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.

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.

Global Metabolites Reprogramming Induced by Spermine Contributing to Salt Tolerance in Creeping Bentgrass.

Soil salinization has become a serious challenge to modern agriculture worldwide. The purpose of the study was to reveal salt tolerance induced by spermine (Spm) associated with alterations in water and redox homeostasis, photosynthetic performance, and global metabolites reprogramming based on analyses of physiological responses and metabolomics in creeping bentgrass (Agrostis stolonifera). Plants pretreated with or without 0.5 mM Spm were subjected to salt stress induced by NaCl for 25 days in controlled growth chambers. Results showed that a prolonged period of salt stress caused a great deal of sodium (Na) accumulation, water loss, photoinhibition, and oxidative damage to plants. However, exogenous application of Spm significantly improved endogenous spermidine (Spd) and Spm contents, followed by significant enhancement of osmotic adjustment (OA), photosynthesis, and antioxidant capacity in leaves under salt stress. The Spm inhibited salt-induced Na accumulation but did not affect potassium (K) content. The analysis of metabolomics demonstrated that the Spm increased intermediate metabolites of γ-aminobutyric acid (GABA) shunt (GABA, glutamic acid, and alanine) and tricarboxylic acid (TCA) cycle (aconitic acid) under salt stress. In addition, the Spm also up-regulated the accumulation of multiple amino acids (glutamine, valine, isoleucine, methionine, serine, lysine, tyrosine, phenylalanine, and tryptophan), sugars (mannose, fructose, sucrose-6-phosphate, tagatose, and cellobiose), organic acid (gallic acid), and other metabolites (glycerol) in response to salt stress. These metabolites played important roles in OA, energy metabolism, signal transduction, and antioxidant defense under salt stress. More importantly, the Spm enhanced GABA shunt and the TCA cycle for energy supply in leaves. Current findings provide new evidence about the regulatory roles of the Spm in alleviating salt damage to plants associated with global metabolites reprogramming and metabolic homeostasis.

Metabolic Regulation and Lipidomic Remodeling in Relation to Spermidine-induced Stress Tolerance to High Temperature in Plants.

Beneficial effects of spermidine (Spd) on alleviating abiotic stress damage have been explored in plants for hundreds of years, but limited information is available about its roles in regulating lipids signaling and metabolism during heat stress. White clover (Trifolium repens) plants were pretreated with 70 µM Spd and then subjected to high temperature (38/33 °C) stress for 20 days. To further investigate the effect of Spd on heat tolerance, transgenic Arabidopsisthaliana overexpressing a TrSAMS encoding a key enzyme involved in Spd biosynthesis was exposed to high temperature (38/33 °C) stress for 10 days. A significant increase in endogenous Spd content in white clover by exogenous application of Spd or the TrSAMS overexpression in Arabidopsisthaliana could effectively mitigate heat-induced growth retardation, oxidative damage to lipids, and declines in photochemical efficiency and cell membrane stability. Based on the analysis of metabolomics, the amino acids and vitamins metabolism, biosynthesis of secondary metabolites, and lipids metabolism were main metabolic pathways regulated by the Spd in cool-season white clover under heat stress. Further analysis of lipidomics found the TrSAMS-transgenic plants maintained relatively higher accumulations of total lipids, eight phospholipids (PC, phosphatidylcholine; PG, phosphatidylglycerol; PS, phosphatidylserine; CL, cardiolipin; LPA, lysophosphatidic acid; LPC, lyso phosphatidylcholine; LPG, lyso phosphatidylglycerol; and LPI, lyso phosphatidylinositol), one glycoglycerolipid (DGDG, digalactosyl diacylglycerol), and four sphingolipids (Cer, ceramide; CerG2GNAc1, dihexosyl N-acetylhexosyl ceramide; Hex1Cer, hexosyl ceramide; and ST, sulfatide), higher ratio of DGDG: monogalactosyl diacylglycerol (MGDG), and lower unsaturation level than wild-type Arabidopsisthaliana in response to heat stress. Spd-induced lipids accumulation and remodeling could contribute to better maintenance of membrane stability, integrity, and functionality when plants underwent a long period of heat stress. In addition, the Spd significantly up-regulated PIP2 and PA signaling pathways, which was beneficial to signal perception and transduction for stress defense. Current findings provide a novel insight into the function of Spd against heat stress through regulating lipids signaling and reprograming in plants.

