Identification and validation of stable reference genes for RT-qPCR analyses of Kobresia littledalei seedlings.

BACKGROUND: Kobreisa littledalei, belonging to the Cyperaceae family is the first Kobresia species with a reference genome and the most dominant species in Qinghai-Tibet Plateau alpine meadows. It has several resistance genes which could be used to breed improved crop varieties. Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) is a popular and accurate gene expression analysis method. Its reliability depends on the expression levels of reference genes, which vary by species, tissues and environments. However, K.littledalei lacks a stable and normalized reference gene for RT-qPCR analysis. RESULTS: The stability of 13 potential reference genes was tested and the stable reference genes were selected for RT-qPCR normalization for the expression analysis in the different tissues of K. littledalei under two abiotic stresses (salt and drought) and two hormonal treatments (abscisic acid (ABA) and gibberellin (GA)). Five algorithms were used to assess the stability of putative reference genes. The results showed a variation amongst the methods, and the same reference genes showed tissue expression differences under the same conditions. The stability of combining two reference genes was better than a single one. The expression levels of ACTIN were stable in leaves and stems under normal conditions, in leaves under drought stress and in roots under ABA treatment. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was stable in the roots under the control conditions and salt stress and in stems exposed to drought stress. Expression levels of superoxide dismutase (SOD) were stable in stems of ABA-treated plants and in the roots under drought stress. Moreover, RPL6 expression was stable in the leaves and stems under salt stress and in the stems of the GA-treated plants. EF1-alpha expression was stable in leaves under ABA and GA treatments. The expression levels of 28 S were stable in the roots under GA treatment. In general, ACTIN and GAPDH could be employed as housekeeping genes for K. littledalei under different treatments. CONCLUSION: This study identified the best RT-qPCR reference genes for different K. littledalei tissues under five experimental conditions. ACTIN and GAPDH genes can be employed as the ideal housekeeping genes for expression analysis under different conditions. This is the first study to investigate the stable reference genes for normalized gene expression analysis of K. littledalei under different conditions. The results could aid molecular biology and gene function research on Kobresia and other related species.

Insights into the Impact of Trans-Zeatin Overproduction-Engineered Sinorhizobium meliloti on Alfalfa (Medicago sativa L.) Tolerance to Drought Stress.

Plant growth-promoting rhizobacteria have been shown to enhance plant tolerance to drought stress through various mechanisms. However, there is limited research on improving drought resistance in alfalfa by genetically modifying PGPR to produce increased levels of cytokinins. Herein, we employed synthetic biology approaches to engineer two novel strains of Sinorhizobium meliloti capable of overproducing trans-Zeatin and investigated their potential in enhancing drought tolerance in alfalfa. Our results demonstrate that alfalfa plants inoculated with these engineered S. meliloti strains exhibited reduced wilting and yellowing while maintaining higher relative water content under drought conditions. The engineered S. meliloti-induced tZ activated the activity of antioxidant enzymes and the accumulation of osmolytes. Additionally, the increased endogenous tZ content in plants alleviated the impact of drought stress on the alfalfa photosynthetic rate. However, under nondrought conditions, inoculation with the engineered S. meliloti strains had no significant effect on alfalfa biomass and nodule formation.

Genome assembly and multi-omic analyses reveal the mechanisms underlying flower color formation in Torenia fournieri.

Torenia fournieri Lind. is an ornamental plant that is popular for its numerous flowers and variety of colors. However, its genomic evolutionary history and the genetic and metabolic bases of flower color formation remain poorly understood. Here, we report the first T. fournieri reference genome, which was resolved to the chromosome scale and was 164.4 Mb in size. Phylogenetic analyses clarified relationships with other plant species, and a comparative genomic analysis indicated that the shared ancestor of T. fournieri and Antirrhinum majus underwent a whole genome duplication event. Joint transcriptomic and metabolomic analyses identified many metabolites related to pelargonidin, peonidin, and naringenin production in rose (TfR)-colored flowers. Samples with blue (TfB) and deep blue (TfD) colors contained numerous derivatives of petunidin, cyanidin, quercetin, and malvidin; differences in the abundances of these metabolites and expression levels of the associated genes were hypothesized to be responsible for variety-specific differences in flower color. Furthermore, the genes encoding flavonoid 3-hydroxylase, anthocyanin synthase, and anthocyanin reductase were differentially expressed between flowers of different colors. Overall, we successfully identified key genes and metabolites involved in T. fournieri flower color formation. The data provided by the chromosome-scale genome assembly establish a basis for understanding the differentiation of this species and will facilitate future genetic studies and genomic-assisted breeding.

