Reaction intensification for biocatalytic production of polyphenolic natural product di-C-β-glucosides

Polyphenolic aglycones featuring two sugars individually attached via C-glycosidic linkage (di-C-glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di-C-glycosides limits their use-inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction-intensified production of the di-C-β-glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl-trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two-fold C-glycosylation of the respective aglycone, supplied as inclusion complex with 2-hydroxypropyl β-cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di-C-glycosyltransferase (di-CGT), and it uses UDP-Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C-glycosylation as rate-limiting (0.4–0.5 mmol/L/min) for the di-C-glucoside production. With internal supply from sucrose keeping the UDP-Glc at a constant steady-state concentration (≥50% of the UDP added) during the reaction, the di-C-glycosylation is driven to completion (≥95% yield). Contrary to the mono-C-glucoside intermediate which is stable, the di-C-glucoside requires the addition of reducing agent (10 mmol/L 2-mercaptoethanol) to prevent its decomposition during the synthesis. Both di-C-glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di-C-β-glucoside synthesis from expedient substrates.     

Impacts of iron on phosphate starvation-induced root hair growth in Arabidopsis

In Arabidopsis, phosphate starvation (-Pi)-induced responses of primary root and lateral root growth are documented to be correlated with ambient iron (Fe) status. However, whether and how Fe participates in -Pi-induced root hair growth (RHG) remains unclear. Here, responses of RHG to different Fe concentrations under Pi sufficiency/deficiency were verified. Generally, distinct dosage effects of Fe on RHG appeared at both Pi levels, due to the generation of reactive oxygen species. Following analyses using auxin mutants and the phr1 mutant revealed that auxin and the central regulator PHR1 are required for Fe-triggered RHG under −Pi. A further proteomic study indicated that processes of vesicle trafficking and auxin synthesis and transport were affected by Fe under −Pi, which were subsequently validated by using a vesicle trafficking inhibitor, brefeldin A, and an auxin reporter, R2D2. Moreover, vesicle trafficking-mediated recycling of PIN2, an auxin efflux transporter, was notably affected by Fe under -Pi. Correspondingly, root hairs of pin2 mutant displayed attenuated responses to Fe under -Pi. Together, we propose that Fe affects auxin signalling probably by modulating vesicle trafficking, chiefly the PIN2 recycling, which might work jointly with PHR1 on modulating -Pi-induced RHG.     

A meta-analysis on morphological,physiological and biochemical responses of plants with PGPR inoculation under drought stress

Plant growth-promoting rhizobacteria (PGPR) can help plants to resist drought stress. However, the mechanisms of how PGPR inoculation affect plant status under drought remain incompletely understood. We performed a meta-analysis of plant response to PGPR inoculation by compiling data from 57 PGPR-inoculation studies, including 2, 387 paired observations on morphological, physiological and biochemical parameters under drought and well-watered conditions. We compare the PGPR effect on plants performances among different groups of controls and treatments. Our results reveal that PGPR enables plants to restore themselves from drought-stressed to near a well-watered state, and that C4 plants recover better from drought stress than C3 plants. Furthermore, PGPR is more effective underdrought than well-watered conditions in increasing plant biomass, enhancing photosynthesis and inhibiting oxidant damage, and the responses of C4 plants to the PGPR effect was stronger than that of C3 plants under drought conditions. Additionally, PGPR belonging to different taxa and PGPR with different functional traits have varying degrees of drought-resistance effects on plants. These results are important to improve our understanding of the PGPR beneficial effects on enhanced drought-resistance of plants.     

Rhizosphere effect of nanoscale zero-valent iron on mycorrhiza-dependent maize assimilation

Rhizosphere effect of nanoscale zero-valent iron (nZVI) is crucial but little reported. Maize seeds were dressed with four nZVI concentrations (0, 1.0, 1.5, 2 g kg⁻¹) and inoculated with arbuscular mycorrhizal fungus (AMF) (Funneliformis mosseae). The SEM images illuminated that excessive nZVI particles (2 g kg⁻¹) were agglomerated on the surface of hyphae and spore, causing severe deformation and inactivation of AMF symbionts and thereafter inhibiting water uptake in maize seedlings. This restrained the scavenging effects of enzymatic (superoxide dismutase, peroxidase) and non-enzymatic compounds (proline & malondialdehyde) on ROS, and leaf photoreduction activity and gas exchange ability (p < 0.05). Interestingly, the inoculation with AMF effectively alleviated above negative effects. In contrast, appropriate dose of nZVI, that is, ≤1.5 g kg⁻¹, can be evenly distributed on the hyphae surface and form the ordered symbionts with AMF. This help massively to enhance hyphae growth and water and nutrient uptake. The enhanced mycorrhizal infection turned to promote rhizosphere symbiont activity and leaf Rubisco and Rubisco activase activity. Light compensation point was massively lowered, which increased photosynthetic carbon supply for AMF symbionts. Particularly, such priming effects were evidently enhanced by drought stress. Our findings provided a novel insight into functional role of nZVI in agriculture and AMF-led green production.     

