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.

BANFF: bending of bilayer membranes by amphiphilic α-helices is necessary for form and function of organelles 1.

By binding to and inserting into the lipid bilayer, amphiphilic α-helices of proteins are involved in the curvature of biological membranes in all organisms. In particular, they are involved in establishing the complex membrane architecture of intracellular organelles like the endoplasmatic reticulum, Golgi apparatus, mitochondria, and chloroplasts. Thus, amphiphilic α-helices are essential for maintenance of cellular metabolism and fitness of organisms. Here we focus on the structure and function of membrane-intrinsic proteins, which are involved in membrane curvature by amphiphilic α-helices, in mitochondria and chloroplasts of the eukaryotic model organisms yeast and Arabidopsis thaliana. Further, we propose a model for transport of fatty acids and lipid compounds across the envelope of chloroplasts in which amphiphilic α-helices might play a role.

Thylakoid Membrane Architecture in Synechocystis Depends on CurT, a Homolog of the Granal CURVATURE THYLAKOID1 Proteins.

Photosynthesis occurs in thylakoids, a highly specialized membrane system. In the cyanobacterium Synechocystis sp PCC 6803 (hereafter Synechocystis 6803), the thylakoids are arranged parallel to the plasma membrane and occasionally converge toward it to form biogenesis centers. The initial steps in PSII assembly are thought to take place in these regions, which contain a membrane subcompartment harboring the early assembly factor PratA and are referred to as PratA-defined membranes (PDMs). Loss of CurT, the Synechocystis 6803 homolog of Arabidopsis thaliana grana-shaping proteins of the CURVATURE THYLAKOID1 family, results in disrupted thylakoid organization and the absence of biogenesis centers. As a consequence, PSII is less efficiently assembled and accumulates to only 50% of wild-type levels. CurT induces membrane curvature in vitro and is distributed all over the thylakoids, with local concentrations at biogenesis centers. There it forms a sophisticated tubular network at the cell periphery, as revealed by live-cell imaging. CurT is part of several high molecular mass complexes, and Blue Native/SDS-PAGE and isoelectric focusing demonstrated that different isoforms associate with PDMs and thylakoids. Moreover, CurT deficiency enhances sensitivity to osmotic stress, adding a level of complexity to CurT function. We propose that CurT is crucial for the differentiation of membrane architecture, including the formation of PSII-related biogenesis centers, in Synechocystis 6803.

Plant-Specific Preprotein and Amino Acid Transporter Proteins Are Required for tRNA Import into Mitochondria.

A variety of eukaryotes, in particular plants, do not contain the required number of tRNAs to support the translation of mitochondria-encoded genes and thus need to import tRNAs from the cytosol. This study identified two Arabidopsis (Arabidopsis thaliana) proteins, Tric1 and Tric2 (for tRNA import component), which on simultaneous inactivation by T-DNA insertion lines displayed a severely delayed and chlorotic growth phenotype and significantly reduced tRNA import capacity into isolated mitochondria. The predicted tRNA-binding domain of Tric1 and Tric2, a sterile-α-motif at the C-terminal end of the protein, was required to restore tRNA uptake ability in mitochondria of complemented plants. The purified predicted tRNA-binding domain binds the T-arm of the tRNA for alanine with conserved lysine residues required for binding. T-DNA inactivation of both Tric proteins further resulted in an increase in the in vitro rate of in organello protein synthesis, which was mediated by a reorganization of the nuclear transcriptome, in particular of genes encoding a variety of proteins required for mitochondrial gene expression at both the transcriptional and translational levels. The characterization of Tric1/2 provides mechanistic insight into the process of tRNA import into mitochondria and supports the theory that the tRNA import pathway resulted from the repurposing of a preexisting protein import apparatus.

The extreme Albino3 (Alb3) C terminus is required for Alb3 stability and function in Arabidopsis thaliana.