A 3-Ketoacyl-CoA Synthase 10 (KCS10) Homologue from Alfalfa Enhances Drought Tolerance by Regulating Cuticular Wax Biosynthesis.

Cuticular wax, forming the first line of defense against adverse environmental stresses, comprises very long-chain fatty acids (VLCFAs) and their derivatives. 3-Ketoacyl-CoA synthase (KCS) is a rate-limiting enzyme for VLCFA biosynthesis. In this study, we isolated KCS10, a KCS gene from alfalfa, and analyzed the effect of gene expression on wax production and drought stress in transgenic plants. MsKCS10 overexpression increased compact platelet-like crystal deposition and promoted primary alcohol biosynthesis through acyl reduction pathways in alfalfa leaves. Overexpression of MsKCS10 induced the formation of coiled-rodlet-like crystals and increased n-alkane content through decarbonylation pathways in tobacco and tomato fruits. Overexpression of MsKCS10 enhanced drought tolerance by limiting nonstomatal water loss, improving photosynthesis, and maintaining osmotic potential under drought stress in transgenic tobacco. In summary, MsKCS10 plays an important role in wax biosynthesis, wax crystal morphology, and drought tolerance, although the mechanisms are different among the plant species. MsKCS10 can be targeted in future breeding programs to improve drought tolerance in plants.

Photosynthesis, Water Status and K+/Na+ Homeostasis of Buchoe dactyloides Responding to Salinity.

Soil salinization is one of the most serious abiotic stresses restricting plant growth. Buffalograss is a C4 perennial turfgrass and forage with an excellent resistance to harsh environments. To clarify the adaptative mechanisms of buffalograss in response to salinity, we investigated the effects of NaCl treatments on photosynthesis, water status and K+/Na+ homeostasis of this species, then analyzed the expression of key genes involved in these processes using the qRT-PCR method. The results showed that NaCl treatments up to 200 mM had no obvious effects on plant growth, photosynthesis and leaf hydrate status, and even substantially stimulated root activity. Furthermore, buffalograss could retain a large amount of Na+ in roots to restrict Na+ overaccumulation in shoots, and increase leaf K+ concentration to maintain a high K+/Na+ ratio under NaCl stresses. After 50 and 200 mM NaCl treatments, the expressions of several genes related to chlorophyll synthesis, photosynthetic electron transport and CO2 assimilation, as well as aquaporin genes (BdPIPs and BdTIPs) were upregulated. Notably, under NaCl treatments, the increased expression of BdSOS1, BdHKT1 and BdNHX1 in roots might have helped Na+ exclusion by root tips, retrieval from xylem sap and accumulation in root cells, respectively; the upregulation of BdHAK5 and BdSKOR in roots likely enhanced K+ uptake and long-distance transport from roots to shoots, respectively. This work finds that buffalograss possesses a strong ability to sustain high photosynthetic capacity, water balance and leaf K+/Na+ homeostasis under salt stress, and lays a foundation for elucidating the molecular mechanism underlying the salt tolerance of buffalograss.

Osmotic stress-induced lignin synthesis is regulated at multiple levels in alfalfa (Medicago sativa L.).

Alfalfa is an important forage crop. Yield and quality are frequently threatened by extreme environments such as drought and salt stress. As a component of the cell wall, lignin plays an important role in the abiotic stress response, the mechanisms of which have not been well clarified. In this study, we combined physiological, transcriptional, and metabolic analyses to reveal the changes in lignin content in alfalfa under mannitol-induced osmotic stress. Osmotic stress enhanced lignin accumulation by increasing G and S units, which was associated with increases in enzyme activities and decreases in 8 intermediate metabolites. Upon combined analysis of the transcriptome and metabolome, we identified five key structural genes and several coexpressed transcription factors, such as MYB and WRKY, which may play a core role in regulating lignin content and composition under osmotic stress. In addition, lignin synthesis was positively regulated by ABA but negatively regulated by ethylene under osmotic stress. These results provide new insight into the regulatory mechanism of lignin synthesis under abiotic stress.

