Circadian rhythm response and its effect on photosynthetic characteristics of the Lhcb family genes in tea plant.

BACKGROUND: The circadian clock, also known as the circadian rhythm, is responsible for predicting daily and seasonal changes in the environment, and adjusting various physiological and developmental processes to the appropriate times during plant growth and development. The circadian clock controls the expression of the Lhcb gene, which encodes the chlorophyll a/b binding protein. However, the roles of the Lhcb gene in tea plant remain unclear. RESULTS: In this study, a total of 16 CsLhcb genes were identified based on the tea plant genome, which were distributed on 8 chromosomes of the tea plant. The promoter regions of CsLhcb genes have a variety of cis-acting elements including hormonal, abiotic stress responses and light response elements. The CsLhcb family genes are involved in the light response process in tea plant. The photosynthetic parameter of tea leaves showed rhythmic changes during the two photoperiod periods (48 h). Stomata are basically open during the day and closed at night. Real-time quantitative PCR results showed that most of the CsLhcb family genes were highly expressed during the day, but were less expressed at night. CONCLUSIONS: Results indicated that CsLhcb genes were involved in the circadian clock process of tea plant, it also provided potential references for further understanding of the function of CsLhcb gene family in tea plant.

[Effects of Governor Vessel electroacupuncture on chloridion homeostasis in the cortex of rats with limb spasm after cerebral ischemia-reperfusion].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of Governor Vessel on chloridion (Cl-) homeostasis and the expression of γ-aminobutyric acid (GABA) and Na+-K+-Cl- cotransporter 1 (NKCC1) in the cerebral cortex of cerebral ischemia-reperfusion injury (CIRI) model rats, so as to explore its mechanism underl-ying alleviating limb spasm after stroke. METHODS: Forty-five male SD rats were randomly divided into normal, sham-operation, model, EA and baclofen groups, with 9 rats in each group. The CIRI model was established by occlusion of the middle cerebral artery and reperfusion. EA(100 Hz) was applied to "Dazhui" (GV14), "Jizhong"(GV6) and "Houhui" for 30 min. Rats of the baclofen group received gavage of baclofen solution (0.4 mg/kg, 1 mL/100 g), once daily for 7 consecutive days. Neurological deficit score was assessed according to Zea Longa's method. The muscular tone of quadriceps femoris of the limb was evaluated by modified Ashworth scale and electrophysiological recor-ding methods, separately. TTC staining was used to detect cerebral infarction volume, and the brain water content of rats in each group was determined by wet and dry weight method. The contents of Cl- and GABA in the cerebral cortex were detected by colorimetric method, and the expression levels of NKCC1 mRNA and protein in the cerebral cortex were detected by quantitative real-time PCR and Western blot, separately. RESULTS: No significant differences were found between the normal and sham-operation groups in all the indexes. Compared with the normal and sham-operation groups, the neurological deficit score, modified Ashworth muscle tone score, brain water content, cerebral infarct volu-me percent, Cl- content and expression levels of NKCC1 mRNA and protein were all evidently increased (P<0.01), and muscle tone of electrophyiological electromyogram (EMG) signal and GABA content were strikingly decreased (P<0.01) in the model group. Compared with the model group, both EA and baclofen groups had an obvious increase in EMG signal displayed muscle tone, and GABA content (P<0.05, P<0.01), and a marked decrease in the neurological deficit score, modified Ashworth score, brain water content, cerebral infarct percent, Cl- content and expression levels of NKCC1 mRNA and protein (P<0.05, P<0.01). CONCLUSION: EA stimulation of acupoints of the Governor Vessel can improve the degree of limb spasm and reduce the degree of cerebral edema and infarction in rats with stroke, which may be related to its functions in protecting Cl- homeostasis, up-regulating GABA concentration, and down-regulating the expression of NKCC1 protein and mRNA in the cerebral cortex.

Molecular markers in tea plant (Camellia sinensis): Applications to evolution, genetic identification, and molecular breeding.

