Quantitative proteomics of a B12 -dependent alga grown in coculture with bacteria reveals metabolic tradeoffs required for mutualism

The unicellular green alga Lobomonas rostrata requires an external supply of vitamin B12 (cobalamin) for growth, which it can obtain in stable laboratory cultures from the soil bacterium Mesorhizobium loti in exchange for photosynthate. We investigated changes in protein expression in the alga that allow it to engage in this mutualism. We used quantitative isobaric tagging (iTRAQ) proteomics to determine the L. rostrata proteome grown axenically with B12 supplementation or in coculture with M. loti. Data are available via ProteomeXchange (PXD005046). Using the related Chlamydomonas reinhardtii as a reference genome, 588 algal proteins could be identified. Enzymes of amino acid biosynthesis were higher in coculture than in axenic culture, and this was reflected in increased amounts of total cellular protein and several free amino acids. A number of heat shock proteins were also elevated. Conversely, photosynthetic proteins and those of chloroplast protein synthesis were significantly lower in L. rostrata cells in coculture. These observations were confirmed by measurement of electron transfer rates in cells grown under the two conditions. The results indicate that, despite the stability of the mutualism, L. rostrata experiences stress in coculture with M. loti, and must adjust its metabolism accordingly

Over-expression of a tomato N-acetyl-L-glutamate synthase gene (SlNAGS1) in Arabidopsis thaliana results in high ornithine levels and increased tolerance in salt and drought stresses

A single copy of the N-acetyl-L-glutamate synthase gene (SlNAGS1) has been isolated from tomato. The deduced amino acid sequence consists of 604 amino acids and shows a high level of similarity to the predicted Arabidopsis NAGS1 and NAGS2 proteins. Furthermore, the N-terminus ArgB domain and the C-terminus ArgA domain found in SlNAGS1 are similar to the structural arrangements that have been reported for other predicted NAGS proteins. SlNAGS1 was expressed at high levels in all aerial organs, and at basic levels in seeds, whereas it was not detected at all in roots. SlNAGS1 transcript accumulation was noticed transiently in tomato fruit at the red-fruit stage. In addition, an increase of SlNAGS1 transcripts was detected in mature green tomato fruit within the first hour of exposure to low oxygen concentrations. Transgenic Arabidopsis plants have been generated expressing the SlNAGS1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Three homozygous transgenic lines expressing the transgene (lines 1-7, 3-8, and 6-5) were evaluated further. All three transgenic lines showed a significant accumulation of ornithine in the leaves with line 3-8 exhibiting the highest concentration. The same lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to 250 mM NaCl. Similarly, mature plants of all three transgenic lines displayed a higher tolerance to salt and drought stress compared to WT plants. Under most experimental conditions, transgenic line 3-8 performed best, while the responses obtained from lines 1-7 and 6-5 depended on the applied stimulus. To our knowledge, this is the first plant NAGS gene to be isolated, characterized, and genetically modified