Saline-Alkali Tolerance in Rice: Physiological Response, Molecular Mechanism, and QTL Identification and Application to Breeding

Salinity-alkalinity is incipient abiotic stress that impairs plant growth and development.Rice(Oryza sativa)is a major food crop greatly affected by soil salinity and alkalinity,requiring tolerant varieties in the saline-alkali prone areas.Understanding the molecular and physiological mechanisms of saline-alkali tolerance paves the base for improving saline-alkali tolerance in rice and leads to progress in breeding.This review illustrated the physiological consequences,and molecular mechanisms especially signaling and function of regulating genes for saline-alkali tolerance in rice plants.We also discussed QTLs regarding saline-alkali tolerance accordingly and ways of deployment for improvement.More efforts are needed to identify and utilize the identified QTLs for saline-alkali tolerance in rice.

Research Progress on the Mechanism of Crop Saline-alkali Tolerance and Mitigation Measures

The wide distribution of saline-alkali land in China is a restrictive factor for the sustainable development of agriculture.Saline-alkaline soil inhibits the growth and development of crops,reducing its yield and quality.In this article,we summarized the germination status,physiological characteristics,response mechanisms and mitigation measures of different crops under saline-alkali stress in recent years,aiming to provide important reference for the study of saline-alkali tolerance mechanism in crops,cultivation of crop varieties tolerant to salts and alkalis and improvement of the utilization rate of saline-alkali land,and put forward suggestions for future development trend of saline-alkali land crops and mitigation measures.

Overexpression of vacuolar proton pump ATPase(V-H^+-ATPase) subunits B, C and H confers tolerance to salt and saline-alkali stresses in transgenic alfalfa(Medicago sativa L.)

The vacuolar proton pump ATPase（V-H^＋-ATPase）, which is a multi-subunit membrane protein complex, plays a major role in the activation of ion and nutrient transport and has been suggested to be involved in several physiological processes, such as cell expansion and salt tolerance. In this study, three genes encoding V-H^＋-ATPase subunits B（Sc VHA-B, Gen Bank： JF826506）, C（Sc VHA-C, Gen Bank： JF826507） and H（Sc VHA-H, Gen Bank： JF826508） were isolated from the halophyte Suaeda corniculata. The transcript levels of Sc VHA-B, Sc VHA-C and Sc VHA-H were increased by salt, drought and saline-alkali treatments. V-H^＋-ATPase activity was also examined under salt, drought and saline-alkali stresses. The results showed that V-H^＋-ATPase activity was correlated with salt, drought and saline-alkali stress. Furthermore, V-H^＋-ATPase subunits B, C and H（Sc VHA-B, Sc VHA-C and Sc VHA-H） from S. corniculata were introduced separately into the alfalfa genome. The transgenic alfalfa was verified by Southern and Northern blot analysis. During salt and saline-alkali stresses, transgenic lines carrying the B, C and H subunits had higher germination rates than the wild type（WT）. More free proline, higher superoxide dismutase（SOD） activity and lower malondialdehyde（MDA） levels were detected in the transgenic plants under salt and saline-alkali treatments. Moreover, the Sc VHA-B transgenic lines showed greater tolerance to salt and saline-alkali stresses than the WT. These results suggest that overexpression of Sc VHA-B, Sc VHA-C and Sc VHA-H improves tolerance to salt and saline-alkali stresses in transgenic alfalfa.

Compositional and Hollow Engineering of Silicon Carbide/Carbon Microspheres as High-Performance Microwave Absorbing Materials with Good Environmental Tolerance

Microwave absorbing materials(MAMs)characterized by high absorption efficiency and good environmental tolerance are highly desirable in practical applications.Both silicon carbide and carbon are considered as stable MAMs under some rigorous conditions,while their composites still fail to produce satisfactory microwave absorption performance regardless of the improvements as compared with the individuals.Herein,we have successfully implemented compositional and structural engineering to fabricate hollow Si C/C microspheres with controllable composition.The simultaneous modulation on dielectric properties and impedance matching can be easily achieved as the change in the composition of these composites.The formation of hollow structure not only favors lightweight feature,but also generates considerable contribution to microwave attenuation capacity.With the synergistic effect of composition and structure,the optimized SiC/C composite exhibits excellent performance,whose the strongest reflection loss intensity and broadest effective absorption reach-60.8 dB and 5.1 GHz,respectively,and its microwave absorption properties are actually superior to those of most SiC/C composites in previous studies.In addition,the stability tests of microwave absorption capacity after exposure to harsh conditions and Radar Cross Section simulation data demonstrate that hollow SiC/C microspheres from compositional and structural optimization have a bright prospect in practical applications.

