Pharmaceutical analysis is a discipline based on chemical, physical, biological, and information technologies. At present, biotechnological analysis is a short branch in pharmaceutical analysis; however, bioanalysis is the basis and an important part of medicine. Biotechnological approaches can provide information on biological activity and even clinical efficacy and safety, which are important characteristics of drug quality. Because of their advantages in reflecting the overall biological effects or functions of drugs and providing visual and intuitive results, some biotechnological analysis methods have been gradually applied to pharmaceutical analysis from raw material to manufacturing and final product analysis, including DNA super-barcoding, DNA-based rapid detection, multiplex ligation-dependent probe amplification, hyperspectral imaging combined with artificial intelligence, 3D biologically printed organoids, omics-based artificial intelligence, microfluidic chips, organ-on-a-chip, signal transduction pathway-related reporter gene assays, and the zebrafish thrombosis model. The applications of these emerging biotechniques in pharmaceutical analysis have been discussed in this review.

Salicylic acid (SA) is a key defense hormone associated with wheat resistance against Fusarium head blight, which is a severe disease mainly caused by Fusarium graminearum. Although F. graminearum can metabolize SA, it remains unclear how this metabolic activity affects the wheat–F. graminearum interaction. In this study, we identified a salicylate hydroxylase gene (FG05_08116; FgNahG) in F. graminearum. This gene encodes a protein that catalyzes the conversion of SA to catechol. Additionally, FgNahG was widely distributed within hyphae. Disrupting the FgNahG gene (ΔFgNahG) led to enhanced sensitivity to SA, increased accumulation of SA in wheat spikes during the early infection stage and inhibited development of head blight symptoms. However, FgNahG did not affect mycotoxin production. Re-introducing a functional FgNahG gene into the ΔFgNahG mutant recovered the wild-type phenotype. Moreover, the expression of FgNahG in transgenic Arabidopsis thaliana decreased the SA concentration and the resistance of leaves to F. graminearum. These results indicate that the endogenous SA in wheat influences the resistance against F. graminearum. Furthermore, the capacity to metabolize SA is an important factor affecting the ability of F. graminearum to infect wheat plants.

ATP-binding cassette (ABC) transporters hydrolyze ATP to transport a wide range of substrates. Fusarium graminearum is a major causal agent of Fusarium head blight, which is a severe disease in wheat worldwide. FgABCC9 (FG05_07325) encodes an ABC-C (ABC transporter family C) transporter in F. graminearum, which was highly expressed during the infection in wheat and was up-regulated by the plant defense hormone salicylic acid (SA) and the fungicide tebuconazole. The predicted tertiary structure of the FgABCC9 protein was consistent with the schematic of the ABC exporter. Deletion of FgABCC9 resulted in decreased mycelial growth, increased sensitivity to SA and tebuconazole, reduced accumulation of deoxynivalenol (DON), and less pathogenicity towards wheat. Re-introduction of a functional FgABCC9 gene into ΔFgABCC9 recovered the phenotypes of the wild type strain. Transgenic expression of FgABCC9 in Arabidopsis thaliana increased the accumulation of SA in its leaves without activating SA signaling, which suggests that FgABCC9 functions as an SA exporter. Taken together, FgABCC9 encodes an ABC exporter, which is critical for fungal exportation of SA, response to tebuconazole, mycelial growth, and pathogenicity towards wheat.

Phytohormones are key regulators of plant growth, development, and signalling networks involved in responses to diverse biotic and abiotic stresses. Transcriptional reference maps of hormone responses have been reported for several model plant species such as Arabidopsis thaliana, Oryza sativa, and Brachypodium distachyon. However, because of species differences and the complexity of the wheat genome, these transcriptome data are not appropriate reference material for wheat studies. We comprehensively analysed the transcriptomic responses in wheat spikes to seven phytohormones, including indole acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA), ethylene (ET), cytokinin (CK), salicylic acid (SA), and methyl jasmonic acid (MeJA). A total of 3386 genes were differentially expressed at 24 h after the hormone treatments. Furthermore, 22.7% of these genes exhibited overlapping transcriptional responses for at least two hormones, implying there is crosstalk among phytohormones. We subsequently identified genes with expression levels that were significantly and differentially induced by a specific phytohormone (i.e., hormone-specific responses). The data for these hormone-responsive genes were then compared with the transcriptome data for wheat spikes exposed to biotic (Fusarium head blight) and abiotic (water deficit) stresses. Our data were used to develop a transcriptional reference map of hormone responses in wheat spikes.

Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) is initiated upon PAMP recognition by pattern recognition receptors (PRR). PTI signals are transmitted through activation of mitogen-activated protein kinases (MAPKs), inducing signaling and defense processes such as reactive oxygen species (ROS) production and callose deposition. Here, we examine mutants for two Arabidopsis thaliana genes encoding homologs of UBIQUITIN-ASSOCIATED DOMAIN-CONTAINING PROTEIN 2 (UBAC2), a conserved endoplasmic reticulum (ER) protein implicated in ER protein quality control. The ubac2 mutants were hypersusceptible to a type III secretion-deficient strain of the bacterial pathogen Pseudomonas syringae, indicating a PTI defect. The ubac2 mutants showed normal PRR biogenesis, MAPK activation, ROS burst, and PTI-associated gene expression. Pathogen- and PAMP-induced callose deposition, however, was compromised in ubac2 mutants. UBAC2 proteins interact with the plant-specific long coiled-coil protein PAMP-INDUCED COILED COIL (PICC), and picc mutants were compromised in callose deposition and PTI. Compromised callose deposition in the ubac2 and picc mutants was associated with reduced accumulation of the POWDERY MILDEW RESISTANT 4 (PMR4) callose synthase, which is responsible for pathogen-induced callose synthesis. Constitutive overexpression of PMR4 restored pathogen-induced callose synthesis and PTI in the ubac2 and picc mutants. These results uncover an ER pathway involving the conserved UBAC2 and plant-specific PICC proteins that specifically regulate pathogen-induced callose deposition in plant innate immunity.