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Cone thermogenesis is a widespread phenomenon in cycads and may function to promote volatile emissions that affect pollinator behavior. Given their large population size and intense and durable heat-producing effects, cycads are important organisms for comprehensive studies of plant thermogenesis. However, knowledge of mitochondrial morphology and function in cone thermogenesis is limited. Therefore, we investigated these mitochondrial properties in the thermogenic cycad species Cycas revoluta Male cones generated heat even in cool weather conditions. Female cones produced heat, but to a lesser extent than male cones. Ultrastructural analyses of the two major tissues of male cones, microsporophylls and microsporangia, revealed the existence of a population of mitochondria with a distinct morphology in the microsporophylls. In these cells, we observed large mitochondria (cross-sectional area of 2 μm2 or more) with a uniform matrix density that occupied >10% of the total mitochondrial volume. Despite the size difference, many nonlarge mitochondria (cross-sectional area

The majority of genes encoding photosynthesis-associated proteins in the nucleus are induced by light during photomorphogenesis, allowing plants to establish photoautotrophic growth. Therefore, optimizing the protein import apparatus of plastids, designated as the translocon at the outer and inner envelope membranes of chloroplast (TOC–TIC) complex, upon light exposure is a prerequisite to the import of abundant nuclear-encoded photosynthesis-associated proteins. However, the mechanism that coordinates the optimization of the TOC–TIC complex with the expression of nuclear-encoded photosynthesis-associated genes remains to be characterized in detail. To address this question, we investigated the mechanism by which plastid protein import is regulated by light during photomorphogenesis in Arabidopsis. We found that the albino plastid protein import2 (ppi2) mutant lacking Toc159 protein import receptors have active photoreceptors, even though the mutant fails to induce the expression of photosynthesis-associated nuclear genes upon light illumination. In contrast, many TOC and TIC genes are rapidly induced by blue light in both WT and the ppi2 mutant. We uncovered that this regulation is mediated primarily by cryptochrome 1 (CRY1). Furthermore, deficiency of CRY1 resulted in the decrease of some TOC proteins in vivo. Our results suggest that CRY1 plays key roles in optimizing the content of the TOC–TIC apparatus to accommodate the import of abundant photosynthesis-associated proteins during photomorphogenesis.

Arabidopsis (Arabidopsis thaliana) GOLDEN2-LIKE (GLK) transcription factors promote chloroplast biogenesis by regulating the expression of photosynthesis-related genes. Arabidopsis GLK1 is also known to participate in retrograde signaling from chloroplasts to the nucleus. To elucidate the mechanism by which GLK1 is regulated in response to plastid signals, we biochemically characterized Arabidopsis GLK1 protein. Expression analysis of GLK1 protein indicated that GLK1 accumulates in aerial tissues. Both tissue-specific and Suc-dependent accumulation of GLK1 were regulated primarily at the transcriptional level. In contrast, norflurazon- or lincomycin-treated gun1-101 mutant expressing normal levels of GLK1 mRNA failed to accumulate GLK1 protein, suggesting that plastid signals directly regulate the accumulation of GLK1 protein in a GUN1-independent manner. Treatment of the glk1glk2 mutant expressing functional GFP-GLK1 with a proteasome inhibitor, MG-132, induced the accumulation of polyubiquitinated GFP-GLK1. Furthermore, the level of endogenous GLK1 in plants with damaged plastids was partially restored when those plants were treated with MG-132. Collectively, these data indicate that the ubiquitin-proteasome system participates in the degradation of Arabidopsis GLK1 in response to plastid signals.

To improve the photosynthetic performance of C3 plants, installing cyanobacterial bicarbonate transporters to the chloroplast inner envelope membrane (IEM) has been proposed for years. In our previous study, we successfully introduced chimeric cyanobacterial sodium-dependent bicarbonate transporters, BicA or SbtA, to the chloroplast IEM of Arabidopsis. However, the installation of authentic BicA and SbtA to the chloroplast IEM has not been achieved yet. In this study, we examined whether or not tobacco etch virus (TEV) protease targeted within chloroplasts can cleave chimeric proteins and produce authentic bicarbonate transporters. To this end, we constructed a TEV protease that carried the transit peptide and expressed it with chimeric BicA or SbtA proteins containing a TEV cleavage site in planta. Chimeric proteins were cleaved only when the TEV protease was co-expressed. The authentic forms of hemagglutinin-tagged BicA and SbtA were detected in the chloroplast IEM. In addition, cleavage of chimeric proteins at the TEV recognition site seemed to occur after the targeting of chimeric proteins to the chloroplast IEM. We conclude that the cleavage of chimeric proteins within chloroplasts is an efficient way to install authentic bicarbonate transporters to the chloroplast IEM. Furthermore, a similar approach can be applied to other bacterial plasma membrane proteins.

In this protocol, we describe a method to design chimeric proteins for specific targeting to the inner envelope membrane (IEM) of Arabidopsis chloroplasts and the confirmation of their localization by biochemical analysis. Specific targeting to the chloroplast IEM can be achieved by fusing the protein of interest with a transit peptide and an IEM targeting signal. This protocol makes it possible to investigate the localization of chimeric proteins in chloroplasts using a small number of transgenic plants by using a modified method of chloroplast isolation and fractionation. IEM localization of chimeric proteins can be further assessed by trypsin digestion and alkaline extraction. Here, the localization of the chimeric bicarbonate transporter, designated as SbtAII, is detected by Western blotting using antibodies against Staphylococcal protein A. This protocol is adapted from Uehara et al., 2016 .

