通讯机构:
[Yanlin Li] I;[Xingyao Xiong] A;Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China<&wdkj&>College of Horticulture, Hunan Agricultural University, Changsha 410128, China<&wdkj&>Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China<&wdkj&>Engineering Research Center for Horticultural Crop Germplasm Creation and New Variety Breeding, Ministry of Education, Changsha 410128, China<&wdkj&>Hunan Mid-Subtropical Quality Plant Breeding and Utilization Engineering Technology Research Center, Changsha 410128, China<&wdkj&>College of Horticulture, Hunan Agricultural University, Changsha 410128, China<&wdkj&>Kunpeng Institute of Modern Agriculture, Foshan 528200, China<&wdkj&>School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
摘要:
Abstract: This study employed a combination of ultraviolet spectrophotometry, LC-ESI-MS/MS system, and RNA-sequencing technology; the extracts and isolation of total RNA from the red and yellow leaf strains of red maple (Acer rubrum L.) at different developmental stages were subjected to an intercomparison of the dynamic content of chlorophyll and total anthocyanin, flavonoid metabolite fingerprinting, and gene expression. The metabonomic results indicated that one hundred and ninety-two flavonoids were identified, which could be classified into eight categories in the red maple leaves. Among them, 39% and 19% were flavones and flavonols, respectively. The metabolomic analysis identified 23, 32, 24, 24, 38, and 41 DAMs in the AR1018r vs. AR1031r comparison, the AR1018r vs. AR1119r comparison, the AR1031r vs. AR1119r comparison, the AR1018y vs. AR1031y comparison, the AR1018y vs. AR1119y comparison, and the AR1031y vs. AR1119y comparison, respectively. In total, 6,003 and 8,888 DEGs were identified in AR1018r vs. AR1031r comparison and in the AR1018y vs. AR1031y comparison, respectively. The GO and KEGG analyses showed that the DEGs were mainly involved in plant hormone signal transduction, flavonoid biosynthesis, and other metabolite metabolic processes. The comprehensive analysis revealed that caffeoyl-CoA 3-O-methyltransferase (Cluster-28704.45358 and Cluster-28704.50421) was up-regulated in the red strain but down-regulated in the yellow strain, while Peonidin 3-O-glucoside chloride and Pelargonidin 3-O-beta-D-glucoside were up-regulated in both the red and yellow strains. By successfully integrating the analyses on the behavior of pigment accumulation, dynamics of flavonoids, and differentially expressed genes with omics tools, the regulation mechanisms underlying leaf coloring in red maple at the transcriptomic and metabolomic levels were demonstrated, and the results provide valuable information for further research on gene function in red maple. Keywords: Acer rubrum L.; transcriptomics; metabolomics; flavonoid; coloring
摘要:
The protein levels in a diet are correlated with immunity but the long-term intake of excessive protein can compromise various aspects of health. L-theanine regulates immunity and protein metabolism; however, how its regulatory immunity effects under a high-protein diet are unclear. We used proteomics, metabonomics, and western blotting to analyze the effects of diets with different protein levels on immune function in rats to determine the role of L-theanine in immunity under a high-protein diet. The long-term intake of high-protein diets (>= 40% protein) promoted oxidative imbalance and inflammation. These were alleviated by L-theanine. High-protein diets inhibited peroxisome proliferator-activated receptor (PPAR)alpha expression through the inter-leukin (IL)-6/signal transducer and activator of transcription (STAT)3 pathway and mediated inflammation. L-theanine downregulated anti-fatty acid-binding protein 5 (FABP5), inhibited the IL-6/STAT3 axis, and reduced high-protein diet-induced PPAR alpha inhibition. Therefore, L-theanine alleviates the adverse effects of high-protein diets via the FABP5/IL-6/STAT3/PPAR alpha pathway and regulates the immunity of normally fed rats through the epoxide hydrolase (EPHX)2/nuclear factor-kappa B inhibitor (I kappa B)alpha/triggering receptor expressed on myeloid cells (TREM)1 axis.
