Identification and quality evaluation of Lushan Yunwu tea from different geographical origins based on metabolomics.

The relationship between the chemical composition and quality of Lushan Yunwu tea (LYT) from different geographical origins is not clear. Sensory evaluation, metabolomics analyses combined with chemometrics were conducted on LYT from 8 different geographical origins, and altitude was identified as the main factor responsible for the differences among LYT. A total of 32 non-volatile and 27 volatile compounds were identified as marker metabolites to distinguish the origins of high altitudes from those of low altitudes. LYT samples from higher altitude areas contained more free amino acids, sugars, and organic acids, and less catechins, which may contribute to the reduction of bitterness and astringency and the enhancement of umami. The contents of geranylacetone, ethyl hexanoate, ethyl caprylate, 3-carene, d-cadinene, linalool, nerol, and nerolidol in high altitude areas were higher than those in low altitude areas, indicating that LYT from high altitude had strong floral and fruity aroma. The altitudes were positively correlated with pH value, total flavonoids, soluble protein, total free amino acids, and the antioxidant capacities of the LYT. This study provided a theoretical basis for the study of the effect of altitude on tea quality.

Identification and characterization of polyamine metabolism in citrus in response to 'Candidatus Liberibacter asiaticus' infection.

Citrus Huanglongbing, one of the most devastating citrus diseases, is caused by Candidatus Liberibacter asiaticus (CLas). Polyamines are aliphatic nitrogen-containing compounds that play important roles in disease resistance. However, the role of polyamine metabolism in the tolerance of citrus to infection with CLas have not been extensively studied. We used HPLC and UPLC-Q/TOF-MS to detect the contents of nine polyamine metabolism-related compounds (PMRCs) in six citrus cultivars with varying levels of tolerance to CLas. Moreover, we systematically detected the changes of PMRC and H2O2 contents, and compared the gene expression levels and activities of enzymes involved in the polyamine metabolic pathway among healthy, asymptomatic, and symptomatic leaves of Newhall navel oranges infected with CLas. The tolerant and moderately tolerant varieties showed higher PMRC levels than those of susceptible varieties. Compared with the healthy group, the symptomatic group showed significantly increased contents of arginine, ornithine, γ-aminobutyric acid, and putrescine by approximately 180, 19, 1.5, and 0.2 times, respectively, and up-regulated expression of biosynthetic genes. Arginase and ornithine decarboxylase enzyme activities were the highest in the symptomatic group, while arginine decarboxylase and agmatine deiminase enzyme activities were the highest in the asymptomatic group. The two polyamine biosynthetic pathways showed different trends with the increase of the titer of CLas, indicating that polyamines were mainly synthesized through the arginine decarboxylase pathway in the asymptomatic leaves, and were synthesized via the ornithine decarboxylase pathway in symptomatic leaves. These findings provide new insight into the changes of polyamine metabolism in citrus infected with CLas.

Flavonoid profile of Anoectochilus roxburghii (Wall.) Lindl. Under short-term heat stress revealed by integrated metabolome, transcriptome, and biochemical analyses.

Global warming severely threatens plant growth, and could lead to yield reduction. Although findings suggest that flavonoids play important roles in biological process in plants, their response to heat stress in Anoectochilus roxburghii (Wall.) Lindl. remains unclear. Here, we aimed to examine the flavonoid profile of A. roxburghii under heat stress and assess the effect of exogenous application of quercetin on heat stress tolerance. Metabolome analysis showed that quercetin, tricetin, isorhamnetin, scutellarein, and 4',7-Isoflavandiol were the main upregulated flavonoids in A. roxburghii, based on variable importance in the projection >1 and with fold change >2. Determination of the concentrations of the flavonoids using a standard curve revealed that quercetin, kaempferol, and isorhamnetin contents increased by 8.24-, 7.55-, and 5.01-fold, respectively, during heat stress, whereas rutin concentration decreased from 83.04 to 80.89 mg/kg (dry weight). Additionally, transcriptome analysis indicated increased expression of several genes in flavonoid biosynthesis pathways, including phenylalanine ammonia-lyase and chalcone synthase. Moreover, exogenous application of quercetin improved the antioxidant capacity and physiological parameters, including photosynthetic rate and chlorophyll content, of A. roxburghii under heat stress. Overall, the flavonoid profile of A. roxburghii under short-term heat stress was characterized based on integrated metabolomic, transcriptomic, and biochemical analyses, providing new insights for improving the biological value of A. roxburghii.

