Guanosine tetraphosphate (ppGpp) accumulation inhibits chloroplast gene expression and promotes super grana formation in the moss Physcomitrium (Physcomitrella) patens.

The nucleotides guanosine tetraphosphate and pentaphosphate (or (p)ppGpp) are implicated in the regulation of chloroplast function in plants. (p)ppGpp signalling is best understood in the model vascular plant Arabidopsis thaliana in which it acts to regulate plastid gene expression to influence photosynthesis, plant development and immunity. However, little information is known about the conservation or diversity of (p)ppGpp signalling in other land plants. We studied the function of ppGpp in the moss Physcomitrium (previously Physcomitrella) patens using an inducible system for triggering ppGpp accumulation. We used this approach to investigate the effects of ppGpp on chloroplast function, photosynthesis and growth. We demonstrate that ppGpp accumulation causes a dramatic drop in photosynthetic capacity by inhibiting chloroplast gene expression. This was accompanied by the unexpected reorganisation of the thylakoid system into super grana. Surprisingly, these changes did not affect gametophore growth, suggesting that bryophytes and vascular plants may have different tolerances to defects in photosynthesis. Our findings point to the existence of both highly conserved and more specific targets of (p)ppGpp signalling in the land plants that may reflect different growth strategies.

AC-202, a highly effective fluorophore for the visualization of lipid droplets in green algae and diatoms.

BACKGROUND: Lipid-specific live cell dyes are an important tool for the study of algal lipid metabolism, the monitoring of lipid production, and the identification of algal strains with high lipid yields. Nile Red and BODIPY have emerged as the principal dyes for these purposes. However, they suffer from a number of shortcomings including for specificity, penetration, interference from chlorophyll autofluorescence, and incompatibility with widely used genetically encoded reporters in the green and blue regions of the spectrum such as the green fluorescent protein and the red fluorescent protein. RESULTS: We tested a new blue fluorescent dye, AC-202, in both the green algae Chlamydomonas reinhardtii and the pennate diatom Phaeodactylum tricornutum. We show that AC-202 is effective in both algae and that after minimal sample preparation, it can label lipid droplets induced by nitrogen starvation or by inhibitors of the TOR (target of rapamycin) kinase. We found that AC-202 benefits from a low background signal and is therefore more sensitive than BODIPY for semiquantitative in vivo fluorescence measurements. Finally, a co-staining experiment indicated that AC-202 can be used for multicolor imaging in combination with both red and green fluorophores. CONCLUSIONS: AC-202 is an alternative and highly effective fluorophore for algal research that resolves drawbacks encountered with other neutral lipid dyes. AC-202 can be used to rapidly and sensitively visualize lipid droplets, and will contribute to the identification of metabolic and signaling pathways involved in lipid droplet formation, monitoring lipid production, and in the development of screens for algal strains suitable for biofuel production.

Guanosine tetraphosphate modulates salicylic acid signalling and the resistance of Arabidopsis thaliana to Turnip mosaic virus.

Chloroplasts can act as key players in the perception and acclimatization of plants to incoming environmental signals. A growing body of evidence indicates that chloroplasts play a critical role in plant immunity. Chloroplast function can be regulated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp]. In plants, (p)ppGpp levels increase in response to abiotic stress and to plant hormones which are involved in abiotic and biotic stress signalling. In this study, we analysed the transcriptome of Arabidopsis plants that over-accumulate (p)ppGpp, and unexpectedly found a decrease in the levels of a broad range of transcripts for plant defence and immunity. To determine whether (p)ppGpp is involved in the modulation of plant immunity, we analysed the susceptibility of plants with different levels of (p)ppGpp to Turnip mosaic virus (TuMV) carrying a green fluorescent protein (GFP) reporter. We found that (p)ppGpp accumulation was associated with increased susceptibility to TuMV and reduced levels of the defence hormone salicylic acid (SA). In contrast, plants with lower (p)ppGpp levels showed reduced susceptibility to TuMV, and this was associated with the precocious up-regulation of defence-related genes and increased SA content. We have therefore demonstrated a new link between (p)ppGpp metabolism and plant immunity in Arabidopsis.

Cel5I, a SLH-Containing Glycoside Hydrolase: Characterization and Investigation on Its Role in Ruminiclostridium cellulolyticum.

Ruminiclostridium cellulolyticum (Clostridium cellulolyticum) is a mesophilic cellulolytic anaerobic bacterium that produces a multi-enzymatic system composed of cellulosomes and non-cellulosomal enzymes to degrade plant cell wall polysaccharides. We characterized one of the non-cellulosomal enzymes, Cel5I, composed of a Family-5 Glycoside Hydrolase catalytic module (GH5), a tandem of Family-17 and -28 Carbohydrate Binding Modules (CBM), and three S-layer homologous (SLH) modules, where the latter are expected to anchor the protein on the cell surface. Cel5I is the only putative endoglucanase targeting the cell surface as well as the only putative protein in R. cellulolyticum containing CBM17 and/or CBM28 modules. We characterized different recombinant structural variants from Cel5I. We showed that Cel5I has an affinity for insoluble cellulosic substrates through its CBMs, that it is the most active endoglucanase on crystalline cellulose of R. cellulolyticum characterized to date and mostly localized in the cell envelope of R. cellulolyticum. Its role in vivo was analyzed using a R. cellulolyticum cel5I mutant strain. Absence of Cel5I in the cell envelope did not lead to a significant variation of the phenotype compared to the wild type strain. Neither in terms of cell binding to cellulose, nor for its growth on crystalline cellulose, thus indicating that the protein has a rather subtle role in tested conditions. Cel5I might be more important in a natural environment, at low concentration of degradable glucose polymers, where its role might be to generate higher concentration of short cellodextrins close to the cell surface, facilitating their uptake or for signalization purpose.

Imaging type VI secretion-mediated bacterial killing.

In the environment, bacteria compete with each other for nutrient availability or to extend their ecological niche. The type VI secretion system contributes to bacterial competition by the translocation of antibacterial effectors from predators into prey cells. The T6SS assembles a dynamic structure-the sheath-wrapped around a tube constituted of the Hcp protein. It has been proposed that by cycling between extended and contracted conformations the sheath acts as a crossbow to propel the Hcp tube toward the target cell. While the sheath dynamics have been studied in monocultures, the activity of the T6SS has not been recorded in presence of the prey. Here, time-lapse fluorescence microscopy of cocultures demonstrates that prey cells are killed upon contact with predator cells. Additional experiments provide evidence that sheath contraction correlates with nearby cell fading and that prey lysis occurs within minutes after sheath contraction. The results support a model in which T6SS dynamics are responsible for T6SS effectors translocation into recipient cells.