Genome-wide identification of Aux/IAA gene family in white clover (Trifolium repens L.) and functional verification of TrIAA18 under different abiotic stress.

BACKGROUND: White clover (Trifolium repens L.) is an excellent leguminous cool-season forage with a high protein content and strong nitrogen-fixing ability. Despite these advantages, its growth and development are markedly sensitive to environmental factors. Indole-3-acetic acid (IAA) is the major growth hormone in plants, regulating plant growth, development, and response to adversity. Nevertheless, the specific regulatory functions of Aux/IAA genes in response to abiotic stresses in white clover remain largely unexplored. RESULTS: In this study, we identified 47 Aux/IAA genes in the white clover genome, which were categorized into five groups based on phylogenetic analysis. The TrIAAs promoter region co-existed with different cis-regulatory elements involved in developmental and hormonal regulation, and stress responses, which may be closely related to their diverse regulatory roles. Collinearity analysis showed that the amplification of the TrIAA gene family was mainly carried out by segmental duplication. White clover Aux/IAA genes showed different expression patterns in different tissues and under different stress treatments. In addition, we performed a yeast two-hybrid analysis to investigate the interaction between white clover Aux/IAA and ARF proteins. Heterologous expression indicated that TrIAA18 could enhance stress tolerance in both yeast and transgenic Arabidopsis thaliana. CONCLUSION: These findings provide new scientific insights into the molecular mechanisms of growth hormone signaling in white clover and its functional characteristics in response to environmental stress.

Spermidine or spermine pretreatment regulates organic metabolites and ions homeostasis in favor of white clover seed germination against salt toxicity.

White clover is widely cultivated as a leguminous forage or ground cover plant worldwide. However, soil salinization decreases its yield and quality. Aims of the present experiment were to elucidate the impact of seed pretreatment with spermidine (Spd) or spermine (Spm) on amylolysis, Na+/K+ accumulation, and metabolic homeostasis during germination. Seed was soaked in distilled water (control), Spd or Spm solution and then germinated under optimal or salt stress conditions for 7 days. Results showed that germination vigor, germination percentage, or seed vigour index of seeds pretreatment with Spd increased by 7%, 11%, or 70% when compared with water-pretreated seeds under salt stress, respectively. Germination percentage or seed vigour index of seeds pretreatment with Spm increased by 17% or 78% than water-pretreated seeds under saline condition, respectively. In response to salt stress, accelerated amylolysis via activation of ß-amylase activity was induced by Spd or Spm pretreatment. Spd or Spm pretreatment also significantly enhanced accumulation of diverse amino acids, organic acids, sugars, and other metabolites (putrescine, myo-inositol, sorbitol, daidzein etc.) associated with enhanced osmotic adjustment, antioxidant capacity, and energy supply during germination under salt stress. In addition, Spd or Spm pretreatment not only significantly reduced salt-induced K+ loss and overaccumulation of Na+, but also improved the ratio of K+ to Na+, contributing to Na+ and K+ balance in seedlings. In response to salt stress, seeds pretreatment with Spd or Spm up-regulated transcription level of NHX2 related to enhancement in compartmentation of Na+ from cytoplasm to vacuole, thus reducing Na+ toxicity in cytoplasm. Spm priming also uniquely up-regulated transcription levels of SKOR, HKT1, and HAL2 associated with K+ and Na + homeostasis and decline in cytotoxicity under salt stress.

Optimization of Protocols for the Induction of Callus and Plant Regeneration in White Clover (Trifolium repens L.).

White clover is a widely grown temperate legume forage with high nutritional value. Research on the functional genomics of white clover requires a stable and efficient transformation system. In this study, we successfully induced calluses from the cotyledons and leaves of 10 different white clover varieties. The results showed that the callus formation rate in the cotyledons did not vary significantly among the varieties, but the highest callus formation rate was observed in 'Koala' leaves. Subsequently, different concentrations of antioxidants and hormones were tested on the browning rate and differentiation ability of the calluses, respectively. The results showed that the browning rate was the lowest on MS supplemented with 20 mg L-1 AgNO3 and 25 mg L-1 VC, respectively, and the differentiation rate was highest on MS supplemented with 1 mg L-1 6-BA, 1 mg L-1 KT and 0.5 mg L-1 NAA. In addition, the transformation system for Agrobacterium tumefaciens-mediated transformation of 4-day-old leaves was optimized to some extent and obtained a positive callus rate of 8.9% using green fluorescent protein (GFP) as a marker gene. According to our data, by following this optimized protocol, the transformation efficiency could reach 2.38%. The results of this study will provide the foundation for regenerating multiple transgenic white clover from a single genetic background.