关键词:
White tea;Rucheng Baimao;Drying treatment;Aroma
摘要:
Rucheng Baimao (Camellia pubescens) is an excellent raw material for manufacturing white tea. Drying is the last step in white tea processing and is essential for white tea's aroma. In this study, aroma sensory evaluation combined with volatile compound analysis reveals the characteristic aroma differences of Rucheng Baimao white tea with oven drying (OD), air drying (AD), and sun drying (SD) treatments. Sensory results showed that OD samples had a stronger floral and grassy aroma, AD samples exhibited a more pronounced fresh and pekoe aroma, and SD samples exhibited a unique sunshine odor. Thirty compounds were screened as differential aroma-active compounds of the aroma difference of Rucheng Baimao white tea with three drying treatments using multivariate statistical analysis and relative odor activity value (ROAV >= 1) analysis. The weighted correlation network analysis of the differential compounds revealed the potential association between volatiles and aroma. The results showed that 17 compounds (such as hexanal and p-cymene) were significantly positively correlated with the grassy aroma and 21 compounds (such as linalool and benzeneacetaldehyde) were significantly posi-tively correlated with the floral aroma. Most of these compounds were abundant in OD samples, contributed to its aroma characteristics.
摘要:
Abstract: Pepper is a horticultural crop that does not tolerate low temperatures. To investigate how the grafted pepper responds to low temperature stress in the short term, transcriptome analysis was performed on grafted seedlings treated with low temperature for 1 h, 4 h, 12 h and 24 h compared with those treated for 0 h. The results showed that genes related to CAM4, MPK8, RbohD and OXI1 might be related to the response of grafted seedlings to low temperature stress in the short term. To investigate how low temperature tolerant rootstocks can improve the low temperature tolerance of grafted peppers, morphological and physiological indices of self-rooted and grafted seedlings were analyzed under low temperature conditions for different days. The results showed that the degree of wilting, REL and MDA content of grafted seedlings were significantly lower than those of self-rooted seedlings, and the antioxidant enzyme activities were significantly higher than those of self-rooted seedlings under low temperature stress. The results indicated that grafted pepper would activate ROS-related genes in a short period of time after low temperature stress and produce a large amount of ROS in response to the low temperature stress. When ROS accumulated to a certain level, the grafted pepper could increase the enzyme activity of antioxidant system to remove the ROS produced in the body, and help the pepper seedlings adapt to low temperature stress through osmoregulation mechanism, so as to resist the damage caused by low temperature. The results of the study provide ideas for growing pepper in low temperature environment. Keywords: pepper; grafting; low-temperature tolerance; enzyme activity; osmotic substances; functional genes
摘要:
BACKGROUND: Heat stress (HS) damages the intestines, disrupting gut microbiota and immune balance. L-theanine (LTA), found in tea, alleviates oxidative stress and cell apoptosis under HS; however, its effects on gut microbiota and immunity under HS remain unclear. To investigate this, we administered LTA doses of 100, 200, and 400 mg·kg(-1) ·d(-1) to C57BL/6J mice. On day 44, the model group and LTA intervention group were subjected to continuous 7-day HS treatment for 2 h per day. RESULTS: The results demonstrated that LTA intervention improved food intake, body weight, and intestinal epithelium, and reduced water intake of heat-stressed mice. It increased the abundance of Turicibacter, Faecalibaculum, Bifidobacterium, and norank_f_Muribaculaceae, while reducing that of Lachnoclostridium and Desulfovibrio. Additionally, LTA intervention increased the concentrations of amino acid and lipid metabolites, regulated macrophage differentiation stimulated by gut microbiota and metabolites, reduced the antigen presentation by macrophages to the specific immune system, promoted B-cell differentiation and sIgA secretion, inhibited pro-inflammatory factors, and enhanced intestinal defense. Mechanistically, LTA downregulated heat shock protein 70 expression and the TLR4/NF-κB/p38 MAPK signaling pathway, restoring gut microbiota and immune balance. CONCLUSION: We suggest that LTA can alleviate HS by modulating gut microbiota, metabolites, and immunity, indicating its potential as a natural active ingredient for anti-HS food products. This article is protected by copyright. All rights reserved.
