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.

Betulinic acid (BA), a potential anticancer compound, could induce apoptosis in human cervical cancer (HeLa) cells, but its mechanism has yet to be fully elucidated. The present study was focused on deciphering the detailed molecular mechanism of BA-induced apoptosis. In the present study, results indicated that BA was highly effective against HeLa cells via induction of time-dependent apoptosis, and the authors demonstrated that the BA treatment acted through downregulating a phosphatidylinositol 3-kinase (PI3K) subunit and suppressing the Akt phosphorylation at Thr308 and Ser473 after increasing the generation of intracellular reactive oxygen species. Then, BA induced cell cycle arrest at the G0/G1 phase, which was consistent with the cell cycle-related protein results in which BA significantly enhanced the expression of p27Kip and p21Waf1/Cip1 in HeLa cells. This target-specific inhibition was associated with mitochondrial apoptosis, as reflected by the increased expression of Bad and caspase-9, the generation of reactive oxygen species (ROS) and the decline in mitochondrial membrane potential. Moreover, preincubation of the cells with glutathione (antioxidant) blocked the process of apoptosis, prevented the phosphorylation of downstream substrates. These results established that ROS acted as a key factor to effect apoptosis by BA treatment in HeLa cells. Therefore, these findings demonstrated that BA induced apoptosis in HeLa cells by downregulating the expression of PI3K/Akt signaling molecules via ROS, and triggering a mitochondrial pathway.

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.

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.