Qiuying Pang

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.

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.

Functional specification of Arabidopsis isopropylmalate isomerases in glucosinolate and leucine biosynthesis.

The Arabidopsis genome encodes one potential isopropylmalate isomerase (IPMI) large subunit and three potential IPMI small subunits, which in bacteria and archaea form heterodimers to catalyze the isomerization of 2-isopropylmalate to 3-isopropylmalate in leucine biosynthesis. We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. Tissue-specific expression analysis revealed that the patterns of AtLeuD1 and AtLeuD2 expression are similar, but distinct from that of AtLeuD3. This result indicates functional redundancy of AtLeuD1 and AtLeuD2, and functional specification of AtLeuD3. Reverse genetics and metabolite profiling showed that AtLeuD1 and AtLeuD2 function redundantly in aliphatic glucosinolate biosynthesis, but AtLeuD3 is not likely to be involved in this pathway. The lethal phenotype of the atleud3 mutant suggests functional specification of AtLeuD3 in leucine biosynthesis. A defect in female gametophyte development was found to contribute to the mutant lethality, suggesting the important role of AtLeuD3 in female gametophyte development.

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.

Proteomic analysis of sugar beet apomictic monosomic addition line M14.

Apomixis in plants holds great promise for agriculture because of its vigor associated with heterozygosity and superior genotype. Despite the significance of apomictic reproductive process, our knowledge of proteins and their functions in apomictic development is limited. Here we report a comparative proteomic and transcriptomic analysis of sexual and apomictic processes in sugar beet. A total of 71 differentially expressed protein spots were successfully identified in the course of apomictic reproductive development using high-resolution 2-DE and MS analysis. The differentially expressed proteins were involved in several processes that might work cooperatively to lead to apomictic reproduction. This study has generated potential protein markers important for apomictic development.

Structural and Functional Evolution of Isopropylmalate Dehydrogenases in the Leucine and Glucosinolate Pathways of Arabidopsis thaliana

The methionine chain-elongation pathway is required for aliphatic glucosinolate biosynthesis in plants and evolved from leucine biosynthesis. In Arabidopsis thaliana, three 3-isopropylmalate dehydrogenases (AtIPMDHs) play key roles in methionine chain-elongation for the synthesis of aliphatic glucosinolates (e.g. AtIPMDH1) and leucine (e.g. AtIPMDH2 and AtIPMDH3). Here we elucidate the molecular basis underlying the metabolic specialization of these enzymes. The 2.25 Å resolution crystal structure of AtIPMDH2 was solved to provide the first detailed molecular architecture of a plant IPMDH. Modeling of 3-isopropylmalate binding in the AtIPMDH2 active site and sequence comparisons of prokaryotic and eukaryotic IPMDH suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function. Site-directed mutagenesis of Phe-137 to a leucine in AtIPMDH1 (AtIPMDH1-F137L) reduced activity toward 3-(2′-methylthio)ethylmalate by 200-fold, but enhanced catalytic efficiency with 3-isopropylmalate to levels observed with AtIPMDH2 and AtIPMDH3. Conversely, the AtIPMDH2-L134F and AtIPMDH3-L133F mutants enhanced catalytic efficiency with 3-(2′-methylthio)ethylmalate ∼100-fold and reduced activity for 3-isopropylmalate. Furthermore, the altered in vivo glucosinolate profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could be restored to wild-type levels by constructs expressing AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by AtIPMDH1-F137L. These results indicate that a single amino acid substitution results in functional divergence of IPMDH in planta to affect substrate specificity and contributes to the evolution of specialized glucosinolate biosynthesis from the ancestral leucine pathway.

Integrated proteomics and metabolomics of Arabidopsis acclimation to gene-dosage dependent perturbation of isopropylmalate dehydrogenases.

Maintaining metabolic homeostasis is critical for plant growth and development. Here we report proteome and metabolome changes when the metabolic homeostasis is perturbed due to gene-dosage dependent mutation of Arabidopsis isopropylmalate dehydrogenases (IPMDHs). By integrating complementary quantitative proteomics and metabolomics approaches, we discovered that gradual ablation of the oxidative decarboxylation step in leucine biosynthesis caused imbalance of amino acid homeostasis, redox changes and oxidative stress, increased protein synthesis, as well as a decline in photosynthesis, which led to rearrangement of central metabolism and growth retardation. Disruption of IPMDHs involved in aliphatic glucosinolate biosynthesis led to synchronized increase of both upstream and downstream biosynthetic enzymes, and concomitant repression of the degradation pathway, indicating metabolic regulatory mechanisms in controlling glucosinolate biosynthesis.

Dihydroxyacid dehydratase is important for gametophyte development and disruption causes increased susceptibility to salinity stress in Arabidopsis

Highlight Molecular characterization of dihydroxyacid dehydratase in Arabidopsis reveals its important roles in gametophyte and root development, as well as involvement in salinity stress resistance.