Xiufeng Yan

Regulation of plant glucosinolate metabolism.

Glucosinolates and their degradation products are known to play important roles in plant interaction with herbivores and micro-organisms. In addition, they are important for human life. For example, some degradation products are flavor compounds and some exhibit anticarcinogenic properties. Recent years have seen great progress made in the understanding of glucosinolate biosynthesis in Arabidopsis thaliana. The core glucosinolate biosynthetic pathway has been revealed using biochemical and reverse genetics approaches. Future research needs to focus on questions related to regulation and control of glucosinolate metabolism. Here we review current status of studies on the regulation of glucosinolate metabolism at different levels, and highlight future research towards elucidating the signaling and metabolic network that control glucosinolate metabolism.

Comparative Proteomics of Salt Tolerance in Arabidopsis thaliana and Thellungiella halophila

Salinity is a major abiotic stress affecting plant cultivation and productivity. Thellungiella halophila is a halophyte and has been used as a model for studying plant salt tolerance. Understanding the molecular mechanisms of salinity tolerance will facilitate the generation of salt tolerant crops. Here we report comparative leaf proteomics of Arabidopsis, a glycophyte, and its close relative Thellungiella, a halophyte, under different salt stress conditions. Proteins from control and NaCl treated Arabidopsis and Thellungiella leaf samples were extracted and separated by two-dimensional gel electrophoresis. A total of 88 protein spots from Arabidopsis gels and 37 protein spots from Thellungiella gels showed significant changes. Out of these spots, a total of 79 and 32 proteins were identified by mass spectrometry in Arabidopsis and Thellungiella, respectively. Most of the identified proteins were involved in photosynthesis, energy metabolism, and stress response in Arabidopsis and Thellungiella. As a complementary approach, isobaric tag for relative and absolute quantification (iTRAQ) LC-MS was used to identify crude microsomal proteins. A total of 31 and 32 differentially expressed proteins were identified in Arabidopsis and Thellungiella under salt treatment, respectively. Overall, there were more proteins changed in abundance in Arabidopsis than in Thellungiella. Distinct patterns of protein changes in the two species were observed. Collectively, this work represents the most extensive proteomic description of salinity responses of Arabidopsis and Thellungiella and has improved our knowledge of salt tolerance in glycophytes and halophytes.

Functional Differentiation of Brassica napus Guard Cells and Mesophyll Cells Revealed by Comparative Proteomics

Guard cells are highly specialized cells that form tiny pores called stomata on the leaf surface. The opening and closing of stomata control leaf gas exchange and water transpiration as well as allow plants to quickly respond and adjust to new environmental conditions. Mesophyll cells are specialized for photosynthesis. Despite the phenotypic and obvious functional differences between the two types of cells, the full protein components and their functions have not been explored but are addressed here through a global comparative proteomics analysis of purified guard cells and mesophyll cells. With the use of isobaric tags for relative and absolute quantification (iTRAQ) tagging and two-dimensional liquid chromatography mass spectrometry, we identified 1458 non-redundant proteins in both guard cells and mesophyll cells of Brassica napus leaves. Based on stringent statistical criteria, a total of 427 proteins were quantified, and 74 proteins were found to be enriched in guard cells. Proteins involved in energy (respiration), transport, transcription (nucleosome), cell structure, and signaling are preferentially expressed in guard cells. We observed several well characterized guard cell proteins. By contrast, proteins involved in photosynthesis, starch synthesis, disease/defense/stress, and other metabolisms are preferentially represented in mesophyll cells. Of the identified proteins, 110 have corresponding microarray data obtained from Arabidopsis guard cells and mesophyll cells. About 72% of these proteins follow the same trend of expression at the transcript and protein levels. For the rest of proteins, the correlation between proteomics data and the microarray data is poor. This highlights the importance of quantitative profiling at the protein level. Collectively this work represents the most extensive proteomic description of B. napus guard cells and has improved our knowledge of the functional specification of guard cells and mesophyll cells.

Desiccation tolerance mechanism in resurrection fern-ally Selaginella tamariscina revealed by physiological and proteomic analysis.