Proteomic and Metabolomic Profilings Reveal Crucial Functions of γ-Aminobutyric Acid in Regulating Ionic, Water, and Metabolic Homeostasis in Creeping Bentgrass under Salt Stress.

The global emergence of soil salinization poses a serious challenge to many countries and regions. γ-Aminobutyric acid (GABA) is involved in systemic regulation of plant adaptation to salt stress but the underlying molecular and metabolic mechanism still remains largely unknown. The elevated endogenous GABA level by the application of exogenous GABA improved salt tolerance associated with the enhancement of antioxidant capacity, photosynthetic characteristics, osmotic adjustment (OA), and water use efficiency in creeping bentgrass. GABA strongly upregulated transcript levels of AsPPa2, AsATPaB2, AsNHX2/4/6, and AsSOS1/20 in roots involved in enhanced capacity of Na+ compartmentalization and mitigation of Na+ toxicity in the cytosol. Significant downregulation of AsHKT1/4 expression could be induced by GABA in leaves in relation to maintenance of the significantly lower Na+ content and higher K+/Na+ ratio. GABA-depressed aquaporin expression and accumulation induced declines in stomatal conductance and transpiration, thereby reducing water loss in leaves during salt stress. For metabolic regulation, GABA primarily enhanced sugar and amino acid accumulation and metabolism, largely contributing to improved salt tolerance through maintaining OA and metabolic homeostasis. Other major pathways could be related to GABA-induced salt tolerance including increases in antioxidant defense, heat shock proteins, and myo-inositol accumulation in leaves. Integrative analyses of molecular, protein, metabolic, and physiological changes reveal systemic functions of GABA in regulating ionic, water, and metabolic homeostasis in nonhalophytic creeping bentgrass under salt stress.

Spermine Regulates Water Balance Associated with Ca2+-Dependent Aquaporin (TrTIP2-1, TrTIP2-2 and TrPIP2-7) Expression in Plants under Water Stress.

Spermine (Spm) regulates water balance involved in water channel proteins, aquaporins (AQPs), in plants. An increase in endogenous Spm content via exogenous Spm application significantly improved cell membrane stability, photosynthesis, osmotic adjustment (OA) and water use efficiency (WUE) contributing to enhanced tolerance to water stress in white clover. Spm upregulated TrTIP2-1, TrTIP2-2 and TrPIP2-7 expressions and also increased the abundance of TIP2 and PIP2-7 proteins in white clover under water stress. Spm quickly activated intracellular Ca2+ signaling and Spm-induced TrTIP2-2 and TrPIP2-7 expressions could be blocked by Ca2+ channel blockers and the inhibitor of Ca2+-dependent protein kinase in leaves of white clover. TrSAMS in relation to Spm biosynthesis was first cloned from white clover and the TrSAMS was located in the nucleus. Transgenic Arabidopsis overexpressing the TrSAMS had significantly higher endogenous Spm content and improved cell membrane stability, photosynthesis, OA, WUE and transcript levels of AtSIP1-1, AtSIP1-2, AtTIP2-1, AtTIP2-2, AtPIP1-2, AtPIP2-1 and AtNIP2-1 than wild type in response to water stress. Current findings indicate that Spm regulates water balance via an enhancement in OA, WUE and water transport related to Ca2+-dependent AQP expression in plants under water stress.

Nitric Oxide Signal, Nitrogen Metabolism, and Water Balance Affected by γ-Aminobutyric Acid (GABA) in Relation to Enhanced Tolerance to Water Stress in Creeping Bentgrass.

