This study aims to develop the pre-column derivatization high performance liquid chromatography(HPLC) method for the determination of 16 kinds of amino acids in Eucommia ulmoides leaves, and compare the content of amino acids in the leaves harvested at different time and under leaf-oriented cultivation mode(LCM) and arbor forest mode(AFM). The HPLC conditions are as below: phenyl isothiocyanate(PITC) as pre-column derivatization agent, Agilent ZORBAX C_(18 )column(4.6 mm×250 mm, 5 μm), mobile phase A of acetonitrile-water(80∶20), mobile phase B of 0.1 mol·L~(-1) sodium acetate solution-acetonitrile(94∶6), gradient elution, flow rate of 1.0 mL·min~(-1), injection volume of 5 μL, column temperature of 40 ℃, and detection wavelength of 254 nm. The HPLC profile indicated well separation of 16 kinds of amino acids and the amino acid content in E. ulmoides leaves was up to 16.26%. In addition, the amino acid content in leaves of E. ulmoides under LCM was higher than under AFM. The amino acid content varied with the harvesting time. Through orthogonal partial least squares discriminant analysis, the amino acids of E. ulmoides under LCM and AFM were compared, which can distinguish the leaves under LCM from those under AFM. Principal component analysis was applied to comprehensively score the amino acids of E. ulmoides leaves. The results showed that the score of leaves under LCM was higher than that under AFM. Nutritional evaluation results indicated that the proteins in E. ulmoides leaves belonged to high-quality vegetable proteins. The established method for the determination of amino acid content is reliable. With the amino acid content as index, the leaf quality of E. ulmoides under LCM is better than that under AFM. This study lays a theoretical basis for the promotion of LCM for E. ulmoides and the development of medicinal and edible products from E. ulmoides leaves.
Amino Acids/metabolism* ; Eucommiaceae/chemistry* ; Chromatography, High Pressure Liquid/methods* ; Plant Leaves/chemistry*

GI (GIGANTEA) is one of the output key genes for circadian clock in the plant. The JrGI gene was cloned and its expression in different tissues was analyzed to facilitate the functional research of JrGI. RT-PCR (reverse transcription-polymerase chain reaction) was used to clone JrGI gene in present study. This gene was then analyzed by bioinformatics, subcellular localization and gene expression. The coding sequence (CDS) full length of JrGI gene was 3 516 bp, encoding 1 171 amino acids with a molecular mass of 128.60 kDa and a theoretical isoelectric point of 6.13. It was a hydrophilic protein. Phylogenetic analysis showed that JrGI of 'Xinxin 2' was highly homologous to GI of Populus euphratica. The result of subcellular localization showed that JrGI protein was located in nucleus. The JrGI, JrCO and JrFT genes in female flower buds undifferentiated and early differentiated of 'Xinxin 2' were analyzed by RT-qPCR (real-time quantitative PCR). The results showed that the expression of JrGI, JrCO and JrFT genes were the highest on morphological differentiation, implying the temporal and special regulation of JrGI in the differential process of female flower buds of'Xinxin 2'. In addition, RT-qPCR analysis showed that JrGI gene was expressed in all tissues examined, whereas the expression level in leaves was the highest. It is suggested that JrGI gene plays a key role in the development of walnut leaves.
Juglans/genetics* ; Phylogeny ; Plant Leaves ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism*