Drought is one of the most severe limitations to plant growth and productivity. Resurrection plants have evolved a unique capability to tolerate desiccation in vegetative tissues. Fern-ally Selaginella tamariscina (Beauv.) is one of the most primitive vascular resurrection plants, which can survive a desiccated state and recover when water becomes available. To better understand the mechanism of desiccation tolerance, we have applied physiological and proteomic analysis. Samples of S. tamariscina were water-deprived for up to seven days followed by 12 h of rewatering. Our results showed that endogenous abscisic acid (ABA) increased to regulate dehydration-responsive genes/proteins and physiological processes. In the course of dehydration, the contents of osmolytes represented by soluble sugars and proline were increased to maintain cell structure integrity. The activities of four antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione reductase (GR)) also increased. In contrast, both the rate of photosynthesis and the chlorophyll content decreased, and plasma membrane integrity was lost. We identified 138 desiccation-responsive two-dimensional electrophoresis (2-DE) spots, representing 103 unique proteins. Hierarchical clustering analysis revealed that 83% of the proteins were down-regulated upon dehydration. They were mainly involved in photosynthesis, carbohydrate and energy metabolism, stress and defense, protein metabolism, signaling, membrane/transport, cell structure, and cell division. The dynamic expression changes of the desiccation-responsive proteins provide strong evidence that cell structure modification, photosynthesis reduction, antioxidant system activation, and protein post-transcriptional/translational modifications are essential to the poikilochlorophyllous fern-ally S. tamariscina in response to dehydration. In addition, our comparative analysis of dehydration-responsive proteins in vegetative tissues from 19 desiccation tolerant and nontolerant plant species suggests that resurrection S. tamariscina has developed a specific desiccation tolerant mechanism. To our knowledge, this study constitutes the first detailed investigation of the protein complement in fern/fern-allies.

Proteomic identification of differentially expressed proteins in Arabidopsis in response to methyl jasmonate.

Jasmonates (JAs) are the well characterized fatty acid-derived cyclopentanone signals involved in the plant response to biotic and abiotic stresses. JAs have been shown to regulate many aspects of plant metabolism, including glucosinolate biosynthesis. Glucosinolates are natural plant products that function in defense against herbivores and pathogens. In this study, we applied a proteomic approach to gain insight into the physiological processes, including glucosinolate metabolism, in response to methyl jasmonate (MeJA). We identified 194 differentially expressed protein spots that contained proteins that participated in a wide range of physiological processes. Functional classification analysis showed that photosynthesis and carbohydrate anabolism were repressed after MeJA treatment, while carbohydrate catabolism was up-regulated. Additionally, proteins related to the JA biosynthesis pathway, stress and defense, and secondary metabolism were up-regulated. Among the differentially expressed proteins, many were involved in oxidative tolerance. The results indicate that MeJA elicited a defense response at the proteome level through a mechanism of redirecting growth-related metabolism to defense-related metabolism.

Proteomics and Metabolomics of Arabidopsis Responses to Perturbation of Glucosinolate Biosynthesis

To understand plant molecular networks of glucosinolate metabolism, perturbation of aliphatic glucosinolate biosynthesis was established using inducible RNA interference (RNAi) in Arabidopsis. Two RNAi lines were chosen for examining global protein and metabolite changes using complementary proteomics and metabolomics approaches. Proteins involved in metabolism including photosynthesis and hormone metabolism, protein binding, energy, stress, and defense showed marked responses to glucosinolate perturbation. In parallel, metabolomics revealed major changes in the levels of amino acids, carbohydrates, peptides, and hormones. The metabolomics data were correlated with the proteomics results and revealed intimate molecular connections between cellular pathways/processes and glucosinolate metabolism. This study has provided an unprecedented view of the molecular networks of glucosinolate metabolism and laid a foundation towards rationale glucosinolate engineering for enhanced defense and quality.

Proteomic investigation into betulinic acid-induced apoptosis of human cervical cancer HeLa cells.

Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3β and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes 14-3-3β and 14-3-3ε. Furthermore, it was found that the antiapoptotic bcl-2 gene was down-regulated while the proapoptotic bax gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.