γ-Aminobutyric acid (GABA) plays an important role in regulating stress tolerance in plants. Purposes of this study was to determine the effect of an exogenous supply of GABA on tolerance to water stress in creeping bentgrass (Agrostis stolonifera), and further reveal the GABA-induced key mechanisms related to water balance, nitrogen (N) metabolism and nitric oxide (NO) production in response to water stress. Plants were pretreated with or without 0.5 mM GABA solution in the roots for 3 days, and then subjected to water stress induced by -0.52 MPa polyethylene glycol 6000 for 12 days. The results showed that water stress caused leaf water deficit, chlorophyll (Chl) loss, oxidative damage (increases in superoxide anion, hydrogen peroxide, malondialdehyde, and protein carbonyl content), N insufficiency, and metabolic disturbance. However, the exogenous addition of GABA significantly increased endogenous GABA content, osmotic adjustment and antioxidant enzyme activities (superoxide dismutase, catalase, dehydroascorbate reductase, glutathione reductase and monodehydroascorbate reductase), followed by effectively alleviating water stress damage, including declines in oxidative damage, photoinhibition, and water and Chl loss. GABA supply not only provided more available N, but also affected N metabolism through activating nitrite reductase and glutamine synthetase activities under water stress. The supply of GABA did not increase glutamate content and glutamate decarboxylase activity, but enhanced glutamate dehydrogenase activity, which might indicate that GABA promoted the conversion and utilization of glutamate for maintaining Chl synthesis and tricarboxylic acid cycle when creeping bentgrass underwent water stress. In addition, GABA-induced NO production, depending on nitrate reductase and NO-associated protein pathways, could be associated with the enhancement of antioxidant defense. Current findings reveal the critical role of GABA in regulating signal transduction and metabolic homeostasis in plants under water-limited condition.

γ-Aminobutyric Acid Enhances Heat Tolerance Associated with the Change of Proteomic Profiling in Creeping Bentgrass.

γ-Aminobutyric acid (GABA) participates in the regulation of adaptability to abiotic stress in plants. The objectives of this study were to investigate the effects of GABA priming on improving thermotolerance in creeping bentgrass (Agrostis stolonifera) based on analyses of physiology and proteome using iTRAQ technology. GABA-treated plants maintained significantly higher endogenous GABA content, photochemical efficiency, performance index on absorption basis, membrane stability, and osmotic adjustment (OA) than untreated plants during a prolonged period of heat stress (18 days), which indicated beneficial effects of GABA on alleviating heat damage. Protein profiles showed that plants were able to regulate some common metabolic processes including porphyrin and chlorophyll metabolism, glutathione metabolism, pyruvate metabolism, carbon fixation, and amino acid metabolism for heat acclimation. It is noteworthy that the GABA application particularly regulated arachidonic acid metabolism and phenylpropanoid biosynthesis related to better thermotolerance. In response to heat stress, the GABA priming significantly increased the abundances of Cu/ZnSOD and APX4 that were consistent with superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities. The GABA-upregulated proteins in relation to antioxidant defense (Cu/ZnSOD and APX4) for the reactive oxygen species scavenging, heat shock response (HSP90, HSP70, and HSP16.9) for preventing denatured proteins aggregation, stabilizing abnormal proteins, promoting protein maturation and assembly, sugars, and amino acids metabolism (PFK5, ATP-dependent 6-phosphofructokinase 5; FK2, fructokinase 2; BFRUCT, ß-fructofuranosidase; RFS2, galactinol-sucrose galactosyltransferase 2; ASN2, asparagine synthetase 2) for OA and energy metabolism, and transcription factor (C2H2 ZNF, C2H2 zinc-finger protein) for the activation of stress-defensive genes could play vital roles in establishing thermotolerance. Current findings provide an illuminating insight into the new function of GABA on enhancing adaptability to heat stress in plants.

Metabolomics and physiological analyses reveal ß-sitosterol as an important plant growth regulator inducing tolerance to water stress in white clover.

