The structural components of the Azotobacter vinelandii iron-only nitrogenase, AnfDKG, form a protein complex within the plant mitochondrial matrix.

A long-held goal of synthetic biology has been the transfer of a bacterial nitrogen-fixation pathway into plants to reduce the use of chemical fertiliser on crops such as rice, wheat and maize. There are three classes of bacterial nitrogenase, named after their metal requirements, containing either a MoFe-, VFe- or FeFe-cofactor, that converts N2 gas to ammonia. Relative to the Mo-nitrogenase the Fe-nitrogenase is not as efficient for catalysis but has less complex genetic and metallocluster requirements, features that may be preferable for engineering into crops. Here we report the successful targeting of bacterial Fe-nitrogenase proteins, AnfD, AnfK, AnfG and AnfH, to plant mitochondria. When expressed as a single protein AnfD was mostly insoluble in plant mitochondria, but coexpression of AnfD with AnfK improved its solubility. Using affinity-based purification of mitochondrially expressed AnfK or AnfG we were able to demonstrate a strong interaction of AnfD with AnfK and a weaker interaction of AnfG with AnfDK. This work establishes that the structural components of the Fe-nitrogenase can be engineered into plant mitochondria and form a complex, which will be a requirement for function. This report outlines the first use of Fe-nitrogenase proteins within a plant as a preliminary step towards engineering an alternative nitrogenase into crops.

Profiling of epididymal small non-protein-coding RNAs.

BACKGROUND: Our understanding of epididymal physiology and function has been transformed over the three decades in which the International Meeting Series on the Epididymis has been hosted. This transformation has occurred along many fronts, but among the most significant advances has been the unexpected discovery of the diversity of small non-protein-coding RNAs (sRNAs) expressed in the epididymal epithelium and differentially accumulated in the luminal population of spermatozoa. OBJECTIVES: Here we survey recent literature pertaining to profiling the sRNA landscape of the mammalian epididymis with the goal of demonstrating the contribution that these key regulatory elements, and their associated pathways, make to epididymal physiology and sperm maturation. RESULTS AND DISCUSSION: High throughput sequencing strategies have fueled an unprecedented advance in our understanding of RNA biology. In the last decade, such high throughput profiling tools have been increasingly applied to study the mammalian epididymis, presaging the discovery of diverse classes of sRNA expressed along the length of the tract. Among the best studied sRNA classes are the microRNAs (miRNA), a sRNA species shown to act in concert with endocrine signals to fine-tune the segmental patterning of epididymal gene expression. In addition to performing this homeostatic role, epithelial cell-derived sRNAs also selectively accumulate into the epididymosomes and spermatozoa that occupy the duct lumen. This exciting discovery alludes to a novel form of intracellular communication that contributes to the establishment of the sperm epigenome and its modification under conditions of paternal stress. CONCLUSION: Compelling literature has identified sRNAs as a crucial regulatory tier that allows the epididymis to fulfill its combined roles of sperm transport, maturation, and storage. Continued research in this emerging field will contribute to our growing understanding of the etiology of male factor infertility and potentially allow for the future design of rational therapeutic options for these individuals.