Iron nanoparticles induced the growth and physio-chemical changes in Kobresia capillifolia seedlings.

Iron nanoparticles (NPs) priming is known to affect the seed germination and seedling growth in many plants. However, whether it has an important role in stimulating the growth of perennial Qinghai-Tibet Plateau plants remains unclear. In this study, the effects of seed priming with different concentrations of nFe2O3 and FeCl3 (10, 50, 100, 500, and 1000 mg L-1) on seed germination, plant growth, photosystem, antioxidant enzyme activities, root morphology, and biomass distribution of Kobresia capillifolia were evaluated under laboratory conditions. The results showed that compared with treatment materials, concentration had more significant effects on K. capillifolia development. There was no significant impact on germination rate were discovered under all treatments, but decreased the seed mildew rate at 100 mg L-1 nFe2O3. Compare with control, Fe-based priming significantly decreased root biomass. All Fe-based treatments increased rubisco activity of leaves, and significantly enhanced Pn at ranged from 10 to 100 mg L-1. Meanwhile, chlorophyll contents were decreased, the chloroplasts were swollen, and thylakoids were disorganized under all Fe treatments. Iron-based priming significantly enhanced SOD, POD, and CAT activities in Kobresia roots. In conclusion, the thick cuticle-covered seed coat of K. capillifolia postponed the penetration of FeNPs into seeds, so FeNPs priming had a weak impact on seed germination. The sustainable release of Fe ions from FeNPs and the uptake of Fe ions by roots affected the physiology, biochemistry and morphology of K. capillifolia. The findings of this study provide an in-depth understanding of how FeNPs impact the alpine meadow plant, K. capillifolia.

Identification of Competing Endogenous RNAs (ceRNAs) Network Associated with Drought Tolerance in Medicago truncatula with Rhizobium Symbiosis.

Drought, bringing the risks of agricultural production losses, is becoming a globally environmental stress. Previous results suggested that legumes with nodules exhibited superior drought tolerance compared with the non-nodule group. To investigate the molecular mechanism of rhizobium symbiosis impacting drought tolerance, transcriptome and sRNAome sequencing were performed to identify the potential mRNA-miRNA-ncRNA dynamic network. Our results revealed that seedlings with active nodules exhibited enhanced drought tolerance by reserving energy, synthesizing N-glycans, and medicating systemic acquired resistance due to the early effects of symbiotic nitrogen fixation (SNF) triggered in contrast to the drought susceptible with inactive nodules. The improved drought tolerance might be involved in the decreased expression levels of miRNA such as mtr_miR169l-5p, mtr_miR398b, and mtr_miR398c and its target genes in seedlings with active nodules. Based on the negative expression pattern between miRNA and its target genes, we constructed an mRNA-miR169l-ncRNA ceRNA network. During severe drought stress, the lncRNA alternative splicings TCONS_00049507 and TCONS_00049510 competitively interacted with mtr_miR169l-5p, which upregulated the expression of NUCLEAR FACTOR-Y (NF-Y) transcription factor subfamily NF-YA genes MtNF-YA2 and MtNF-YA3 to regulate their downstream drought-response genes. Our results emphasized the importance of SNF plants affecting drought tolerance. In conclusion, our work provides insight into ceRNA involvement in rhizobium symbiosis contributing to drought tolerance and provides molecular evidence for future study.

Proteomic Analysis of Alfalfa (Medicago sativa L.) Roots in Response to Rhizobium Nodulation and Salt Stress.