MAIN CONCLUSION: ß-sitosterol influences amino acids, carbohydrates, organic acids, and other metabolite metabolism and homeostasis largely contributing to better tolerance to water stress in white clover. ß-sitosterol (BS) could act as an important plant growth regulator when plants are subjected to harsh environmental conditions. Objective of this study was to examine effects of BS on growth and water stress tolerance in white clover based on physiological responses and metabolomics. White clover was pretreated with or without BS and then subjected to water stress for 7 days in controlled growth chambers. Physiological analysis demonstrated that exogenous application of BS (120 µM) could significantly improve stress tolerance associated with better growth performance and photosynthesis, higher leaf relative water content, and less oxidative damage in white clover in response to water stress. Metabolic profiling identified 78 core metabolites involved in amino acids, organic acids, sugars, sugar alcohols, and other metabolites in leaves of white clover. For sugars and sugar alcohol metabolism, the BS treatment enhanced the accumulation of fructose, glucose, maltose, and myo-inositol contributing to better antioxidant capacity, growth maintenance, and osmotic adjustment in white clover under water stress. The application of BS was inclined to convert glutamic acid into proline, 5-oxoproline, and chlorophyll instead of going to pyruvate and alanine; the BS treatment did not significantly affect intermediates of tricarboxylic acid cycle (citrate, aconitate, and malate), but promoted the accumulation of other organic acids including lactic acid, glycolic acid, glyceric acid, shikimic acid, galacturonic acid, and quinic acid in white clover subjected to water stress. In addition, cysteine, an important antioxidant metabolite, was also significantly improved by BS in white clover under water stress. These altered amino acids and organic acids metabolism could play important roles in growth maintenance and modulation of osmotic and redox balance against water stress in white clover. Current findings provide a new insight into BS-induced metabolic homeostasis related to growth and water stress tolerance in plants.

Establishing and evaluating an auto-verification system of thalassemia gene detection results.

The manual verification of gene tests is time-consuming and error prone. In this study, we try to explore a high-efficiency, clinically useful auto-verification system for gene detection of thalassemia. A series of verification elements were rooted in the auto-verification system. Consistency check was applied initially as one of the essential elements in our study. One hundred twenty-four archived cases were used to choose the consistency-check rules' indices from routine blood examination and hemoglobin electrophoresis by the receiver operating characteristic curves. Rule 1 and rule 2 established by the chosen indices were compared by their passing rate, consistency with manual validation, and error rate. Finally, 748 cases were used for verifying the system's feasibility by evaluating the passing rate, turn-around time (TAT), and error rate. The rule 2 had a higher passing rate (67.7% vs. 50.8%) and consistency (0.623 vs. 0.364) than the rule 1 with an error rate of zero. In a "live" valuation, the auto-verification system can reduce the TAT and error rate of verification by 51.5% and 0.13%, respectively, with a high passing rate of 82.8%. The auto-verification system for gene detection of thalassemia in this study can shorten the validation time, reduce errors, and enhance efficiency.

Nitric oxide, γ-aminobutyric acid, and mannose pretreatment influence metabolic profiles in white clover under water stress.

Nitric oxide (NO), γ-aminobutyric acid (GABA), and mannose (MAS) could be important regulators of plant growth and adaptation to water stress. The application of sodium nitroprusside (SNP, a NO donor), GABA, and MAS improved plant growth under water-sufficient conditions and effectively mitigated water stress damage to white clover. The metabonomic analysis showed that both SNP and GABA application resulted in a significant increase in myo-inositol content; the accumulation of mannose was commonly regulated by SNP and MAS; GABA and MAS induced the accumulation of aspartic acid, quinic acid, trehalose, and glycerol under water deficit. In addition, citric acid was uniquely up-regulated by SNP associated with tricarboxylic acid (TCA) cycle under water stress. GABA specially induced the accumulation of GABA, glycine, methionine, and aconitic acid related to GABA shunt, amino acids metabolism, and TCA cycle in response to water stress. MAS uniquely enhanced the accumulation of asparagine, galactose, and D-pinitol in association with amino acids and sugars metabolism under water stress. SNP-, GABA-, and MAS-induced changes of metabolic profiles and associated metabolic pathways could contribute to enhanced stress tolerance via involvement in the TCA cycle for energy supply, osmotic adjustment, antioxidant defense, and signal transduction for stress defense in white clover.