Tea plants have a long cultivation history in the world, and the beverage (tea) made from its leaves is well known in the world. Due to the characteristics of self-incompatibility, long-term natural and artificial hybridization, tea plants have a very complex genetic background, which make the classification of tea plants unclear. Molecular marker, one type of genetic markers, has the advantages of stable inheritance, large amount of information, and high reliability. The development of molecular marker has facilitated the understanding of complex tea germplasm resources. So far, molecular markers had played important roles in the study of the origin and evolution, the preservation and identification of tea germplasms, and the excellent cultivars breeding of tea plants. However, the information is scattered, making it difficult to understand the advance of molecular markers in tea plants. In this paper, we summarized the development process and types of molecular markers in tea plants. In addition, the application advance of these molecular markers in tea plants was reviewed. Perspectives of molecular markers in tea plants were also systematically provided and discussed. The elaboration of molecular markers in this paper should help us to renew understanding of its application in tea plants.

Exogenous methyl jasmonate and cytokinin antagonistically regulate lignin biosynthesis by mediating CsHCT expression in Camellia sinensis.

Tea plant, an important beverage crop, is cultivated worldwide. Lignification can improve the hardness of tea plant, which is of great significance for tea quality. Jasmonates (JAs) and cytokinin are plant hormones that control processes of plant development and secondary metabolite accumulation. Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) is primarily involved in lignin biosynthesis. The effects of exogenous application of JAs and cytokinin on lignin biosynthesis and related HCT gene expression profiles in tea plants are still unclear. In order to investigate the effects of exogenous JAs and cytokinin on lignin accumulation, anatomical structures, and CsHCT gene profiles in tea plants, we treated tea plants with methyl jasmonate (MeJA) and cytokinin (6-BA). MeJA and 6-BA treatments triggered the lignification at 6 and 12 d in tea leaves. The combined treatment resulted in an increase in lignin content at 6 d, which was 1.32 times of that at 0 d for 'Mengshan 9.' The CsHCTs in clade 2 (CsHCT5, CsHCT6, CsHCT7, and CsHCT8) were mainly expressed in leaves. We found that exogenous MeJA and cytokinin might be able to antagonistically regulate tea plant lignin accumulation through the mediation of CsHCT expression. This study revealed that HCTs play potential important roles involved in lignin biosynthesis of tea plant development and hormonal stimuli.

[Effect of electroacupuncture of Governor Vessel on expression of GAD67 and GABA-T in cerebral cortex of rats with post-stroke limb spasticity].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of Governor Vessel (GV) on the expressions of glutamate decarboxylase 67 (GAD67) and γ-aminobutyric acid transaminase (GABA-T) in the cerebral cortex of rats with post-stroke limb spasticity, so as to explore its mechanism underlying improvement of limb spasticity. METHODS: Twenty four male SD rats were randomly and equally divided into control, sham operation, model, and EA groups. The cerebral ischemia model was established by occlusion of the middle cerebral artery (MCAO). EA (100 Hz, 1-3 mA) was applied to "Dazhui"(GV14), "Jizhong"(GV6) and "Houhui"( anteromedial of transverse process of the sixth lumbar vertebra) for 30 min, once daily for 7 consecutive days. The neurologic deficit score (0-5 points) was evaluated according to Zea Longa's method, and the muscular tension severity (0-5 points) was assessed according to the modified Ashworth muscle tone rating scale, and the tension signals of the quadriceps ferroris of the affected limb were recorded using tonotransducer and BL-420F electrophysiological recorder. The expression levels of GAD67 and GABA-T proteins and mRNAs in the cerebral cortex were detected by immunohistochemistry, fluorescence quantitative real-time PCR and Western blot, separately. RESULTS: After modeling, the neurological deficit score, muscle tone score, and the expression levels of GABA-T mRNA and protein in cerebral cortex were significantly increased (P<0.01, P<0.05), tension signal value and the expression levels of GAD67 mRNA and protein in cerebral cortex were significantly decreased (P<0.01) in the model group relevant to the control and sham operation groups. Following the intervention, the neurological deficit score, muscle tone score, and expression levels of GABA-T mRNA and protein in cerebral cortex were significantly decreased (P<0.01), tension signal value and the expression levels of GAD67 mRNA and protein in cerebral cortex were significantly increased (P<0.01, P<0.05) in the EA group in contrast to the model group. CONCLUSION: EA stimulation of Governor Vessel can ameliorate the limb spasticity symptom in MCAO rats, which may be associated with its functions in increasing the expressions of GAD67 protein and mRNA and inhibiting the expressions of GABA-T protein and mRNA, thereby playing the inhibitory role of GABA.