Phytoremediation plays an important role in the treatment of heavy metal pollution in soil. In order to elucidate the mechanism of salicylic acid (SA) on copper absorption, seedlings from Xuzhou (with strong Cu-tolerance) and Weifang Helianthus tuberosus cultivars (with weak Cu-tolerance) were selected for pot culture experiments. 1 mmol/L SA was sprayed upon 300 mg/kg soil copper stress, and the photosynthesis, leaf antioxidant system, several essential mineral nutrients and the changes of root upon copper stress were analyzed to explore the mechanism of copper resistance. The results showed that Pn, Tr, Gs and Ci upon copper stress decreased significantly compared to the control group. Meanwhile, chlorophyll a, chlorophyll b and carotenoid decreased with significant increase in initial fluorescence (F₀), maximum photochemical quantum yield of PSⅡ (Fv/Fm), electron transfer rate (ETR) and photochemical quenching coefficient (qP) content all decreased. The ascorbic acid (AsA) content was decreased, the glutathione (GSH) value was increased, the superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activity in the leaves were decreased, and the peroxidase (POD) activity was significantly increased. SA increased the Cu content in the ground and root system, and weakened the nutrient uptake capacity of K, Ca, Mg, and Zn in the root stem and leaves. Spray of exogenous SA can maintain the opening of leaf stomata, improve the adverse effect of copper on photosynthetic pigment and PSⅡ reaction center. Mediating the SOD and APX activity started the AsA-GSH cycle process, effectively regulated the antioxidant enzyme system in chrysanthemum taro, significantly reduced the copper content of all parts of the plant, and improved the ion exchange capacity in the body. External SA increased the content of the negative electric group on the root by changing the proportion of components in the root, promoted the absorption of mineral nutrient elements and the accumulation of osmoregulatory substances, strengthened the fixation effect of the root on metal copper, and avoided its massive accumulation in the H. tuberosus body, so as to alleviate the inhibitory effect of copper on plant growth. The study revealed the physiological regulation of SA upon copper stress, and provided a theoretical basis for planting H. tuberosus to repair soil copper pollution.
Antioxidants ; Copper ; Helianthus/metabolism* ; Salicylic Acid/pharmacology* ; Chlorophyll A/pharmacology* ; Spectroscopy, Fourier Transform Infrared ; Chlorophyll/pharmacology* ; Ascorbic Acid ; Superoxide Dismutase/metabolism* ; Photosynthesis ; Glutathione ; Plant Leaves ; Stress, Physiological ; Seedlings

This paper aimed to investigate the effect of total flavonoids of buckwheat flower and leaf on myocardial cell apoptosis and Wnt/β-catenin/peroxisome proliferator-activated receptor γ(PPARγ) pathway in arrhythmic rats. SD rats were randomly divided into a control group, a model group, a low-dose(20 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a medium-dose(40 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a high-dose(80 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a propranolol hydrochloride(2 mg·kg~(-1)) group, with 12 rats in each group. Except the control group, rats in other groups were prepared as models of arrhythmia by sublingual injection of 1 mL·kg~(-1) of 0.002% aconitine. After grouping and intervention with drugs, the arrhythmia, myocardial cells apoptosis, myocardial tissue glutathione peroxidase(GSH-Px), catalase(CAT), malondialdehyde(MDA), serum interleukin-6(IL-6), prostaglandin E2(PGE2) levels, myocardial tissue apoptosis, and Wnt/β-catenin/PPARγ pathway-related protein expression of rats in each group were measured. As compared with the control group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA levels in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels increased significantly in the model group, whereas the GSH-Px and CAT levels, and Bcl-2 and PPARγ protein expression levels in myocardial tissues reduced significantly. As compared with the model group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA leve in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels reduced in the drug intervention groups, whereas the GSH-Px and CAT levels and Bcl-2 and PPARγ protein expression levels in myocardial tissues increased. The groups of total flavonoids of buckwheat flower and leaf were in a dose-dependent manner. There was no significant difference in the levels of each index in rats between the propranolol hydrochloride group and the high-dose group of total flavonoids of buckwheat flower and leaf. The total flavonoids of buckwheat flower and leaf inhibit the activation of Wnt/β-catenin pathway, up-regulate the expression of PPARγ, reduce oxidative stress and inflammatory damage in myocardial tissues of arrhythmic rats, reduce myocardial cell apoptosis, and improve the symptoms of arrhythmia in rats.
Rats ; Animals ; PPAR gamma/metabolism* ; Fagopyrum/genetics* ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; beta Catenin/metabolism* ; Interleukin-6 ; Flavonoids/pharmacology* ; Propranolol/pharmacology* ; Ventricular Fibrillation ; Dinoprostone ; Wnt Signaling Pathway ; Plant Leaves/metabolism* ; Flowers/metabolism* ; Apoptosis ; Cardiac Complexes, Premature