Pretreatment with NaCl Promotes the Seed Germination of White Clover by Affecting Endogenous Phytohormones, Metabolic Regulation, and Dehydrin-Encoded Genes Expression under Water Stress.

This study was designed to examine the effects of NaCl pretreatment on the seed germination of white clover (Trifolium repens cv. Ladino) under water stress induced by 19% polyethylene glycol (PEG) 6000. Lower concentrations of NaCl (0.5, 1, and 2.5 mM) pretreatment significantly alleviated stress-induced decreases in germination percentage, germination vigor, germination index, and radicle length of seedlings after seven days of germination under water stress. The soaking with 1 mM of NaCl exhibited most the pronounced effects on improving seed germination and alleviating stress damage. NaCl-induced seeds germination and growth could be associated with the increases in endogenous gibberellic acid (GA) and indole-3-acetic acid (IAA) levels through activating amylases leading to improved amylolysis under water stress. Seedlings pretreated with NaCl had a significantly lower osmotic potential than untreated seedlings during seed germination, which could be related to significantly higher soluble sugars and free proline content in NaCl-treated seedlings under water stress. For antioxidant metabolism, NaCl pretreatment mainly improved superoxide dismutase, peroxidase, ascorbate peroxidase, and glutathione reductase activities, transcript levels of FeSOD, APX, and DHAR, and the content of ascorbic acid, reduced glutathione, and oxidized glutathione during seed germination under water stress. The results indicated that seeds soaking with NaCl could remarkably enhance antioxidant metabolism, thereby decreasing the accumulation of reactive oxygen species and membrane lipid peroxidation during germination under water stress. In addition, NaCl-upregulated dehydrin-encoded genes SK2 expression could be another important mechanism of drought tolerance during seeds germination of white clover in response to water stress.

The γ-Aminobutyric Acid (GABA) Alleviates Salt Stress Damage during Seeds Germination of White Clover Associated with Na⁺/K⁺ Transportation, Dehydrins Accumulation, and Stress-Related Genes Expression in White Clover.

The objective of this study was to determine the effect of soaking with γ-aminobutyric acid (GABA) on white clover (Trifolium repens cv. Haifa) seed germination under salt stress induced by 100 mM NaCl. Seeds soaking with GABA (1 µM) significantly alleviated salt-induced decreases in endogenous GABA content, germination percentage, germination vigor, germination index, shoot and root length, fresh and dry weight, and root activity of seedling during seven days of germination. Exogenous application of GABA accelerated starch catabolism via the activation of amylase and also significantly reduced water-soluble carbohydrate, free amino acid, and free proline content in seedlings under salt stress. In addition, improved antioxidant enzyme activities (SOD, GPOX, CAT, APX, DHAR, GR and MDHR) and gene transcript levels (Cu/ZnSOD, FeSOD, MnSOD, CAT, GPOX, APX, MDHR, GPX and GST) was induced by seeds soaking with GABA, followed by decreases in O2∙-, H2O2, and MDA accumulation during germination under salt stress. Seeds soaking with GABA could also significantly improve Na⁺/K⁺ content and transcript levels of genes encoding Na⁺/K⁺ transportation (HKT1, HKT8, HAL2, H⁺-ATPase and SOS1) in seedlings of white clover. Moreover, exogenous GABA significantly induced the accumulation of dehydrins and expression of genes encoding dehydrins (SK2, Y2K, Y2SK, and dehydrin b) in seedlings under salt stress. These results indicate that GABA mitigates the salt damage during seeds germination through enhancing starch catabolism and the utilization of sugar and amino acids for the maintenance of growth, improving the antioxidant defense for the alleviation of oxidative damage, increasing Na⁺/K⁺ transportation for the osmotic adjustment, and promoting dehydrins accumulation for antioxidant and osmotic adjustment under salt stress.