(1) Background: Alfalfa is an important legume forage throughout the world. Although alfalfa is considered moderately tolerant to salinity, its production and nitrogen-fixing activity are greatly limited by salt stress. (2) Methods: We examined the physiological changes and proteomic profiles of alfalfa with active nodules (NA) and without nodules (NN) under NaCl treatment. (3) Results: Our data suggested that NA roots showed upregulation of the pathways of abiotic and biotic stress responses (e.g., heat shock proteins and pathogenesis-related proteins), antioxidant enzyme synthesis, protein synthesis and degradation, cell wall degradation and modification, acid phosphatases, and porin transport when compared with NN plants under salt stress conditions. NA roots also upregulated the processes or proteins of lipid metabolism, heat shock proteins, protein degradation and folding, and cell cytoskeleton, downregulated the DNA and protein synthesis process, and vacuolar H+-ATPase proteins under salt stress. Besides, NA roots displayed a net H+ influx and low level of K+ efflux under salt stress, which may enhance the salt tolerance of NA plants. (4) Conclusions: The rhizobium symbiosis conferred the host plant salt tolerance by regulating a series of physiological processes to enhance stress response, improve antioxidant ability and energy use efficiency, and maintain ion homeostasis.

Distinct rhizosphere soil responses to nitrogen in relation to microbial biomass and community composition at initial flowering stages of alfalfa cultivars.

In the long-term growth process, alfalfa rhizosphere forms specific microbiome to provide nutrition for its growth and development. However, the effects of different perennial alfalfa cultivars on changes in the rhizosphere soil characteristics and microbiome are not well understood. In this study, 12 perennial alfalfa cultivars were grown continuously for eight years. Rhizosphere samples were tested using Illumina sequencing of the 16S rRNA gene coupled with co-occurrence network analysis to explore the relationship between alfalfa (biomass and crude protein content), soil properties, and the microbial composition and diversity. Redundancy analysis showed SOC and pH had the greatest impact on the composition of the rhizosphere microbial community. Moreover, microbial diversity also contributes to microbial composition. Soil properties (AP, EC, SOC and pH) exhibited a significant positive correlation with soil bacterial communities, which was attributed to the differences between plant cultivars. Partial least squares path modeling (PLS-PM) revealed that microbial biomass and community composition rather than diversity, are the dominant determinants in the rhizosphere soil nitrogen content of perennial alfalfa. Our findings demonstrate that the soil microbial biomass and composition of rhizosphere bacterial communities are strongly affected by cultivar, driving the changes in soil nitrogen content, and variances in the selective capacities of plants.

Effects of MiR-214-3p Regulation of SERCA2a Expression on Contractility of Cardiomyocytes in Heart Failure Model.

This study aimed to explore the targeted regulation of microRNA-214-3p (miR-214-3p) on sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) and its mechanism on heart failure (HF). In this study, a rat model of HF was established by injecting isoproterenol to detect the changes in heart function. Then the primary rat cardiomyocytes were extracted and cultured. The cells were divided into the normal group, HF model group, miR-214-3p mimic group, and inhibitor group according to treatment methods. The expression differences of SERCA2a in each group were detected. The binding sites of miR-214-3p and SERCA2a were predicted, wild-type or mutant SERCA2a was prepared and co-transfected into cardiomyocytes with mimic, and the targeting effect was detected by the dual-luciferase reporter gene. Finally, the systolic function of each group was detected by a single-cell systolic dynamic edge detection system. The results showed that cardiac output and left ventricular ejection fraction of HF rats were significantly lower than those of normal rats (P<0.05). The results of the cell test showed that messenger ribonucleic acid (mRNA) and protein expression levels of SERCA2a in the model group and the mimic group were significantly lower than those in the mimic group (P<0.05), but there were no differences between normal group and inhibitor group (P>0.05). Target prediction revealed that miR-214-3p had a complementary pairing of 6 bases with the SERCA2a 3'non-coding region. After co-transfection with miR-214-3p mimic and wild-type SERCA2a expression vector, the dual-luciferase activity was significantly decreased (P<0.05). The percentage of maximal contraction amplitude, peak contraction time, and 50% diastolic time of cells in the model group and mimic group decreased significantly. The mimic group was significantly smaller (P<0.05), but there were no differences between the normal group and the inhibitor group (P>0.05). These results indicated that SERCA2a expression was significantly reduced in HF cells, and miR-214-3p could inhibit SERCA2a expression by targeting the SERCA2a 3'UTR region. Inhibition of miR-214-3p could promote the expression of SERCA2a, which in turn promoted the contractile function of HF rat cardiomyocytes.

Green nanopriming: responses of alfalfa (Medicago sativa L.) seedlings to alfalfa extracts capped and light-induced silver nanoparticles.