This study aimed to investigate the biological effects and underlying mechanisms of the total ginsenosides from Panax ginseng stems and leaves on lipopolysaccharide(LPS)-induced acute lung injury(ALI) in mice. Sixty male C57BL/6J mice were randomly divided into a control group, a model group, the total ginsenosides from P. ginseng stems and leaves normal administration group(61.65 mg·kg~(-1)), and low-, medium-, and high-dose total ginsenosides from P. ginseng stems and leaves groups(15.412 5, 30.825, and 61.65 mg·kg~(-1)). Mice were administered for seven continuous days before modeling. Twenty-four hours after modeling, mice were sacrificed to obtain lung tissues and calculate lung wet/dry ratio. The number of inflammatory cells in bronchoalveolar lavage fluid(BALF) was detected. The levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in BALF were detected. The mRNA expression levels of IL-1β, IL-6, and TNF-α, and the levels of myeloperoxidase(MPO), glutathione peroxidase(GSH-Px), superoxide dismutase(SOD), and malondialdehyde(MDA) in lung tissues were determined. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in lung tissues. The gut microbiota was detected by 16S rRNA sequencing, and gas chromatography-mass spectrometry(GC-MS) was applied to detect the content of short-chain fatty acids(SCFAs) in se-rum. The results showed that the total ginsenosides from P. ginseng stems and leaves could reduce lung index, lung wet/dry ratio, and lung damage in LPS-induced ALI mice, decrease the number of inflammatory cells and levels of inflammatory factors in BALF, inhibit the mRNA expression levels of inflammatory factors and levels of MPO and MDA in lung tissues, and potentiate the activity of GSH-Px and SOD in lung tissues. Furthermore, they could also reverse the gut microbiota disorder, restore the diversity of gut microbiota, increase the relative abundance of Lachnospiraceae and Muribaculaceae, decrease the relative abundance of Prevotellaceae, and enhance the content of SCFAs(acetic acid, propionic acid, and butyric acid) in serum. This study suggested that the total ginsenosides from P. ginseng stems and leaves could improve lung edema, inflammatory response, and oxidative stress in ALI mice by regulating gut microbiota and SCFAs metabolism.
Mice ; Male ; Animals ; Ginsenosides/pharmacology* ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6 ; Panax/genetics* ; Lipopolysaccharides/adverse effects* ; Gastrointestinal Microbiome ; RNA, Ribosomal, 16S ; Mice, Inbred C57BL ; Acute Lung Injury/genetics* ; Lung/metabolism* ; Superoxide Dismutase/metabolism* ; Plant Leaves/metabolism* ; RNA, Messenger

A field experiment was conducted to measure the physiological characteristics, yield, active ingredient content, and other indicators of Carthamus tinctorius leaves undergoing 13 sowing date treatments. The principal component analysis(PCA) and redundancy analysis were used to analyze the correlation between these indicators to explore the effect of sowing date on the yield and active ingredient content of C. tinctorius in Liupanshan of Ningxia. The results illustrated that the early sowing in autumn and spring had significant effects on leaf photosynthetic parameters, SPAD value, antioxidant enzyme activity, nitrogen metabolism enzyme activity, filament yield, grain yield, and hydroxy safflower yellow A(HYSA) of C. tinctorius. Sowing in mid-November and late March had the best effect. Leaf transpiration rate, stomatal conductance, nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase increased by 44.9%, 52.4%, 15.9%, 60.8%, 10.3%, and 38.3%, respectively. The activities of superoxide dismutase, peroxidase, and catalase decreased by 10.8%, 4.1%, and 20.9%, respectively. The improvement of photosynthetic physiological characteristics promoted the dry matter accumulation and reproductive growth of C. tinctorius. The yield of filaments and seeds increased by 15.5% and 11.7%, and the yield of HYSA and kaempferol increased by 17.9% and 20.0%. In short, the suitable sowing date can promote the growth and development of C. tinctorius in Liupanshan of Ningxia, and significantly improve the yield and quality, which is conducive to the high quality and efficient production of C. tinctorius.
Carthamus tinctorius ; Seeds ; Peroxidase/metabolism* ; Plant Leaves/metabolism* ; Antioxidants