Abiotic stress treatment reveals expansin like A gene OfEXLA1 improving salt and drought tolerance of Osmanthus fragrans by responding to abscisic acid

Sweet osmanthus(Osmanthus fragrans) is a having general approval aromatic tree in China that is widely applied to landscaping and gardening. However, the evergreen tree adaptability is limited by many environmental stresses. Currently, limited information is available regarding the genetic analysis and functional identification of expansin genes in response to abiotic stress in sweet osmanthus. In this study, a total of 29 expansin genes were identified and divided into four groups by genome-wide analysis from the sweet osmanthus genome. Transcriptome and quantitative Real-time PCR analysis showed that the cell wall-localized protein expansin-like A(OfEXLA1) gene was significantly induced by salt and drought treatment. Histochemical GUS staining of transgenic Arabidopsis lines in which GUS activity was driven with the OfEXLA1 promoter, GUS activity was significantly induced by salt, drought, and exogenous abscisic acid(ABA). In yeast, we found OfEXLA1overexpression significantly improved the population of cells compared with wild-type strains after NaCl and polyethylene glycol(PEG)treatment. Additionally, OfEXLA1 overexpression not only promoted plant growth, but also improved the salt and drought tolerance in Arabidopsis. To gain insight into the role of ABA signaling in the regulation of OfEXLA1 improving abiotic tolerance in sweet osmanthus, four differentially expressed ABA Insensitive 5(ABI5)-like genes(OfABL4, OfABL5, OfABL7, and OfABL8) were identified from transcriptome, and dualluciferase(dual-LUC) and yeast one hybrid(Y1H) assay showed that OfABL4 and OfABL5 might bind to OfEXLA1 promoter to accumulate the OfEXLA1 expression by responding to ABA signaling to improve abiotic tolerance in sweet osmanthus. These results provide the information for understanding the molecular functions of expansin-like A gene and molecular breeding of sweet osmanthus in future.

MIR1868 negatively regulates rice cold tolerance at both the seedling and booting stages

Low temperature causes rice yield losses of up to 30%–40%,therefore increasing its cold tolerance is a breeding target.Few genes in rice are reported to confer cold tolerance at both the vegetative and reproductive stages.This study revealed a rice-specific 24-nt miRNA,miR1868,whose accumulation was suppressed by cold stress.Knockdown of MIR1868 increased seedling survival,pollen fertility,seed setting,and grain yield under cold stress,whereas its overexpression conferred the opposite phenotype.Knockdown of MIR1868 increased reactive oxygen species(ROS)scavenging and soluble sugar content under cold stress by increasing the expression of peroxidase genes and sugar metabolism genes,and its overexpression produced the opposite effect.Thus,MIR1868 negatively regulated rice cold tolerance via ROS scavenging and sugar accumulation.

PHD17 acts as a target of miR1320 to negatively control cold tolerance via JA-activated signaling in rice

Plant Homeo Domain(PHD)proteins are involved in diverse biological processes during plant growth.However,the regulation of PHD genes on rice cold stress response remains largely unknown.Here,we reported that PHD17 negatively regulated cold tolerance in rice seedlings as a cleavage target of miR1320.PHD17 expression was greatly induced by cold stress,and was down-regulated by miR1320 overexpression and up-regulated by miR1320 knockdown.Through 5'RACE and dual luciferase assays,we found that miR1320 targeted and cleaved the 3'UTR region of PHD17.PHD17 was a nuclearlocalized protein and acted as a transcriptional activator in yeast.PHD17 overexpression reduced cold tolerance of rice seedlings,while knockout of PHD17 increased cold tolerance,partially via the CBF cold signaling.By combining transcriptomic and physiological analyses,we demonstrated that PHD17 modulated ROS homeostasis and flavonoid accumulation under cold stress.K-means clustering analysis revealed that differentially expressed genes in PHD17 transgenic lines were significantly enriched in the jasmonic acid(JA)biosynthesis pathway,and expression of JA biosynthesis and signaling genes was verified to be affected by PHD17.Cold stress tests applied with MeJA or IBU(JA synthesis inhibitor)further suggested the involvement of PHD17 in JA-mediated cold signaling.Taken together,our results suggest that PHD17 acts downstream of miR1320 and negatively regulates cold tolerance of rice seedlings through JA-mediated signaling pathway.

