Aquaporin homologues in Norway spruce (Picea abies)

Sammanfattning: The recent discovery of aquaporins - proteins that mediate bidirectional passage of water over membranes - provides a molecular basis for the underlying processes in plant water relations. In plants, aquaporins are found in plasma membranes and in vacuolar membranes (tonoplasts) and are suggested to participate in dynamic operations such as cell elongation, osmotic regulation, and long-distance transport.Focusing on the role of aquaporins or homologous proteins during seed development, the gene expression patterns and the accumulation of their products were investigated in the conifer Norway spruce (Picea abies (L.) Karst), which belongs to a plant group not studied before in this respect.In plant propagation programs, also the germination of seeds or somatic embryos derived from tissue cultures, as well as the early plant establishment are critical steps as regards to water limiting conditions. Involvement of aquaporins or homologues also in these processes was therefor investigated.Using a broad-selective antiserum raised against a plant seed aquaporin, immunoblot analyses indicated the presence of at least two isoforms in seeds during development. One isoform was abundant in mature seeds and another was detected in extracts of young seedlings, particularly in the roots. The protein from mature seeds was purified and partially sequenced. That information was used in primer design for PCR-based methods and resulted in the identification of full-length genes in both seeds (mipfg) and roots (mipr). These two genes showed high homology to the tonoplast sub-class of aquaporins. In addition, in mature seeds, immunolocalization experiments gave a high labelling in the tonoplast of protein storage vesicles (PSVs).Contrary, in the early stages of seeds, before PSV formation, labelling was seen in the plasmamembrane of the jacket cells lining the archegonia in young female gametophytes. In situ hybridization experiments agreed with these results, as strong expression signals were present in the jacket cells as well as in the maturing seeds. In the latter the hybridization signal was found in the entire female gametophyte.A strong signal was also seen in the spongy tissue surrounding the female gametophyte of early ovules. The high signal corresponded well with results from Northern analyses. Similarly a strong hybridization signal was seen in root tips from plants of various ages, including lateral roots and adventitious roots formed on hypocotyl cuttings. The columella cells of the root cap stained intensely, as did guard cells of stomata in needles. A gene transcript identical to mipfg was also identified in somatic embryos of Norway spruce. The in situ hybridization staining patterns in plantlets derived from somatic embryos were similar to the staining signals seen in seedlings grown from seeds.The results indicate important roles of aquaporin function in Norway spruce, in regulating transmembrane water and solute transport and in response to stress reactions.