Mulberry (Morus alba L.) leaf is a well-established traditional Chinese botanical and culinary resource. It has found widespread application in the management of diabetes. The bioactive constituents of mulberry leaf, specifically mulberry leaf flavonoids (MLFs), exhibit pronounced potential in the amelioration of type 2 diabetes (T2D). This potential is attributed to their ability to safeguard pancreatic β cells, enhance insulin resistance, and inhibit α-glucosidase activity. Our antecedent research findings underscore the substantial therapeutic efficacy of MLFs in treating T2D. However, the precise mechanistic underpinnings of MLF's anti-T2D effects remain the subject of inquiry. Activation of brown/beige adipocytes is a novel and promising strategy for T2D treatment. In the present study, our primary objective was to elucidate the impact of MLFs on adipose tissue browning in db/db mice and 3T3-L1 cells and elucidate its underlying mechanism. The results manifested that MLFs reduced body weight and food intake, alleviated hepatic steatosis, improved insulin sensitivity, and increased lipolysis and thermogenesis in db/db mice. Moreover, MLFs activated brown adipose tissue (BAT) and induced the browning of inguinal white adipose tissue (IWAT) and 3T3-L1 adipocytes by increasing the expressions of brown adipocyte marker genes and proteins such as uncoupling protein 1 (UCP1) and beige adipocyte marker genes such as transmembrane protein 26 (Tmem26), thereby promoting mitochondrial biogenesis. Mechanistically, MLFs facilitated the activation of BAT and the induction of WAT browning to ameliorate T2D primarily through the activation of AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) signaling pathway. These findings highlight the unique capacity of MLF to counteract T2D by enhancing BAT activation and inducing browning of IWAT, thereby ameliorating glucose and lipid metabolism disorders. As such, MLFs emerge as a prospective and innovative browning agent for the treatment of T2D.
Mice ; Animals ; Adipose Tissue, Brown ; Sirtuin 1/pharmacology* ; Diabetes Mellitus, Type 2/metabolism* ; AMP-Activated Protein Kinases/metabolism* ; Morus/metabolism* ; Flavonoids/metabolism* ; Prospective Studies ; Signal Transduction ; Adipose Tissue, White ; Plant Leaves ; Uncoupling Protein 1/metabolism* ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism*

Grape (Vitis vinifera L.) in production is frequently exposed to inadequate light, which significantly affects its agronomic traits via inhibiting their physiological, metabolic and developmental processes. To explore the mechanism how the grape plants respond to the weak light stress, we used 'Yinhong' grape and examined their physiology-biochemistry characteristics and transcriptional profile under different levels of weak light stress. The results showed that grape seedlings upon low intensity shading treatments were not significantly affected. As the shading stress intensity was strengthened, the epidermis cells, palisade tissue, and spongy tissue in the leaves were thinner, the intercellular space between the palisade tissue and spongy tissue was larger compared with that of the control, and the activities of superoxide dismutase, catalase and peroxidase were decreased gradually. Additionally, the soluble protein content increased and the free proline content decreased gradually. Compared with the control, significant changes in plant photosynthetic characteristics and physiology-biochemistry characteristics were observed under high intensity of shading (80%). RNA-seq data showed that the differentially expressed genes between CK and T2, CK and T4, T2 and T4 were 13 913, 13 293 and 14 943, respectively. Most of the enrichment pathways were closely related with the plant's response to stress. Several signaling pathways in response to stress-resistance, e.g. JA/MYC2 pathway and MAPK signal pathway, were activated under weak light stress. The expression level of a variety of genes related to antioxidation (such as polyphenol oxidase and thioredoxin), photosynthesis (such as phytochrome) was altered under weak light stress, indicating that 'Yinhong' grape may activate the antioxidation related pathways to cope with reactive oxygen species (ROS). In addition, it may activate the expression of photosynthetic pigment and light reaction structural protein to maintain the photosynthesis activity. This research may help better understand the relevant physiological response mechanism and facilitate cultivation of grape seedlings under weak light.
Vitis/metabolism* ; Gene Expression Regulation, Plant ; Photosynthesis/genetics* ; Plant Leaves ; Light ; Seedlings/metabolism*