A Data Intrusion Tolerance Model Based on an Improved Evolutionary Game Theory for the Energy Internet

Malicious attacks against data are unavoidable in the interconnected,open and shared Energy Internet(EI),Intrusion tolerant techniques are critical to the data security of EI.Existing intrusion tolerant techniques suffered from problems such as low adaptability,policy lag,and difficulty in determining the degree of tolerance.To address these issues,we propose a novel adaptive intrusion tolerance model based on game theory that enjoys two-fold ideas:(1)it constructs an improved replica of the intrusion tolerance model of the dynamic equation evolution game to induce incentive weights;and (2)it combines a tournament competition model with incentive weights to obtain optimal strategies for each stage of the game process.Extensive experiments are conducted in the IEEE 39-bus system,whose results demonstrate the feasibility of the incentive weights,confirm the proposed strategy strengthens the system’s ability to tolerate aggression,and improves the dynamic adaptability and response efficiency of the aggression-tolerant system in the case of limited resources.

The ZOS7-MYB60 module confers drought-stress tolerance in rice

Shanlan upland rice is an important landrace resource with high drought stress(DS)tolerance.Despite its importance,genes responsible for yield in Shanlan upland rice have yet to be discovered.Our previous study identified a drought-responsive zinc finger protein,ZOS7,as highly expressed in Shanlandao upland rice.However,the function of this gene in controlling drought tolerance remains largely unexplored.In this study,we found that overexpressing ZOS7,a drought-responsive zinc finger protein,in rice increased biomass and yield under drought stress.Co-overexpressing ZOS7 and MYB60,encoding a protein with which ZOS7 interacted,intensified the yield increase.ZOS7 and MYB60 appear to form a module that confers drought tolerance by regulating stomatal density and wax biosynthesis.The ZOS7-MYB60module could be used in molecular breeding for drought tolerance in rice.

Assessment of molecular markers and marker-assisted selection for drought tolerance in barley(Hordeum vulgare L.)

This review updates the present status of the field of molecular markers and marker-assisted selection(MAS),using the example of drought tolerance in barley.The accuracy of selected quantitative trait loci(QTLs),candidate genes and suggested markers was assessed in the barley genome cv.Morex.Six common strategies are described for molecular marker development,candidate gene identification and verification,and their possible applications in MAS to improve the grain yield and yield components in barley under drought stress.These strategies are based on the following five principles:(1)Molecular markers are designated as genomic‘tags’,and their‘prediction’is strongly dependent on their distance from a candidate gene on genetic or physical maps;(2)plants react differently under favourable and stressful conditions or depending on their stage of development;(3)each candidate gene must be verified by confirming its expression in the relevant conditions,e.g.,drought;(4)the molecular marker identified must be validated for MAS for tolerance to drought stress and improved grain yield;and(5)the small number of molecular markers realized for MAS in breeding,from among the many studies targeting candidate genes,can be explained by the complex nature of drought stress,and multiple stress-responsive genes in each barley genotype that are expressed differentially depending on many other factors.

The UDP-glycosyltransferase OsUGT706D2 positively regulates cold and submergence stress tolerance in rice

In a genome-wide association study,we identified a rice UDP-glycosyltransferase gene,OsUGT706D2,whose transcription was activated in response to cold and submergence stress and to exogenous abscisic acid(ABA).OsUGT706D2 positively regulated the biosynthesis of tricin-4’-O-(syringyl alcohol)ether-7-O-glucoside at both the transcriptional and metabolic levels.OsUGT706D2 mediated cold and submergence tolerance by modulating the expression of stress-responsive genes as well as the abscisic acid(ABA)signaling pathway.Gain of function of OsUGT706D2 increased cold and submergence tolerance and loss of function of OsUGT706D2 reduced cold tolerance.ABA positively regulated OsUGT706D2-mediated cold tolerance but reduced submergence tolerance.These findings suggest the potential use of OsUGT706D2 for improving abiotic stress tolerance in rice.