Plastids are DNA-containing organelles and can have unique differentiation states depending on age, tissue, and environment. Plastid biogenesis is optimized by bidirectional communication between plastids and the nucleus. Import of nuclear-encoded proteins into plastids serves as anterograde signals and vice versa, plastids themselves send retrograde signals to the nucleus, thereby controlling de novo synthesis of nuclear-encoded plastid proteins. Recently, it has become increasingly evident that the ubiquitin–proteasome system regulates both the import of anterograde plastid proteins and retrograde signaling from plastids to the nucleus. Targets of ubiquitin–proteasome regulation include unimported chloroplast precursor proteins in the cytosol, protein translocation machinery at the chloroplast surface, and transcription factors in the nucleus. This review will focus on the mechanism through which the ubiquitin–proteasome system optimizes plastid biogenesis and plant development through the regulation of nuclear–plastid interactions.

Floral thermogenesis has been found in dozens of primitive seed plants and the reproductive organs in these plants produce heat during anthesis. Thus, characterization of the molecular mechanisms underlying flowering is required to fully understand the role of thermogenesis, but this aspect of thermogenic plant development is largely unknown. In this study, extensive database searches and cloning experiments suggest that thermogenic skunk cabbage (Symplocarpus renifolius), which is a member of the family Araceae, possesses two genes encoding phosphatidyl ethanolamine-binding proteins (PEBP), FLOWERING LOCUS T (SrFT) and MOTHER OF FT AND TFL1 (SrMFT). Functional analyses of SrFT and SrMFT in Arabidopsis indicate that SrFT promotes flowering, whereas SrMFT does not. In S. renifolius, the stage- and tissue-specific expression of SrFT was more evident than that of SrMFT. SrFT was highly expressed in flowers and leaves and was mainly localized in fibrovascular tissues. In addition, microarray analysis revealed that, within floral tissues, SrFT was co-regulated with the genes associated with cellular respiration and mitochondrial function, including ALTERNATIVE OXIDASE gene proposed to play a major role in floral thermogenesis. Taken together, these data suggest that, among the PEBP genes, SrFT plays a role in flowering and floral development in the thermogenic skunk cabbage.

Installation of cyanobacterial bicarbonate transporters to the inner envelope membrane (IEM) of chloroplasts in C3 plants has been thought to improve photosynthetic performance. However, the method to deliver cyanobacterial bicarbonate transporters to the chloroplast IEM remains to be established. In this study, we provide evidence that the cyanobacterial bicarbonate transporters, BicA and SbtA, can be specifically installed into the chloroplast IEM using the chloroplast IEM targeting signal in conjunction with the transit peptide. We fused the transit peptide and the mature portion of Cor413im1, whose targeting mechanism to the IEM has been characterized in detail, to either BicA or SbtA isolated from Synechocystis sp. PCC6803. Among the seven chimeric constructs tested, we confirmed that four chimeric bicarbonate transporters, designated as BicAI, BicAII, SbtAII, and SbtAIII, were expressed in Arabidopsis. Furthermore, these chimeric transporters were specifically targeted to the chloroplast IEM. They were also resistant to alkaline extraction but can be solubilized by Triton X-100, indicating that they are integral membrane proteins in the chloroplast IEM. One of the transporters, BicA, could reside in the chloroplast IEM even after removal of the IEM targeting signal. Taken together, our results indicate that the addition of IEM targeting signal, as well as the transit peptide, to bicarbonate transporters allows us to efficiently target nuclear-encoded chimeric bicarbonate transporters to the chloroplast IEM.

Highlight text This work demonstrated that the targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments including the indispensable transmembrane segment., The inner envelope membrane (IEM) of the chloroplast plays crucial roles in forming an osmotic barrier and controlling metabolite exchange between the organelle and the cytosol. The IEM therefore harbours a number of membrane proteins and requires the import and integration of these nuclear-encoded proteins for its biogenesis. Recent studies have demonstrated that the transmembrane segment of single-spanning IEM proteins plays key roles in determining their IEM localization. However, few studies have focused on the molecular mechanisms by which polytopic membrane proteins are targeted to the IEM. In this study, we investigated the targeting mechanism of polytopic IEM proteins using the protein Cor413im1 as a model substrate. Cor413im1 does not utilize a soluble intermediate for its targeting to the IEM. Furthermore, we show that the putative fifth transmembrane segment of Cor413im1 is necessary for its targeting to the IEM. The C-terminal portion containing this transmembrane segment is also able to deliver Cor413im1 protein to the IEM. However, the fifth transmembrane segment of Cor413im1 itself is insufficient to target a fusion protein to the IEM. These data suggest that the targeting of polytopic membrane proteins to the chloroplast IEM in vivo involves multiple transmembrane segments and that chloroplasts have evolved a unique mechanism for the integration of polytopic proteins to the IEM.