A cytokinin response factor PtCRF1 is involved in the regulation of wood formation in poplar.

Wood formation is a complex developmental process under the control of multiple levels of regulatory transcriptional network and hormone signals in trees. It is well known that cytokinin (CK) signaling plays an important role in maintaining the activity of the vascular cambium. The CK response factors (CRFs) encoding a subgroup of AP2 transcription factors have been identified to mediate the CK-dependent regulation in different plant developmental processes. However, the functions of CRFs in wood development remain unclear. Here, we characterized the function of PtCRF1, a CRF transcription factor isolated from poplar, in the process of wood formation. The PtCRF1 is preferentially expressed in secondary vasculature, especially in vascular cambium and secondary phloem, and encodes a transcriptional activator. Overexpression of PtCRF1 in transgenic poplar plants led to a significant reduction in the cell layer number of vascular cambium. The development of wood tissue was largely promoted in the PtCRF1-overexpressing lines, while it was significantly compromised in the CRISPR/Cas9-generated double mutant plants of PtCRF1 and its closest homolog PtCRF2. The RNA sequencing (RNA-seq) and quantitative reverse transcription PCR (RT-qPCR) analyses showed that PtCRF1 repressed the expression of the typical CK-responsive genes. Furthermore, bimolecular fluorescence complementation assays revealed that PtCRF1 competitively inhibits the direct interactions between histidine phosphotransfer proteins and type-B response regulator by binding to PtHP protein. Collectively, these results indicate that PtCRF1 negatively regulates CK signaling and is required for woody cell differentiation in poplar.

The microRNA7833-AUX6 module plays a critical role in wood development by modulating cellular auxin influx in Populus tomentosa.

The critical role of auxin on secondary vascular development in woody plants has been demonstrated. The concentration gradient of endogenous indole-3-acetic acid and the cellular and molecular pathways contributing to the auxin-directed vascular organization and wood growth have been uncovered in recent decades. However, our understanding of the roles and regulations of auxin influx in wood formation in trees remains limited. Here, we reported that a microRNA, miR7833, participates in the negative regulation of stem cambial cell division and secondary xylem development in Populus tomentosa. The miR7833 is mainly expressed in the vascular cambium during stem radical growth and specifically targets and represses two AUX/LAX family auxin influx carriers, AUX5 and AUX6, in poplar. We further revealed that poplar AUX6, the most abundant miR7833 target in the stem, is preferentially enriched in the developing xylem and is a positive regulator for cell division and differentiation events during wood formation. Moreover, inhibition of auxin influx carriers by 1-naphthoxyacetic acids abolished the regulatory effects of miR7833 and AUX6 on secondary xylem formation in poplar. Our results revealed the essential roles of the miR7833-AUX6 module in regulating cellular events in secondary xylem development and demonstrated an auxin influx-dependent mechanism for wood formation in poplar.

[Nitrogen metabolism and secondary metabolism regulation of Atropa belladonna by exogenous NO under NaCl stress].

Atropa belladonna seedlings were used as experimental materials and cultivated by soil culture method. Different concentrations(0,0.05,0.1,0.2,0.5 mmol·L~(-1))of NO donor sodium nitroprusside(SNP) were sprayed on the leaves. The effects of different concentrations of SNP and different treatment time(4,8,12,16 d) on nitrogen metabolism, secondary metabolite content, precursor content of tropane alkaloid synthesis pathway and expression of key enzyme genes under 100 mmol·L~(-1) NaCl stress were studied. The results showed that with the prolongation of salt stress, the nitrogen metabolism and the accumulation of secondary metabolites of A. belladonna were inhibited to some extent. After treatment with different concentrations of exogenous SNP, the ammonium nitrogen content decreased dramatically, and the contents of nitrate nitrogen, free amino acid, soluble protein and the activities of key enzymes of nitrogen metabolism(NR, GS, GDH) were all greatly improved; the contents of precursor amino acids(ornithine, arginine) and polyamines(Put, Spd, Spm) in the secondary metabolic pathway have increased to varying degrees. The qRT-PCR analysis showed that exogenous SNP treatment can effectively promote the high expression of key enzyme genes PMT, TRⅠ and H6H in the secondary metabolic pathway of A. belladonna, and the production of hyoscyamine and scopolamine were increased notably. In summary, the application of appropriate concentration of SNP can effectively alleviate the inhibition of salt stress on the nitrogen metabolism and secondary metabolism of Atropa belladonna, and enhance its salt tolerance. Overall, 0.1 mmol·L~(-1) and 0.2 mmol·L~(-1) SNP treatment achieved the most remarkable effect.