Identification of genes involved in cadmium-ion tolerance in evolutionary Synechocystis sp.PCC 6803 tolerant to both cadmium and high light

Photosynthetic cyanobacteria have shown great potential as“autotrophic cell factories”for the synthesis of fuels and chemicals.However,poor tolerance to various environmental stressors such as high light and heavy metals is an important factor limiting their economic viability.While numerous studies have focused on the tolerance mechanism of cyanobacteria to individual stressors,their response to simultaneous stresses remains to be recovered.To investigate the mechanism of cross tolerance to heavymetal Cd^(2+) and high light,the model cyanobacterium Synechocystis sp.PCC 6803 tolerant to both Cd^(2+) and high light was obtained via about 800 days’cross-adaptive laboratory evolution.Three evolutionary strains capable of tolerating both 5.5 μmol·L^(-1) Cd^(2+) and 600 μmol·m^(-2)·s^(-1) high light were successfully obtained,achieving about 83%enhancement of Cd^(2+) tolerance compared with the parent strain.The different response of parent and evolutionary strains to Cd^(2+) was elucidated via metabolomics.Furthermore,a total of 15 genes that were mutated during evolution were identified by whole-genome re-sequencing.Finally,by single-gene knockout and complementation analysis,four genes including ssl2615,sll1732,ssr1480,and sll1659 involved in the improvement of Cd^(2+) tolerance under high-light condition were successfully identified.This work explored the tolerance mechanism of Synechocystis sp.PCC 6803 to cadmium under high-light condition and provided valuable reference for deciphering multitolerance mechanism of cyanobacteria in the future.

Heterogeneous expression of stearoyl-acyl carrier protein desaturase genes SAD1 and SAD2 from Linum usitatissimum enhances seed oleic acid accumulation and seedling cold and drought tolerance in Brassica napus

Flax(Linum usitatissimum L.)is a versatile crop and its seeds are a major source of unsaturated fatty acids.Stearoyl-acyl carrier protein desaturase(SAD)is a dehydrogenase enzyme that plays a key role in oleic acid biosynthesis as well as responses to biotic and abiotic stresses.However,the function of SAD orthologs from L.usitatissimum has not been assessed.Here,we found that two LuSAD genes,LuSAD1 and LuSAD2,are present in the genome of L.usitatissimum cultivar‘Longya 10’.Heterogeneous expression of either LuSAD1 or LuSAD2 in Arabidopsis thaliana resulted in higher contents of total fatty acids and oleic acid in the seeds.Interestingly,ectopic expression of LuSAD2 in A.thaliana caused altered plant architecture.Similarly,the overexpression of either LuSAD1 or LuSAD2 in Brassica napus also resulted in increased contents of total fatty acids and oleic acid in the seeds.Furthermore,we demonstrated that either LuSAD1 or LuSAD2 enhances seedling resistance to cold and drought stresses by improving antioxidant enzyme activity and nonenzymatic antioxidant levels,as well as reducing membrane damage.These findings not only broaden our knowledge of the LuSAD functions in plants,but also offer promising targets for improving the quantity and quality of oil,and the abiotic stress tolerance of oil-producing crops,through molecular manipulation.

Flavonol synthase gene MsFLS13 regulates saline-alkali stress tolerance in alfalfa