A Trifolium repens flavodoxin-like quinone reductase 1 (TrFQR1) improves plant adaptability to high temperature associated with oxidative homeostasis and lipids remodeling.

Maintenance of stable mitochondrial respiratory chains could enhance adaptability to high temperature, but the potential mechanism was not elucidated clearly in plants. In this study, we identified and isolated a TrFQR1 gene encoding the flavodoxin-like quinone reductase 1 (TrFQR1) located in mitochondria of leguminous white clover (Trifolium repens). Phylogenetic analysis indicated that amino acid sequences of FQR1 in various plant species showed a high degree of similarities. Ectopic expression of TrFQR1 protected yeast (Saccharomyces cerevisiae) from heat damage and toxic levels of benzoquinone, phenanthraquinone and hydroquinone. Transgenic Arabidopsis thaliana and white clover overexpressing TrFQR1 exhibited significantly lower oxidative damage and better photosynthetic capacity and growth than wild-type in response to high-temperature stress, whereas AtFQR1-RNAi A. thaliana showed more severe oxidative damage and growth retardation under heat stress. TrFQR1-transgenic white clover also maintained better respiratory electron transport chain than wild-type plants, as manifested by significantly higher mitochondrial complex II and III activities, alternative oxidase activity, NAD(P)H content, and coenzyme Q10 content in response to heat stress. In addition, overexpression of TrFQR1 enhanced the accumulation of lipids including phosphatidylglycerol, monogalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and cardiolipin as important compositions of bilayers involved in dynamic membrane assembly in mitochondria or chloroplasts positively associated with heat tolerance. TrFQR1-transgenic white clover also exhibited higher lipids saturation level and phosphatidylcholine:phosphatidylethanolamine ratio, which could be beneficial to membrane stability and integrity during a prolonged period of heat stress. The current study proves that TrFQR1 is essential for heat tolerance associated with mitochondrial respiratory chain, cellular reactive oxygen species homeostasis, and lipids remodeling in plants. TrFQR1 could be selected as a key candidate marker gene to screen heat-tolerant genotypes or develop heat-tolerant crops via molecular-based breeding.

Primary plant nutrients modulate the reactive oxygen species metabolism and mitigate the impact of cold stress in overseeded perennial ryegrass.

Overseeded perennial ryegrass (Lolium perenne L.) turf on dormant bermudagrass (Cynodon dactylon Pers. L) in transitional climatic zones (TCZ) experience a severe reduction in its growth due to cold stress. Primary plant nutrients play an important role in the cold stress tolerance of plants. To better understand the cold stress tolerance of overseeded perennial ryegrass under TCZ, a three-factor and five-level central composite rotatable design (CCRD) with a regression model was used to study the interactive effects of nitrogen (N), phosphorus (P), and potassium (K) fertilization on lipid peroxidation, electrolyte leakage, reactive oxygen species (ROS) production, and their detoxification by the photosynthetic pigments, enzymatic and non-enzymatic antioxidants. The study demonstrated substantial effects of N, P, and K fertilization on ROS production and their detoxification through enzymatic and non-enzymatic pathways in overseeded perennial ryegrass under cold stress. Our results demonstrated that the cold stress significantly enhanced malondialdehyde, electrolyte leakage, and hydrogen peroxide contents, while simultaneously decreasing ROS-scavenging enzymes, antioxidants, and photosynthetic pigments in overseeded perennial ryegrass. However, N, P, and K application mitigated cold stress-provoked adversities by enhancing soluble protein, superoxide dismutase, peroxide dismutase, catalase, and proline contents as compared to the control conditions. Moreover, N, P, and, K application enhanced chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids in overseeded perennial ryegrass under cold stress as compared to the control treatments. Collectively, this 2-years study indicated that N, P, and K fertilization mitigated cold stress by activating enzymatic and non-enzymatic antioxidants defense systems, thereby concluding that efficient nutrient management is the key to enhanced cold stress tolerance of overseeded perennial ryegrass in a transitional climate. These findings revealed that turfgrass management will not only rely on breeding new varieties but also on the development of nutrient management strategies for coping cold stress.

A Heat Shock Transcription Factor TrHSFB2a of White Clover Negatively Regulates Drought, Heat and Salt Stress Tolerance in Transgenic Arabidopsis.

