Multi-Omics Analysis Reveals the Resistance Mechanism and the Pathogens Causing Root Rot of Coptis chinensis.

Coptis chinensis is a traditional Chinese medicinal herb used for more than 2,000 years. Root rot in C. chinensis can cause brown discoloration (necrosis) in the fibrous roots and rhizomes, leading to plants wilting and dying. However, little information exists about the resistance mechanism and the potential pathogens of the root rot of C. chinensis plants. As a result, in order to investigate the relationship between the underlying molecular processes and the pathogenesis of root rot, transcriptome and microbiome analyses were performed on healthy and diseased C. chinensis rhizomes. This study found that root rot can lead to the significant reduction of medicinal components of Coptis, including thaliotrine, columbamine, epiberberin, coptisine, palmatine chloride, and berberine, affecting its efficacy quality. In the present study, Diaporthe eres, Fusarium avenaceum, and Fusarium solani were identified as the main pathogens causing root rot in C. chinensis. At the same time, the genes in phenylpropanoid biosynthesis, plant hormone signal transduction, plant-pathogen interaction, and alkaloid synthesis pathways were involved in the regulation of root rot resistance and medicinal component synthesis. In addition, harmful pathogens (D. eres, F. avenaceum and F. solani) also induce the expression of related genes in C. chinensis root tissues to reduce active medicinal ingredients. These results provide insights into the root rot tolerance study and pave the way for process disease resistance breeding and quality production of C. chinensis. IMPORTANCE Root rot disease significantly reduces the medicinal quality of Coptis chinensis. In the present study, results found that the C. chinensis fibrous and taproot have different tactics in response to rot pathogen infection. Diaporthe eres, Fusarium avenaceum, and Fusarium solani were isolated and identified to cause different degrees of C. chinensis root rot. These results are helpful for researchers to further explore the mechanism of resistance to rhizoma Coptis root rot.

First report of stem rot of Scrophularia ningpoensis caused by Fusarium commune in China.