Alfalfa(Medicago sativa L.) is one of the most extensively grown leguminous forage worldwide.Environmental saline-alkali stress significantly influences the growth,development,and yield of alfalfa,posing a threat to its agricultural production.However,little is known about the potential mechanisms by which alfalfa responds to saline-alkali stress.Here,we investigated these mechanisms by cloning a saline-alkali-induced flavonol synthase gene(Ms FLS13) from alfalfa,which was previously reported to be significantly upregulated under saline-alkali stress,and examining its function in the saline-alkali response.Overexpression of Ms FLS13 in alfalfa promoted plant tolerance to saline-alkali stress by enhancing flavonol accumulation,antioxidant capacity,osmotic balance,and photosynthetic efficiency.Conversely,Ms FLS13 inhibition using RNA interference reduced flavonol synthase activity and inhibited hairy root growth under saline-alkali stress.Yeast one-hybrid and dual-luciferase reporter assays indicated that the R2R3-MYB Ms MYB12 transcription factor activates Ms FLS13 expression by binding to the MBS motif in the Ms FLS13 promoter.Further analysis revealed that abscisic acid mediates the salinealkali stress response partially by inducing Ms MYB12 and Ms FLS13 expression,which consequently increases flavonol levels and maintains antioxidant homeostasis in alfalfa.Collectively,our findings highlight the crucial role of Ms FLS13 in alfalfa in response to saline-alkali stress and provide a novel genetic resource for creating saline-alkali-resistant alfalfa through genetic engineering.

Morphological and Molecular Characterizations of Country Bean (Lablab purpureus L.) Genotypes for Drought Tolerance

Country bean, Lablab purpureus (L.) is considered one of the most important leguminous crops, but their cultivation under drought stress condition encounters challenges. In this study, an experiment has been conducted among 30 genotypes under drought condition to explore morphological diversity of qualitative and quantitative, biochemical, molecular analysis. The study identified significant variations in eight traits among the genotypes examined, with phenotypic variance exceeding genotypic variance, indicating both genetic and environmental influences. High heritability and genetic advance were observed in primary, secondary, and tertiary branch lengths, suggesting these traits are likely controlled by additive gene effects, making them effective targets for selection. Principal component analysis identified three components that made a substantial contribution, accounting for approximately 73.06% of the overall quantitative variations. Among the quantitative traits, the highest coefficient of variation (CV%) has been found in number of flowers (55.05%). While number of primary branches, primary branch length, number of secondary branches, secondary branch length, number of tertiary branches, tertiary branch length has individually more than 20% of CV%. The genotypes have been grouped into three clusters based on quantitative traits. Analysis of protein reveals that the genotypes of DS28 and DS29 have higher protein content than other genotypes. Dehydrogenase responsive genotypes have been found on DS28 and DS29 from the molecular analysis. The results suggest that the genotypes DS28 and DS29 could contribute as genetic resource of high protein content and DREB responsive, and the eight quantitative traits of 30 genotypes could be used for further breeding programme.

Cardiac Tolerance of Hydroalcoholic Extract of Bark of Terminalia mantaly H. Perrier (HAEBTM) in Wistar Rats

Terminalia mantaly H. Perrier is a plant used in traditional medicine for the treatment of various pathologies. However, Terminalia mantaly H. Perrier could present potential health effects on patients. In order to determine the possible cardiotoxic effects of the hydro-alcoholic extract of the bark of Terminalia mantaly H. Perrier, (HAEBTM) forty (40) rats distributed randomly into 4 groups, including 10 animals per group (5 males and 5 females) were used. Animals in group 1 received distilled water and were used as a control group. On the other hand, groups 2, 3, 4 received oral administration a volume of the hydroalcoholic extract of Terminalia mantaly H. Perrier corresponding to 1 mL/100g of body weight at 150 mg/kg, 300 mg/kg, 600 mg/kg, respectively. The extract was administered daily at the same time for 28 days and serum was collected once a week to evaluate cardiac biochemical markers using spectrophotometric methods using a Cobas C311 HITACHI biochemistry system. After one month of study, all rats were euthanized by overdose of ether, and the hearts of the rats were removed for gross morphological and histopathological analysis. Results were analysed using variance analysis (ANOVA) to compare outcomes as a function of doses administered and treatment times. The biochemical parameters ALT, LDH, CPK, CPKMB showed no significant change (p Terminalia mantaly showed no lesions, edema and necrosis. These results suggest that the hydroalcoholic extract of Terminalia mantaly did not interfere with the functioning or alter the integrity of the heart.