Heat shock transcription factors (HSF) are divided into classes A, B and C. Class A transcription factors are generally recognized as transcriptional activators, while functional characterization of class B and C heat shock transcription factors have not been fully developed in most plant species. We isolated and characterized a novel HSF transcription factor gene, TrHSFB2a (a class B HSF) gene, from the drought stress-sensitive forage crop species, white clover (Trifolium repens). TrHSFB2a was highly homologous to MtHSFB2b, CarHSFB2a, AtHSFB2b and AtHSFB2a. The expression of TrHSFB2a was strongly induced by drought (PEG6000 15% w/v), high temperature (35 °C) and salt stresses (200 mM L-1 NaCl) in white clover, while subcellular localization analysis showed that it is a nuclear protein. Overexpression of the white clover gene TrHSFB2a in Arabidopsis significantly reduced fresh and dry weight, relative water contents (RWC), maximum photosynthesis efficiency (Fv/Fm) and performance index on the absorption basis (PIABS), while it promoted leaf senescence, relative electrical conductivity (REC) and the contents of malondialdehyde (MDA) compared to a wild type under drought, heat and salt stress conditions of Arabidopsis plants. The silencing of its native homolog (AtHSFB2a) by RNA interference in Arabidopsis thaliana showed opposite trends by significantly increasing fresh and dry weights, RWC, maximum photosynthesis efficiency (Fv/Fm) and performance index on the absorption basis (PIABS) and reducing REC and MDA contents under drought, heat and salt stress conditions compared to wild type Arabidopsis plants. These phenotypic and physiological indicators suggested that the TrHSFB2a of white clover functions as a negative regulator of heat, salt and drought tolerance. The bioinformatics analysis showed that TrHSFB2a contained the core B3 repression domain (BRD) that has been reported as a repressor activator domain in other plant species that might repress the activation of the heat shock-inducible genes required in the stress tolerance process in plants. The present study explores one of the potential causes of drought and heat sensitivity in white clover that can be overcome to some extent by silencing the TrHSFB2a gene in white clover.

Enhanced Adaptability to Limited Water Supply Regulated by Diethyl Aminoethyl Hexanoate (DA-6) Associated With Lipidomic Reprogramming in Two White Clover Genotypes.

Membrane lipid reprogramming is one of the most important adaptive strategies in plant species under unfavorable environmental circumstances. Therefore, the present experiment was conducted to elucidate the effect of diethyl aminoethyl hexanoate (DA-6), a novel synthetic plant growth regulator, on oxidative damage, photosynthetic performance, changes in lipidomic profile, and unsaturation index of lipids in two white clover (Trifolium repens) cultivars (drought-sensitive "Ladino" and drought-resistant "Riverdel") under PEG-6000-induced water-deficit stress. Results revealed that water-deficit stress significantly enhanced oxidative damage and decreased photosynthetic functions in both cultivars. However, the damage was less in Riverdel. In addition, water-deficit stress significantly decreased the relative content of monogalactocyl-diacylglycerols (MGDG), sulfoquinovosyl-diacylglycerols (SQDG), phosphatidic acisd (PA), phosphatidyl-ethanolamines (PE), phosphatidyl-glycerols (PG), phosphatidyl-serines (PS), ceramides (Cer), hexosylmonoceramides (Hex1Cer), sphingomyelins (SM), and sphingosines (Sph) in both cultivars, but a more pronounced decline was observed in Ladino. Exogenous application of DA-6 significantly increased the relative content of digalactocyl-diacylglycerols (DGDG), monogalactocyl-diacylglycerolsabstra (MGDG), sulfoquinovosyl-diacylglycerols (SQDG), phosphatidic acids (PA), phosphatidyl-ethanolamines (PE), phosphatidyl-glycerols (PG), phosphatidyl-inositols (PI), phosphatidyl-serines (PS), ceramides (Cer), hexosylmonoceramides (Hex1Cer), neutral glycosphingolipids (CerG2GNAc1), and sphingosines (Sph) in the two cultivars under water-deficit stress. DA-6-treated Riverdel exhibited a significantly higher DGDG:MGDG ratio and relative content of sphingomyelins (SM) than untreated plants in response to water deficiency. Furthermore, the DA-6-pretreated plants increased the unsaturation index of phosphatidic acids (PA) and phosphatidylinositols (PI) in Ladino, ceramides (Cer) and hexosylmonoceramides (Hex1Cer) in Riverdel, and sulfoquinovosyl-diacylglycerols (SQDG) in both cultivars under water stress. These results suggested that DA-6 regulated drought resistance in white clover could be associated with increased lipid content and reprogramming, higher DGDG:MGDG ratio, and improved unsaturation index of lipids, contributing to enhanced membrane stability, integrity, fluidity, and downstream signaling transduction.

Metabolites Reprogramming and Na+/K+ Transportation Associated With Putrescine-Regulated White Clover Seed Germination and Seedling Tolerance to Salt Toxicity.