Metabolic Profiling of Oryza sativa L. Triggered by Chilling Stress Using Ultraperformance Liquid Chromatography Coupled with Quadrupole/Time-of-Flight Mass Spectrometry (UPLC-QTOF-MS) with Transcriptome Analysis.

Low temperature, a major abiotic stress, often causes molecular changes in crops, which leads to metabolic disturbances and probably affects crop yield. In this study, chilling stress induced distinct metabolic profiles associated with transcriptome regulation, exhibiting great metabolic differences between Qiutianxiaoting (japonica) and 93-11 (indica). In total, 41 and 58 differential metabolites were screened and identified in Qiutianxiaoting and 93-11, respectively. Five key metabolites were screened in response to chilling stress, which were involved or related to different metabolic pathways. Moreover, starch and sucrose metabolism, aminoacyl-tRNA biosynthesis, and phenylpropanoid biosynthesis were significantly enriched in Qiutianxiaoting to maintain cellular homeostasis. Aminoacyl-tRNA biosynthesis and antioxidation metabolism were significantly enriched in 93-11, but disorders of the metabolome and transcriptome occurred at recovery stage. The results could provide some useful information for in-depth understanding of cold-resistant mechanisms, as well as reference for the selection and breeding of rice varieties.

Early detection of Huanglongbing with EESI-MS indicates a role of phenylpropanoid pathway in citrus.

Huanglongbing (HLB), a devastating disease for citrus worldwide, is caused by Candidatus Liberibacter asiaticus (CLas). In this study, we employed a novel extractive electrospray ionization-mass spectrometry (EESI-MS) method to analyze the metabolites in leaves of uninfected and HLB-infected Newhall navel orange. The results showed that uninfected and HLB-infected leaves could be readily distinguished based on EESI-MS combined by multivariable analysis. Nine phenolic compounds involved in phenylpropanoid pathway, such as p-coumaric acid, naringin, and apigenin, were principal components to distinguish the leaves of uninfected and HLB-infected Newhall navel orange. Gene expression was also conducted to further explore the molecular mechanism of phenylpropanoid branch pathway in HLB. The expression of genes (4CL, HCT, CHI, CHS, CYP, and C12R) involved in phenylpropanoid branch pathway was increased in asymptomatic and early period of HLB-infected leaves, while decreased in later period of HLB-infected leaves. This study provides a novel method for early detection of citrus HLB and suggests the regulation mechanism of phenylpropanoid pathway in the interaction between citrus and CLas.

Differential Antioxidant Compounds and Activities in Seedlings of Two Rice Cultivars Under Chilling Treatment.

Variations in antioxidant compounds were examined in seedlings of two rice cultivars (Qiutianxiaoting and 93-11) exposed to low temperature (4°C) for 0, 12, 36, and 48 h. Antioxidant activity was identified by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. The concentrations of total phenols, flavonoids, chlorophyll, and anthocyanins (ACNs) were determined by spectrophotometry. In addition, high-performance liquid chromatography (HPLC) was used to reveal the changes in phenolic compound concentrations in rice seedlings under chilling treatment. Results showed that antioxidant concentrations and antioxidant activity after chilling treatment were higher in 93-11 compared to Qiutianxiaoting, reaching the highest level at 36 h chilling treatment in 93-11. Phenolic compounds in Qiutianxiaoting decreased between 12 and 36 h but then increased at 48 h, whereas the corresponding levels in 93-11 increased as chilling time increased. Moreover, 10 phenolic compounds were detected and quantified by HPLC, of which gallic acid and caffeic acid tended to only exist in 93-11, whereas rutin was observed only in Qiutianxiaoting. The results of this study could be leveraged to optimize the antioxidant potential of rice in the context of healthy food choices.

Metabolic response of Citrus limon to Asian citrus psyllid infestation revealed by EESI-MS and HPLC.

Asian citrus psyllid (ACP) causes direct and indirect damage to the citrus industry. Extractive electrospray ionization mass spectrometry (EESI-MS) and high performance liquid chromatography (HPLC) were used to detect the metabolites of C. limon leaves at 0, 12, 24, and 72 h after ACP treatment. The EESI-MS results showed that ACP infestation significantly affected metabolites within a short feeding duration with 8 metabolites identified. The metabolites in leaves of these four groups could be distinguished, with 55 peaks showing significant differences including methyl N-methylanthranilate, caffeic acid, and syringic acid. The quantification of 15 phenolic compounds with HPLC-UV method in C. limon leaves after ACP infestation showed that the total content of them reached a peak of 3504.69 µg g-1 at 12 h, with 9 phenolic compounds changing significantly (P < 0.05). A total of 21 metabolites identified in this study were involved in the biosynthesis pathways of flavonoid, flavone and flavonol, isoflavonoid and phenylpropanoid, and the degradation of aminobenzoate. Contents of epicatechin and caffeic acid increased with the feeding time of ACP as detected by both EESI-MS and HPLC. This may be related to plant defense. This study provides novel insights into the biochemical relationship of ACP and its host plants.

