ENDOGENOUS HAEMOGLOBINS AND HETEROLOGOUS VITREOSCILLA HAEMOGLOBIN IN HYBRID ASPEN, ACTA UNIVERSITATIS OULUENSIS A Scientiae Rerum Naturalium 572
|Kustantaja:||Oulun yliopisto|| |
|Sijainti:||Print Tietotalo|| |
|Tekijät:||JOKIPII-LUKKARI SOILE || |
In plants, there are at least three types of haemoglobins (Hbs): symbiotic, non-symbiotic andtruncated. Symbiotic Hbs are known to participate in nitrogen fixation, but the roles of the lattertwo groups are more obscure. Previous reports have connected both plant non-symbiotic andtruncated Hbs to the scavenging of an important signal molecule, nitric oxide (NO). The aim ofthe present thesis was to study the effects of a bacterial Hb of Vitreoscilla sp. (VHb) on a woodymodel organism, hybrid aspen (Populus tremula x tremuloides), and the role of endogenous hybridaspen Hbs. To store the produced hybrid aspen lines, the suitability of different cryopreservationmethods was also tested.
VHb-expressing hybrid aspens were generated by Agrobacterium-mediated gene transfer. Theeffects of VHb expression were examined in standard greenhouse conditions, under elevated UV-B light as well as during culture with ectomycorrhizal fungi. Both slow cooling and vitrificationmethods were applied in cryostoring samples of the different genetic backgrounds. Hybrid aspennon-symbiotic and truncated Hb genes PttHb1 and PttTrHb, respectively, were also isolated. Thefunction of the genes and corresponding proteins PttHb1 and PttTrHb were studied using non-transgenic and VHb hybrid aspen lines as well as a mutant yeast (Saccharomyces cerevisiae)defective in NO resistance.
VHb expression did not improve the general growth of hybrid aspen but resulted in enhancedstarch accumulation in chloroplasts, pointing to changes in energy metabolism. Of the studiedcryopreservation protocols, the slow cooling of dormant in vivo buds proved to be the mostfeasible way of cryostoring hybrid aspen lines. The culture with the ectomycorrhizal fungus wasshown to increase the expression of both PttHb1 and PttTrHb in the roots of non-transgenic lines.However, the fungi did not up-regulate the hybrid aspen Hb genes in the VHb lines. Therefore, itis hypothesized that endogenous Hbs may contribute to the growth of roots and that VHb maycompensate this function. When expressed alone in the mutant yeast, the recombinant PttHb1 andPttTrHb did not protect cells against the toxicity of NO. Subsequently, a novel mRNA transcriptof the heterotrophic ferredoxin NADP+ oxidoreductase gene PtthFNR was found. The absence ofa plastid presequence in the transcript suggests targeting of the encoded protein into cytosol. Thecoexpression of PttHb1 and cytosolic PtthFNR partially rescued the mutant yeast during NOtreatment, demonstrating for the first time that plant Hb1 with an applicable reductase scavengesNO in vivo at a physiologically relevant rate.
This thesis extends current knowledge about plant Hbs and the effects of VHb on a phenotypeof a tree. It also provides new information about plant ferredoxin reductase genes.