QTL mapping by GWAS and functional analysis of OsbZIP72 for cold tolerance at rice seedling stage

Rice is a major crop susceptible to chilling stress.The identification of quantitative trait loci and genes for cold tolerance is crucial for the rice breeding.Of 30 quantitative-trait loci affecting seedling cold tolerance identified in a genome-wide association study of 540 rice accessions,OsbZIP72 was assigned as the causative gene for one,qCTS9.1.A single-nucleotide polymorphism in its promoter accounted for variation in expression between indica and japonica subspecies.The favorable haplotype of OsbZIP72 originated in wild rice and contributed to the expansion of japonica rice to colder habitats.OsbZIP72 positively regulates genes coding reactive oxygen species(ROS)-scavenging proteins and maintains intracellular ROS homeostasis.These findings not only enhanced our understanding of environmental adaptation but also provide novel genetic resources and potential targets for molecular design breeding for cold tolerance in rice.

Genome-wide identification,molecular evolution,and functional characterization of fructokinase gene family in apple reveal its role in improving salinity tolerance

Fructokinase(FRK)is a regulator of fructose signaling in plants and gateway proteins that catalyze the initial step in fructose metabolism through phosphorylation.Our previous study demonstrated that MdFRK2 protein exhibit not only high affinity for fructose,but also high enzymatic activity due to sorbitol.However,genome-wide identification of the MdFRK gene family and their evolutionary dynamics in apple are yet to be reported.A systematic genome-wide analysis in this study identified a total of nine MdFRK gene members,which could phylogenetically be clustered into seven groups.Chromosomal location and synteny analysis of MdFRKs revealed that their expansion in the apple genome is primarily driven by tandem and segmental duplication events.Divergent expression patterns of MdFRKs were observed in four source-sink tissues and at five different apple fruit developmental stages,which suggested their potential crucial roles in the apple fruit development and sugar accumulation.Reverse transcription-quantitative PCR(RT-qPCR)identified candidate NaCl or drought stress responsive MdFRKs,and transgenic apple plants overexpressing MdFRK2 exhibited considerably enhanced salinity tolerance.Our results will be useful for understanding the functions of MdFRKs in the regulation of apple fruit development and salt stress response.

Leaf Morphology Genes SRL1 and RENL1 Co-Regulate Cellulose Synthesis and Affect Rice Drought Tolerance

The morphological development of rice(Oryza sativa L.)leaves is closely related to plant architecture,physiological activities,and resistance.However,it is unclear whether there is a co-regulatory relationship between the morphological development of leaves and adaptation to drought environment.In this study,a drought-sensitive,roll-enhanced,and narrow-leaf mutant(renl1)was induced from a semi-rolled leaf mutant(srl1)by ethyl methane sulfonate(EMS),which was obtained from Nipponbare(NPB)through EMS.Map-based cloning and functional validation showed that RENL1 encodes a cellulose synthase,allelic to NRL1/OsCLSD4.The RENL1 mutation resulted in reduced vascular bundles,vesicular cells,cellulose,and hemicellulose contents in cell walls,diminishing the water-holding capacity of leaves.In addition,the root system of the renl1 mutant was poorly developed and its ability to scavenge reactive oxygen species(ROS)was decreased,leading to an increase in ROS after drought stress.Meanwhile,genetic results showed that RENL1 and SRL1 synergistically regulated cell wall components.Our results revealed a theoretical basis for further elucidating the molecular regulation mechanism of cellulose on rice drought tolerance,and provided a new genetic resource for enhancing the synergistic regulation network of plant type and stress resistance,thereby realizing simultaneous improvement of multiple traits in rice.

Histone Acetyltransferase GCN5 Regulates Rice Growth and Development and Enhances Salt Tolerance

Histone acetylation is indispensable in the process of crops resisting abiotic stress,which is jointly catalyzed by histone acetyltransferases and deacetylases.However,the mechanism of regulating salt tolerance through histone acetyltransferase GCN5 is still unclear.We revealed that GCN5 can catalyze the acetylation of canonical H3 and H4 lysine residues both in vivo and in vitro in rice.The knockout mutants and RNA interference lines of Os GCN5 exhibited severe growth inhibition and defects in salt tolerance,while the over-expression of Os GCN5 enhanced the salt tolerance of rice seedlings,indicating that Os GCN5 positively regulated the response of rice to salt stress.RNA-seq analysis suggested Os GCN5 may positively regulate the salt tolerance of rice by inhibiting the expression of Os HKT2;1 or other salt-responsive genes.Taken together,our study indicated that GCN5 plays a key role in enhancing salt tolerance in rice.