Cloning and functional analysis of the DXR gene and promoter region in Osmanthus fragrans var. semperflorens.

The precise biological function and activity of the deoxylulose-5-phosphate reductoisomerase (DXR) gene and its promoter in Osmanthus fragrans var. semperflorens remain unclear, even though OfDXR is known as the crucial enzyme involved in plant terpenoid synthesis. This study aimed to shed light on the role and activity of the OfDXR gene and its promoter in O. fragrans var. semperflorens by employing RACE-PCR and Hi-TAIL-PCR techniques for the cloning of the gene and promoter sequence from the petal tissue. Subsequently, genetic transformation and histochemical staining methods were utilized to analyze their function and activity. The OfDXR gene exhibited a DNA sequence length of 5241 bp, encompassing 12 exons and 11 introns. The corresponding cDNA sequence of the OfDXR gene was 1629 bp, encoding 474 amino acid residues. Expression analysis revealed that the OfDXR gene was predominantly active in the petals during the early full blooming stage. Overexpression of the OfDXR gene in Arabidopsis plants at the primary or full blooming stage led to an augmentation in the total terpenoid content. Furthermore, the promoter sequence of the OfDXR gene spanned a length of 1174 bp and contained conserved regulatory/response elements, demonstrating functional activity. These findings indicate that the OfDXR gene plays a pivotal role in terpenoid synthesis, while its promoter exhibits robust activity.

Transcriptomic analysis reveals the significant effects of fertilization on the biosynthesis of sesquiterpenes in Phoebe bournei.

Phoebe bournei is a potential medicinal plant. Its essential oils (Eos) are mainly composed of sesquiterpenes that has potential activities of anti-bacteria and anti-tumors. In this study, we evaluated the effects of compost and compound fertilizer on the total amount and main components of Eos in P. bournei, we also studied the molecular mechanism undergoing this process by deep sequencing the genes involved in the biosynthesis of sesquiterpenes. Fertilization enhanced the total amount of main components in Eos from both leaves and twigs. Bicyclogermacrene, the primary sesquiterpene in the leaf EO, was significantly increased under compost treatment, while bicyclogermacrene and δ-cadinene (the second most abundant sesquiterpene) were decreased under compound fertilizer treatment. The two fertilizers had no significant effect on the abundance of the primary (+) - δ-cadinene in the twig EO, but had a positive effect on the second most abundant sesquiterpene copaene. Significant differences were observed in the number of differentially expressed genes (DEGs) with the leaves showing greater number of DEGs as compared to the twigs after compost treatment. Terpenoid backbone biosynthesis (TBB) is a key pathway of sesquiterpenes synthesis. The expression of genes regulating several important enzymes in TBB was altered after fertilization. After the compost treatment, the expression of the leaf DXS gene (ACQ66107.1), being closely related to the sesquiterpene biosynthesis in P. bournei leaves, was decreased. Compost and compound fertilizer altered the expression of the two important branch-point enzymes (FPPS and GGPPS) genes (ART33314.1 and ATT59265.1), which contributed to the changes of the total amount and components of P. bournei sesquiterpenes. This study provides a new insight into the future use of P. bournei for Eos.

Integrated Transcriptomic and Metabolomic Analysis Reveals the Mechanism of Gibberellic acid Regulates the Growth and Flavonoid Synthesis in Phellodendron chinense Schneid Seedlings.

The phytohormone gibberellic acids (GAs) play a crucial role in the processes of growth, organ development, and secondary metabolism. However, the mechanism of exogenous GA3 regulating the growth and flavonoid synthesis in Phellodendron chinense Schneid (P. chinense Schneid) seedlings remains unclear. In this study, the physicochemical properties, gene expression level, and secondary metabolite of P. chinense Schneid seedlings under GA3 treatment were investigated. The results showed that GA3 significantly improved the plant height, ground diameter, fresh weight, chlorophyll content, soluble substance content, superoxide dismutase, and peroxidase activities. This was accompanied by elevated relative expression levels of Pc(S)-GA2ox, Pc(S)-DELLA, Pc(S)-SAUR50, Pc(S)-PsaD, Pc(S)-Psb 27, Pc(S)-PGK, Pc(S)-CER3, and Pc(S)-FBA unigenes. Conversely, a notable reduction was observed in the carotenoid content, catalase activity and the relative expression abundances of Pc(S)-KAO, Pc(S)-GID1/2, and Pc(S)-GH 3.6 unigenes in leaves of P. chinense Schneid seedlings (p < 0.05). Furthermore, GA3 evidently decreased the contents of pinocembrin, pinobanksin, isosakuranetin, naringin, naringenin, (-)-epicatechin, tricetin, luteolin, and vitexin belonged to flavonoid in stem bark of P. chinense Schneid seedlings (p < 0.05). These results indicated that exogenous GA3 promoted growth through improving chlorophyll content and gene expression in photosynthesis and phytohormone signal pathway and inhibited flavonoid synthesis in P. chinense Schneid seedlings.