Chinese figwort (Scrophularia ningpoensis Hemsl.) is an important annual herb and its dried root tubers are used as a traditional Chinese herbal medicine. In May 2021, a disease with stem rot symptoms on S. ningpoensis was observed at three randomly selected fields (~0.67 ha per field) in Nanchuan district (28.93°N, 107.27°E) of Chongqing, China. Disease incidence was estimated between 10% and 17% based on calculating the proportion of symptomatic plants. Initially, watery dark brown spots appeared on the epidermis of the stem. Then the spots expanded into spindle or strip shape, and the center of lesions were sunken, constricted and rotted finally (Figure 1A and Figure 1B). Leaves turned yellow and the plants wilted (Figure 1C). The infected parts of the stem broke easily and became brittle. The number of daughter buds used for reproduction was reduced by more than 24% and the production of root tubers decreased by more than 3%. Twelve stems with typical rot symptoms were sampled from the three fields for further investigation. Infested tissue fragments (4×4 mm) were surface sterilized with 75% ethanol for 30s and 2% sodium hypochlorite for 2 minutes in turn, finally, were rinsed 4 times with sterilized water. The disinfected tissue were air-dried and transferred onto potato dextrose agar (PDA) in the dark for 6 days at 25℃. The resulting fungal colonies were isolated by the single-spore isolation technique (Fang. 1998). Six different fungal colonies were isolated (X1-X6) and Koch's postulates were conducted to verify the pathogenicity of individual isolates. The stem surfaces of 8 months old plants were sterilized with 75% ethanol for 30 s, rinsed three times with sterilized water, and stabbed with a sterilized needle. Conidial from the fungal colonies grown on PDA plate were harvested by filtration through five layers of sterilized absorbent gauze. Conidial concentration was then adjusted to 106 conidia per mL. 10 µL of conidial suspension was sprayed on stems injured with a sterile syringe. For each isolate, 6 plants were inoculated. Stems inoculated with sterilized water were used as a blank control. All plants were all put in a growth chamber at 28℃ with 75 to 80% relative humidity under a 12 h photoperiod for 15 days. The pathogenicity test was repeated once. After 13 days, the stems inoculated with X3 showed the same rot symptoms as we observed in the fields (Figure 1D) whereas the control stems remained symptomless (Figure 1E). The fungus re-isolated from the plants showing 100% symptoms had a similar morphology than X3 as described below. At the same time, the stems inoculated with X1, X2, X4, X5 and X6 showed no sign of rot. After culturing on PDA for 9 days under 25℃ in dark, isolate X3 grew all over the dish with white or pale pink pigmentation in the center (Figure 1F). Macroconidia were produced on synthetic low nutrient agar (SNA) plates, which showed sickle or spindle, 3 septate, straight to slightly curved with a foot-shaped basal cell, ranging from 17.595~44.88 × 2.04~3.315 µm (n=30). Microconidia were oval, elliptical or reniform, 0 to 1 septate, 3.06~12.75 ×1.785~2.805 µm (n=30) in size (Figure 1G). Phialides of conidiophores were cylindrical, short and monophialides or polyphialides (Figure 1H). Chlamydospores were found terminal or cluster with round or oblong (Figure 1I). These morphological characteristics described as Fusarium commone (Skovgaard et al. 2003). For molecular identification, the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF-1α), RNA polymerase II subunit 1 (RPB1), the largest subunit of RNA polymerase Ⅱ gene sequences (RPB2) and the mitochondrial small subunit rDNA (mtSSU) genes were amplified with primers V9G /ITS4 (Hoog et al. 1998; White et al. 1990), EF1-668F /EF1-1251R (Alves et al. 2008), Fa/G2R (O'Donnell et al. 2010), 5f2/7cr (Liu et al. 1999; O'Donnell et al. 2010) and NMS1/NMS2 (Li et al. 1994). The sequences of isolate X3 were deposited in GenBank (MZ571935 (ITS), MZ576201 (EF-1α), MZ882396 (RPB1), MZ882397 (RPB2) and MZ867716 (mtSSU)). All sequences were revealed more than 99.8% sequence identity with reported sequences of Fusarium commune (GenBank accession No: KY630717, JF740838, KU171680, KU171700 and MK439851). Based on the optimal nucleotide replacement model SYM of multi-gene series sequence matrix, the system development tree was constructed. Results showed the strain X3 and those of F. commune (Isolates numbers were NRRL 28387, MRC 2566, MRC 2564 and CZ3-5-6) were clustered into the same evolutionary branch with a post-mortem probability of 0.996 (Figure 2). According to the morphology, molecular identification and phylogenetic analysis based on the concatenated of EF-1α and RPB2 genes sequences, the isolated X3 was identified as F. commune. The ITS sequences of X1, X2, X4, X5 and X6 showed homology exceeding 97.1% to Fusarium tricinctum (MH931273), Plectosphaerella cucumerina (MH858371), Sordariomycetes sp. (JX179237), Whalleya microplace (EF026129) and Pestalotiopsis maculiformans (EU552147), respectively, suggested the five strains to be these species possibly. GeneBank accession number of X1, X2, X4, X5 and X6 was OM074010, OM074011, OM074013, OM074015 and OM074018, respectively. To our best knowledge, this is the first report of F. commune infecting S. ningpoensis in China. Stem rot caused by F. commune is a severe threat to Chinese figwort cultivation, and identification of this pathogen is important for effective disease management and control.

[Effect of drying processing methods on different specifications of Sophorae Flos based on comprehensive statistical analysis].

To investigate the effects of six common drying methods on the quality of different specifications of Sophorae Flos, in order to select their suitable drying methods. According to appearance and morphology, Sophorae Flos was divided into the following three specifications: flower bud type(HL), half-open type(BK) and blooming type(SK). All specifications of samples were treated with shade-drying method(25 ℃, natural temperature), sun-drying method, hot-air-drying method(60, 105 ℃), and drying method(60 ℃) after steaming. The contents of total flavonoids, rutin, narcissus, quercetin, isorhamnetin, and Fe~(3+) reducing ability, DPPH free radical scavenging ability, ABTS free radical scavenging ability and fluorescence recovery after photobleaching(FRAP) were detected by UV, HPLC and colorimetry, respectively. Principal component analysis(PCA), cluster analysis(CA) and correlation analysis were used to comprehensively evaluate the quality of samples. According to the results, there were significant differences in the effect of drying methods on different specifications of samples. The drying method(60 ℃) after steaming was suitable for HL and BK, while the hot-air-drying method(60 ℃) was suitable for SK. When the fresh medicinal materials could not be treated in time, they should be spread out in a cool and ventilated place. Under high and low temperature conditions, the quality of three specifications of Sophorae Flos would be reduced. The hot-air-drying method(105 ℃) and shade-drying method(25 ℃) were not suitable for the treatment of fresh flowers and flower buds of Sophora japonicus. There were obviously differences of chemical compositions and antioxidant activities among the three specifications of samples. Therefore, the specifications of medicinal materials should be controlled to ensure the uniform quality. The study provided the abundant data reference for the selection of appropriate drying methods for the three specifications of Sophorae Flos, and useful exploration for the classification and processing of medicinal materials of flowers.

