Effects of multiple abiotic stresses on gene expression in Saccharina latissima (Phaeophyceae)

Sandra.Heinrich [ at ] awi.de


Marine macroalgae are globally distributed on rocky coastal shores, from tropical to polar regions. They are important marine coastal primary producers, and of particular importance to the function of many ecosystems. Kelps, brown algae of the order Laminariales, dominate rocky shores of cold-temperate regions. There, they help to structure the biodiversity of coastal ecosystems by forming huge forests, which provide habitats and nurseries for various marine organisms. The distribution of kelps is constrained by abiotic factors like light including UV radiation and temperature. Future global environmental changes could therefore have a potentially significant impact on geographic distribution patterns, vertical zonation, and primary productivity of kelp. The basic physiological and ecophysiological characteristics of kelps are well studied. Several physiological studies have been performed on kelp, primarily on the effects of single abiotic stressors, e.g. UV radiation and temperature. Only a few projects have focused on the interactive effects of multiple stresses. So far, no study is available on the molecular processes underlying physiological acclimation to abiotic stress factors in these important organisms. This thesis represents the first large-scale transcriptomic study of acclimation to abiotic stress in a kelp species, and aims on investigating molecular mechanisms underlying physiological acclimation to multiple abiotic stresses in Saccharina latissima from the Arctic (Spitsbergen). Young sporophytes of Saccharina latissima were exposed in multifactorial experiments to different combinations of photosynthetically active radiation, UV radiation and temperature for durations of 8h, 24h and 2 weeks. In order to observe the degree of photoinhibition in response to different exposure conditions, maximum quantum yield of PS II (Fv/Fm) was measured at the beginning and at the end of the experiments. A cDNA library from RNA sampled under various light and temperature regimes was constructed for subsequent functional genomic studies on the mechanisms and pathways involved in stress acclimation to multiple stressors. Gene expression profiles under abiotic stress were assessed by microarray hybridizations. Thereby two different stress exposure durations, 24hours and 2 weeks, were applied to distinguish molecular mechanisms of short-term versus long-term acclimation to stress. Finally, a comparative approach investigating gene expression profiles in both laboratory and field grown sporophytes was carried out to elucidate interactive effects of UVR, temperature and growth conditions. The established cDNA library consists of 400,503 ESTs, which were assembled to 28,803 contigs. Sequence comparison by BLASTx, Interpro protein-motif annotation, and Gene Ontology (GO) yielded in putative functions or orthology relationships for over 10,000 contigs. Comparative analysis with the genome of E. siliculosus revealed high functional genomic coverage of 70% of the cDNA library. The cDNA library is representative of the S. latissima transcriptome under the tested conditions and displays a rather complete gene catalogue of the species. It therefore constitutes an excellent basis for subsequent functional genomic studies on molecular acclimation to multiple stresses in Saccharina latissima. S. latissima responds to abiotic stress with a multitude of transcriptional changes. Temperature had a smaller influence on metabolic processes than light. Two main temperature effects were observed. On the one hand, induction of genes associated with the glycine, serine and threonine metabolism in response to low temperature, and on the other hand repression of transcripts encoding carbohydrate biosynthetic and catabolic processes at high temperature. General stress responses observed in sporophytes subjected to high PAR include induction of catabolic processes for energy supply, heat shock proteins and antioxidant enzymes. The combination of the stress factors high PAR, UVR and temperature caused interactive effects on photosynthesis and gene expression. Thereby excessive light at 17°C was the most destructive stress condition for S. latissima, resulting in a strong repression of several crucial metabolic processes, e.g. photosynthesis, carbohydrate metabolism and amino acid metabolism. Acclimation to high irradiance at low temperatures includes enhanced induction of glycine, serine and threonine metabolism, potentially as a consequence of a higher demand of glutathione (GSH), a reducing co-factor for several enzymes involved in reactive oxygen species (ROS) detoxification. Reactive oxygen species formation (ROS) displays a central element of abiotic stress response. The observed regulation of various ROS scavenging enzymes in response to temperature, high PAR and UVR stress demonstrates the crucial role of ROS metabolism in acclimation to abiotic stress in S. latissma. Interestingly, gene expression data bear evidence for the existence of compartment specific ROS scavenging mechanisms in S. latissma. Furthermore, sophisticated regulation of Hsps was observed, which is involved in acclimation not only to temperature but also to combined environmental stresses such as high PAR in combination with high temperature. Short- and long-term acclimation to UVR includes enhanced regulation of photosynthetic components, e.g. light harvesting complex proteins, thylakoid proteins and proteins associated with both photosystems. Gene expression analysis showed that photosystem II exhibits a higher susceptibility towards UV radiation than photosystem I. Furthermore, repair of UV damaged PS II reaction centre seems to function by increasing the transcript pool for transcripts associated with PS II. The observed induction of vitamin B6 biosynthesis after all short- and long-term UVR treatments seems to be a crucial component of UVR acclimation in Saccharin latissima. Only short acclimation to UVR caused enhanced regulation of DNA replication and DNA repair. Three different DNA repair processes, photoreactivation, homologous recombination, and nucleotide excision repair were detected, indicating a sophisticated regulation of different DNA repair processes. As no enhanced regulation of DNA metabolism was detected after the 2 weeks UVR exposure experiments, S. latissima seems to be able to acclimate to UVR radiation and to overcome the negative effects of UV radiation on DNA. Finally, comparisons of gene expression profiles in field and cultivated sporophytes were conducted. Large differences in gene expression between cultured and field material were observed. Principal effects of UVR, targeting mostly photosynthesis and DNA, were similar in cultured and field sporophytes, demonstrating laboratory experiments being well suited to investigate basic molecular mechanisms of acclimation to abiotic stresses. The study revealed that field sporophytes exhibit a higher susceptibility to UVR and a higher oxidative stress level at 12°C, whereas cultivated sporophytes in contrast must make stronger efforts to acclimate to UVR at 2°C. These findings are mostly due to the different growth temperatures of between -3°C and 1°C for field sporophytes versus 10°C for cultivated sporophytes. However, the results indicate that cold acclimation of S. latissima from the field caused metabolic alterations to increase stress performance at low temperatures, which concurrently led to higher susceptibility at 12°C. This thesis presents an initial idea on the complexity of molecular acclimation to abiotic stress in Saccharina latissima. The molecular data obtained by this study improve our understanding on stress response in a kelp species and provide a useful platform for future research regarding molecular approaches in kelp. This project furthermore demonstrates the importance of research on interactions of abiotic stresses on both the physiological as well as on the molecular level. The results of the comparative gene expression study in cultured and field sporophytes highlight the influence of growth conditions on molecular acclimation to stress and underscore the importance of conducting experiments with field material, when aiming to predict effects of changing abiotic factors in the field.

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Heinrich, S. (2012): Effects of multiple abiotic stresses on gene expression in Saccharina latissima (Phaeophyceae) , PhD thesis, Universität Bremen.

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