To explore the mechanism of tea albino variation and high theanine formation, 'Fuyun 6' and a new theanine-rich tea cultivar 'Fuhuang 2' were as materials in this study, pigment content, metabolome and transcriptome of the two cultivars were analyzed by ultramicroelectron microscopy, widely targeted metabolomics, targeted metabolomics and transcriptomics. The results showed that five catechins, theobromine, caffeine, and 20 free amino acids, including theanine, glutamine, arginine, etc., were identified by targeted metabolomics. The amino acid content of 'Fuhuang 2' was significantly higher than that of 'Fuyun 6', and the theanine content was as high as 57.37 mg/g in 'Fuhuang 2'. The ultrastructure of leaves showed that the chloroplast cell structure of 'Fuhuang 2' was fuzzy, most of the grana lamellae were arranged in disorder, with large gaps, and the thylakoids were filiform. The determination of pigments showed that compared with 'Fuyun 6', the contents of chlorophyll A and B, carotenoids, flavonoids and other pigments of 'Fuhuang 2' decreased significantly, some important pigment-related-genes, such as chlorophyllase (CLH), 9-cis-epoxycarotenoid dioxygenase (NCED), flavonoid 3β-hydroxylase (F3H) and flavonoid 3', 5'-hydroxylase (F3'5'H) were significantly changed. Compared with 'Fuyun 6', 'Fuhuang 2' identified 138 significantly changed metabolites (SCMs) and 658 differentially expressed genes (DEGs). KEGG enrichment analysis showed that SCMs and DEGs were significantly enriched in amino acid biosynthesis, glutathione metabolism and TCA cycle. In general, the albino phenotype of 'Fuhuang 2' may be caused by a deficiency in photosynthetic proteins, chlorophyll metabolism genes and chlorophyll content. The accumulation of high theanine in 'Fuhuang 2' may be due to the low nitrogen consumption in yellowed leaves and the lack of carbon skeleton, amino and nitrogen resources are stored more effectively, resulting in the up regulation of metabolites and related gene expression in the amino acid synthesis pathway, theanine has become a significant accumulation of nitrogen-containing compounds in yellowed leaves.
Camellia sinensis/genetics* ; Chlorophyll A/metabolism* ; Plant Proteins/genetics* ; Plant Leaves/chemistry* ; Chlorophyll/metabolism* ; Transcriptome ; Flavonoids/metabolism* ; Amino Acids/genetics* ; Tea ; Mixed Function Oxygenases/metabolism* ; Nitrogen/metabolism*

Light is the main source for plants to obtain energy.Asarum forbesii is a typical shade medicinal plant, which generally grows in the shady and wet place under the bushes or beside the ditches.It can grow and develop without too much light intensity.This experiment explores the effects of shading on the growth, physiological characteristics and energy metabolism of A.forbesii, which can provide reference and guidance for its artificial planting.In this experiment, A.forbesii was planted under 80%, 60%, 40%, 20% and no shade.During the vigorous growth period, the photosynthetic physiological characteristics such as fluorescence parameters, photosynthetic parameters, photosynthetic pigment content and ultrastructure, as well as the content of mitochondrial electron transport chain(ETC) synthase and nutrients were measured.The results showed that the photosynthetic pigment content, chlorophyll fluorescence parameters and net photosynthesis rate(P_n) decreased with the decrease of shading.Under 20%-40% shading treatment, the plants had damaged ultrastructure, expanded and disintegrated chloroplast, disordered stroma lamella and grana lamella, and increased osmiophi-lic granules and starch granules.The activities of nicotinamide adenine dinucleotide dehydrogenase(NADH), succinate dehydrogenase(SDH), cytochrome C oxidoreductase(CCO) and adenosine triphosphate(ATP) synthasewere positively related to light intensity.With the reduction of shading, the content of total sugar and protein in nutrients increased first and then decreased, and the content was the highest under 60% shade.In conclusion, under 60%-80% shading treatment, the chloroplast and mitochondria had more complete structure, faster energy metabolism, higher light energy-conversion efficiency, better absorption and utilization of light energy and more nutrient synthesis, which was more suitable for the growth and development of A.forbesii.
Asarum ; Chlorophyll/metabolism* ; Chloroplasts ; Energy Metabolism ; Photosynthesis/physiology* ; Plant Leaves/metabolism*