Cilostazol prevents the degradation of collagen type II in human chondrocytes.

The alteration of extracellular matrix (ECM) in cartilage during the pathological development of Osteoarthritis (OA) changes the biomechanical environment of chondrocytes, which further drives the progression of the disease in the presence of inflammation. Healthy cartilage matrix mainly contains collagen type II, which is degraded by matrix metalloproteinase13 (MMP13), an important molecules responsible for joint damage in OA. Cilostazol (6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)-quinolinone) is a medication approved by the US Food and Drug Administration and used in the alleviation of the symptom of intermittent claudication in individuals with peripheral vascular disease. In this study, we reported that cilostazol is able to suppress the degradation of type II collagen in human chondrocytes induced by IL-1ß. Mechanistically, cilostazol treatment leads to inhibiting the expression of IRF-1, thereby prevents the induction of MMP-13. Signal transducers and activator of transcription 1 (STAT1) has been reported to play an essential role in regulating the activation of IRF-1. Our results indicated that cilostazol suppresses the activation of STAT1 by mitigating the phosphorylation of STAT1 at Ser727 and tyrosine phosphorylation of STAT1 at position 701 (Tyr701).

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.

Development of two protocols for Agrobacterium-mediated transformation of white clover (Trifolium repens) via the callus system.

White clover (Trifolium repens) is one of the most widely cultivated livestock forage plants whose persistence is severely affected by abiotic stresses. For the white clover, efficient regeneration systems is still a great necessity. In this study, inoculating 4-day-old cotyledons into MS media fortified with 0.4 mg·L-1 6-BA and 2 mg·L-1 2,4-D significantly increased the callus induction rate. Roots and cotyledons proved to be better explants, followed by hypocotyls, leaves, and petioles for callus induction. The development of differentiated structures occurred effectively on MS supplemented with 1 mg·L-1 6-BA and 0.1 mg·L-1 NAA. To increase transformation, we investigated various factors affecting the Agrobacterium tumefaciens transformation in white clover. The optimal conditions for root-derived callus and 4-day-old cotyledons were as follows: Agrobacterium suspension density with OD600 of 0.5, 20 mg·L-1 AS, and 4 days of co-cultivation duration. Subsequently, we developed two transformation protocols: transformation after callus induction from 4-day-old roots (Protocol A) and transformation before initiation of callus from cotyledons (Protocol B). The transformation frequencies varied from 1.92 to 3.17% in Protocol A and from 2.76 to 3.47% in Protocol B. We report the possibility to regenerate multiple transgenic white clover plants from a single genetic background. Our research may also contribute to successful genetic manipulation and genome editing in white clover. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03591-2.

Turf Quality and Physiological Responses to Summer Stress in Four Creeping Bentgrass Cultivars in a Subtropical Zone.

Cool-season creeping bentgrass (Agrostis stolonifera) has the ability to form fine sports turf, but high temperatures result in summer bentgrass decline (SBD), especially in transitional and subtropical zones. Physiological responses in combination with the alteration in turf quality (TQ) will contribute to a better understanding of SBD in a subtropical zone. Field experiments were conducted from 2017 to 2019 to test the adaptability to summer stress among four cultivars (13M, Penncross, Seaside II, and PA-1). A constant ambient high temperature above 30 °C significantly decreased the TQ of the four cultivars during the summer months in 2017, 2018, and 2019. Significant declines in the chlorophyll content, photochemical efficiency of photosystem II (Fv/Fm and PIABS), leaf relative water content (RWC), and osmotic potential (OP) were induced by summer stress, whereas gradual increases in water-soluble carbohydrates, proline, hydrogen peroxide (H2O2), malondialdehyde (MDA), and electrolyte leakage (EL) were observed in the four cultivars during the summer months. The 13M and Penncross cultivars exhibited better performance than Seaside II and PA-1 in response to summer stress from 2017 to 2019, which is associated with better maintenance of photosynthesis, water status, WSC and proline accumulation, and cell membrane stability. The 13M and Penncross cultivars could be used as potential candidates for turf establishment in a subtropical zone. Physiological responses together with alterations in TQ also provided critical information for the breeding and development of germplasm with heat tolerance in creeping bentgrass species.