通讯机构:
[Ai-qing Zhao] S;[Zhong-hua Liu; Ai-ling Liu] N;National Research Center of Engineering and Technology for Utilization of Botanical Functional Ingredients, Co-Innovation Centre of Education Ministry for Utilization of Botanical Functional Ingredients, Hunan Agricultural University, Changsha, Hunan, China<&wdkj&>College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan, China<&wdkj&>Shaanxi Engineering Laboratory for Food Green Processing and Safety Control, and Shaanxi Key Laboratory for Hazard Factors Assessment in Processing and Storage of Agricultural Products, College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi'an, Shaanxi, China<&wdkj&>National Research Center of Engineering and Technology for Utilization of Botanical Functional Ingredients, Co-Innovation Centre of Education Ministry for Utilization of Botanical Functional Ingredients, Hunan Agricultural University, Changsha, Hunan, China<&wdkj&>Key Laboratory of Tea Science of Ministry of Education, Hunan Agricultural University, Changsha, Hunan, China
摘要:
Polyphenol oxidase (PPO) is a metalloenzyme with a type III copper core that is abundant in nature. As one of the most essential enzymes in the tea plant (Camellia sinensis), the further regulation of PPO is critical for enhancing defensive responses, cultivating high-quality germplasm resources of tea plants, and producing tea products that are both functional and sensory qualities. Due to their physiological and pharmacological values, the constituents from the oxidative polymerization of PPO in tea manufacturing may serve as functional foods to prevent and treat chronic non-communicable diseases. However, current knowledge of the utilization of PPO in the tea industry is only available from scattered sources, and a more comprehensive study is required to reveal the relationship between PPO and tea obviously. A more comprehensive review of the role of PPO in tea was reported for the first time, as its classification, catalytic mechanism, and utilization in modulating tea flavors, compositions, and nutrition, along with the relationships between PPO-mediated enzymatic reactions and the formation of functional constituents in tea, and the techniques for the modification and application of PPO based on modern enzymology and synthetic biology are summarized and suggested in this article.
摘要:
Abstract: As a most significant cereal crop, maize provides vital nutritional components to humans and livestock. Drought stress curtails maize growth and yield by impairing several morphological, physiological, and biochemical functions. The rising threats of drought stress significantly affect global food security and increase the ratio of hunger and starvation. The use of molecular breeding techniques has enabled maize researchers to deeply examine the genetic control of drought tolerance and the genetic differences between genotypes to drought stress. Despite the significant progress in molecular genetics, the drought tolerance mechanism is still not fully understood. With the advancements in molecular research, researchers have identified several molecular factors associated with maize tolerance to drought stress. Quantitative trait loci (QTL) mapping and genome-wide association study (GWAS) analysis have led to identifying QTL, and genes linked to drought tolerance in maize that can be further exploited for their possible breeding applications. Transcriptome and transcription factors (TFs) analysis has revealed the documentation of potential genes and protein groups that might be linked to drought tolerance and accelerate the drought breeding program. Genetic engineering has been used to develop transgenic maize cultivars that are resistant to drought stress. Clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) is a new ray of hope to edit the gene of interest to enhance drought tolerance in maize and save both time and cost in cultivar development. In the current review article, we have tried to present an updated picture of the advancements of drought tolerance in maize and its future prospects. These organized pieces of information can assist future researchers in understanding the basis of drought tolerance to adopt a potential breeding tool for breeding drought-tolerant maize cultivars. Keywords: maize; drought; tolerance; genes; QTL; genetic mechanism; cultivars
摘要:
The most important physiological processes in insects are those related to reproduction and development. Ecdysone is an essential hormone in insects that controls various physiological processes, including reproduction and development. E74A, a subtype of the essential ecdysone-induced transcription factor E74, affects the reproductive systems of many insects. Uncertainty exists regarding the molecular mechanism of E74A in non-model insect reproduction processes. Using bioinformatics analysis, we determined that Chilo suppressalis E74A shared the highest homology with E74 in Ostrinia furnacalis which belongs to the ETS superfamily. By characterizing the spatiotemporal expression profile of CsE74A from different developmental stages and tissues, we found that CsE74A expression levels were highest in female pupae on the 4th day and in the head of female pupa. Knockdown of CsE74A resulted in delayed oocyte maturation and reduced yolk deposition. Additionally, the expression level of vitellogenin (Vg), beta FTZ-F1, and E93, which are associated with vitellogenesis and the ecdysone pathway, were also downregulated in the E74A silencing group. Collectively, our findings demonstrate that CsE74A not only plays a critical role in the reproductive processes of C. suppressalis but may also participate in the transcriptional regulation of genes involved in the ecdysone pathway.