Ectopic Expression of Poplar ABC Transporter PtoABCG36 Confers Cd Tolerance in Arabidopsis thaliana.

Cadmium (Cd) is one of the most toxic heavy metals for plant growth in soil. ATP-binding cassette (ABC) transporters play important roles in biotic and abiotic stresses. However, few ABC transporters have been characterized in poplar. In this study, we isolated an ABC transporter gene PtoABCG36 from Populus tomentosa. The PtoABCG36 transcript can be detected in leaves, stems and roots, and the expression in the root was 3.8 and 2 times that in stems and leaves, respectively. The PtoABCG36 expression was induced and peaked at 12 h after exposure to Cd stress. Transient expression of PtoABCG36 in tobacco showed that PtoABCG36 is localized at the plasma membrane. When overexpressed in yeast and Arabidopsis, PtoABCG36 could decrease Cd accumulation and confer higher Cd tolerance in transgenic lines than in wild-type (WT) lines. Net Cd2+ efflux measurements showed a decreasing Cd uptake in transgenic Arabidopsis roots than WT. These results demonstrated that PtoABCG36 functions as a cadmium extrusion pump participating in enhancing tolerance to Cd through decreasing Cd content in plants, which provides a promising way for making heavy metal tolerant poplar by manipulating ABC transporters in cadmium polluted areas.

[Cloning and expression analysis of calmodulin gene from Pinellia ternate].

According to the data of Pinellia ternate transcriptome,two calmodulin genes were cloned and named as Pt Ca M1 and PtCa M2 respectively. The results of bioinformatics analysis showed that Pt Ca Ms genes contained a 450 bp open reading frame,encoding149 amino acids.The identity of the coding sequences was 80%,and the identity of amino acids sequence was 91%. Pt Ca Ms genes contained EF-hand structure domain,belonging to the Ca M families. The Real-time PCR analysed the expression patterns of Pt Ca Ms in different tissues and different treatments. RESULTS:: showed that Pt Ca M1 and Pt Ca M2 gene were the highest expression level in tuber. Under Ca Cl2 treatment,the expressions of Pt Ca Ms were significantly higher than the control. Under EGTA,La Cl3 and TFP treatments,the expression level of Pt Ca Ms decreased gradually. In this study,the Pt Ca Ms gene were successfully cloned from P. ternate,which laid a foundation for the functional characteristic of Pt Ca Ms gene and the synthesis of alkaloids from P. ternata for further study.

[Effects of methyl jasmonate on metabolism of topical alkaloids and expression of relate genes in Atropa belladonna].

Hyoscyamine and scopolamine are important secondary metabolites of tropane alkaloid in Atropa belladonna with pharmacological values in many aspects.In this study, the seedlings of A.belladonna were planted by soil culture and treated with different concentrations of methyl jasmonate (MeJA). The contents of hyoscyamine and scopolamine,the upstream products in alkaloid synthesis,and the expression levels of key enzyme genes PMT, TR Ⅰ and H6H in secondary metabolites of A. belladonna seedlings were measured to clarify the mechanism of MeJA regulating alkaloids synthesis.The results showed that MeJA(200 µmol·L⁻¹) treatment was more favorable for the accumulation of alkaloids.The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine,the both increased first and then decreased with the increased MeJA concentration and the content of putrescine reached the highest at 200 µmol·L⁻¹ MeJA.Further detection of gene expression of PMT, TR Ⅰ and H6H in TAs synthesis pathway showed that no significant trend in PMT gene expression levels.The expression levels of TR Ⅰ and H6H in leaves and roots under 200 µmol·L⁻¹ MeJA were the highest.It can be speculated that the regulation of the formation of hyoscyamine and scopolamine by MeJA mainly through affecting the expression of key enzyme genes.Appropriate concentration of MeJA increased the gene expression of TR Ⅰ in both leaves and roots as well as H6H in roots,promoting the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine.