Soil salinization is a serious challenge to many countries worldwide. Putrescine (Put) is related to the improvement of seed germination under salt stress, but molecular and metabolic mechanisms are still not fully understood. The objectives of this study were to determine the effect of seed soaking with Put on germination characteristics under salt stress induced by 100 mm sodium chloride (NaCl) and to further analyze subsequent stress tolerance associated with amylolysis, oxidative damage, sodium (Na+)/ potassium (K+) accumulation and transportation, and metabolic homeostasis in white clover (Trifolium repens cv. Haifa) seedlings. The results showed that seed soaking with Put significantly alleviated salt-induced decreases in the endogenous Put content, germination rate, germination vigor, germination index, Rl/SL, and fresh/dry weight of seedlings. Put application also significantly promoted starch metabolism through activating α-amylase and ß-amylase activities under salt stress. The metabolomic analysis showed that seed soaking with Put significantly increased the accumulation of polyamines (Put and spermidine), amino acids (γ-aminobutyric acid, glutamate, alanine, proline, citrulline, etc.), organic acids (ketopentanic acid, malonic acid, malic acid, ketopentanic acid, cis-sinapinic acid, etc.), lipids and fatty acids (glycerol, stearic acid, linoleic acid, palmitic acid, etc.), sugars (levoglucosan, fucose, and anhydro-D-galactose), alcohols (myo-inositol, allo-inositol, hexadecanol, and threitol), and other metabolites (thymine, xanthine, adenine, guanine, and glycerol 1-phosphate, etc.) associated with enhanced tricarboxylic acid (TCA) cycle and γ-aminobutyric acid (GABA) shunt contributing to better osmotic adjustment, cell membrane stability, energy supply, and metabolic homeostasis when seeds germinated under salt stress. In addition, Put significantly up-regulated the AsSOS1, NHX6, SKOR, HKT1, and HKT8 expression levels which played critical roles in Na+ rejection and K+ retention resulting in higher K+/Na+ ratio during seed germination under salt stress. The Put-induced up-regulation of HAL2 transcription level could reduce the toxicity of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) in cells. Current findings will provide an integrative understanding of Put-induced salt tolerance associated with amylolysis, metabolic regulation, and ionic homeostasis during seed germination.

Polyamines Metabolism Interacts with γ-Aminobutyric Acid, Proline and Nitrogen Metabolisms to Affect Drought Tolerance of Creeping Bentgrass.

Due to increased global warming and climate change, drought has become a serious threat to horticultural crop cultivation and management. The purpose of this study was to investigate the effect of spermine (Spm) pretreatment on metabolic alterations of polyamine (PAs), γ-aminobutyric acid (GABA), proline (Pro), and nitrogen associated with drought tolerance in creeping bentgrass (Agrostis stolonifera). The results showed that drought tolerance of creeping bentgrass could be significantly improved by the Spm pretreatment, as demonstrated by the maintenance of less chlorophyll loss and higher photosynthesis, gas exchange, water use efficiency, and cell membrane stability. The Spm pretreatment further increased drought-induced accumulation of endogenous PAs, putrescine, spermidine, and Spm, and also enhanced PAs metabolism through improving arginine decarboxylases, ornithine decarboxylase, S-adenosylmethionine decarboxylase, and polyamine oxidase activities during drought stress. In addition, the Spm application not only significantly improved endogenous GABA content, glutamate content, activities of glutamate decarboxylase and α-ketoglutarase, but also alleviated decline in nitrite nitrogen content, nitrate reductase, glutamine synthetase, glutamate synthetase, and GABA aminotransferase activities under drought stress. The Spm-pretreated creeping bentgrass exhibited significantly lower ammonia nitrogen content and nitrite reductase activity as well as higher glutamate dehydrogenase activity than non-pretreated plants in response to drought stress. These results indicated beneficial roles of the Spm on regulating GABA and nitrogen metabolism contributing towards better maintenance of Tricarboxylic acid (TCA) cycle in creeping bentgrass. Interestingly, the Spm-enhanced Pro metabolism rather than more Pro accumulation could be the key regulatory mechanism for drought tolerance in creeping bentgrass. Current findings provide a comprehensive understanding of PAs interaction with other metabolic pathways to regulate drought tolerance in grass species.

Effect of different planting pattern arrangements on soil organic matter and soil nitrogen content under a maize/soybean strip relay intercropping system.

