Analysis of Nitrogenase Fe Protein Activity in Transplastomic Tobacco

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.

A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation

“…Regarding the functional assessment of Nif proteins expressed in yeast and plants, we have observed that many of the essential Nif components have poor solubility, especially NifH and NifB 22 , 24 , 27 , 47 . This is critical as the structural components (NifH and NifDK) are needed at very high levels during nitrogen fixation.…”

Functional expression of the nitrogenase Fe protein in transgenic rice

“…3 ). To minimize O 2 exposure produced during photosynthesis and maximize the isolation of functional Os NifH Ht , plants were Fe-fertilized and harvested before onset of light at the end of the dark period, following a procedure previously shown to be successful to obtain active NifH Av and NifH Ht from tobacco chloroplasts and mitochondria 15 , 23 . Purified Os NifH Ht was mostly soluble and could be isolated from plant line Ht200 with yields of 0.5 mg kg −1 fresh weight (1-month-old plants) or 0.25 mg kg −1 fresh weight (2-month-old plants).…”

“…The heat treatment precipitated apo-NifH protein lacking [4Fe-4S] clusters, and therefore enriched the holo-protein, increasing the specific activity of the remaining NifH protein. The study suggested that the endogenous [Fe-S] cluster biosynthesis system in plastids was, like that in N. benthamiana mitochondria, insufficient for complete NifH maturation 23 . Similar results were reported by the transient expression of soluble A. vinelandii NifH, NifM, NifU and NifS in N. benthamiana chloroplasts 14 .…”

“…However, active Fe protein was only detected when the plants were incubated in a 10% O 2 atmosphere 13 . The same transplastomic plants were later grown in normal air, and Fe protein activity was detected when leaves were collected at the end of the dark period and when the protein extract was heated 23 . The heat treatment precipitated apo-NifH protein lacking [4Fe-4S] clusters, and therefore enriched the holo-protein, increasing the specific activity of the remaining NifH protein.…”