The response of nutrient cycle, microbial community abundance and metabolic function to nitrogen fertilizer in rhizosphere soil of Phellodendron chinense Schneid seedlings.

Nitrogen (N) as an essential macronutrient affects the soil nutrient cycle, microbial community abundance, and metabolic function. However, the specific responses of microorganisms and metabolic functions in rhizosphere soil of Phellodendron chinense Schneid seedlings to N addition remain unclear. In this study, four treatments (CK, N5, N10 and N15) were conducted, and the soil physicochemical properties, enzyme activities, microbial community abundances and diversities, metabolism, and gene expressions were investigated in rhizosphere soil of P. chinense Schneid. The results showed that N addition significantly decreased rhizosphere soil pH, among which the effect of N10 treatment was better. N10 treatment significantly increased the contents of available phosphorus (AP), available potassium (AK), ammonium nitrogen (NH4+-N), nitrate nitrogen (NO3--N) and sucrase (SU) activity, as well as fungal diversity and the relative expression abundances of amoA and phoD genes in rhizosphere soil, but observably decreased the total phosphorus (TP) content, urease (UR) activity and bacterial diversity, among which the pH, soil organic matter (SOM), AP, NH4+-N and NO3--N were the main environmental factors for affecting rhizosphere soil microbial community structure based on RDA and correlation analyses. Meanwhile, N10 treatment notably enhanced the absolute abundances of the uracil, guanine, indole, prostaglandin F2α and γ-glutamylalanine, while reduced the contents of D-phenylalanine and phenylacetylglycine in rhizosphere soil of P. chinense Schneid seedlings. Furthermore, the soil available nutrients represented a significant correlation with soil metabolites and dominant microorganisms, suggesting that N10 addition effectively regulated microbial community abundance and metabolic functions by enhancing nutrient cycle in the rhizosphere soil of P. chinense Schneid seedlings.

Effects of exogenous 6-BA and NAA on growth and contents of medicinal ingredient of Phellodendron chinense seedlings.

Using Phellodendron chinense seedlings as material, and treated with different concentrations of exogenous 6-Benzylaminopurine (6-BA) and α-naphthyacetic acid (NAA), then observed the growth status. Furthermore, we detected the contents of chlorophyll and soluble sugar, the activities of antioxidases by spectrophotometry, and determined the contents of secondary metabolite by high performance liquid chromatograph. The results showed that different concentrations of exogenous 6-BA increases the fresh weights and plant heights of Phellodendron chinense seedlings, and enhances the contents of chlorophyll and soluble sugar. NAA promoted growth, but deduced the contents of soluble sugar. Compared with control, culturing for 40 d, proper concentrations 6-BA enhanced the activity levels of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), proper concentrations NAA increased the activity levels of SOD and CAT, but decreased the levels of POD compared with CK. Suitable concentrations 6-BA enhanced contents of berberine, phellodendrine and palmatine in stems, proper concentrations NAA increased contents of berberine and phellodendrine, but deduced contents of palmatine compared with CK. Based on these results, we concluded that the exogenous 6-BA and NAA had key regulation on the growth and contents of medicinal ingredient of Phellodendron chinense seedlings.

High-efficient extraction of principal medicinal components from fresh Phellodendron bark (cortex phellodendri).

There are three key medicinal components (phellodendrine, berberine and palmatine) in the extracts of Phellodendron bark, as one of the fundamental herbs of traditional Chinese medicine. Different extraction methods and solvent combinations were investigated to obtain the optimal technologies for high-efficient extraction of these medicinal components. RESULTS: The results showed that combined solvents have higher extracting effect of phellodendrine, berberine and palmatine than single solvent, and the effect of ultrasonic extraction is distinctly better than those of distillation and soxhlet extraction. CONCLUSION: The hydrochloric acid/methanol-ultrasonic extraction has the best effect for three medicinal components of fresh Phellodendron bark, providing an extraction yield of 103.12 mg/g berberine, 24.41 mg/g phellodendrine, 1.25 mg/g palmatine.

Knockout of OsNramp5 using the CRISPR/Cas9 system produces low Cd-accumulating indica rice without compromising yield.

Rice grain with excessive cadmium (Cd) is a major source of dietary Cd intake and a serious threat to health for people who consume rice as a staple food. The development of elite rice cultivars with consistently low Cd content is challenging for conventional breeding approaches, and new strategies urgently need to be developed. Here, we report the development of new indica rice lines with low Cd accumulation and no transgenes by knocking out the metal transporter gene OsNramp5 using CRISPR/Cas9 system. Hydroponic culture showed that Cd concentrations in shoots and roots of osnramp5 mutants were dramatically decreased, resulting in rescue of impaired growth in high Cd condition. Cd-contaminated paddy field trials demonstrated that Cd concentration in osnramp5 grains was consistently less than 0.05 mg/kg, in contrast to high Cd concentrations from 0.33 mg/kg to 2.90 mg/kg in grains of Huazhan (the wild-type indica rice). In particular, the plant yield was not significantly affected in osnramp5 mutants. Furthermore, we developed promising hybrid rice lines with extremely low Cd content in grains. Our work supplies a practical approach to developing Cd pollution-safe indica rice cultivars that minimizes Cd contamination risk in grains.