Isolation and Characterization of an LBD Transcription Factor CsLBD39 from Tea Plant (Camellia sinensis) and Its Roles in Modulating Nitrate Content by Regulating Nitrate-Metabolism-Related Genes.

Nitrate nitrogen is an important nitrogen source for tea plants' growth and development. LBD transcription factors play important roles in response to the presence of nitrate in plants. The functional study of LBD transcription factors in tea plants remains limited. In this study, the LBD family gene CsLBD39 was isolated and characterized from tea plants. Sequence analysis indicated that CsLBD39 contained a highly conserved CX2CX6CX3CX domain. The phylogenetic tree assay showed that CsLBD39 belonged to class II subfamily of the LBD family. CsLBD39 was highly expressed in flowers and root; we determined that its expression could be induced by nitrate treatment. The CsLBD39 protein was located in the nucleus and has transcriptional activation activity in yeast. Compared with the wild type, overexpression of CsLBD39 gene in Arabidopsis resulted in smaller rosettes, shorter main roots, reduced lateral roots and lower plant weights. The nitrate content and the expression levels of genes related to nitrate transport and regulation were decreased in transgenic Arabidopsis hosting CsLBD39 gene. Compared with the wild type, CsLBD39 overexpression in transgenic Arabidopsis had smaller cell structure of leaves, shorter diameter of stem cross section, and slender and compact cell of stem longitudinal section. Under KNO3 treatment, the contents of nitrate, anthocyanins, and chlorophyll in leaves, and the content of nitrate in roots of Arabidopsis overexpressing CsLBD39 were reduced, the expression levels of nitrate transport and regulation related genes were decreased. The results revealed that CsLBD39 may be involved in nitrate signal transduction in tea plants as a negative regulator and laid the groundwork for future studies into the mechanism of nitrate response.

[Clinical observation on wrist-ankle acupuncture for shoulder-hand syndrome phaseâ after stroke].

OBJECTIVE: To compare the clinical efficacy between wrist-ankle acupuncture and conventional acupuncture on shoulder-hand syndrome (SHS) phaseⅠafter stroke. METHODS: A total of 64 patients with SHS phaseⅠafter stroke were randomized into a wrist-ankle acupuncture group and a conventional acupuncture group, 32 cases in each group. On the basis treatment of internal medicine and conventional rehabilitation, wrist-ankle acupuncture was applied at upper 4 area, upper 5 area and upper 6 area on the affected side in the wrist-ankle acupuncture group, while acupuncture was applied at Jianyu (LI 15), Quchi (LI 11), Shousanli (LI 10), etc. on the affected side in the conventional acupuncture group. The treatment was given 30 min each time, once a day, 5 days a week for 3 weeks in both groups. Before and after treatment, the visual analogue scale (VAS) score, degree of hand swelling, shoulder-hand syndrome scale (SHSS) score, Fugl-Meyer assessment for upper extremity (FMA-UE) score and modified Barthel index (MBI) score were observed, and the clinical therapeutic effect was evaluated in both groups. RESULTS: After treatment, the VAS scores, degree of hand swelling and SHSS scores were decreased (P<0.05), and the FMA-UE scores and MBI scores were increased (P<0.05) compared before treatment in both groups; in the wrist-ankle acupuncture group, the VAS score, degree of hand swelling and SHSS score were lower (P<0.05), and the FMA-UE score and MBI score were higher (P<0.05) than those in the conventional acupuncture group. The total effective rate was 96.9% (31/32) in the wrist-ankle acupuncture group, which was superior to 90.6% (29/32) in the conventional acupuncture group (P<0.05). CONCLUSION: Wrist-ankle acupuncture can effectively relieve pain and hand swelling, improve motor function of upper extremity and self-care ability of daily life in patients with shoulder-hand syndrome phaseⅠafter stroke, the therapeutic effect is superior to conventional acupuncture.

[Characteristics of phosphorus fractions and their driving factors in forest soils with different parent materials in the mid-subtropics, China]. / ä¸­äºšçƒ­å¸¦ä¸åŒæ¯è´¨å‘è‚²æ£®æž—åœŸå£¤ç£·ç»„åˆ†ç‰¹å¾åŠå ¶å½±å“å› ç´ .