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.

Spermine-mediated metabolic homeostasis improves growth and stress tolerance in creeping bentgrass (Agrostis stolonifera) under water or high-temperature stress.

Plants have developed diverse defense strategies to reduce the detrimental effects of a wide range of environmental stresses. The objectives of this study were to explore the function of spermine (Spm) on mediating growth and physiological changes in water homeostasis, photosynthetic performance, and oxidative damage and to further examine the regulatory mechanism of Spm on global metabolites reprogramming and associated metabolic pathways in horticultural creeping bentgrass (Agrostis stolonifera) under water and heat stresses. The 21-days-old plants were pretreated with or without 100 µM Spm for 3 days and then subjected to water stress (17% polyethylene glycol 6000), high-temperature stress (40/35°C, day/night), or normal condition (control without water stress and heat stress) for 18 days. Results demonstrated that exogenous application of Spm could significantly increase endogenous polyamine (PAs), putrescine (Put), spermidine (Spd), and Spm contents, followed by effective alleviation of growth retardant, water imbalance, photoinhibition, and oxidative damage induced by water and heat stress. Metabolites' profiling showed that a total of 61 metabolites were differentially or commonly regulated by Spm in leaves. Spm upregulated the accumulation of mannose, maltose, galactose, and urea in relation to enhanced osmotic adjustment (OA), antioxidant capacity, and nitrogen metabolism for growth maintenance under water and heat stress. Under water stress, Spm mainly induced the accumulation of sugars (glucose-1-phosphate, sucrose-6-phosphate, fructose, kestose, maltotriose, and xylose), amino acids (glutamic acid, methionine, serine, and threonine), and organic acids (pyruvic acid, aconitic acid, and ketoglutaric acid) involved in the respiratory pathway and myo-inositol associated with energy production, the ROS-scavenging system, and signal transduction. In response to heat stress, the accumulation of alanine, glycine, gallic acid, malic acid, or nicotinic acid was specifically enhanced by Spm contributing to improvements in antioxidant potency and metabolic homeostasis. This study provides novel evidence of Spm-induced,tolerance to water and heat stresses associated with global metabolites reprogramming in favor of growth maintenance and physiological responses in horticultural plants.

Seed Priming with Chitosan Improves Germination Characteristics Associated with Alterations in Antioxidant Defense and Dehydration-Responsive Pathway in White Clover under Water Stress.

Water stress decreases seed-germination characteristics and also hinders subsequent seedling establishment. Seed priming with bioactive compounds has been proven as an effective way to improve seed germination under normal and stressful conditions. However, effect and mechanism of seed priming with chitosan (CTS) on improving seed germination and seedling establishment were not well-understood under water-deficit conditions. White clover (Trifolium repens) seeds were pretreated with or without 5 mg/L CTS before being subjected to water stress induced by 18% (w/v) polyethylene glycol 6000 for 7 days of germination in a controlled growth chamber. Results showed that water stress significantly decreased germination percentage, germination vigor, germination index, seed vigor index, and seedling dry weight and also increased mean germination time and accumulation of reactive oxygen species, leading to membrane lipid peroxidation during seed germination. These symptoms could be significantly alleviated by the CTS priming through activating superoxide dismutase, catalase, and peroxidase activities. In addition, seeds pretreated with CTS exhibited significantly higher expression levels of genes encoding dehydration-responsive transcription factors (DREB2, DREB4, and DREB5) and dehydrins (Y2K, Y2SK, and SK2) than those seeds without the CTS priming. Current findings indicated that the CTS-induced tolerance to water stress could be associated with the enhancement in dehydration-responsive pathway during seed germination.