First Report of Root Rot Caused by Fusarium avenaceum on Coptis chinensis Franchet in Chongqing, China.

Coptis chinensis Franchet, is a perennial herb used as a traditional Chinese medicine. Annual production of Coptis is about 3000 tons in Shizhu, Chongqing. In recent years, root rot has become a serious and widespread disease on Coptis in Shizhu with an average incidence of 40%, and yield losses up to 67%. Infected plants were easy to pull from the soil, and most of the fibrous roots and main roots were brown or black compared to healthy roots that were yellow. Severely infected plants were wilted and necrotic. In October 2019, 33 diseased roots were collected from Shizhu (30°18'N, 108°30'E), and small samples (0.5 cm in length) were cut from the border between diseased and healthy tissue, successively sterilized with 75% ethanol and 2% sodium hypochlorite, rinsed 3 times with sterilized water, dried on sterilized filter paper, and transferred onto PDA, and incubated at 25°C for 7 days in dark. Eighteen distinct fungal isolates (H1-H18) were isolated and Koch's postulates were conducted to verify the pathogenicity of individual isolates. The rhizosphere soil of healthy 2-year-old Coptis plants was inoculated by pouring 5 mL of conidial suspension (106 conidia/mL) scraped from a culture of each isolate on PDA. Sterilized water was used to mock inoculate. For each isolate, 6 plants were inoculated. After 20 days, the roots of all plants inoculated with H15 or H18 were dark brown and rotten, while mock inoculated plants were healthy. The isolates H15 and H18 were re-isolated from symptomatic plants. Isolate H15 formed abundant white mycelium on PDA and produced rose pigment in the agar. Conidia were long and slender, straight to slightly curved, with 1-3 septate. The apical cells were tapering and bent, and the foot cells were distinctly notched. Conidiogenous cells were monophialides and polyphialides. No chlamydospores were observed (Figure S1). Isolate H18 formed white sparse mycelium on PDA and produced no pigment in the agar. Conidia were relatively wide, straight and stout, with 3-5 septate. The apical cells were blunt and rounded, and the foot cells were barely notched. Conidiogenous cells were long monophialides. Chlamydospores were formed intercalary in the hyphae (Figure S2). For further identification, the internal transcribed spacer (ITS), ß-tubulin, translation elongation factor 1ɑ (EF1ɑ) and RNA polymerase second largest subunit (RPB2) gene regions were amplified with ITS1/ITS4, Bt2a/Bt2b, EF1/EF2 and 5f2/7cr (White et al. 1990; Glass and Donaldson, 1995; O'Donnell et al. 2010). GenBank accession numbers of H15 and H18 were MT463390 and MT463389 for the ITS region, MT465656 and MT465654 for ß-tubulin, MT653321 and MT465651 for EF1ɑ, and MT653323 and MT653322 for RPB2. BLAST results showed the ITS, ß-tubulin, EF1ɑ, and RPB2 sequences revealed 100% (533/533 base pairs), 100% (265/265 base pairs), 98% (622/632 base pairs), and 99% (936/947 base pairs) homology respectively with those of Fusarium avenaceum (MN186746.1, MH791368.1, KU238140.1, and MK185027.1), and 100% (537/537 base pairs), 100% (227/227 base pairs), 100% (688/688 base pairs), and 99.03% (918/927 base pairs) with F. solani in GenBank (MH857319.1, MN692929.1, KP674211.1, and MH300549.1), respectively. Thus, H15 and H18 were identified as F. avenaceum and F. solani based on its morphological and molecular characteristics. To our knowledge, F. solani has been previously reported as a pathogen on Coptis (Luo et al. 2014), and this is the first report of root rot on Coptis caused by F. avenaceum in the world. Identification of the pathogens is important for effective disease management and control.

Berberine inhibits cancer cells growth by suppressing fatty acid synthesis and biogenesis of extracellular vesicles.

