Abstract
Integration of prokaryotic nitrogen fixation (nif) genes into the plastid genome for expression of functional nitrogenase components could render plants capable of assimilating atmospheric N2 making their crops less dependent of nitrogen fertilizers. The nitrogenase Fe protein component (NifH) has been used as proxy for expression and targeting of Nif proteins within plant and yeast cells. Here we use tobacco plants with the Azotobacter vinelandii nifH and nifM genes integrated into the plastid genome. NifH and its maturase NifM were constitutively produced in leaves, but not roots, during light and dark periods. Nif protein expression in transplastomic plants was stable throughout development. Chloroplast NifH was soluble, but it only showed in vitro activity when isolated from leaves collected at the end of the dark period. Exposing the plant extracts to elevated temperatures precipitated NifM and apo-NifH protein devoid of [Fe4S4] clusters, dramatically increasing the specific activity of remaining NifH protein. Our data indicate that the chloroplast endogenous [Fe-S] cluster biosynthesis was insufficient for complete NifH maturation, albeit a negative effect on NifH maturation due to excess NifM in the chloroplast cannot be excluded. NifH and NifM constitutive expression in transplastomic plants did not affect any of the following traits: seed size, germination time, germination ratio, seedling growth, emergence of the cotyledon and first leaves, chlorophyll content and plant height throughout development.
Highlights
Under optimal climate and water availability, crop yields correlate directly with chemical fertilizer inputs
Accumulation of the nitrogenase NifH protein polypeptide was analyzed in leaf and root tissues of transplastomic tobacco plants
The protein migrated to native A. vinelandii NifH in SDS-PAGE gels and was almost exclusively detected in the soluble fraction of leaf extracts, with only a faint band being associated to the membrane fraction after centrifugation (Figure 1A)
Summary
Under optimal climate and water availability, crop yields correlate directly with chemical fertilizer inputs. Nitrogen (N) fertilizers are critical to maximize commercial crop yields (Mueller et al, 2012). Conversion of dinitrogen gas (N2) into ammonia (NH3) by the Haber-Bosh process is energetically costly and uses fossil fuels. Once applied in the field, NH3 is converted to nitrate (NO−3 ) by nitrification in the soil. This causes N runoff into ground waters and aquatic systems and is often followed by toxic algal and cyanobacterial blooms. Some of the NO−3 is denitrified into the potent greenhouse gas nitrous oxide (N2O). As the world’s population is expected to reach 9.7 billion by 2050 and as the agricultural yield overall should increase with 60% from 2007 levels to meet coming food demand (Alexandratos and Bruinsma, 2012), new technologies could be important tools to reduce future agricultural impact on the environment
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