Direct identification and metabolomic analysis of Huanglongbing associated with Candidatus Liberibacter spp. in navel orange by MALDI-TOF-MS.

Citrus Huanglongbing (HLB) is one of the most destructive citrus diseases worldwide, and is associated with the phloem-limited plant pathogenic bacteria Candidatus Liberibacter species. However, there is a latency period during which newly infected trees do not show symptoms, creating challenges for the early detection of HLB. In order to establish a method for rapid detection and to assess the metabolite differences between healthy and HLB-affected Newhall navel oranges, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to study asymptomatic and symptomatic leaf extracts compared with healthy leaves. The results showed that healthy, asymptomatic, and symptomatic leaves were distinguishable using MALDI-TOF-MS combined with multivariable analysis. Discriminant analysis (DA) results showed that the accuracy of discrimination of the training set and the success rate of cross-validation analysis were both 100%. Blind testing revealed that nine validation samples were correctly classified to their corresponding group. In addition, 32 MS peaks of metabolites had significant differences between healthy and different HLB-affected navel orange leaves. Among 32 MS peaks, the relative content of nine substances showed an upward trend, and 19 metabolites showed a downward trend, including a significant decrease in malate and citrate, with an increase in HLB bacterial populations. Malate and citrate were found to be specifically reduced in infected leaves regardless of whether symptoms appeared. Thus, they could be used as potential biomarkers for early detection of HLB, reducing false-negative results caused by uneven distribution of the HLB bacterial pathogen. This novel method can efficiently differentiate HLB-affected samples from the host population and provide new information for early detection of HLB in citrus. The entire process from metabolite extraction to the collection of mass spectra of citrus leaves was a simple operation, with no need for sterile conditions. We believe this strategy has significant implications for rapid and effective HLB screening. Graphical abstract.

Chemical Composition and Antioxidant Activity of Essential Oil of Chinese Propolis.

The chemical composition and in vitro antioxidant activity of the essential oil of propolis (EOP) collected from 25 locations in China was investigated. Steam-distillation extraction was used to extract the EOP, and chemical composition was identified by GC/MS. The antioxidant activities of EOP were also measured. The result showed that a total of 406 compounds were detected in EOP. The major compounds of Chinese EOP were cedrol, γ-eudesmol, benzyl alcohol, phenethyl alcohol, 2-methoxy-4-vinylphenol, 3,4-dimethoxystyrene and guaiol. Principal component analysis revealed the significant correlation between EOP compositions and their origins, and certain correlation was detected between EOP and their color. Linear discriminant analysis showed that 88 % and 84 % of the propolis samples were predicted correctly as the groupings identified by climatic zone and the color, respectively. Furthermore, the differences of antioxidant activities of EOP were significant. EOP of Shandong had the strongest antioxidant activities, whereas EOP of Guangdong, Yunnan and Hunan showed the poorest.

In Situ Study of Metabolic Response of Arabidopsis thaliana Leaves to Salt Stress by Neutral Desorption-Extractive Electrospray Ionization Mass Spectrometry.

Salt stress is one of the most common factors limiting plant cultivation. In this study, metabolic responses to salt stress in Arabidopsis thaliana (A. thaliana) leaves were analyzed in situ by neutral desorption-extractive electrospray ionization mass spectrometry (ND-EESI-MS) without any sample pretreatment. Metabolic changes of A. thaliana leaves were observed in response to salt stress conditions, including the levels of serine, glutamic acid, arginine, cinnamic acid, ferulic acid, caffeic acid, protocatechuic acid, epicatechin, morin, myricetin, apigravin, and ß-cotonefuran. The content of serine increased under 50, 100, and 200 mM NaCl salt stress, reaching the highest level at 200 mM NaCl, but decreased under the maximum concentration of 300 mM NaCl. A similar phenomenon was observed for arginine, glutamic acid, cinnamic acid, caffeic acid, ferulic acid, and epicatechin, respectively, involved in the metabolic pathway of shikimate-phenylpropanoid. Both principal component analysis (PCA) and partial least-squares discrimination analysis (PLS-DA) showed that the salt stress treatment groups of the higher concentrations (200 and 300 mM) could be well distinguished from those of the lower concentrations (50 and 100 mM) and the control. Marker metabolites, like m/z 261 (apigravin) and m/z 305 (ß-cotonefuran), were assistantly selected from the fingerprints by variable importance for the projection (VIP). Our results indicated the potential of the ND-EESI-MS method for the rapid recognition of metabolic conditions in plant leaves under salt stress.