[Mechanism exploration on nitrogen metabolism and secondary metabolism in Atropa belladonna hairy roots treated with yeast extract].

In order to study the mechanism of nitrogen metabolism and secondary metabolism in Atropa belladonna hairy roots treated with yeast extract, yeast extract（YE） was added to the culture medium. Then the changes of physiological and biochemical indexes of A. belladonna hairy roots after treatment with YE were detected. The results are as follows，the activity of key enzymes of nitrogen metabolism changed differently. Compared with the control group (CK), the activity of nitrate reductase (NR) and glutamine synthetase (GS) were significantly increased, while the activity of glutamate dehydrogenase (GDH) was not changed significantly. The content of nitrate nitrogen, ammonium nitrogen had a significant decrease,but the content of soluble protein, free amino acid, total nitrogen are significantly more than CK. Moreover, YE treatment led to the increase of the content precursor amino acids (ornithine and arginine) and precursor putrescine in secondary metabolic pathways of A. belladonna. The expression level of gene putrescine N-methyl transferase (pmt), tropinone reductase-I (trI) and hyoscyamine 6-ß-hydroxylase(h6h) all increased in a different rate caused by YE treatment, which eventually led to the increase of the yield of tropane alkaloids. The yield of hyoscyamine and scopolamine were 3.09 and 1.85 folds than that of CK after 16 days treatment time. The results indicated that YE can induce more synthesis of tropane alkaloids by increasing the activity of key enzymes in nitrogen metabolism to provide more synthetic materials for secondary metabolism, meanwhile it regulated the expression level of some genes of key metabolic enzyme to accelerate secondary metabolism.

[Effects of nitrogen form on accumulation of alkaloids and expression of relative genes in Atropa belladonna].

Hyoscyamine and scopolamine are two main alkaloids in Atropa belladonna with great medicinal value. In this paper, the contents of hyoscyamine and scopolamine, the upstream products in alkaloid synthesis, and the expression levels of key enzyme genes PMT, TRⅠ and H6H in secondary metabolism of A. belladonna seedlings were measured to clarify the mechanism of nitrogen forms regulating alkaloids synthesis.The results showed that the 50/50 (NH⁺4/NO⁻3) treatment was more favorable for the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine. The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine, they both increased with the rise of ammonium ratio, reaching the highest at 75/25 (NH⁺4/NO⁻3). The detection of signaling molecule nitric oxide (NO) showed that the NO concentration decreased with the decrease of nitrate proportion. Further detection of gene expression levels of PMT, TRⅠ and H6H in TAs synthesis pathway showed that a certain amount of ammonium promoted the expression of PMT and H6H in roots. When the ratio of ammonium to nitrate was 50/50, PMT, TRⅠ and H6H in leaves and roots had higher expression levels. It can be speculated that the regulation of the formation of hyoscyamine to scopolamine by nitrogen forms mainly through affecting the expression of key enzyme genes. 50/50 (NH⁺4/NO⁻3) treatment increased the gene expression of TRⅠ in both leaves and roots as well as PMT and H6H in roots, promoting the synthesis of putrescine to hyoscyamine and the conversion of hyoscyamine to scopolamine.

[Effects of Ca2+ on photosynthetic parameters of Pinellia ternata and accumulations of active components in heat stress].

OBJECTIVE: To study the effect of exogenous Ca2+ on photosynthetic parameters of Pinellia ternate and accumulations of active components under high temperature stress. METHOD: The pigment contents of P. ternata leaves, photosynthesis parameters and chlorophyll fluorescence parameters of P. ternata leaves, the contents of guanosine, adenosine and polysaccharide in P. ternata tubers were measured based on different concentrations of exogenous Ca2+ in heat stress when the plant height of P. ternata was around 10 cm. RESULT: The contents of total chlorophyll and ratio of chlorophyll a/b were relatively higher by spaying Ca2+. Compared with the control, spaying 6 mmol x L(-1) Ca2+ significantly enhanced the net photosynthetic rate (Pn), transpiration (Tr) and stomatal limitation (L8), but reduced intercellular CO2 concentration (C) in P. ternata leaves. With the increase of Ca2+ concentration, maximal PS II efficiency (Fv/Fm), actual photosynthetic efficiency (Yield) and photochemical quenching coefficient (qP) initially increased and then decreased, however, minimal fluorescence (Fo) and non-photochemical quenching coefficient (NPQ) went down first and then went up. The contents of guanosine and polysaccharide and dry weight of P. ternata tubers showed a tendency of increase after decrease, and the content of adenosine increased with the increase of Ca2+ concentration. The content of guanosine and polysaccharide in P. ternata tubers and its dry weight reached maximum when spaying 6 mmol x L(-1) Ca2+. CONCLUSION: With the treatment of calcium ion, the inhibition of photosynthesis and the damage of PS II system were relieved in heat stress, which increased the production of P. ternata tubers.

