Expression of the Vitreoscilla Hemoglobin (VHb)-Encoding Gene in Transgenic White Poplar: Plant Growth and Biomass Production, Biochemical Characterization and Cell Survival under Submergence, Oxidative and Nitrosative Stress Conditions

Abstract

Expression of the vhb gene, encoding the hemoglobin protein from Vitreoscilla spp. (VHb), has been shown to increase cell growth and protein synthesis, modify the oxygen-dependent product biosynthesis and the susceptibility to oxidative and nitrosative stresses in several host microrganisms, and to improve plant tolerance to flooding-submergence. A chimeric construct consisting of the CaMV35S promoter fused to the vhb gene and nopaline synthase terminator was transferred into white poplar (Populus alba L.) via Agrobacterium tumefaciens in order to test the generality of these phenomena. The presence of the vhb gene was demonstrated by Southern blot analysis. Accumulation of the vhb transcript and protein was detected in all the selected transgenic poplar lines. In vitro growth bioassays revealed that the vhb gene expression in transgenic poplar plants did not significantly affect their growth pattern. One out of the six selected transgenic lines showed significantly higher values for plant height and stem biomass in greenhouse conditions and exhibited enhancement of root biomass production and stem diameter when compared to the wild-type plants. However, no significant differences in chlorophyll a, b, total carotenoid and protein contents were observed. Two selected transgenic lines were characterized in more detail for tolerance to submergence, oxidative and nitrosative stresses. Under in vitro and in vivo submergence conditions, growth parameters and total protein content of transgenic VHb poplars were similar to those observed in the wild-type plants. In addition, leaf discs from the transgenic plants maintained in standard growth conditions did not reveal increased tolerance to oxidative stress by hydrogen peroxide compared to wild-type plants. In a parallel study, cell suspension cultures obtained from both wild-type and VHb transgenic lines were evaluated for growth and survival in the presence of oxidative and nitrosative stresses. No significant differences were observed between the tested VHb and wild-type poplar lines. Our results show that VHb expression in plants can have erratic effects since the enhancement of plant growth and biomass production and the tolerance to submergence, oxidative and nitrosative stresses are not consistently dependent on the presence of this specific function. Consequently, the genetic manipulation of plant oxygen metabolism must be carefully evaluated and extensive biochemical, molecular and cellular investigations are required to assess the real value of the final products.