摘要:
Eurotium is the teleomorph genus associated with the section Aspergillus. Eurotium comprises approximately 20 species, which are widely distributed in nature and human environments. Eurotium is usually the key microorganism for the fermentation of traditional food, such as Fuzhuan brick tea, Liupao tea, Meju, and Karebushi; thus, Eurotium is an important fungus in the food industry. Eurotium has been extensively studied because it contains a series of interesting, structurally diverse, and biologically important secondary metabolites, including anthraquinones, benzaldehyde derivatives, and indol diketopiperazine alkaloids. These secondary metabolites have shown multiple biological activities, including antioxidative, antimicrobial, cytotoxic, antitumor, insecticidal, antimalarial, and anti-inflammatory activities. This study presents an up-to-date review of the phytochemistry and biological activities of all Eurotium species. This review will provide recent advances on the secondary metabolites and their bioactivities in the genus Eurotium for the first time and serve as a database for future research and drug development from the genus Eurotium.
摘要:
This research mainly focused on the leaf color change and photosystem function differ-entiation between Loropetalum chinense and its variety L. chinense var. rubrum under heat stress, which were tightly concerned about their ornamental traits and growth. L. chinense 'Xiangnong Xiangyun' (X) and L. chinense var. rubrum 'Xiangnong Fendai' (F) and L. chinense var. rubrum 'Hei Zhenzhu' (H) were chosen to be experimented on to investigate whether leaf color morphology and pigment composition could influence the adaptability of plants to high temperature in order to select foliage plants which posses stable leaf color and better adaptability for hot regions. The plants were cultured in hot environment (40 degrees C/33 degrees C, day/night) and normal environment (25 degrees C/18 degrees C, day/night). Phenotype and anatomic observation of three cultivars were made and leaf color indices and pigment contents were measured. During the experiment, H and F gradually turned green, total anthocyanins contents significantly decreased in them, however, chlorophyll b contents increased in all three cultivars. In addition, the initial fluorescence (Fo) decreased in X, while increased in H and F. For the maximum fluorescence (Fm) and maximum photochemical efficiency of PSII (Fv/Fm), they only increased in H and decreased in both F and X. The non-photochemical chlorophyll fluorescence quenching (NPQ) also increased in H and decreased in F. For X, it increased at first then gradually decreased. The coefficient of photochemical quenching all increased at first then gradually decreased. Correlation analysis between showed that there was relatively strong connection between anthocyanins, flavonoids and chlorophyll fluorescence parameters, especially NPQ, proved anthocyanins and flavonoids might not only involved in enriching leaf color, but also interfered with the protection of photosystem. Generally speaking, we found higher anthocyanin and flavonoids content level not only dramatically enriched the leaf color of L. chinense var. rubrum cultivars, but also offered more potential antioxidant to keep their normal growth when encountered heat stress.
通讯机构:
[Yang, S ; Ou, LJ] H;Hunan Agr Univ, Coll Hort, Engn Res Ctr Educ, Key Lab Vegetable Biol Hunan Prov,Minist Germplasm, Changsha 410125, Peoples R China.
摘要:
Light quality and intensity can have a significant impact on plant health and crop productivity. Chlorophylls and carotenoids are classes of plant pigments that are responsible for harvesting light energy and protecting plants from the damaging effects of intense light. Our understanding of the role played by plant pigments in light sensitivity has been aided by light-sensitive mutants that change colors upon exposure to light of variable intensity. In this study, we conducted transcriptomic, metabolomic, and hormone analyses on a novel yellowing mutant of pepper (yl1) to shed light on the molecular mechanism that regulates the transition from green to yellow leaves in this mutant upon exposure to high-intensity light. Our results revealed greater accumulation of the carotenoid precursor phytoene and the carotenoids phytofluene, antheraxanthin, and zeaxanthin in yl1 compared with wild-type plants under high light intensity. A transcriptomic analysis confirmed that enzymes involved in zeaxanthin and antheraxanthin biosynthesis were upregulated in yl1 upon exposure to high-intensity light. We also identified a single basic helix-loop-helix (bHLH) transcription factor, bHLH71-like, that was differentially expressed and positively correlated with light intensity in yl1. Silencing of bHLH71-like in pepper plants suppressed the yellowing phenotype and led to reduced accumulation of zeaxanthin and antheraxanthin. We propose that the yellow phenotype of yl1 induced by high light intensity could be caused by an increase in yellow carotenoid pigments, concurrent with a decrease in chlorophyll accumulation. Our results also suggest that bHLH71-like functions as a positive regulator of carotenoid biosynthesis in pepper.