[Expression pattern of genes involved in tropane alkaloids biosynthesis and tropane alkaloids accumulation in Atropa belladonna].

Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids (TAs) including scopolamine and hyoscyamine, which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results, the digital expression patterns of UniGenes representing 9 structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, TRII) involved in TAs biosynthesis were constructed, and simultaneously expression analysis of 4 released genes in NCBI (PMT, CYP80F1, H6H, TRII) for verification was performed using qPCR, as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC, ADC, AIH and CPA involved in the upstream pathway of TAs, and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT, CYP80F1, H6H were only expressed in secondary roots and primary roots, mainly in secondary roots. The qPCR detection results of PMT, CYP80F1 and H6H were consistent with the digital expression patterns, but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems (3.364 mg x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x g(-1)). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root, and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.

[Enhanced biosynthesis of scopolamine in transgenic Atropa belladonna by overexpression of h6h gene].

Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering. A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A. belladonna via Agrobacterium-mediation. The transgenic plants were culturing till fruiting through micropropogating and acclimating. The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. Analysis of the difference of plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight was carried out using SPSS software. The content of hyoscyamine and scopolamine in roots, stems, leaves and fruits was determined by HPLC. The investigation of the expression levels of Hnh6h by qPCR. Both Kan(r) and Hnh6h genes were detected in five transgenic lines of A. belladonna plants (A8, A11, A12, C8 and C19), but were not detected in the controls. The plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones, and furthermore some agronomic characters of transgenic plants were better than those of the controls. The highest level of scopolamine was found in leaves of transgenic A. belladonna, and the content of scopolamine was also higher than that of hyoscyamine in leaves. The contents of scopolamine of leaves in different transgenic lines were listed in order: C8 > A12 > C19 > A11 > A8, especially, the content of scopolamine in transgenic line C8 was 2.17 mg x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x g(-1) DW). The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control. The highest level of Hnh6h expression was found in transgenic leaves. Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis, and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants. As a result, transgenic plants of A. belladonna with higher level of scopolamine were developed.

[Response of isozyme and stress indexes of Coptis chinensis to UV-B radiation].

OBJECTIVE: To study the physiological mechanism of anti-stress of Coptic chinensis and provide theoretical basis for its cultivation and promoting its quality. METHODS: Different degrees of the range of time and intensity of UV-B radiation were set in the experiment. Used the technique of polyacrylamide gelatin vertical board electrophoresis (PAGE) to analyse the isozyme and related stress index. RESULTS: The isoenzymic bands of SOD1 (Rf = 0.125), SOD2 (Rf = 0.312), CAT1 (Rf = 0.428), POD3 (Rf = 0.290), POD4 (Rf = 0.636) were induced by UV-B radiation after 3 hours, with the increase of the time of UV-B radiation, those isoenzymic bands was going to vanish or became unclear. Moreover, isoenzymic bands of CAT1 (Rf = 0.428), POD3 (Rf = 0.290) disappeared in advance under heavy intensity of UV-B radiation. Furthermore, the contents of MDA, soluble sugar, proline were higher dramatically than those of control group under UV-B radiation. However, excluding the increases of proline in UL group, the content of MDA, soluble sugar, proline of other groups commenced to decrease slowly and isoenzymic bands of soluble protein increase after 7 hours of UV-B radiation. CONCLUSION: The increase of the expression of antioxidase isozyme, accumulation of soluble sugar, soluble protein and other antioxidase matter is induced by the short-time UV-B radiation, which can protect Coptis chinensis from being harmed by UV-B radiation. However, regulation system of Coptis chinensis are broken, metabolism is disordered, the bands of antioxidase isozyme vanish or weaken, the bands of soluble protein are increased and widened, these phenomenon is caused by 7 hours of UV-B radiation.