AIMS: Berberine is an isoquinoline alkaloid extracted from the root, rhizome and stem bark of Coptidis Rhizoma. Previous studies have revealed the anti-tumor potential of berberine against various types of cancer cells. However, the underlying mechanisms are not yet fully understood. In this study, we focused on the effects of berberine on fatty acid synthesis and extracellular vesicles formation in cancer cells, and revealed the internal mechanism of berberine inhibition on cancer cell proliferation. MATERIALS AND METHODS: Anti-proliferative activity of berberine was determined by cell counting and microscope observation and cell cycle analysis. Activities of AMPK and ACC, expression of extracellular vesicles markers were detected by western blotting. 13C labeling metabolic flux analysis was used for determination of de novo synthesis of fatty acids. The excreted extracellular vesicles in culture mediums were separated by both polyethylene glycol enrichment of extracellular vesicles and differential centrifugation separation. KEY FINDINGS: Among our early experiments, 5-10 µmol/L berberine exhibited the substantial anti-proliferative effect against human colon cancer cell line HCT116, cervical cancer cell line HeLa and other cancer cells. It was also revealed that, through activating AMPK, berberine inhibited ACC activity then suppressed intracellular fatty acid synthesis, finally decreased the biogenesis of extracellular vesicles. Moreover, supplement with citrate acid, palmitic acid, as well as exogenous extracellular vesicles, could rescue the inhibitory effect of berberine on cell proliferation, suggesting that inhibited ACC activity, suppressed fatty acid synthesis and decreased extracellular vesicles production were important mechanisms account for berberine inhibiting cancer cell proliferation. SIGNIFICANCE: Our study indicates that berberine suppresses cancer cell proliferation through inhibiting the synthesis of fatty acids and decreasing biogenesis and secretion of extracellular vesicles, suggests that berberine is a promising candidate for the development of new therapies for cancer.

Variations in the Components and Antioxidant and Tyrosinase Inhibitory Activities of Styphnolobium japonicum (L.) Schott Extract during Flower Maturity Stages.

Styphnolobium japonicum (L.) Schott is widely cultivated in China, and its flowers and flower buds (FFB-SJ) are commonly used as traditional Chinese medicine. This work aimed to assess variations in the chemical components and antioxidant and tyrosinase inhibitory activities of S. japonicum extract during five flower maturity stages (ES1-ES5). The results showed that the contents of total flavonoids, rutin, and narcissin were highest at ES1, whereas the contents of quercetin and isorhamnetin were highest at ES3. ES1 presented considerable antioxidant activities in terms of reducing power (RP) and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH. ) and hydroxyl radical (. OH) scavenging capacity, whereas ES3 showed excellent tyrosinase inhibitory activity and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS.+ )- and O2 .- -scavenging capacity. Rutin and quercetin are the main bioactive components of FFB-SJ with antioxidant and tyrosinase inhibition, and the immature flower buds of S. japonicum (S2 and S3) with excellent biological activities and relatively high extract yields were the best for product development.

[Research for dynamic changes of growth and alkaloids of Coptis chinensis].

With Coptis chinensis in high-yielding soil as the object,the growth regularity of plant and dynamic change of alkaloid content was studied. The plant growth model of C. chinensis was constructed. The plant height equation was y=3.030 9+0.732 6x-0.009 6x²,the number of leaves equation was y=111.882 6-2 234.881 7/x+15 218.960 8/x²-31 740.960 8/x³,the leaf area equation was y=-217.136 1+30.552 2x-0.359 0x²,the roots talk biomass equation was y=-2.748 8+0.210 3x+0.006 4x²,the number of rootstalk equation was y=-1.246 0+0.192 6x+0.000 8x²,the fibrous root biomass equation was y=-4.973 5+0.589 4x -0.002 6x². The results indicated that the number of leaves and leaf area were increasing continuously after seedling transplanting,the leaf area of 3-year-old C. chinensis reached a maximum value of 425.83 cm²/plant,after declining.The number of leave of 5-year-old C. chinensis reached a maximum value of 70.91. With the increasing of years of growth, the number of rootstalk and rootstalk biomass of C. chinensis was increasing continuously. The biomass growth of 3-year-old and 4-year-old rootstalk was the fastest in the whole development stage of C. chinensis,the annual increase of more than 300%. The change curve of rootstalk number, rootstalk biomass and fibrous root biomass in the whole growth stage was a s-type.The dry matter partition of leafwas the highest in 1-year-old C. chinensis, and then gradually decreased,the change trend of dry matter partition of rootstalk was just the opposite, the dry matter partition of fibrous root increases with the increase of the growing year, reaching the maximum value in 3-year-old, then gradually lower trend. The root-shootratio of 1-year-old C. chinensis was the smallest, then gradually increases, the growth center gradually shifted to the roots from stems and leaves, The weight of underground part of 3-year-old C. chinensis exceeded the aboveground part, the 5-year-old C. chinensis root-shoot ratio reached the maximum value of 1.91:1.With the increasing of years of growth, the contents of coptisine, berberine, epiberberine and palmatine in rootstalk was increasing continuously. The jatrorrhizine content in 2-year-old C. chinensis was significantly lower than that in other years, the content was no significant change after that. The columbamine content reached a maximum value in 3-year-old C. chinensis,then the decreased gradually. The content of magnoflorine gradually increased and reached maximum value in 5-year-old C. chinensis.