The application of nanotechnology in agriculture can remarkably improve the cultivation and growth of crop plants. Many studies showed that nanoparticles (NPs) made plants grow more vigorously. Light can make NPs aggregated, leading to the reduction of the NPs toxicity. In addition, treatment with NPs had a "hormesis effect" on plants. In this study, light-induced silver nanoparticles (AgNPs) were synthesized by using the alfalfa (Medicago sativa L.) extracts, and then the optimal synthetic condition was determined. Light-induced AgNPs were aggregated, spherical and pink, and they were coated with esters, phenols, acids, terpenes, amino acids and sugars, which were the compositions of alfalfa extracts. The concentration of free Ag+ was less than 2 % of the AgNPs concentration. Through nanopriming, Ag+ got into the seedlings and caused the impact of AgNPs on alfalfa. Compared with the control group, low concentration of light-induced AgNPs had a positive effect on the photosynthesis. It was also harmless to the leaf cells, and there was no elongation effect on shoots. Although high concentration of AgNPs was especially beneficial to root elongation, it had a slight toxic effect on seedlings due to the accumulation of silver. With the increase of AgNPs concentration, the content of silver in the seedlings increased and the silver enriched in plants was at the mg/kg level. Just as available research reported the toxicity of NPs can be reduced by using suitable synthesis and application methods, the present light induction, active material encapsulation and nanopriming minimized the toxicity of AgNPs to plants, enhancing the antioxidant enzyme system.

MDP25 mediates the fine-tuning of microtubule organization in response to salt stress.

Microtubules are dynamic cytoskeleton structures playing fundamental roles in plant responses to salt stress. The precise mechanisms by which microtubule organization is regulated under salt stress are largely unknown. Here, we report that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN 25 (MDP25; also known as PLASMA MEMBRANE-ASSOCIATED CATION-BINDING PROTEIN 1 (PCaP1)) helps regulate microtubule organization. Under salt treatment, elevated cytosolic Ca2+ concentration caused MDP25 to partially dissociate from the plasma membrane, promoting microtubule depolymerization. When Ca2+ signaling was blocked by BAPTA-AM or LaCl3 , microtubule depolymerization in wild-type and MDP25-overexpressing cells was slower, while there was no obvious change in mdp25 cells. Knockout of MDP25 improved microtubule reassembly and was conducive to microtubule integrity under long-term salt treatment and microtubule recovery after salt stress. Moreover, mdp25 seedlings exhibited a higher survival rate under salt stress. The presence microtubule-disrupting reagent oryzalin or microtubule-stabilizing reagent paclitaxel differentially affected the survival rates of different genotypes under salt stress. MDP25 promoted microtubule instability by affecting the catastrophe and rescue frequencies, shrinkage rate and time in pause phase at the microtubule plus-end and the depolymerization rate at the microtubule minus-end. These findings reveal a role for MDP25 in regulating microtubule organization under salt treatment by affecting microtubule dynamics.

Ming-Mu-Di-Huang-Pill Activates SQSTM1 via AMPK-Mediated Autophagic KEAP1 Degradation and Protects RPE Cells from Oxidative Damage.

Oxidative stress and diminished autophagy in the retinal pigment epithelium (RPE) play crucial roles in the pathogenesis of age-related macular degeneration (AMD). Enhancing autophagy has recently been identified as an important strategy to protect RPE cells from oxidative damage. Ming-Mu-Di-Huang-Pill (MMDH pill) is a traditional herbal medicine used to treat AMD, and its molecular mechanism is not well understood. The aim of the present study was to investigate whether the MMDH pill relieved acute oxidative damage by activating autophagy in an in vitro and in vivo model of sodium iodate (NaIO3). The results showed that NaIO3 induced cell death and inhibited proliferation. The MMDH pill increased cell viability, restored the activities of antioxidant enzymes, and reduced reactive oxygen species (ROS) fluorescence intensity. The MMDH pill mediated Kelch-like ECH-associated protein 1 (Keap1) degradation and decreased oxidative damage, which was blocked in autophagy inhibitor (chloroquine) or sequestosome-1 (SQSTM1) siRNA-treated RPE cells. Furthermore, we indicated that the MMDH pill could promote adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and autophagy adaptor-SQSTM1 expression, which could stimulate autophagic degradation of Keap1. In addition, the MMDH pill increased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) nuclear translocation in a SQSTM1-dependent manner and induced the expression of the downstream antioxidant factors heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate quinone dehydrogenase 1 (NQO1). In conclusion, MMDH pill plays a protective role in relieving NaIO3-induced oxidative stress by activating the AMPK/SQSTM1/Keap1 pathway. The MMDH pill may be useful to treat AMD by maintaining redox homeostasis and autophagy.