[Effects of 6-benzylaminopurine and α-naphthaleneacetic acid on growth and isoflavone contents of Pueraria phaseoloides hairy roots].

In order to study the effect of phytohormone on growth and isoflavones contents of Pueraria phaseoloides hairy roots, we cultured the hairy roots with different concentrations of 6-benzylaminopurine (6-BA) alone or in combination with α-naphthaleneacetic acid (NAA). Then we determined the effects of 6-BA alone or in combination with NAA on the growth and the contents of isoflavones compounds and levels of antioxidase activities of hairy roots by spectrophotometry. The results show that 6-BA inhibited the growth, and decreased biomass and total isoflavones compounds of P. phaseoloides hairy roots. Furthermore, the inhibition was increased with the concentrations of 6-BA. Compared with the controls, different concentrations of 6-BA in combination with NAA 2.0 mg/L could inhibit the growth of hairy roots and decrease the content of total isoflavone compounds, and also significantly enhanced the contents of soluble protein and levels of peroxidase (POD) activities, but decreased the activities of superoxide dismutase (SOD). DNA ladders detected by agarose gel electrophoresis can be observed after hairy roots of P. phaseoloides were cultured with 6-BA alone for 30 days, but can appear on the 20th day after culture with 6-BA in combination with NAA 2.0 mg/L. This result indicates that 6-BA or 6-BA in combination with NAA can both stimulate appearance of programmed cell death (PCD), and NAA may play a synergistic role on PCD.

Two homologous putative protein tyrosine phosphatases, OsPFA-DSP2 and AtPFA-DSP4, negatively regulate the pathogen response in transgenic plants.

Protein phosphatases, together with protein kinases, regulate protein phosphorylation and dephosphorylation, and play critical roles in plant growth and biotic stress responses. However, little is known about the biological functions of plant protein tyrosine dual-specificity phosphatase (PFA-DSP) in biotic stresses. Here, we found that OsPFA-DSP2 was mainly expressed in calli, seedlings, roots, and young panicles, and localized in cytoplasm and nucleus. Ectopic overexpression of OsPFA-DSP2 in rice increased sensitivity to Magnaporthe grisea (M. grisea Z1 strain), inhibited the accumulation of hydrogen peroxide (H(2)O(2)) and suppressed the expression of pathogenesis-related (PR) genes after fungal infection. Interestingly, transgenic Arabidopsis plants overexpressing AtPFA-DSP4, which is homologous to OsPFA-DSP2, also exhibited sensitivity to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), reduced accumulation of H(2)O(2) and decreased photosynthesic capacity after infection compared with Col-0. These results indicate that OsPFA-DSP2 and AtPFA-DSP4 act as negative regulators of the pathogen response in transgenic plants.

[Effects of sucrose and light on the growth and production of secondary metabolites in Pueraria phaseoloides hairy roots].

Effects of sucrose concentrations and light on the growth and production of total isoflavones and puerarin in Pueraria phaseoloides hairy roots cultured onto solid MS media supplemented with 1%, 3%, 5%, 7% and 9% sucrose, respectively, were investigated. The results showed that among the sucrose concentrations tested, 3% sucrose in the medium enhanced the growth and stimulated accumulation of total isoflavones and puerarin in P. phaseoloides hairy roots, After cultured for 20 days, the biomass of hairy roots reached 0.48 g (DW dry weight)/flask and its contents of total isoflavones and puerarin were 25.44 mg/g (DW) and 11.64 mg/g (DW), respectively. In comparison with 3% sucrose, the dry weight proliferation of hairy roots cultured with 5% sucrose was increased by 7.0%, while cultured with 1%, 7% and 9% sucrose, the dry weight proliferation of hairy roots was decreased by 62.4%, 42.8% and 65.3%, their total isoflavones content was decreased by 57.4%, 13% and 33.4% and their puerarin content was decreased by 47.9%, 15.8% and 35.1%; but their content of total soluble sugars was increased 0.52, 1.45 and 1.54 times, respectively. Compared with hairy roots in blue light and white light, the biomass of hairy roots cultured in the dark for 30 days was 0.83 g (DW)/flask and was increased by 37.1% and 23.3%, respectively. The content of total isoflavones in hairy roots cultured in white light was as much as 1.15 times and 1.19 times that in blue light and in the dark, respectively. It was also observed that hairy roots cultured in blue light and white light partly became light green and that blue light could inhibit accumulation of puerarin in hairy roots and the puerarin content in hairy root cultured in white light and in the dark were 1.61 times and 1.52 times that in blue light, respectively.