Research Article: Bioinformatic analysis of molecular network of glucosinolate biosynthesis

Glucosinolates constitute a major group of secondary metabolites in Arabidopsis, which play an important role in plant interaction with pathogens and insects. Advances in glucosinolate research have defined the biosynthetic pathways. However, cross-talk and interaction between glucosinolate pathway and other molecular pathways are largely unknown. Here three bioinformatics tools were used to explore novel components and pathway connections in glucosinolate network. Although none of the software tools were prefect to predict glucosinolate genes, combination of results generated by all the tools led to successful prediction of all known glucosinolate genes. This approach was used to predict new genes in glucosinolate network. A total of 330 genes were found with high potential to relate to glucosinolate biosynthesis. Among them 64 genes were selected to construct glucosinolate network because their individual connection to at least one known glucosinolate gene was predicted by all the software tools. Microarray data of candidate gene mutants were used for validation of the results. The mutants of nine genes predicted by glucosinolate seed genes all exhibited changes in the expression of glucosinolate genes. Four of the genes have been well-known to functionally interact with glucosinolate biosynthesis. These results indicate that the approach we took provides a powerful way to reveal new players in glucosinolate networks. Creation of an in silico network of glucosinolate biosynthesis will allow the generation of many testable hypotheses and ultimately enable predictive biology.

Characterization of glucosinolate—myrosinase system in developing salt cress Thellungiella halophila

Glucosinolates are specialized plant metabolites derived from amino acids. They can be hydrolyzed by myrosinases into different degradation products, which have a variety of biological activities. In this study, the compositions and contents of glucosinolates in salt cress (Thellungiella halophila) at different developmental stages were analyzed by high performance liquid chromatography and mass spectrometry (HPLC-MS). Myrosinase activities were also measured. Seven glucosinolates were identified in T. halophila throughout its life cycle. The glucosinolate profiles varied significantly among different tissues. The roots at stage 4 contained the highest concentrations of total, aromatic and indole glucosinolates among all tissues. Whereas roots, flowers and siliques contained all seven glucosinolates, seeds contained only four aliphatic glucosinolates. During development, the concentrations also displayed significant changes. From seeds to cotyledons and from stage 4 roots to stage 5 roots, there were dramatic declines of glucosinolates, which correlated well with changes in myrosinase activities. In other tissues, myrosinase activity alone could not explain the glucosinolate concentration changes. Certain tissues of T. halophila contained Arabidopsis myrosinase TGG1 and TGG2 orthologs. The molecular basis and functional significance of our findings are discussed here.

Development and validation of a RP-HPLC method with fluorescence detection for simultaneous determination of 10-methoxycamptothecin and its metabolite 10-hydroxycamptothecin in rat plasma

Both 10-methoxycamptothecin (MCPT) and 10-hydroxycamptothecin (HCPT) are the natural bioactive derivatives of camptothecin (CPT) isolated from Camptotheca acuminata, and have been confirmed to possess high anti-cancer properties. In the present study, HCPT was identified as the major metabolite of MCPT in rat plasma through HPLC/photodiode array detection (PDA) and LC-MS/MS analysis. A sensitive and reliable RP-HPLC method with fluorescence detection was developed and validated for the simultaneous analysis of MCPT and HCPT in rat plasma. The parental CPT was used as an internal standard (IS). A piecewise linear function was used over lower and higher concentrations, respectively. The calibration curves were linear (r² > 0.999) over concentrations from 1.25 to 20 ng/mL and 20 to 320 ng/mL for both MCPT and HCPT. The method had an accuracy of 92.24-113.90%, and the intra- and inter-day precision (RSD%) were 10.05% or less for MCPT and HCPT. The stability data showed no significant degradation occurred under the experimental conditions. The mean recoveries at concentrations of 2.5, 40 and 160 ng/mL were 95.09±3.94%, 98.67±1.40% and 95.65±2.15% for MCPT and 84.06±4.39%, 84.85±3.10% and 81.03±4.44% for HCPT, respectively. The lower limit of quantification (LLOQ) using 0.1 mL of plasma was 1.25 ng/mL for both MCPT and HCPT. This method was successfully applied to the pharmacokinetic study of MCPT and its metabolite HCPT in rat plasma after intravenous administration.