Analysis of risk factors for early death in patients with acute promyelocytic leukaemia treated with arsenic trioxide.

To date, no specific studies have evaluated early death (ED) in patients with acute promyelocytic leukaemia (APL) homogeneously treated with arsenic trioxide induction therapy and investigated according to the white blood cell (WBC) count at onset. Such patients were retrospectively analysed in this study, including 314 patients with a WBC count ≤ 10 × 109/L (standard-risk (SR) group) and 144 with a WBC count > 10 × 109/L (high-risk (HR) group). The baseline clinical characteristics and risk factors for ED were compared between the two groups. The incidence of fibrinogen < 1.0 g/L and elevated serum uric acid, aspartate aminotransferase and creatinine levels were higher in the HR group than in the SR group (P = 0.001; P < 0.001; P < 0.001; P = 0.044, respectively). The ED rate was significantly higher in the HR group than in the SR group (29.17% vs. 10.83%, P < 0.001). The main cause of ED was bleeding, followed by infection and differentiation syndrome (DS) in the HR group, while it was bleeding, followed by DS and infection in the SR group. Male sex, age > 50 years old, and fibrinogen < 1.0 g/L were independent risk factors for ED in the SR group. Increased serum creatinine levels, decreased albumin levels, and fibrinogen < 1.0 g/L were independent risk factors for ED in the HR group. Overall, the incidence of ED was higher in the HR group, and the baseline clinical characteristics, causes, times, and predictors of ED in the HR group differed from those in the SR group.

Experimental and Theoretical Study of the Effects of Rare Earth Elements on Growth and Chlorophyll of Alfalfa (Medicago sativa L.) Seedling.

Rare earth elements (REEs) of low concentration are usually beneficial to plant growth, while they are toxic at high concentrations. The effects of treatment with lanthanum (La) (10 and 20 µM), cerium (Ce) (10 and 20 µM), and terbium (Tb) (10 and 20 µM) on seedling growth of alfalfa (Medicago sativa L.), which is one of the most important perennial leguminous forages in the world, were studied. The results showed that all three REE treatments quickened the germination of seeds. The length of shoot under La (20 µM) treatment was significantly shortened (P < 0.05). In addition, treatment with La, Ce, and Tb had a "hormesis effect" on root length. There was a significant decrease in chlorophyll content on treatment with the three REEs, and the degree of decline was in the order of La < Ce < Tb, under the same concentration. In vitro experiments and quantum chemical calculations were further performed to explain why the treatments with REEs reduced the chlorophyll content. In vitro experiments showed that La, Ce, and Tb treatments reduced the absorbance of chlorophyll, and the decrease followed in the order of La > Ce > Tb. Quantum chemical calculations predicted that the decrease in absorption intensity was caused by the reactions between La, Ce, Tb, and chlorophyll, which formed lanthanides-chlorophyll; and there were five types of stable lanthanides-chlorophyll. In conclusion, the decrease in chlorophyll content on treatment with REEs was caused by the change in chlorophyll structure.

Composition and Protein Precipitation Capacity of Condensed Tannins in Purple Prairie Clover (Dalea purpurea Vent.).