[Transcriptome analysis reveals candidate genes involved in flavonols biosyhthesis in Sophora japonica].

In order to study the pathways of biosynthesis of flavonoids in Sophora japonica, 113 797 unigenes were obtained by Trinity software, with an average length of 803 bp, of which 72 752 unigenes were obtained from the database by high-throughput sequencing, and a total of 38 891 SSR loci were searched. Through the metabolic pathway analysis, we found that there were 135 unigenes involved in the biosynthesis of flavonoids and 959 unigene involved in other secondary metabolic pathways. Further analysis of genes involved in rutin biosynthesis revealed that 24 were associated with CHS, 52 were associated with FLS, and 11 were associated with UFGT. The obtained data of S. japonica transcriptome lays the foundation for studying the pathways of biosynthesis of flavonoids in S. japonica and provides theoretical basis for the formation of the quality of S. japonica.

Composition and diversity of rhizosphere fungal community in Coptis chinensis Franch. continuous cropping fields.

In this study, effects of continuous cropping on soil properties, enzyme activities, and relative abundance, community composition and diversity of fungal taxa were investigated. Rhizosphere soil from field continuously cropped for one-year, three-year and five-year by Coptis chinensis Franch. was collected and analyzed. Illumina high-throughput sequencing analysis showed that continuous cropping of C. chinensis resulted in a significant and continuous decline in the richness and diversity of soil fungal population. Ascomycota, Zygomycota, Basidiomycota, and Glomeromycota were the dominant phyla of fungi detected in rhizosphere soil. Fungal genera such as Phoma, Volutella, Pachycudonia, Heterodermia, Gibberella, Cladosporium, Trichocladium, and Sporothrix, were more dominant in continuously cropped samples for three-year and five-year compared to that for one-year. By contrast, genera, such as Zygosaccharomyces, Pseudotaeniolina, Hydnum, Umbelopsis, Humicola, Crustoderma, Psilocybe, Coralloidiomyces, Mortierella, Polyporus, Pyrenula, and Monographella showed higher relative abundance in one-year samples than that in three-year and five-year samples. Cluster analysis of the fungal communities from three samples of rhizosphere soil from C. chinensis field revealed that the fungal community composition, diversity, and structure were significantly affected by the continuous cropping. Continuous cropping of C. chinensis also led to significant declines in soil pH, urease, and catalase activities. Redundancy analysis showed that the soil pH had the most significant effect on soil fungal population under continuous cropping of C. chinensis.

[Identification and phylogenetic analysis of endophytic fungi isolated from Scrophularia ningpoensis].

The endophytic fungi from root, main stem, branch and leaf of Scrophularia ningpoensis were isolated and identified from Wulong and Chongqing, and the population diversity analysis and phylogenetic analysis were followed. The result indicated that, as to population diversity index, S. ningpoensis from Wulong： leaf>main stem=branch>root, branch from Chongqing>branch from Wulong. Fifty-eight endophytic fungi were obtained, most of which were the pathogens of the plant. Colletotrichum was the prevailing genus, of which C. gloeosporioides and C. boninense were the prevailing strains. Leaf and seedlings might be the main path of infection. Endophytic fungi and pathogen might convert to each other, influenced by such factors as environment, genotype et al.

[Research on bacteria microecology in root rot rhizosphere soil of Coptis chinensis produced in Shizhu city].