MAIN CONCLUSION: The extreme Alb3 C terminus is important for Alb3 stability in a light dependent manner, but is dispensable for LHCP insertion or D1 synthesis. YidC/Oxa1/Alb3 dependent insertion of membrane proteins is evolutionary conserved among bacteria, mitochondria and chloroplasts. Chloroplasts are challenged by the need to coordinate membrane integration of nuclear encoded, post-translationally targeted proteins into the thylakoids as well as of proteins translated on plastid ribosomes. The pathway facilitating post-translational targeting of the light-harvesting chlorophyll a/b binding proteins involves the chloroplast signal recognition particle, cpSRP54 and cpSRP43, as well as its membrane receptor FtsY and the translocase Alb3. Interaction of cpSRP43 with Alb3 is mediated by the positively charged, stromal exposed C terminus of Alb3. In this study, we utilized an Alb3 T-DNA insertion mutant in Arabidopsis thaliana lacking the last 75 amino acids to elucidate the function of this domain (alb3∆C). However, the truncated Alb3 protein (Alb3∆C) proved to be unstable under standard growth conditions, resulting in a reduction of Alb3∆C to 20 % of wild-type levels. In contrast, accumulation of Alb3∆C was comparable to wild type under low light growth conditions. Alb3∆C mutants grown under low light conditions were only slightly paler than wild type, accumulated almost wild-type levels of light harvesting proteins and were not affected in D1 synthesis, therefore showing that the extreme Alb3 C terminus is dispensable for both, co- and post-translational, protein insertion into the thylakoid membrane. However, reduction of Alb3∆C levels as observed under standard growth conditions resulted not only in a severely diminished accumulation of all thylakoid complexes but also in a strong defect in D1 synthesis and membrane insertion.

Initial steps of photosystem II de novo assembly and preloading with manganese take place in biogenesis centers in Synechocystis.

In the cyanobacterium Synechocystis sp PCC 6803, early steps in thylakoid membrane (TM) biogenesis are considered to take place in specialized membrane fractions resembling an interface between the plasma membrane (PM) and TM. This region (the PratA-defined membrane) is defined by the presence of the photosystem II (PSII) assembly factor PratA (for processing-associated TPR protein) and the precursor of the D1 protein (pD1). Here, we show that PratA is a Mn(2+) binding protein that contains a high affinity Mn(2+) binding site (K(d) = 73 µM) and that PratA is required for efficient delivery of Mn(2+) to PSII in vivo, as Mn(2+) transport is retarded in pratA(-). Furthermore, ultrastructural analyses of pratA(-) depict changes in membrane organization in comparison to the wild type, especially a semicircle-shaped structure, which appears to connect PM and TM, is lacking in pratA(-). Immunogold labeling located PratA and pD1 to these distinct regions at the cell periphery. Thus, PratA is necessary for efficient delivery of Mn(2+) to PSII, leading to Mn(2+) preloading of PSII in the periplasm. We propose an extended model for the spatial organization of Mn(2+) transport to PSII, which is suggested to take place concomitantly with early steps of PSII assembly in biogenesis centers at the cell periphery.

The cytosolic kinases STY8, STY17, and STY46 are involved in chloroplast differentiation in Arabidopsis.

In Arabidopsis (Arabidopsis thaliana), transit peptides for chloroplast-destined preproteins can be phosphorylated by the protein kinases STY8, STY17, and STY46. In this study, we have investigated the in vitro properties of these plant-specific kinases. Characterization of the mechanistic functioning of STY8 led to the identification of an essential threonine in the activation segment, which is phosphorylated by an intramolecular mechanism. STY8 is inhibited by specific tyrosine kinase inhibitors, although it lacked the ability to phosphorylate tyrosine residues in vitro. In vivo analysis of sty8, sty17, and sty46 Arabidopsis knockout/knockdown mutants revealed a distinct function of the three kinases in the greening process and in the efficient differentiation of chloroplasts. Mutant plants displayed not only a delayed accumulation of chlorophyll but also a reduction of nucleus-encoded chloroplast proteins and a retarded establishment of photosynthetic capacity during the first 6 h of deetiolation, supporting a role of cytosolic STY kinases in chloroplast differentiation.

Chloroplast differentiation in the growing leaves of Arabidopsis thaliana.

Here, we describe the development of chloroplasts and the buildup of the thylakoid membranes in growing Arabidopsis leaves. Organelles were analyzed from three distinct positions, namely, at the tip, the upper leaf margin, and the base from leaves 1, 3, 5, and 7 of 14-day-old plants. Clear developmental gradients are described within a given leaf and between leaves of different age. Chloroplasts at the tip of every leaf are always the most matured within a given leaf, while already at the upper leaf margin a differentiation gradient can be observed from the edge of the leaf toward the midrib. The data presented here can serve as a standard for a subcellular phenotypic analysis in chloroplast biogenesis mutants.