Two wrongs make a right: heat stress reversion of a male-sterile Brassica napus line.

Male-sterile lines play important roles in plant breeding to obtain hybrid vigour. The male sterility Lembke (MSL) system is a thermosensitive genic male sterility system of Brassica napus and is one of the main systems used in European rapeseed breeding. Interestingly, the MSL system shows high similarity to the 9012AB breeding system from China, including the ability to revert to fertile in high temperature conditions. Here we demonstrate that the MSL system is regulated by the same restorer of fertility gene BnaC9-Tic40 as the 9012AB system, which is related to the translocon at the inner envelope membrane of chloroplasts 40 (TIC40) from Arabidopsis. The male sterility gene of the MSL system was also identified to encode a chloroplast-localized protein which we call BnChimera; this gene shows high sequence similarity to the sterility gene previously described for the 9012AB system. For the first time, a direct protein interaction between BnaC9-Tic40 and the BnChimera could be demonstrated. In addition, we identify the corresponding amino acids that mediate this interaction and suggest how BnaC9-Tic40 acts as the restorer of fertility. Using an RNA-seq approach, the effects of heat treatment on the male fertility restoration of the C545 MSL system line were investigated. These data demonstrate that many pollen developmental pathways are affected by higher temperatures. It is hypothesized that heat stress reverses the male sterility via a combination of slower production of cell wall precursors in plastids and a slower flower development, which ultimately results in fertile pollen. The potential breeding applications of these results are discussed regarding the use of the MSL system in producing thermotolerant fertile plants.

The OXA2a Insertase of Arabidopsis Is Required for Cytochrome c Maturation.

In yeast (Saccharomyces cerevisiae) and human (Homo sapiens) mitochondria, Oxidase assembly protein1 (Oxa1) is the general insertase for protein insertion from the matrix side into the inner membrane while Cytochrome c oxidase assembly protein18 (Cox18/Oxa2) is specifically involved in the topogenesis of the complex IV subunit, Cox2. Arabidopsis (Arabidopsis thaliana) mitochondria contain four OXA homologs: OXA1a, OXA1b, OXA2a, and OXA2b. OXA2a and OXA2b are unique members of the Oxa1 superfamily, in that they possess a tetratricopeptide repeat (TPR) domain at their C termini. Here, we determined the role of OXA2a by studying viable mutant plants generated by partial complementation of homozygous lethal OXA2a transfer-DNA insertional mutants using the developmentally regulated ABSCISIC ACID INSENSITIVE3 (ABI3) promoter. The ABI3p:OXA2a plants displayed growth retardation due to a reduction in the steady-state abundances of both c-type cytochromes, cytochrome c 1 and cytochrome c The observed reduction in the steady-state abundance of complex III could be attributed to cytochrome c 1 being one of its subunits. Expression of a soluble heme lyase from an organism with cytochrome c maturation system III could functionally complement the lack of OXA2a. This implies that OXA2a is required for the system I cytochrome c maturation of Arabidopsis. Due to the interaction of OXA2a with Cytochrome c maturation protein CcmF C-terminal-like protein (CCMFC) in a yeast split-ubiquitin based interaction assay, we propose that OXA2a aids in the membrane insertion of CCMFC, which is presumed to form the heme lyase component of the cytochrome c maturation pathway. In contrast with the crucial role played by the TPR domain of OXA2b, the TPR domain of OXA2a is not essential for its functionality.

Recurrent evolutionary switches of mitochondrial cytochrome c maturation systems in Archaeplastida.

Mitochondrial cytochrome c maturation (CCM) requires heme attachment via distinct pathways termed systems I and III. The mosaic distribution of these systems in Archaeplastida raises questions about the genetic mechanisms and evolutionary forces promoting repeated evolution. Here, we show a recurrent shift from ancestral system I to the eukaryotic-specific holocytochrome c synthase (HCCS) of system III in 11 archaeplastid lineages. Archaeplastid HCCS is sufficient to rescue mutants of yeast system III and Arabidopsis system I. Algal HCCS mutants exhibit impaired growth and respiration, and altered biochemical and metabolic profiles, likely resulting from deficient CCM and reduced cytochrome c-dependent respiratory activity. Our findings demonstrate that archaeplastid HCCS homologs function as system III components in the absence of system I. These results elucidate the evolutionary trajectory and functional divergence of CCM pathways in Archaeplastida, providing insight into the causes, mechanisms, and consequences of repeated cooption of an entire biological pathway.

Assessment of Mitochondrial Protein Topology and Membrane Insertion.

Analyzing the membrane integrity and topology of a mitochondrial protein is essential for truly understanding its function. In this chapter, we demonstrate how to analyze mitochondrial membrane proteins using both an immunological-based assay and an in vivo self-assembling GFP approach. First, immunological approaches to investigate the solubility or membrane association of a protein within mitochondria are described. With this method, we demonstrate how the topology of soluble domains of a membrane-integrated protein can be determined. Using protein-specific antibodies, the localization of the soluble domains of a protein are analyzed by a proteolytic-cleavage approach using proteinase K in mitochondria, outer membrane-ruptured mitochondria, and solubilized mitochondrial membranes. In a second approach, we determine the topology of plant mitochondrial proteins using an in vivo self-assembling GFP localization approach.