This study aimed to determine the concentration and composition of condensed tannins (CT) in different tissues of purple prairie clover (PPC; Dalea purpurea Vent.) at different maturities and to determine their protein-precipitating capacity. The compositions of CT were elucidated after thiolysis with benzyl mercaptan followed by high-performance liquid-chromatography (HPLC) and 1H-13C heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The results indicated that PPC flowering heads contained the highest CT concentration. Purple prairie clover CT consisted mainly of epicatechin (EC) and epigallocatechin (EGC) subunits. CT in the leaves were composed of more EC and less EGC than CT in stems and flowering heads at both the early flowering (EF) and late flowering (LF) head stages. The mean degree of polymerization was the highest for CT in stems and increased with maturity. CT isolated from PPC leaves at the early flowering head stage exhibited the greatest biological activity in terms of protein precipitation. Overall, the CT in PPC were predominantly procyanidins and the concentration and composition varied among the plant tissues and with maturity.

Addressing the challenge of cold stress resilience with the synergistic effect of Rhizobium inoculation and exogenous melatonin application in Medicago truncatula.

Cold stress is an adverse environmental condition that limits the growth and yield of leguminous plants. Thus, discovering an effective way of ameliorating cold-mediated damage is important for sustainable legume production. In this study, the combined use of Rhizobium inoculation (RI) and melatonin (MT) pretreatment was investigated in Medicago truncatula plants under cold stress. Eight-week-old seedlings were divided into eight groups: (i) CK (no stress, noninoculated, no MT), (ii) RI (Rhizobium inoculated), (iii) MT (75 µM melatonin), (iv) RI+MT, (v) CS (cold stress at 4 °C without Rhizobium inoculation and melatonin), (vi) CS+RI, (vii) CS+MT, and (viii) CS+RI+MT. Plants were exposed to cold stress for 24 hrs. Cold stress decreased photosynthetic pigments and increased oxidative stress. Pretreatment with RI and MT alone or combined significantly improved root activity and plant biomass production under cold stress. Interestingly, chlorophyll contents increased by 242.81% and MDA levels dramatically decreased by 34.22% in the CS+RI+MT treatment compared to the CS treatment. Moreover, RI+MT pretreatment improved the antioxidative ability by increasing the activities of peroxidase (POD; 8.45%), superoxide dismutase (SOD; 50.36%), catalase (CAT; 140.26%), and ascorbate peroxidase (APX; 42.63%) over CS treated plants. Additionally, increased osmolyte accumulation, nutrient uptake, and nitrate reductase activity due to the combined use of RI and MT helped the plants counteract cold-mediated damage by strengthening the nonenzymatic antioxidant system. These data indicate that pretreatment with a combined application of RI and MT can attenuate cold damage by enhancing the antioxidation ability of legumes.

Exploration of the Effect and Mechanism of Fructus Lycii, Rehmanniae Radix Praeparata, and Paeonia lactiflora in the Treatment of AMD Based on Network Pharmacology and in vitro Experimental Verification.

PURPOSE: The aim of this study was to observe the mechanism of Fructus Lycii (FL), Rehmanniae Radix Praeparata (RRP) and Paeonia lactiflora (PL) in treating age-related macular degeneration (AMD) based on network pharmacology and biological experiments. METHODS: Bioactive compounds, potential targets of FL, RRP and PL, and genes related to AMD, were acquired from public databases. Functional and pathway enrichment analyses of the core targets were conducted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Subsequently, the finding was further verified with cell experiments. The MTT assay and flow cytometric analysis were used to assess cell viability and apoptosis. The production of reactive oxygen species (ROS) was analyzed by DCFH-DA staining; the activity of antioxidant enzymes was chemically measured with assay kits. The expression of key proteins was evaluated by Western blot analysis. RESULTS: Fifty-nine active compounds, 182 potential targets, and 2536 AMD-related human genes were identified. A total of 103 key targets of the three herbs on AMD were identified by protein-protein interaction (PPI) analysis. The abovementioned targets were correlated with nuclear receptor activity, oxidative stress, and apoptosis pathways according to the GO and KEGG analyses. MTT assay and flow cytometry demonstrated that pretreatment of ARPE-19 cells with the three herbs significantly increased cell viability and decreased apoptosis induced by H2O2. The three herbs might reduce the intracellular ROS levels and increase the SOD and CAT activities after H2O2. Furthermore, the three herbs significantly inhibited oxidative stress via increasing the expression of Nrf2, HO-1 and NQO1. CONCLUSION: The combined results of network pharmacology and validation experiments showed that FL, RRP and PL reduce oxidative stress and apoptosis in RPE cells to exert its effect in the treatment of AMD.