Norway spruce deploys tissue-specific responses during acclimation to cold

Climate change in the conifer-dominated boreal forest is expected to lead to warmer but more dynamic winter air temperatures, reducing the depth and duration of snow cover and lowering winter soil temperatures. To gain insight into the mechanisms that have enabled conifers to dominate extreme cold environments, we performed genome-wide RNA-Seq analysis from needles and roots of non-dormant two-year Norway spruce (Picea abies (L.) H. Karst), and contrasted these response to herbaceous model Arabidopsis We show that the main transcriptional response of Norway spruce needles exposed to cold was delayed relative to Arabidopsis, and this delay was associated with slower development of freezing tolerance. Despite this difference in timing, Norway spruce principally utilizes early response transcription factors (TFs) belonging to the same gene families as Arabidopsis, indicating broad evolutionary conservation of cold response networks. In keeping with their different metabolic and developmental states, needles and root of Norway spruce showed contrasting results. Regulatory network analysis identified both conserved TFs with known roles in cold acclimation (e.g. homologs of ICE1, AKS3, and of the NAC and AP2/ERF superfamilies), but also a root-specific bHLH101 homolog, providing functional insights into cold stress response strategies in Norway spruce

Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number

Background: Synechocystis sp. PCC 6803 provides a well-established reference point to cyanobacterial metabolic engineering as part of basic photosynthesis research, as well as in the development of next-generation biotechnological production systems. This study focused on expanding the current knowledge on genomic integration of expression constructs in Synechocystis, targeting a range of novel sites in the chromosome and in the native plasmids, together with established loci used in literature. The key objective was to obtain quantitative information on site-specific expression in reference to replicon copy numbers, which has been speculated but never compared side by side in this host. Results: An optimized sYFP2 expression cassette was successfully integrated in two novel sites in Synechocystis chromosome (slr0944; sll0058) and in all four endogenous megaplasmids (pSYSM/slr5037-slr5038; pSYSX/slr6037; pSYSA/slr7023; pSYSG/slr8030) that have not been previously evaluated for the purpose. Fluorescent analysis of the segregated strains revealed that the expression levels between the megaplasmids and chromosomal constructs were very similar, and reinforced the view that highest expression in Synechocystis can be obtained using RSF1010-derived replicative vectors or the native small plasmid pCA2.4 evaluated in comparison. Parallel replicon copy number analysis by RT-qPCR showed that the expression from the alternative loci is largely determined by the gene dosage in Synechocystis, thereby confirming the dependence formerly proposed based on literature. Conclusions: This study brings together nine different integrative loci in the genome of Synechocystis to demonstrate quantitative differences between target sites in the chromosome, the native plasmids, and a RSF1010-based replicative expression vector. To date, this is the most comprehensive comparison of alternative integrative sites in Synechocystis, and provides the first direct reference between expression efficiency and replicon gene dosage in the context. In the light of existing literature, the findings support the view that the small native plasmids can be notably more difficult to target than the chromosome or the megaplasmids, and that the RSF1010-derived vectors may be surprisingly well maintained under non-selective culture conditions in this cyanobacterial host. Altogether, the work broadens our views on genomic integration and the rational use of different integrative loci versus replicative plasmids, when aiming at expressing heterologous genes in Synechocystis.The research was financially supported by the Academy of Finland Centre of Excellence (#307335), NordForsk Nordic Centre of Excellence (#82845) and Jane and Aatos Erkko Foundation (#4605–26422). The work also received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Action—Innovative Training Network 2017 (#764920), and Fundação para a Ciência e a Tecnologia (CEECIND/00259/2017 to CCP)

Sub-aggregator Business Models for Demand Response

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