Olives (Olea europaea L.) are a significant part of the agroindustry in China. Olive leaves, the most abundant by-products of the olive and olive oil industry, contain bioactive compounds that are beneficial to human health. The purpose of this study was to evaluate the phytochemical profiles and antioxidant capacities of olive leaves from 32 cultivars grown in China. A total of 32 phytochemical compounds were identified using high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry, including 17 flavonoids, five iridoids, two hydroxycinnamic acids, six triterpenic acids, one simple phenol, and one coumarin. Specifically, olive leaves were found to be excellent sources of flavonoids (4.92–18.29 mg/g dw), iridoids (5.75–33.73 mg/g dw), and triterpenic acids (15.72–35.75 mg/g dw), and considerable variations in phytochemical content were detected among the different cultivars. All tested cultivars were classified into three categories according to their oil contents for further comparative phytochemicals assessment. Principal component analysis indicated that the investigated olive cultivars could be distinguished based upon their phytochemical profiles and antioxidant capacities. The olive leaves obtained from the low-oil-content (20%) cultivars contained mainly triterpenic acids in their compositions. Correspondingly, the low-oil-content cultivars (OL3, Frantoio selection and OL14, Huaou 5) exhibited the highest ABTS antioxidant activities (758.01 ± 16.54 and 710.64 ± 14.58 mg TE/g dw, respectively), and OL9 (Olea europaea subsp. Cuspidata isolate Yunnan) and OL3 exhibited the highest ferric reducing/antioxidant power assay values (1228.29 ± 23.95 mg TE/g dw and 1099.99 ± 14.30 mg TE/g dw, respectively). The results from this study may be beneficial to the comprehensive evaluation and utilization of bioactive compounds in olive leaves.

Protein phosphorylation plays an important role in mediating signal transduction in cold response in plants. To better understand how plants sense and respond to the early temperature drop, we performed data-independent acquisition (DIA) method-based mass spectrometry analysis to profile the proteome and phosphoproteome of Arabidopsis seedlings upon cold stress in a time-course manner (10, 30 and 120 min of cold treatments). Our results showed the rapid and extensive changes at the phosphopeptide levels, but not at the protein abundance levels, indicating cold-mediated protein phosphorylation and dephosphorylation events. Alteration of over 1200 proteins at phosphopeptide levels were observed within 2 h of cold treatment, including over 140 kinases, over 40 transcriptional factors and over 40 E3 ligases, revealing the complexity of regulation of cold adaption. We summarized cold responsive phosphoproteins involved in phospholipid signaling, cytoskeleton reorganization, calcium signaling, and MAPK cascades. Cold-altered levels of 73 phosphopeptides (mostly novel cold-responsive) representing 62 proteins were validated by parallel reaction monitoring (PRM). In summary, this study furthers our understanding of the molecular mechanisms of cold adaption in plants and strongly supports that DIA coupled with PRM are valuable tools in uncovering early signaling events in plants.

Sanghuangporus, a medicinal mushroom, has gained significant attention due to its beneficial properties. Phenolic acids are among the major bioactive compounds in Sanghuangporus, known for their antioxidant and anti-inflammatory activities. To precisely quantify the phenolic acid content, we developed a method utilizing ultra-high-performance liquid chromatography with triple quadrupole (UHPLC-QqQ). This study optimized the UHPLC-QqQ conditions to simultaneously separate and detect eight phenolic acids in Sanghuangporus baumii (Pilát) L.W. Zhou and Y.C. Dai, including chlorogenic acid, p-coumaric acid, caffeic acid, cryptochlorogenic acid, protocatechuic acid, ferulic acid, sinapic acid, and syringic acid. The separation process utilized a ZORBAX Eclipse Plus C18 column using 0.01% formic acid and 2 mmol/L ammonium formate in water as the aqueous phase and methanol containing 0.01% formic acid and 2 mmol/L ammonium formate as the organic phase. Calibration curves were constructed using standard solutions to quantitatively determine the phenolic acid content. The results showed significant variation in phenolic acid content among S. baumii fruiting bodies, with Protocatechuic acid, p-coumaric acid, and caffeic acid being the most abundant. This method is valuable for quantifying phenolic acid compounds under different cultivation conditions. It provides excellent sensitivity, selectivity, and reproducibility for the quantification of phenolic acids in Sanghuangporus, contributing to a better understanding of its chemical composition and potential health benefits. This approach represents a novel technical means for the simultaneous analysis of compound phenolic acids in Sanghuangporus fruiting bodies.

A rapid, precise, and dependable method for quantifying flavonoids in the fruiting bodies of Sanghuangporus was established using ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS). Separation was achieved using a ZORBAX Eclipse Plus C18 column (1.8 μm, 3.0 mm × 100 mm) with a 15 min gradient of a mobile phase consisting of 0.01% aqueous formic acid and 2 mm/L ammonium formate (mobile phase A), and 0.01% formic acid and 2 mm/L ammonium formate in methanol (mobile phase B). A mass spectrometry analysis was performed using the multiple reaction monitoring (MRM) mode with an electrospray ion source. This method enabled the simultaneous detection of 10 flavonoids (sakuranetin, quercitrin, myricitrin, kaempferol, luteolin, rutin, hyperoside, kaempferol-3-O-rutinoside, catechin, and catechin gallate) in the fruiting bodies of Sanghuangporus. Additionally, we applied this method to analyze the flavonoid content in fruiting bodies of various Sanghuangporus species. The results revealed substantial variations in flavonoid content, up to a 100-fold difference, among different species, with myricitrin, hyperoside, and rutin identified as the most abundant flavonoids. This protocol serves as a valuable tool for quantifying flavonoid compounds in different Sanghuangporus species or under diverse cultivation conditions, particularly for identifying species with high levels of specific flavonoid compounds.

Haloxyfop-P-methyl (HPM) is widely used for grass weed control in oilseed rape production. However, whether its residues affect subsequent seed germination and the underlying mechanisms remains unclear. In this study, seeds harvested from HPM-treated fields exhibited a significantly reduced germination rate of 36.5%, compared with 100% in the control group, accompanied by abnormal cell morphology. Residue analysis revealed an HPM concentration of 3.36 mg kg⁻¹ in the treated seeds, exceeding the Chinese maximum residue limit (MRL) of 3 mg kg⁻¹ (GB 2763). Given that herbicides often induce oxidative stress, we measured a range of antioxidant markers (H₂O₂, MDA, SOD, POD, GSH, GSSG, AsA, and DHA) during germination (12 h and 24 h) but detected no significant changes, which did not support oxidative stress as the primary mechanism during this phase. Relative electrical conductivity measurements further indicated that membrane integrity was already compromised in dry seeds, suggesting that oxidative injury—if it occurred—may have taken place during seed development rather than during germination. To elucidate the molecular basis, we performed transcriptomic, metabolomic, and lipidomic analyses, which revealed significant downregulation of fatty acid biosynthesis genes and marked alterations in the lipidome profile. Collectively, our multi-stage investigation demonstrates that HPM residue accumulation is associated with impaired seed germination, primarily through metabolic disruption of lipid reserves rather than oxidative damage during germination, highlighting a non-target phytotoxic effect of this herbicide on the crop itself. These findings provide a theoretical basis for understanding the potential toxicity of persistent HPM residues in rapeseed, with implications for pesticide environmental risk assessment and the safe production of rapeseed.