Illumina Hiseq 2500 high-throughput sequencing platform was used to study the bacteria richness and diversity, the soil enzyme activities, nutrients in unplanted soil, root-rot and healthy rhizophere soil of Coptis chinensis for deeply discussing the mechanism of the root-rot of C. chinensis. The high-throughput sequencing result showed that the artificial cultivation effected the bacteria community richness and diversity. The bacteria community richness in healthy and diseased rhizosphere soil showed significant lower than that of in unplanted soil (P<0.05) and declined bacteria diversity. The bacteria community richness in root-rot rhizosphere soil increased significantly than that of health and unplanted soil and the diversity was lower significant than that of unplanted soil (P<0.05). The results of soil nutrients and enzyme activities detected that the pH value, available phosphorus and urease activity decreased and the sucrase activity increased significantly (P<0.05). The content of organic carbon and alkaline hydrolysis nitrogen the catalase and urease activity in root rot soil samples was significantly lower than that of healthy soil samples (P<0.05). However, the contents of available phosphorus and available potassium were significantly in root-rot sample higher than that of healthy soil samples (P<0.05). Comprehensive analysis showed that the artificial cultivation declined the bacteria community richness and diversity. The bacteria community richness decreased significantly and the decreased diversity may be the cause of the root-rot. Meanwhile, the decrease of carbon and the catalase activity may be another cause of the root-rot in C. chinensis produced in Shizhu city, Chongqing province.

[Transcriptome characterization for Scrophularia ningpoensis based on high-throughput sequencing technology and related genes for synthesis of terpenoid compounds].

To investigate the profile of gene function and search for SSR, a new technology of high-throughput Solexa/Illumina sequencing was used to generate the root transcriptome of Scrophularia ningpoensis, and 65 602 036 raw reads were obtained. Based on the bioinformatics analysis and Trinity, 73 983 unigenes were obtained with an average length of 823 bp. The comparison of sequence homology in database showed that 56 389 unigenes had different degrees of homology. A total of 520 metabolic pathways related genes and 191 relDODO transcription factors were identified by the Swiss-Prot, GO, KEGG and COG.The 11 659 SSRs were found by MISA and the highest frequency was AG/CT. In this study, we obtained numerous SSRs to provide references for the study of functional gene cloning and genetic diversity of S. ningpoensis. The key genes involved in the secondary metabolism are the basis for the study of biosynthesis and regulatory mechanism of the secondary metabolites.

[Growth and developmental rhythm of Scrophularia ningpoensisin southwest middle mountain area of China].

Plant samples were collected and investigated periodically. According to the growth of different parts and the characteristics of dry substance accumulation of Scrophularia ningpoensis, the development of S. ningpoensis could be divided into four stages: seeding stage, stem and leaf growth stage, expanding period of root tubers, and dry substance accumulation stage of root tuber. Leaf numbers of S. ningpoensis grew gradually from one at first to 370 at the final stage, main stem leaf were 50 pieces. Leaf size increasesed with the fastest growth at the stem and leaf growth stage, average daily increase amount was 225 cm2. By the middle of August, leaf size reached to 16,270 cm2. Leaf area indexrose sharply in the seeding stage, and remained above 8 among stem and leaf growth stage and expanding period of root tubers, and rapidly reduced to zero in the stage of dry substance accumulation of root tuber. Leaf area ratio has a tendency of obvious dropping. The net assimilation rate had a small change ranges, two small peak were seeding stage and dry substance accumulation of root tuber. The value of specific leaf area was higher in seeding stage, and in the earlier stage of dry substance accumulation of root tuber. Relative growth rate changed with large ranges, higher in seeding stage, rapid decrease in stem and leaf growth stage, rose in expanding period of root tubers, and declined again in the stage of dry substance accumulation of root tuber. Crop growth rate was higher in the first and last stages, and smaller in interim stage. The growth parameters of S. ningpoensis such as relative growth rate, net assimilation rate, leaf area index, leaf area ratio, specific leaf area, crop growth rate changed along with the growth. The rule of dry matter accumulation was as follows: the dry matter increased slowly during the seeding stage and speeded up in the middle and late stages, and in dry substance accumulation of root tuber increased slower, the growth of dry matter all appeared an "S" curve, and accorded with logistic equation. Cultivation technologies of S. ningpoensis and the relevant management methods could be established according to the growth of different parts of S. ningpoensis and the characteristics of dry substance accumulation in different stage.

A 27.368 kDa retinal reductase in New Zealand white rabbit liver cytosol encoded by the peroxisomal retinol dehydrogenase-reductase cDNA: purification and characterization of the enzyme.