摘要:
Ampelopsis grossedentata, commonly known as "Vine Tea" and well-recognized for its rich flavonoid content, is mainly distributed in the southern regions of the Yangtze River basin in China. These regions include Hunan, Hubei, Jiangxi, and Guizhou provinces. Vine Tea is mainly consumed as an herbal tea and has garnered attention for its reported health benefits, including antioxidant, anti-inflammatory, anti-tumor, anti-diabetic, and neuroprotective properties. It has been used to alleviate coughs and sore throats (Zhang et al., 2021; Wang et al., 2017; Gao et al., 2009). In the Zhangjiajie region of Hunan province alone, the Vine Tea planting area reached 7,670.5 hectares and produced commercial goods worth 1.417 billion RMB in 2022. In May 2021, leaf margins and veins fading to yellowing mottling, and crumpling of leaf blades in the shape of a boat symptoms were found in ~16% of Vine Tea plants in the Sanjiakuan Township, Yongding District, Zhangjiajie region (29°15'E, 110°30' N) (Figure 1a, b, c). (Figure 1a, b, c). Phytoplasma-like microbial cells (small oval shaped bacterial cells, around 1000 nm in size) were observed in sieve tube cells in the phloem of diseased leaves using transmission electron microscopy. No such cell was observed in the phloem of healthy leaves (Figure 2a, b). To investigate the potential association between phytoplasma and the observed symptoms of the diseased plants, total DNA was isolated from ten diseasedeaves and compared with ten healthy leaves from the same field using SteadyPure Plant Genomic DNA Extraction Kit. The isolated DNAs were analyzed first in a direct PCR using universal phytoplasma primer pair R16mF2/R16mR1 targeting the 16S rRNA gene (Gundersen and Lee 1996) and specific pair rpF1/rpR1 (Lee et al. 1998) targeting the DNA fragment encoding partial ribosomal proteins (rp) L22 (complete) and S3 and S19 (partial). The initial amplified products were used as templates and further amplified by nested PCR respectively with primer pair R16F2n/R16R2 for the 16S rRNA gene (Lee et al. 1998) and the rpF2/rpR2 primer pair for the rp gene (Martini et al. 2007). No amplification was obtained with DNA from healthy leaf samples using any of the four primer pairs. The amplified fragments from diseased leaves by nested PCR were cloned and sequenced (Qingke Biotech, China). The obtained sequences have been deposited in GenBank with accession numbers OR282806 for the 16S rRNA gene and GenBank OR353012 for the rp gene. BLASTn analysis revealed that the partial 16S rRNA gene sequence in our sample shared 99.4% nucleotide sequence identity with 'Candidatus Phytoplasma sp.' (MW364378) and 'Peony yellows phytoplasma' (KY814723) of the 16SrI group. Similarly, our rp gene sequence shared 99.6% nucleotide identity with the rpI group of phytoplasma such as the 'Balsamine virescence phytoplasma' (JN572890) and 'Paulownia witches'-broom phytoplasma' (HM146079). Phylogenetic analysis of the 16S rRNA and rp sequences using MEGA version 7.0 revealed that the phytoplasma strain associated with A. grossedentata yellow leaf syndrome in our study site belonged to the 16SrI (Candidatus Phytoplasma asteris) group of phytoplasma (Figure 3a, b). Using the interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009), virtual restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene sequences showed our strain having a distinct RFLP map but was closest to that of the onion yellow phytoplasma 16SrI-B subgroup (GenBank accession number: AP006628), with a similarity coefficient of 0.94 (Figure 4a, b). To confirm phytoplasma transmission, healthy plants were inoculated with three scions of infected plants of A. grossedentata. After 16 days, the new leaves of the inoculated A. grossedentata showed yellow leaf symptoms (Figure 5a, b, c), akin to the symptoms originally observed in the field, and the outer contour of the leaf margin appeared chlorotic. After 26 days, primer pairs R16mF2/R16R1 and R16F2n/R16R2 were used for nested PCR detection of phytoplasma in symptomatic A. grossedentata leaves. Phytoplasma was detected in the first and second leaves of symptomatic branches and leaves while negative control showed no amplification. Sequencing of the amplified fragments showed 100% nucleotide identity to the strain from the grafting source. Our results indicated that the pathogen and the disease can be transmitted by tissue grafting, consistent with the biological characteristics of phytoplasma, and further confirmed that the phytoplasma was the pathogen of yellow leaf syndrome of A. grossedentata. Toour knowledge, this is the first report of phytoplasma of group 16SrI affecting A. grossedentata.
