Jijing Luo

As organelles for photosynthesis in green plants, chloroplasts play a vital role in solar energy capture and carbon fixation. The maintenance of normal chloroplast physiological functions is essential for plant growth and development. Low temperature is an adverse environmental stress that affects crop productivity. Low temperature severely affects the growth and development of plants, especially photosynthesis. To date, many studies have reported that chloroplasts are not only just organelles of photosynthesis. Chloroplasts can also perceive chilling stress signals via membranes and photoreceptors, and they maintain their homeostasis and promote photosynthesis by regulating the state of lipid membranes, the abundance of photosynthesis-related proteins, the activity of enzymes, the redox state, and the balance of hormones and by releasing retrograde signals, thus improving plant resistance to low temperatures. This review focused on the potential functions of chloroplasts in fine tuning photosynthesis processes under low-temperature stress by perceiving stress signals, modulating the expression of photosynthesis-related genes, and scavenging excess reactive oxygen species (ROS) in chloroplasts to survive the adverse environment.

Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication.

Ubiquitination modulates nearly all aspects of plant life. Here, we reconstituted the Arabidopsis thaliana ubiquitination cascade in Escherichia coli using a synthetic biology approach. In this system, plant proteins are expressed and then immediately participate in ubiquitination reactions within E. coli cells. Additionally, the purification of individual ubiquitination components prior to setting up the ubiquitination reactions is omitted. To establish the reconstituted system, we co-expressed Arabidopsis ubiquitin (Ub) and ubiquitination substrates with E1, E2 and E3 enzymes in E. coli using the Duet expression vectors. The functionality of the system was evaluated by examining the auto-ubiquitination of a RING (really interesting new gene)-type E3 ligase AIP2 and the ubiquitination of its substrate ABI3. Our results demonstrated the fidelity and specificity of this system. In addition, we applied this system to assess a subset of Arabidopsis E2s in Ub chain formation using E2 conjugation assays. Affinity-tagged Ub allowed efficient purification of Ub conjugates in milligram quantities. Consistent with previous reports, distinct roles of various E2s in Ub chain assembly were also observed in this bacterial system. Therefore, this reconstituted system has multiple advantages, and it can be used to screen for targets of E3 ligases or to study plant ubiquitination in detail.

BACKGROUND: Rice (Oryza sativa L.) is a thermophilic crop vulnerable to chilling stress. However, common wild rice (Oryza rufipogon Griff.) in Guangxi (China) has the ability to tolerate chilling stress. To better understand the molecular mechanisms underlying chilling tolerance in wild rice, iTRAQ-based proteomic analysis was performed to examine CTS-12, a major chilling tolerance QTL derived from common wild rice, mediated chilling and recovery-induced differentially expressed proteins (DEPs) between the chilling-tolerant rice line DC90 and the chilling-sensitive 9311. RESULTS: Comparative analysis identified 206 and 155 DEPs in 9311 and DC90, respectively, in response to the whole period of chilling and recovery. These DEPs were clustered into 6 functional groups in 9311 and 4 in DC90. The majority were enriched in the 'structural constituent of ribosome', 'protein-chromophore linkage', and 'photosynthesis and light harvesting' categories. Short Time-series Expression Miner (STEM) analysis revealed distinct dynamic responses of both chloroplast photosynthetic and ribosomal proteins between 9311 and DC90. CONCLUSION: CTS-12 might mediate the dynamic response of chloroplast photosynthetic and ribosomal proteins in DC90 under chilling (cold acclimation) and recovery (de-acclimation) and thereby enhancing the chilling stress tolerance of this rice line. The identified DEPs and the involvement of CTS-12 in mediating the dynamic response of DC90 at the proteomic level illuminate and deepen the understanding of the mechanisms that underlie chilling stress tolerance in wild rice.