Extensive expression regulation and lack of heterologous enzymatic activity of the Class II trehalose metabolism proteins from Arabidopsis thaliana

Trehalose metabolism has profound effects on plant growth and metabolism, but the mechanisms involved are unclear. In Arabidopsis , 21 putative trehalose biosynthesis genes are classified in three subfamilies based on their similarity with yeast TPS1 (encoding a trehalose-6-phosphate synthase, TPS) or TPS2 (encoding a trehalose-6-phosphate phosphatase, TPP). Although TPS1 (Class I) and TPPA and TPPB (Class III) proteins have established TPS and TPP activity, respectively, the function of the Class II proteins (AtTPS5-AtTPS11) remains elusive. A complete set of promoter- β -glucurinidase/green fluorescent protein reporters demonstrates their remarkably differential tissue-specific expression and responsiveness to carbon availability and hormones. Heterologous expression in yeast furthermore suggests that none of the encoded enzymes displays significant TPS or TPP activity, consistent with a regulatory rather than metabolic function for this remarkable class of proteins.     

Carbon-isotopic analysis of microbial cells sorted by flow cytometry

One of the outstanding current problems in both geobiology and environmental microbiology is the quantitative analysis of in situ microbial metabolic activities. Techniques capable of such analysis would have wide application, from quantifying natural rates of biogeochemical cycling to identifying the metabolic activity of uncultured organisms. We describe here a method that represents one step towards that goal, namely the high‐precision measurement of ¹³C in specific populations of microbial cells that are purified by fluorescence‐activated cell sorting (FACS). Sorted cells are concentrated on a Teflon membrane filter, and their ¹³C content is measured by coupling an isotope ratio mass spectrometer (IRMS) with a home‐built spooling wire microcombustion (SWiM) apparatus. The combined instrumentation provides measurements of δ¹³C in whole cells with precision better than 0.2‰ for samples containing as little as 25 ng of carbon. When losses associated with sample handling are taken into account, isotopic analyses require sorting roughly 10⁴ eukaryotic or 10⁷ bacterial cells per sample. Coupled with ¹³C‐labelled substrate additions, this approach has the potential to directly quantify uptake of metabolites in specific populations of sorted cells. The high precision afforded by SWiM‐IRMS also permits useful studies of natural abundance variations in ¹³C. The approach is equally applicable to specific populations of cells sorted from multicellular organisms.