Transcriptome analyses of Arabidopsis thaliana seedlings grown in space: implications for gravity-responsive genes.

The transcriptome of seedlings was analyzed from experiments performed on the International Space Station to study the interacting effects of light and gravity on plant tropisms (project named TROPI-2; Kiss et al. 2012). Seeds of Arabidopsis were germinated in space, and seedlings were then grown in the European Modular Cultivation System for 4 days at ~1g followed by exposure to a range of gravitational accelerations (from microgravity to 1g) and two light treatments (blue light with or without a 1 h pretreatment with red). At the end of the experiments, the cassettes containing the seedlings were frozen in the minus eighty laboratory freezer and returned to Earth on space shuttle mission STS-131. The RNA was extracted from whole seedlings and used for the transcriptome analyses. A comparison of 1g spaceflight samples with 1g ground controls identified 230 genes that were differentially regulated at least twofold, emphasizing the need for "in situ" tissue fixation on a 1g centrifuge as an important control for spaceflight experiments. A further comparison of all spaceflight samples with ground controls identified approximately 280 genes that were differentially regulated at least twofold. Of these genes, several were involved in regulating cell polarity (i.e., auxin, calcium, lipid metabolism), cell-wall development, oxygen status, and cell defense or stress. However, when the transcriptome of the all g-treated spaceflight samples was compared with microgravity samples, only ~130 genes were identified as being differently regulated (P ≤ 0.01). Of this subset, only 27 genes were at least twofold differently regulated between microgravity and 1g space samples and included putative/pseudo/undefined genes (14), transposable elements (5), an expansin (ATEXP24; At1g21240), a cell-wall kinase (WAK3; At1g21240), a laccase-like flavonoid oxidase (TT10; At5g48100), among others.

Experimental evolution of a facultative thermophile from a mesophilic ancestor.

Experimental evolution via continuous culture is a powerful approach to the alteration of complex phenotypes, such as optimal/maximal growth temperatures. The benefit of this approach is that phenotypic selection is tied to growth rate, allowing the production of optimized strains. Herein, we demonstrate the use of a recently described long-term culture apparatus called the Evolugator for the generation of a thermophilic descendant from a mesophilic ancestor (Escherichia coli MG1655). In addition, we used whole-genome sequencing of sequentially isolated strains throughout the thermal adaptation process to characterize the evolutionary history of the resultant genotype, identifying 31 genetic alterations that may contribute to thermotolerance, although some of these mutations may be adaptive for off-target environmental parameters, such as rich medium. We undertook preliminary phenotypic analysis of mutations identified in the glpF and fabA genes. Deletion of glpF in a mesophilic wild-type background conferred significantly improved growth rates in the 43-to-48°C temperature range and altered optimal growth temperature from 37°C to 43°C. In addition, transforming our evolved thermotolerant strain (EVG1064) with a wild-type allele of glpF reduced fitness at high temperatures. On the other hand, the mutation in fabA predictably increased the degree of saturation in membrane lipids, which is a known adaptation to elevated temperature. However, transforming EVG1064 with a wild-type fabA allele had only modest effects on fitness at intermediate temperatures. The Evolugator is fully automated and demonstrates the potential to accelerate the selection for complex traits by experimental evolution and significantly decrease development time for new industrial strains.