Antibacterial Activity and Mechanisms of Ethanol Extracts from <i>Scutellaria baicalensis</i> Georgi and <i>Magnolia officinalis</i> against <i>Phytophthora nicotianae</i>

Phytophthora nicotianae causes substantial economic losses in most countries where tobacco is produced. At present, the control of P. nicotianae mainly depends on chemical methods, with considerable environmental and health issues. We investigated the effects of ethanol extracts from Scutellaria baicalensis Georgi (SBG) and Magnolia officinalis (MO). On mycelial growth, sporangium formation, and zoospore release of P. nicotianae. Both extracts inhibited the growth of P. nicotianae, with mycelial growth inhibition rates of 88.92% and 93.92%, respectively, at 40 mg/mL, and EC50 values of 5.39 and 5.74 mg/mL, respectively. The underlying mechanisms were the inhibition of sporangium formation, the reduction of zoospore number, and the destruction of the mycelium structure. At an SBG extract concentration of 16.17 mg/mL, the inhibition rates for sporangia and zoospores were 98.66% and 99.39%, respectively. At an MO extract concentration of 2.87 mg/mL, the production of sporangia and zoospores was completely inhibited. The hyphae treated with the two plant extracts showed different degrees of deformation and damage. Hyphae treated with SBG extract showed adhesion and local swelling, whereas treatment with MO extract resulted in broken hyphae. Mixture of the extracts resulted in a good synergistic effect.     

Effects of compound sodium nitrophenol on microspore embryogenesis and plantlet regeneration in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee)

Isolated microspore culture has been implemented in breeding programs to produce doubled haploid (DH) lines and thus accelerates the breeding process. However, low microspore embryogenesis frequency in flowering Chinese cabbage remains a key obstacle to the practical application of this technique. This study aimed to establish an efficient microspore culture protocol for flowering Chinese cabbage that would be applied for heterosis breeding. Microspores of five genotypes, 19AY05, 19AY06, 19AY10, 19AY12, and 19AY15, were successfully induced to produce embryos in NLN-13 medium. Microspores of two genotypes, 19AY05 and 19AY15, were cultivated in NLN-13 medium supplemented with different concentrations (0, 0.01, 0.05, 0.1, or 0.2 mg·L⁻¹) of compound sodium nitrophenol (sodium nitrophenol, 5-nitrophenol) to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. The results showed that 0.05 ~ 0.1 mg· L⁻¹ sodium nitrophenol and 0.01 ~ 0.2 mg· L⁻¹ of 5-nitrophenol significantly promoted the induction of microspore embryogenesis of two genotypes, and the best concentrations required for different genotypes are different. Moreover, 0.1 mg· L⁻¹ sodium nitrophenol can significantly increase the plant regeneration rate of the two genetypes. The 5-nitrophenol at 0.01 mg·L⁻¹ significantly increased rate of embryos directly convert to plant in 19AY15. In addition, the average doubled haploid rates in the five genotypes were close to 63%. Horticultural traits of DH lines from 19AY05 were identified and all of them were self-incompatible lines. They showed a high uniformity and consistency that can be directly used for hybrid breeding. Furthermore, the hybrid combination was prepared with the selected DH lines and the Guangdong nucleus genic sterile line GMS019 to screen the excellent hybrid combination for the flowering Chinese cabbage breeding program. This method accelerates the application of microspore culture in hybrid breeding of flowering Chinese cabbage.

     

Responses of the desert green algae,Chlorella sp. to drought stress

Desert algae are important components of the desert soil crust and play an essential role in desert soil ecosystem development. Owing to their special habitat, desert algae are often exposed to harsh environments, among which drought represents the most common stress. Green algae are considered to have drought tolerance potential; however, only a few studies have investigated this. In this study, we selected the green alga Chlorella sp., which was isolated from desert soil, and studied its physiological response to polyethylene glycol (PEG) 6000-induced drought stress. The results showed that drought stress can affect the photosynthetic efficiency of Chlorella sp., reduce its water retention ability, and destroy its ultrastructure. However, Chlorella sp. can cope with drought stress through a series of physiological regulatory strategies. Protective strategies include quick recovery of photosynthetic efficiency and increased chlorophyll content. In addition, induced synthesis of soluble proteins, lipids, and extracellular polysaccharide (EPS), and accumulation of osmotic regulatory substances, such as sucrose and trehalose, also contribute to improving drought tolerance in Chlorella sp. This study provides insights into the physiological responses of Chlorella sp. to drought stress, which may be valuable for understanding the underlying drought adaptation mechanisms of desert green algae.     

Rapid Analysis of Four Alkaloids in <i>Uncaria rhynchophylla</i> by Core-Shell Column HPLC and Quantitative Analysis of Multi-Components by Single Marker (QAMS)

As a traditional herbal medicine, the major alkaloids in Uncaria rhynchophylla have been proven to have blood pressure-lowering and sedative effects. It is essential to develop an effective method for the determination of the major alkaloids in U. rhynchophylla. In this research, a rapid quantitative analysis involving multi-components analysis by a single marker strategy coupled with core-shell column HPLC was adopted to analyse four alkaloids (corynoxeine, isocorynoxeine, isorhynchophylline, rhynchophylline) in U. rhynchophylla. Isorhynchophylline was selected as the internal reference substance, the content of which was determined by the traditional external standard method. Relative correction factors (RCF) between isorhynchophylline and the other three alkaloids were calculated respectively. The results showed that the QAMS method had good robustness under different HPLC instruments. Nineteen batches of U. rhynchophylla were tested. No significant difference was observed between the results by QAMS and EMS (Correlation coefficient > 0.99, p > 0.05). The QAMS method could be employed as a rapid, effective technique for the quality control of U. rhynchophylla.