[Effects of water stress and nitrogen nutrition on regulation of Catharanthus roseus alkaloids metabolism].

OBJECTIVE: Under various drought conditions and nitrogen application, the content of vindoline, catharanthine, vincristine and vinblastine in the leaf of Catharanthus roseus were illustrated to improve the content of alkaloid theoretically. METHOD: Six groups were set in the experiment, which included: CK (natural control), CN (natural control + nitrogen), LK (low drought), LN (low drought + nitrogen), HK (high drought), HN (high drought + nitrogen) to discuss the change characteristics of total nitrogen, the activity of alkaline POD and TDC, the content of four alkaloids under the different conditions were measured. RESULT: Under LK condition, the activity of POD, TDC were enhanced. In the early stage of stress (0-21 d), vindoline, catharanthine, vincristine and vinblastine accumulated, and reduced in the later stage (28-35 d). For all groups, adding exogenous nitrogen could improve the total content of nitrogen, vindoline and vinblastine, meanwhile the activity of POD and TDC were enhanced as well. The LN, HN treatments were beneficial to accumulating catharanthine and vinblastine. CONCLUSION: Drought stress or additional nitrogen have an influence on both of the activities of POD and TDC, and the four alkaloids were affected as well. Thereinto, the LN condition was the most effective treatment for accumulating the four alkaloids (vindoline, catharanthine, vincristine and vinblastine), which were regulated by improve nitrogen content and enzymatic activity.

[Effect of enhanced UV-B radiation on metabolism and berberine content of Coptis chinensis].

OBJECTIVE: To reveal the response of content berberine in root of Coptis chinensis to different intensity of UV-B radiation, and provide the theory basis for promoting the content of berberine. METHOD: Four groups of UV-B radiation were set in the experiment which included: natural light control (0 W x m(-2)), UL (0.05 W x m(-2)), UM (0.10 W x m(-2)), UH (0.20 W x m(-2)). The special photosynthesis character, PPP pathway in the primary metabolism and lyrosinase activity, the changes of berberine in the root of C. chinensis were measured under different UV-B radiation. RESULT: Photosynthetic pigment, qN, Fo, ETR, activity of glucose-6-phosphate dehydrogenase and the content of berberine in the root of C. chinensis, all of these parameters were lower than other groups under the UH radiation. However, under the UM radiation, C. chinensis protected itself from the light UV-B radiation by promoting the power of photosynthesis and PPP pathway in order to produce more NADPH and secondary metabolites. CONCLUSION: C. chinensis increases its photosynthetic ability and PPP pathway which can furnish more precursor of secondary metabolites and NADPH that are needed in the secondary metabolism. Furthermore, the content of berberine increases correspondingly. The research provide the example for increasing the content of berberine in C. chinensis cultivation.

[Effect of enhanced UV-B radiation on photosynthetic structure and photosynthetic characteristics of Mentha piperita].

OBJECTIVE: To reveal the effects of UV-B radiation on the growth of medical plant Mentha piperita, simulate an enhanced UV-B radiation and evaluate intensity of radiation on the photosynthesis of M. piperita. METHOD: Three different levels of UV-B radiation were set in the experiment which included: natural light control (0 W x m(-2)), light UV-B radiation stress (0.15 W x m(-2)) and heavy UV-B radiation stress (0.35 W x m(-2)). The chloroplast ultrastructure, photosynthesis indexes and chlorophyll fluorescence parameters of the M. piperita were observed under the three treatments. RESULT: Although the chloroplast ultrastructure was destroyed to some degree under the light UV-B radiation stress, F(v)/(F)m, F(v)/F(o), qP, phiPS II and ETR could resume to the comparative level of natural light control. At the same time, qN increased firstly and decreased thereafter. But under the high strength UV-B radiation stress, the photosynthetic structures were badly destroyed, which could not recover through protecting mechanism by itself. CONCLUSION: It was showed that M. piperita was able to protect photosynthetic structures by increasing respiration and dissipation when photosynthetic capacity reduced under light UV-B radiation stress. It is demonstrated that M. piperita has high adaptation to light UV-B radiation stress, which is kind of promising medical plant for area with higher UV-B radiation.