We obtained a full-length cDNA based on a sequence deposited in GenBank (accession No. AB045133), annotated as rabbit peroxisomal NADP(H)-dependent retinol dehydrogenase-reductase (NDRD). The rabbit NDRD gene, like its mouse and human homologs, harbors 2 initiation sites, one of which theoretically encodes a 29.6 kDa protein with 279 amino acids, and the other encodes a 27.4 kDa protein with 260 amino acids. The purification of a rabbit cytosolic retinol oxidoreductase with a subunit molecular mass of 34 kDa and an N terminus that is not completely identical to that of NDRD, has been reported. An enzyme responsible for the all-trans retinal reductase activity in the liver cytosol of New Zealand white rabbit was purified to homogeneity using differential centrifugation and successive chromatographic analyses. The subunit molecular mass of the purified enzyme, revealed by SDS-PAGE, was approximately 27 kDa. The intact molecular mass, measured by MALDI-TOF mass spectrometry, was 27.368 kDa. The 60 kDa relative mobility observed in size-exclusion chromatography indicates that the native protein probably exists as a dimer. The purified enzyme was positively confirmed to be the product of NDRD by peptide mass fingerprinting, tandem mass spectrometry, and N-terminal sequencing. Taken together, the results suggested that the native protein is truncated at the N terminus.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on vitamin A metabolism in mice.

This study aimed to clarify the effects of single and repeated administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the activities or expression of some metabolic enzymes of retinoids and the influence of supplemental vitamin A on changed vitamin A homeostasis by TCDD. In Experiment I, the mice were given a single oral dose of 40 mug TCDD/kg body weight, with or without continuous administration of 2,500 IU vitamin A/kg body weight/day, and were killed on day 1, 3, 7, 14, and 28. In Experiment II, the mice were daily given 0.1 microg TCDD/kg body weight, with or without supplemental 2,000 IU vitamin A/kg body weight, and were killed on day 14, 28, and 42. In both experiments, TCDD significantly decreased the hepatic all-trans-retinol level and increased the hepatic all-trans-retinoic acid (RA) content, increased the mRNA and enzymatic activities of retinal oxidase. In TCDD + vitamin A mice, the all-trans retinol content was significantly higher, and the retinal oxidase mRNA was significantly lower on day 3 or 7 in Experiment I and on day 14 in Experiment II, compared to TCDD-treated mice. The induction of the retinal oxidase may contribute to the decrease in hepatic all-trans-retinol level and the increase in hepatic all-trans-RA caused by TCDD. Supplemental vitamin A might decelerate the effect of TCDD on retinal oxidase mRNA.

Inhibitory effects of vitamin A on TCDD-induced cytochrome P-450 1A1 enzyme activity and expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an extremely potent environmental contaminant that produces a wide range of adverse biological effects, including the induction of cytochrome P450 1A1(CYP1A1) that may enhance the toxic effects of TCDD. Several studies indicated that concurrent supplementation of vitamin A could reduce the toxicity, and potentially inhibit CYP1A1 activity (measured as ethoxyresorufin-O-deethylase [EROD] activity). In the present study, we investigated the in vivo effects of vitamin A on EROD activities and the expression of CYP1A1 in the liver of TCDD-treated mice. In Experiment I, the mice were given a single oral dose of 40 mug TCDD/kg body weight with or without the continuous administration of 2500 IU vitamin A/kg body weight/day, and were killed on day 1, 3, 7, 14, or 28. In Experiment II, the mice were given daily an oral dose of 0.1 mug TCDD/kg body weight with or without supplement of 2000 IU vitamin A/kg body weight, and were killed on day 14, 28, or 42. In both experiments, TCDD caused liver damage and increase in relative liver weights, augmented the EROD activities and CYP1A1 expression, and increased the aryl hydrocarbon receptor (AhR) mRNA expression, but did not alter the AhR nuclear translocator (ARNT) mRNA expression. CYP1A1 mRNA expression and AhR mRNA expression showed a similar time course. The liver damage in TCDD + vitamin A-treated mice was less severe than that in TCDD-treated mice. EROD activities, CYP1A1 expression, and AhR mRNA expression in vitamin A + TCDD-treated mice were lower than those in TCDD-treated mice, indicating that supplementation of vitamin A might attenuate the liver damage caused by TCDD.