regulator of salt and alkali tolerance via an ABA-dependent signaling pathway. The salt and alkali stress-inducible MsGRAS genes identified serve as candidates for the improvement of abiotic stress tolerance in Miscanthus.

Cell Wall Matrix Polysaccharides Contribute to Salt-Alkali Tolerance in Rice.

Salt-alkali stress threatens the resilience to variable environments and thus the grain yield of rice. However, how rice responds to salt-alkali stress at the molecular level is poorly understood. Here, we report isolation of a novel salt-alkali-tolerant rice (SATR) by screening more than 700 germplasm accessions. Using 93-11, a widely grown cultivar, as a control, we characterized SATR in response to strong salt-alkali stress (SSAS). SATR exhibited SSAS tolerance higher than 93-11, as indicated by a higher survival rate, associated with higher peroxidase activity and total soluble sugar content but lower malonaldehyde accumulation. A transcriptome study showed that cell wall biogenesis-related pathways were most significantly enriched in SATR relative to 93-11 upon SSAS. Furthermore, higher induction of gene expression in the cell wall matrix polysaccharide biosynthesis pathway, coupled with higher accumulations of hemicellulose and pectin as well as measurable physio-biochemical adaptive responses, may explain the strong SSAS tolerance in SATR. We mapped SSAS tolerance to five genomic regions in which 35 genes were candidates potentially governing SSAS tolerance. The 1,4-ß-D-xylan synthase gene OsCSLD4 in hemicellulose biosynthesis pathway was investigated in details. The OsCSLD4 function-disrupted mutant displayed reduced SSAS tolerance, biomass and grain yield, whereas the OsCSLD4 overexpression lines exhibited increased SSAS tolerance. Collectively, this study not only reveals the potential role of cell wall matrix polysaccharides in mediating SSAS tolerance, but also highlights applicable value of OsCSLD4 and the large-scale screening system in developing SSAS-tolerant rice.

PsnWRKY70 Negatively Regulates NaHCO3 Tolerance in Populus.

Poplar is an important afforestation and ornamental tree species in Northeast China. The distribution area of saline-alkali land is approximately 765 hm2 in Northeast China. The breeding of saline-alkali-resistant transgenic trees could be an effective method of afforestation in saline-alkali land. WRKY transcription factors play a crucial role in abiotic stress. In this study, we analyzed the genetic stability of the two-year-old PsnWRKY70 transgenic poplars. The results showed that PsnWRKY70 of transgenic poplars had been expressed stably and normally at the mRNA level. The gene interference expression (RE) lines had no significant effect on the growth of PsnWRKY70 under NaHCO3 stress, and the alkali damage index of RE lines was significantly lower than that of WT and overexpression (OE) lines at day 15 under NaHCO3 stress. POD activity was significantly higher in RE lines than in WT. The MDA content of the RE line was lower than that of the WT line. Transcriptome analysis showed that RE lines up-regulated genes enriched in cell wall organization or biogenesis pathway-related genes such as EXPA8, EXPA4, EXPA3, EXPA1, EXPB3, EXP10, PME53, PME34, PME36, XTH9, XTH6, XTH23, CESA1, CESA3, CES9; FLA11, FLA16 and FLA7 genes. These genes play an important role in NaHCO3 stress. Our study showed that the interference expression of the PsnWRKY70 gene can enhance the tolerance of NaHCO3 in poplar.