With the aim to understand the influence degree and mechanism of parent material and forest type on soil phosphorus component, we analyzed soil P fractions, iron and aluminum oxides, microbial biomass, and phosphatase activity in Castanopsis carlesii and Cunninghamia lanceolata forest soils developed from two types of parent materials, sandstone and granite, in Sanming, Fujian Province. The results showed that both parent material and forest type significantly affected the contents of different P fractions. The contents of total P, labile inorganic/organic P, moderately labile inorganic/organic P, and non-labile P from sandstone-developed soils were significantly higher than those from granite-developed soils. Moreover, soil labile organic P, moderately labile inorganic/organic P and non-labile P fraction in sandstone-developed soils of C. carlesii forest were significantly higher than those of C. lanceolata forest, while the corresponding soil P fractions in granite-deve-loped soils had no significant difference between the two types of forests. The activity of acid phosphatase (ACP) in granite-developed soils was significantly higher than that in sandstone-developed soils under two types of forest, while soil microbial biomass carbon (MBC) and P (MBP) in sandstone-developed soils were significantly higher under C. carlesii forest than under C. lanceolata fo-rest. The content of soil P fractions was significantly positively correlated with the content of different forms of iron and aluminum oxides, MBP, and MBC, but negatively correlated with soil pH and acid phosphatase activity (ACP). Our results indicated that parent material and forest types might affect soil P fractions and characteristics mainly through altering soil iron and aluminum oxides, ACP, MBP in mid-subtropical forest soils.

A polysaccharide purified from Radix Adenophorae promotes cell activation and pro-inflammatory cytokine production in murine RAW264.7 macrophages.

Radix Adenophorae, a traditional Chinese medicine, has been reported to have a variety of biological functions. In the present study, a polysaccharide component, Radix Adenophorae Polysaccharide (RAPS), was purified from Radix Adenophorae by decoloring with ADS-7 macroporous adsorption resin, DEAE-52 cellulose ion-exchange chromatography, and Sephacryl S-300HR gel chromatography, with the purity of 98.3% and a molecular weight of 1.8 × 10(4) Da. The cell viability assay and microscopic examination revealed that RAPS promoted the proliferation and activation of macrophages. At 400 µg·mL(-1), RAPS stimulated RAW264.7 cell proliferation by 1.91-fold compared with the control. Meanwhile, RAPS significantly increased the secretion of pro-inflammatory cytokines (TNF-α and IL-6) in a dose-dependent manner in the supernatant of RAW264.7 cell culture as determined by ELISA. At 400 µg·mL(-1), the production of TNF-iα was 20.8-fold higher than that of the control. Simultaneously, the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) were increased in RAW264.7 cells incubated with RAPS, as measured by Griess assay and Western blot analysis. The NO production of cells treated with RAPS (400 µg·mL(-1)) reached 15.8 µmol·L(-1), which was 30.4-fold higher than that of the control (0.53 µmol·L(-1)). These data suggested that RAPS may enhance the immune function and protect against exogenous pathogens by activating macrophages.

A R2R3-MYB transcription factor, GmMYB12B2, affects the expression levels of flavonoid biosynthesis genes encoding key enzymes in transgenic Arabidopsis plants.

Isoflavones play diverse roles in plant-microbe interactions and are potentially important for human nutrition and health. To study the regulation of isoflavonoid synthesis in soybean, the R2R3-MYB transcription factor GmMYB12B2 was isolated and characterized. Yeast expression experiments demonstrated that GmMYB12B2 showed transcriptional activity. GmMYB12B2 was localized in the nucleus when it was transiently expressed in onion epidermal cells. Real-time quantitative PCR analysis revealed that GmMYB12B2 transcription was increased in roots and mature seeds compared with other organs. The gene expression level in immature embryos was consistent with the accumulation of isoflavones. CHS8 is a key enzyme in plant flavonoid biosynthesis. Transient expression experiments in soybean calli demonstrated that CHS8 was regulated by GmMYB12B2 and produced more fluorescence. The expression levels of some key enzymes in flavonoid biosynthesis were examined in transgenic Arabidopsis lines. The results showed that the expression levels of PAL1, CHS and FLS in transgenic plants were significantly higher than those in wild type plants. However, the expression level of DFR was lower, and the expression levels of CHI, F3H and F3'H were the same in all lines. GmMYB12B2 expression caused a constitutive increase in the accumulation of flavonoids in transgenic Arabidopsis lines compared with wild type plants.