Multi-Omics Analysis Reveals Synergistic Enhancement of Nitrogen Assimilation Efficiency via Coordinated Regulation of Nitrogen and Carbon Metabolism by Co-Application of Brassinolide and Pyraclostrobin in Arabidopsis thaliana.

Improving nitrogen (N) assimilation efficiency without yield penalties is important to sustainable food security. The chemical regulation approach of N assimilation efficiency is still less explored. We previously found that the co-application of brassinolide (BL) and pyraclostrobin (Pyr) synergistically boosted biomass and yield via regulating photosynthesis in Arabidopsis thaliana. However, the synergistic effect of BL and Pyr on N metabolism remains unclear. In this work, we examined the N and protein contents, key N assimilatory enzyme activities, and transcriptomic and metabolomic changes in the four treatments (untreated, BL, Pyr, and BL + Pyr). Our results showed that BL + Pyr treatment synergistically improved N and protein contents by 56.2% and 58.0%, exceeding the effects of individual BL (no increase) or Pyr treatment (36.4% and 36.1%). Besides synergistically increasing the activity of NR (354%), NiR (42%), GS (62%), and GOGAT (62%), the BL + Pyr treatment uniquely coordinated N metabolism, carbon utilization, and photosynthesis at the transcriptional and metabolic levels, outperforming the effects of individual BL or Pyr treatments. These results revealed that BL + Pyr treatments could synergistically improve N assimilation efficiency through improving N assimilatory enzyme activities and coordinated regulation of N and carbon metabolism. The identified genes and metabolites also informed potential targets and agrochemical combinations to enhance N assimilation efficiency.

Co-application of Brassinolide and Pyraclostrobin Improved Disease Control Efficacy by Eliciting Plant Innate Defense Responses in Arabidopsis thaliana.

Applying brassinolide (BL, a phytohormone) in combination with pyraclostrobin (Pyr, a fungicide) has shown effective disease control in field trials. However, the mechanism by which BL + Pyr control disease remains uncertain. This work compared the disease control and defense responses of three pretreatments (BL, Pyr, and BL + Pyr) in Arabidopsis thaliana. We found that BL + Pyr improved control against Pyr-sensitive Hyaloperonospora arabidopsidis and Botrytis cinerea by 19 and 17% over Pyr, respectively, and achieved 29% control against Pyr-resistant B. cinerea. Furthermore, BL + Pyr outperformed BL or Pyr in boosting transient H2O2 accumulation, and the activities of POD, APX, GST, and GPX. RNA-seq analysis revealed a more potent activation of defense genes elicited by BL + Pyr than by BL or Pyr. Overall, BL + Pyr controlled disease by integrating the elicitation of plant innate disease resistance with the fungicidal activity of Pyr.

The combined formulation of brassinolide and pyraclostrobin increases biomass and seed yield by improving photosynthetic capacity in Arabidopsis thaliana.

In the context of global food crisis, applying the phytohormone-brassinosteroids (BRs) in combination with the fungicide-pyraclostrobin (Pyr) was beneficial for plant quality and productivity in several field trials. However, in addition to the benefits of disease control due to the innate fungicidal activity of Pyr, it remains to be understood whether the coapplication of BL+ Pyr exerts additional growth-promoting effects. For this purpose, the effects of BL treatment, Pyr treatment, and BL+ Pyr treatment in Arabidopsis thaliana were compared. The results showed that the yield increased at a rate of 25.6% in the BL+Pyr group and 9.7% in the BL group, but no significant change was observed in the Pyr group. Furthermore, the BL+Pyr treatment increased the fresh weight of both the leaves and the inflorescences. In contrast, the Pyr and BL treatments only increased the fresh weight of leaves and inflorescences, respectively. Additionally, the BL + Pyr treatment increased the Pn, Gs, Tr, Vc, max, Jmax, VTPU, ETR, Fv'/Fm', ΦPSII, Rd, AYE and Rubisco enzyme activity by 26%, 38%, 40%, 16%, 19%, 15%, 9%, 10%, 17%, 179%, 18% and 32%, respectively. While, these paraments did not change significantly by the BL or Pyr treatments. Treatment with BL + Pyr and Pyr, rather than BL, improved the chlorophyll a and chlorophyll b contents by upregulating genes related to chlorophyll biosynthesis and downregulating genes related to chlorophyll degradation. Additionally, according to transcriptomic and metabolomic analysis, the BL+ Pyr treatment outperformed the individual BL or Pyr treatments in activating the transcription of genes involved in photosynthesis and increasing sugar accumulation. Our results first validated that the combined usage of BL and Pyr exerted striking synergistic effects on enhancing plant biomass and yield by increasing photosynthetic efficiency. These results might provide new understanding for the agricultural effects by the co-application of BL and Pyr, and it might stimulate the efforts to develop new environment-friendly replacement for Pyr to minimize the ecotoxicology of Pyr.

Comprehensive quantitative analysis of Chinese patent drug YinHuang drop pill by ultra high-performance liquid chromatography quadrupole time of flight mass spectrometry.

YinHuang drop pill (YHDP) is a new preparation, derived from the traditional YinHuang (YH) decoction. Since drop pills are one of the newly developed forms of Chinese patent drugs, not much research has been done regarding the quality and efficacy. This study aims to establish a comprehensive quantitative analysis of the chemical profile of YHDP. ultra high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS/MS) was used to identify 34 non-sugar small molecules including 15 flavonoids, 9 phenolic acids, 5 saponins, 1 iridoid, and 4 iridoid glycosides in YHDP samples, and 26 of them were quantitatively determined. Sugar composition of YHDP in terms of fructose, glucose and sucrose was examined via a high performance liquid chromatography-evaporative light scattering detector on an amide column (HPLC-NH2P-ELSD). Macromolecules were examined by high performance gel permeation chromatography coupled with ELSD (HPGPC-ELSD). The content of the drop pill's skeleton component PEG-4000 was also quantified via ultra-high performance liquid chromatography coupled with charged aerosol detector (UHPLC-CAD). The results showed that up to 73% (w/w) of YHDP could be quantitatively determined. Small molecules accounted for approximately 5%, PEG-4000 represented 68%, while no sugars or macromolecules were found. Furthermore, YHDP showed no significant differences in terms of daily dosage, compared to YinHuang granules and YinHuang oral liquid; however, it has a higher small molecules content compared to YinHuang lozenge.