Assessing the spatial distribution of organic matter and total nitrogen in soil is essential for management and optimum utilization of fertilizers. Therefore, the present field experiment was conducted to evaluate the impact of different planting pattern arrangements on the spatial distribution of soil total nitrogen and organic matter content under a maize/soybean strip relay intercropping system. The planting was arranged in a manner such that soil sampling could be done from continuous maize/soybean relay strip intercropping (MS1), maize/soybean relay strip intercropping in rotation (MS2), traditional maize/soybean intercropping (MS3), sole maize (M), sole soybean (S), and fallow land (FL) from 2018 to 2020. The results showed significant variations for soil organic matter and total nitrogen content under different planting pattern arrangements of maize and soybean in the strip relay intercropping system. Across all systems, the highest soil organic matter (29.19 g/kg) and total nitrogen (10.19 g/kg) were recorded in MS2. In contrast, the lowest soil organic matter (1.69 g/kg) and total nitrogen (0.64 g/kg) were observed in FL. Soil organic matter and total nitrogen in MS2 increased by 186.45% and 164.06%, respectively, when compared with FL. Soil organic matter and total nitrogen in MS2 increased by 186.45% and 164.06%, respectively, when compared with FL. Furthermore, under MS2, the spatial distribution of soil organic matter was higher in both maize and soybean crop rows as compared with other cropping patterns, whereas the soil total nitrogen was higher under soybean rows as compared with maize in all other treatment. However, correlation analysis of the treatments showed variations in organic matter content. It can be concluded that different planting patterns can have varying effects on soil organic matter and total nitrogen distribution under the strip relay intercropping system. Moreover, it is recommended from this study that MS2 is a better planting pattern for the strip relay intercropping system, which can increase the spatial distribution of soil organic matter and total nitrogen, thereby improving soil fertility, C:N ratio, and crop production. This study will serve as a foundation towards the scientific usage of chemical fertilizers in agricultural sector.

γ-Aminobutyric Acid (GABA) Priming Improves Seed Germination and Seedling Stress Tolerance Associated With Enhanced Antioxidant Metabolism, DREB Expression, and Dehydrin Accumulation in White Clover Under Water Stress.

As an important plant growth regulator, the role of γ-aminobutyric acid (GABA) in regulating seeds germination was less well elucidated under water stress. The present study was conducted to investigate the impact of GABA pretreatment on seeds germination of white clover (Trifolium repens) under water deficient condition. Results demonstrated that seeds pretreated with 2µmol/l GABA significantly alleviated decreases in endogenous GABA content, germination percentage, germination potential, germination index, root length, and fresh weight along with marked reduction in mean germination time after 7days of germination under drought stress. In addition, seeds priming with GABA significantly increased the accumulation of soluble sugars, non-enzymatic antioxidants [reduced ascorbate, dehydroascorbic acid, oxidized glutathione (GSSG), and reduced glutathione (GSH)], and enzymes [superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathioe reductase, and monodehydroasorbate reductase (MDHR)] activities involved in antioxidant metabolism, which could be associated with significant reduction in osmotic potential and the accumulation of superoxide anion, hydrogen peroxide, electrical leakage, and malondialdehyde in seeds under drought stress. The GABA-pretreated seeds exhibited significantly higher abundance of dehydrin (DHN, 56 KDa) and expression levels of DHNs encoding genes (SK2, Y2K, Y2SK, and Dehydrin b) and transcription factors (DREB2, DREB3, DREB4, and DREB5) than the untreated seeds during germination under water-limited condition. These results indicated that the GABA regulated improvement in seeds germination associated with enhancement in osmotic adjustment, antioxidant metabolism, and DREB-related DHNs expression. Current study will provide a better insight about the GABA-regulated defense mechanism during seeds germination under water-limited condition.

A novel deletion mutation in EPM2A underlies progressive myoclonic epilepsy (Lafora body disease) in a Pakistani family.

Lafora body disease (MIM-254780), a glycogen storage disease, characterized by Lafora bodies (deformed glycogen molecules) accumulating in multiple organs, is a rare form of myoclonic epilepsy. It manifests in early adolescent years, initially with seizures and myoclonus, followed by dementia and progressive cognitive decline, ultimately culminating in death within 10 years. In Pakistan so far 5 cases have been reported. Here, we report a new case of Lafora body disease belonging to a consanguineous family from Pakistan. Histopathological analysis confirmed presence of lafora bodies in the patient`s skin. Sanger sequencing revealed novel homozygous 5bp deletion mutation (NM_005670.4; c.359_363delGTGTG) in exon 2 of the EPM2A gene, which was truly segregated in the family. These results will increase our understanding regarding the aetiology of this disorder and will further add to the mutation spectrum of EPM2A gene.