Dynamic changes of phenolic compounds during artificial aging of soybean seeds identified by high-performance liquid chromatography coupled with transcript analysis.

Phenolic compounds are important bioactive substances in plants, but study of their alteration during soybean seed aging is still limited. In this study, we conducted artificial aging on soybean seeds, detected the dynamic changes of phenolic compound concentrations using high-performance liquid chromatography, and analyzed the gene expression of key enzymes of phenolic metabolism. A detailed method for detection of 19 phenolic compounds during artificial aging of soybean seeds was constructed, and all of these phenols significantly changed in concentration. The content of protocatechuic acid, rutin, and morin decreased, whereas that of daidzein, glycitein, genistein, and baicalin increased. The concentration of caffeic acid, epicatechin, ferulic acid, daidzin, genistin, and resveratrol first rose and then declined, and the content of ferulic acid was highest after 2 days of artificial aging, with the other five phenolic compounds showing the highest content after 4 days of artificial aging. The total content of the 19 phenolic compounds reached a peak of 2357.43 µg g-1 dry weight at 2 days. Relative expression of PAL1, PAL2, PAL3, CHS7, CHS8, IFS1, IFS2, CHR1, 4CL2, C4H, and CHI2 was mostly downregulated as the duration of artificial aging increased. This study provides novel insights into the storage and use of soybean seed resources. Graphical abstract.

TaMCA1, a regulator of cell death, is important for the interaction between wheat and Puccinia striiformis.

Metacaspase orthologs are conserved in fungi, protozoa and plants, however, their roles in plant disease resistance are largely unknown. In this study, we identified a Triticum aestivum metacaspase gene, TaMCA1, with three copies located on chromosomes 1A, 1B and 1D. The TaMCA1 protein contained typical structural features of type I metacaspases domains, including an N-terminal pro-domain. Transient expression analyses indicated that TaMCA1 was localized in cytosol and mitochondria. TaMCA1 exhibited no caspase-1 activity in vitro, but was able to inhibit cell death in tobacco and wheat leaves induced by the mouse Bax gene. In addition, the expression level of TaMCA1 was up-regulated following challenge with the Puccinia striiformis f. sp. tritici (Pst). Knockdown of TaMCA1 via virus-induced gene silencing (VIGS) enhanced plant disease resistance to Pst, and the accumulation of hydrogen peroxide (H2O2). Further study showed that TaMCA1 decreased yeast cell resistance similar to the function of yeast metacaspase, and there was no interaction between TaMCA1 and TaLSD1. Based on these combined results, we speculate that TaMCA1, a regulator of cell death, is important during the compatible interaction of wheat and Pst.

Transcriptome Analysis Provides Insights into the Mechanisms Underlying Wheat Plant Resistance to Stripe Rust at the Adult Plant Stage.

Stripe rust (or yellow rust), which is caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating wheat diseases worldwide. The wheat cultivar Xingzi 9104 (XZ) is an elite wheat germplasm that possesses adult plant resistance (APR), which is non-race-specific and durable. Thus, to better understand the mechanism underlying APR, we performed transcriptome sequencing of wheat seedlings and adult plants without Pst infection, and a total of 157,689 unigenes were obtained as a reference. In total, 2,666, 783 and 2,587 differentially expressed genes (DEGs) were found to be up- or down-regulated after Pst infection at 24, 48 and 120 hours post-inoculation (hpi), respectively, based on a comparison of Pst- and mock-infected plants. Among these unigenes, the temporal pattern of the up-regulated unigenes exhibited transient expression patterns during Pst infection, as determined through a Gene Ontology (GO) enrichment analysis. In addition, a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that many biological processes, including phenylpropanoid biosynthesis, reactive oxygen species, photosynthesis and thiamine metabolism, which mainly control the mechanisms of lignification, reactive oxygen species and sugar, respectively, are involved in APR. In particular, the continuous accumulation of reactive oxygen species may potentially contribute to the ability of the adult plant to inhibit fungal growth and development. To validate the bioinformatics results, 6 candidate genes were selected for further functional identification using the virus-induced gene silencing (VIGS) system, and 4 candidate genes likely contribute to plant resistance against Pst infection. Our study provides new information concerning the transcriptional changes that occur during the Pst-wheat interaction at the adult stage and will help further our understanding of the detailed mechanisms underlying APR to Pst.