摘要:
Doxorubicin (DOX)-induced cardiotoxicity (DIC) is a major impediment to its clinical application. It is indispensable to explore alternative treatment molecules or drugs for mitigating DIC. WGX50, an organic extract derived from Zanthoxylum bungeanum Maxim, has anti-inflammatory and antioxidant biological activity, however, its function and mechanism in DIC remain unclear. We established DOX-induced cardiotoxicity models both in vitro and in vivo. Echocardiography and histological analyses were used to determine the severity of cardiac injury in mice. The myocardial damage markers cTnT, CK-MB, ANP, BNP, and ferroptosis associated indicators Fe2+, MDA, and GPX4 were measured using ELISA, RT-qPCR, and western blot assays. The morphology of mitochondria was investigated with a transmission electron microscope. The levels of mitochondrial membrane potential, mitochondrial ROS, and lipid ROS were detected using JC-1, MitoSOX™, and C11-BODIPY 581/591 probes. Our findings demonstrate that WGX50 protects DOX-induced cardiotoxicity via restraining mitochondrial ROS and ferroptosis. In vivo, WGX50 effectively relieves doxorubicin-induced cardiac dysfunction, cardiac injury, fibrosis, mitochondrial damage, and redox imbalance. In vitro, WGX50 preserves mitochondrial function by reducing the level of mitochondrial membrane potential and increasing mitochondrial ATP production. Furthermore, WGX50 reduces iron accumulation and mitochondrial ROS, increases GPX4 expression, and regulates lipid metabolism to inhibit DOX-induced ferroptosis. Taken together, WGX50 protects DOX-induced cardiotoxicity via mitochondrial ROS and the ferroptosis pathway, which provides novel insights for WGX50 as a promising drug candidate for cardioprotection.
摘要:
The tea plant is a vital strategic forest resource in China. Dark tea produced from its leaves is an indispensable health-promoting product in western China due to its unique lipid-lowering function. Eurotium cristatum is the dominant strain in Fuzhuan brick tea (a variety of Anhua dark tea) and could produce many functional components, including lovastatin, a lipid-lowering compound. In this study, the lovastatin yield of dark tea was improved by breeding Eurotium cristatum using the protoplast fusion method. The experiments were carried out by inducing a fusion between inactivated Eurotium cristatum JH1205 and Monascus CICC5031. Among the 92 fusants screened the HPLC method, four strains (A4, A36, A54, and A76) with higher lovastatin production (more than three times as high) were obtained. The A76 strain had the highest lovastatin yield, which was 23.93 mu g/mL. The location of the tea forest strongly influenced the lovastatin yield of loose dark tea. The strain bred in this study improved the lovastatin yield of loose dark tea by more than three times when compared to wild Eurotium cristatum. These results are promising for the development of tea forest resources.
摘要:
Protoplast-based engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. Uncaria rhynchophylla is a traditional Chinese medicinal plant with a variety of pharmaceutically important indole alkaloids. In this study, an optimized protocol for U. rhynchophylla protoplast isolation, purification, and transient gene expression was developed. The best protoplast separation protocol was found to be 0.8 M D-mannitol, 1.25% Cellulase R-10, and 0.6% Macerozyme R-10 enzymolysis for 5 h at 26 degrees C in the dark with constant oscillation at 40 rpm/min. The protoplast yield was as high as 1.5 x 10(7) protoplasts/g fresh weight, and the survival rate of protoplasts was greater than 90%. Furthermore, polyethylene glycol (PEG)-mediated transient transformation of U. rhynchophylla protoplasts was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. The U. rhynchophylla protoplast transfection rate was highest (71%) when protoplasts were transfected overnight at 24 degrees C with the 40 mu g of plasmid DNA for 40 min in a solution containing 40% PEG. This highly efficient protoplast-based transient expression system was used for subcellular localization of transcription factor UrWRKY37. Finally, a dual-luciferase assay was used to detect a transcription factor promoter interaction by co-expressing UrWRKY37 with a UrTDC-promoter reporter plasmid. Taken together, our optimized protocols provide a foundation for future molecular studies of gene function and expression in U. rhynchophylla.