Adaptability to High Temperature and Stay-Green Genotypes Associated With Variations in Antioxidant, Chlorophyll Metabolism, and γ-Aminobutyric Acid Accumulation in Creeping Bentgrass Species.

High temperature limits the cultivation and utilization of cool-season plants in many regions worldwide. Recently, extreme hot waves swept across the globe in summer, leading to enormous economic loss. The evaluation and identification of genotypic variation in thermotolerance within species are critical to breeding for environmental adaptation and also provide potential materials to explore thermo-resistant mechanism in plants. Forty-two accessions of creeping bentgrass (Agrostis stolonifera), which is a cool-season perennial grass for turf and ecological remediation, were collected from 15 different countries. Physiological traits, namely, chlorophyll (Chl) content, electrolyte leakage, photochemical efficiency, performance index on absorption basis, leaf relative water content, and osmotic potential were used to evaluate the heat tolerance of these materials in controlled growth chambers and field during summer. Stay-green and early-aging genotypes were selected to further reveal the potential mechanism of tolerance to senescence and heat damage associated with alterations in Chl metabolism, antioxidant and photosynthetic capacity, and endogenous γ-aminobutyric acid (GABA). Findings showed that there were significant genetic variations in physiological traits among 41 materials in response to high temperature stress. The 13M, PROVIDENCE, and LOFTS L-93 were the top three accessions with superior tolerance to heat and summer stress than other materials in terms of laboratory and field tests. In response to heat stress, the stay-green genotype PROVIDENCE exhibited significantly higher photochemical efficiency, net photosynthetic rate, transpiration rate, and water use efficiency than the heat-susceptible W6 6570. Delayed leaf senescence in relation to less Chl loss was detected in the PROVIDENCE associated with maintenance of significantly higher expression levels of Chl-anabolic genes (AsCHLH, AsPBGD, and AsPOR) and lower Chl-catabolic gene AsPPH under heat stress. Genetic attributes, such as better capacity to scavenge reactive oxygen species and higher endogenous GABA content could play positive roles in alleviating heat-induced senescence, oxidative damage, and metabolic disturbance in the PROVIDENCE.

Chitosan (CTS) Alleviates Heat-Induced Leaf Senescence in Creeping Bentgrass by Regulating Chlorophyll Metabolism, Antioxidant Defense, and the Heat Shock Pathway.

Chitosan (CTS) is a deacetylated derivative of chitin that is involved in adaptive response to abiotic stresses. However, the regulatory role of CTS in heat tolerance is still not fully understood in plants, especially in grass species. The aim of this study was to investigate whether the CTS could reduce heat-induced senescence and damage to creeping bentgrass associated with alterations in antioxidant defense, chlorophyll (Chl) metabolism, and the heat shock pathway. Plants were pretreated exogenously with or without CTS (0.1 g L-1) before being exposed to normal (23/18 °C) or high-temperature (38/33 °C) conditions for 15 days. Heat stress induced detrimental effects, including declines in leaf relative water content and photochemical efficiency, but significantly increased reactive oxygen species (ROS) accumulation, membrane lipid peroxidation, and Chl loss in leaves. The exogenous application of CTS significantly alleviated heat-induced damage in creeping bentgrass leaves by ameliorating water balance, ROS scavenging, the maintenance of Chl metabolism, and photosynthesis. Compared to untreated plants under heat stress, CTS-treated creeping bentgrass exhibited a significantly higher transcription level of genes involved in Chl biosynthesis (AsPBGD and AsCHLH), as well as a lower expression level of Chl degradation-related gene (AsPPH) and senescence-associated genes (AsSAG12, AsSAG39, Asl20, and Ash36), thus reducing leaf senescence and enhancing photosynthetic performance under heat stress. In addition, the foliar application of CTS significantly improved antioxidant enzyme activities (SOD, CAT, POD, and APX), thereby effectively reducing heat-induced oxidative damage. Furthermore, heat tolerance regulated by the CTS in creeping bentgrass was also associated with the heat shock pathway, since AsHSFA-6a and AsHSP82 were significantly up-regulated by the CTS during heat stress. The potential mechanisms of CTS-regulated thermotolerance associated with other metabolic pathways still need to be further studied in grass species.

Diethyl Aminoethyl Hexanoate Priming Ameliorates Seed Germination via Involvement in Hormonal Changes, Osmotic Adjustment, and Dehydrins Accumulation in White Clover Under Drought Stress.

Drought is a serious outcome of climate change reducing the productivity of forage species under arid and semi-arid conditions worldwide. Diethyl aminoethyl hexanoate (DA-6), a novel plant growth regulator, has proven to be involved in the amelioration of critical physiological functions in many agricultural crops under various abiotic stresses, but the role of the DA-6 in improving seed germination has never been investigated under drought stress. The present study was carried out to elucidate the impact of the DA-6 priming on seeds germination of white clover under drought stress. Results showed that seed priming with the DA-6 significantly mitigated the drought-induced reduction in germination percentage, germination vigor, germination index, seed vigor index, root length, shoot length, and fresh weight after 7 days of seed germination. The DA-6 significantly increased the endogenous indole-3-acetic acid, gibberellin, and cytokinin content with marked reduction in abscisic acid content in seedlings under drought stress. In addition, the DA-6 significantly accelerated starch catabolism by enhancing the activities of hydrolases contributing toward enhanced soluble sugars, proline content and ameliorated the antioxidant defense system to enhance the ability of reactive oxygen species scavenging under drought stress. Furthermore, exogenous DA-6 application significantly increased dehydrins accumulation and upregulated transcript levels of genes encoding dehydrins (SK2, Y2SK, or DHNb) during seeds germination under water deficient condition. These findings suggested that the DA-6 mediated seeds germination and drought tolerance associated with changes in endogenous phytohormones resulting in increased starch degradation, osmotic adjustment, antioxidants activity, and dehydrins accumulation during seed germination under water deficient condition.

Effects of silicon on heavy metal uptake at the soil-plant interphase: A review.

Silicon (Si) is the second richest element in the soil and surface of earth crust with a variety of positive roles in soils and plants. Different soil factors influence the Si bioavailability in soil-plant system. The Si involves in the mitigation of various biotic (insect pests and pathogenic diseases) and abiotic stresses (salt, drought, heat, and heavy metals etc.) in plants by improving plant tolerance mechanism at various levels. However, Si-mediated restrictions in heavy metals uptake and translocation from soil to plants and within plants require deep understandings. Recently, Si-based improvements in plant defense system, cell damage repair, cell homeostasis, and regulation of metabolism under heavy metal stress are getting more attention. However, limited knowledge is available on the molecular mechanisms by which Si can reduce the toxicity of heavy metals, their uptake and transfer from soil to plant roots. Thus, this review is focused the following facets in greater detail to provide better understandings about the role of Si at molecular level; (i) how Si improves tolerance in plants to variable environmental conditions, (ii) how biological factors affect Si pools in the soil (iii) how soil properties impact the release and capability of Si to decrease the bioavailability of heavy metals in soil and their accumulation in plant roots; (iv) how Si influences the plant root system with respect to heavy metals uptake or sequestration, root Fe/Mn plaque, root cell wall and compartment; (v) how Si makes complexes with heavy metals and restricts their translocation/transfer in root cell and influences the plant hormonal regulation; (vi) the competition of uptake between Si and heavy metals such as arsenic, aluminum, and cadmium due to similar membrane transporters, and (vii) how Si-mediated regulation of gene expression involves in the uptake, transportation and accumulation of heavy metals by plants and their possible detoxification mechanisms. Furthermore, future research work with respect to mitigation of heavy metal toxicity in plants is also discussed.

A 24-generation-old founder mutation impairs splicing of RBBP8 in Pakistani families affected with Jawad syndrome.

Jawad syndrome is a multiple congenital anomaly and intellectual disability syndrome with mutation in RBBP8 reported only in two families. Here, we report on two new families from Pakistan and identified a previously reported variant in RBBP8, NM_002894.3:c.1808-1809delTA. We could show that this mutation impairs splicing resulting in two different abnormal transcripts. Finally, we could verify a shared haplotype among all four families and estimate the founder event to have occurred some 24 generations ago.

Chitosan regulates metabolic balance, polyamine accumulation, and Na+ transport contributing to salt tolerance in creeping bentgrass.

BACKGROUND: Chitosan (CTS), a natural polysaccharide, exhibits multiple functions of stress adaptation regulation in plants. However, effects and mechanism of CTS on alleviating salt stress damage are still not fully understood. Objectives of this study were to investigate the function of CTS on improving salt tolerance associated with metabolic balance, polyamine (PAs) accumulation, and Na+ transport in creeping bentgrass (Agrostis stolonifera). RESULTS: CTS pretreatment significantly alleviated declines in relative water content, photosynthesis, photochemical efficiency, and water use efficiency in leaves under salt stress. Exogenous CTS increased endogenous PAs accumulation, antioxidant enzyme (SOD, POD, and CAT) activities, and sucrose accumulation and metabolism through the activation of sucrose synthase and pyruvate kinase activities, and inhibition of invertase activity. The CTS also improved total amino acids, glutamic acid, and γ-aminobutyric acid (GABA) accumulation. In addition, CTS-pretreated plants exhibited significantly higher Na+ content in roots and lower Na+ accumulation in leaves then untreated plants in response to salt stress. However, CTS had no significant effects on K+/Na+ ratio. Importantly, CTS enhanced salt overly sensitive (SOS) pathways and also up-regulated the expression of AsHKT1 and genes (AsNHX4, AsNHX5, and AsNHX6) encoding Na+/H+ exchangers under salt stress. CONCLUSIONS: The application of CTS increased antioxidant enzyme activities, thereby reducing oxidative damage to roots and leaves. CTS-induced increases in sucrose and GABA accumulation and metabolism played important roles in osmotic adjustment and energy metabolism during salt stress. The CTS also enhanced SOS pathway associated with Na+ excretion from cytosol into rhizosphere, increased AsHKT1 expression inhibiting Na+ transport to the photosynthetic tissues, and also up-regulated the expression of AsNHX4, AsNHX5, and AsNHX6 promoting the capacity of Na+ compartmentalization in roots and leaves under salt stress. In addition, CTS-induced PAs accumulation could be an important regulatory mechanism contributing to enhanced salt tolerance. These findings reveal new functions of CTS on regulating Na+ transport, enhancing sugars and amino acids metabolism for osmotic adjustment and energy supply, and increasing PAs accumulation when creeping bentgrass responds to salt stress.

Structural and functional annotation of PR/SET Domain (PRDM) protein family: In-silico study elaborating role of PRDM12 mutation in congenital insensitivity to pain.

Congenital insensitivity to pain (CIP), classified as a type of hereditary sensory and autonomic neuropathies, is a rare disease in which the affected individuals fail to perceive sensation of pain. One of the PR/SET Domain Proteins, PRDM12, has been identified in recent past as a candidate gene for congenital insensitivity to pain. In the present study, we performed whole exome sequencing in a Pakistani family with CIP phenotype to ascertain the causative mutation. We identified a previously described alanine repeat duplication in PRDM12 (Ala353_Ala359dup) in this family. After this, we performed structural annotations for PR/SET Domain (PRDM) containing protein family to prognosticate the potential hypothetical structure of PRDM proteins with physical and chemical parameters. Out of nineteen members of this family, four members (PRDM5, PRDM8, PRDM12 and PRDM13) were specially focused because of their role in neurological disorders. Predictions about structure and interactions of these proteins revealed novel interacting molecules and pathways. Detailed in silico analysis of PRDM12 was performed to elaborate importance of its domain structure in interaction with other proteins and its role in pain insensitivity phenotype. These results have substantially enhanced our understanding regarding the etiology of congenital pain insensitivity and would stimulate further research on therapy and prevention.

Amelioration of salt induced toxicity in pearl millet by seed priming with silver nanoparticles (AgNPs): The oxidative damage, antioxidant enzymes and ions uptake are major determinants of salt tolerant capacity.

Abiotic stresses in plants reduce crop growth and productivity. Nanoparticles (NPs) are effectively involved in the physiochemical processes of crop plants, especially under the abiotic stresses; whereas, less information is available regarding the role of AgNPs in salt-stressed plants. Therefore, in the current study, we investigated the effects of seed priming with commercially available silver nanoparticles (AgNPs) (size range between 50 and 100 nm) on plant morphology, physiology, and antioxidant defence system of pearl millet (Pennisetum glaucum L.) under different concentrations of salt stress (0, 120 and 150 mM NaCl). The seed priming with AgNPs at different levels (0, 10, 20 and 30 mM) mitigated the adverse impacts of salt stress and improved plant growth and defence system. The results demonstrated that salt-stressed plants had restricted growth and a noticeable decline in fresh and dry weight. Salt stress enhanced the oxidative damage by excessive production of hydrogen peroxide (H2O2), malondialdehyde (MDA) contents in pearl millet leaves. However, seed priming with AgNPs significantly improved the plant height growth related attributes, relative water content, proline contents and ultimately fresh and dry weight at 20 mM AgNPs alone or with salt stress. The AgNPs reduced the oxidative damage by improving antioxidant enzyme activities in the pearl millet leaves under salt stress. Furthermore, sodium (Na+) and Na+/K+ ratio was decreased and potassium (K+) increased by NPs, and the interactive effects between salt and AgNPs significantly impacted the total phenolic and flavonoid content in pearl millet. It was concluded that seed priming with AgNPs could enhance salinity tolerance in crop plants by enhancing physiological and biochemical responses. This might boost global crop production in salt-degraded lands.