Yih-Shan Lo

Structural and functional characterizations of mung bean mitochondrial nucleoids

Mitochondrial nucleoids isolated from mung bean seedlings exhibited a chromatin-like structure associated with a membrane component. A similar structure, which underwent discrete changes during cotyledon development, was identified in situ. Isolated nucleoids consisted of essentially the same phospholipids, including cardiolipin, as whole mitochondria and proteins of inner- and outer-mitochondrial-membrane origin. Actin was consistently found with mitochondrial nucleoids prepared with different detergent concentrations. Formaldehyde cross-linking of cytochalasin B- and proteinase K-treated mitochondria further revealed that actin was associated with DNA in nucleoids. Mitochondrial nucleoids were self-sufficient in directing DNA synthesis in vitro in a pattern mimicking mtDNA synthesis in isolated mitochondria. In pulse-field gel electrophoresis, newly synthesized mtDNA separated into two major components, well-bound and fast-moving forms. Nucleoids DNA synthesis was resistant to aphidicolin but sensitive to N-ethylmaleimide, which indicates that a γ-type DNA polymerase was responsible for this activity. Mitochondrial nucleoids were capable of self-directed RNA transcription in a non-random fashion in vitro. Consistent with and complementary to results from fungi and human cells done mostly in situ, our present work helps to establish the important paradigm that mitochondrial nucleoids in eukaryotes are more than mere mtDNA compaction and segregation entities but are centers of mtDNA maintenance and expression.

Actin in mung bean mitochondria and implications for its function.

This work demonstrates the existence of actin within plant mitochondria, and the import of actin into mitochondria is shown in fluorescent-actin transgenic plants and in wild-type cotyledon mitochondria during germination. The findings suggest that the dynamics of mitochondrial actin can regulate cytochrome C release and, consequently, may initiate programmed cell death of the mung bean cotyledons. Here, a large fraction of plant mitochondrial actin was found to be resistant to protease and high-salt treatments, suggesting it was protected by mitochondrial membranes. A portion of this actin became sensitive to protease or high-salt treatment after removal of the mitochondrial outer membrane, indicating that some actin is located inside the mitochondrial outer membrane. The import of an actin–green fluorescent protein (GFP) fusion protein into the mitochondria in a transgenic plant, actin:GFP, was visualized in living cells and demonstrated by flow cytometry and immunoblot analyses. Polymerized actin was found in mitochondria of actin:GFP plants and in mung bean (Vigna radiata). Notably, actin associated with mitochondria purified from early-developing cotyledons during seed germination was sensitive to high-salt and protease treatments. With cotyledon ageing, mitochondrial actin became more resistant to both treatments. The progressive import of actin into cotyledon mitochondria appeared to occur in concert with the conversion of quiescent mitochondria into active forms during seed germination. The binding of actin to mitochondrial DNA (mtDNA) was demonstrated by liquid chromatography–tandem mass spectrometry analysis. Porin and ADP/ATP carrier proteins were also found in mtDNA-protein complexes. Treatment with an actin depolymerization reagent reduced the mitochondrial membrane potential and triggered the release of cytochrome C. The potential function of mitochondrial actin and a possible actin import pathway are discussed.

The Nucleolar Fibrillarin Protein Is Required for Helper Virus-Independent Long-Distance Trafficking of a Subviral Satellite RNA in Plants

Bamboo mosaic virus satellite RNA can move autonomously in a fibrillarin-dependent manner in the absence of its helper virus in Nicotiana benthamiana. RNA trafficking plays pivotal roles in regulating plant development, gene silencing, and adaptation to environmental stress. Satellite RNAs (satRNAs), parasites of viruses, depend on their helper viruses (HVs) for replication, encapsidation, and efficient spread. However, it remains largely unknown how satRNAs interact with viruses and the cellular machinery to undergo trafficking. Here, we show that the P20 protein of Bamboo mosaic potexvirus satRNA (satBaMV) can functionally complement in trans the systemic trafficking of P20-defective satBaMV in infected Nicotiana benthamiana. The transgene-derived satBaMV, uncoupled from HV replication, was able to move autonomously across a graft union identified by RT-qPCR, RNA gel blot, and in situ RT-PCR analyses. Coimmunoprecipitation experiments revealed that the major nucleolar protein fibrillarin is coprecipitated in the P20 protein complex. Notably, silencing fibrillarin suppressed satBaMV-, but not HV-, phloem-based movement following grafting or coinoculation with HV. Confocal microscopy revealed that the P20 protein colocalized with fibrillarin in the nucleoli and formed punctate structures associated with plasmodesmata. The mobile satBaMV RNA appears to exist as ribonucleoprotein (RNP) complex composed of P20 and fibrillarin, whereas BaMV movement proteins, capsid protein, and BaMV RNA are recruited with HV coinfection. Taken together, our findings provide insight into movement of satBaMV via the fibrillarin-satBaMV-P20 RNP complex in phloem-mediated systemic trafficking.

Characterization of protein synthesis by isolated rice mitochondria.

SummaryBacteria-free mitochondria were isolated from aseptically grown, etiolated and green seedlings of both cytoplasmic male-sterile (WA-type) and male-fertile rice (Oryza sativa L.). Protein synthesis in these isolated mitochondria was characterized by gel electrophoresis/fluorography and by the incorporation of [35S]-methionine into protein. In the presence of cycloheximide, a set of some 25 discrete polypeptides and an electrophoretically unresolved population were synthesized. This pattern of protein synthesis in organello was essentially the same in mitochondria isolated from both male-fertile and malesterile cytoplasms. Our data does not preclude the possibility, however, that the WA-type CMS possesses a tissue-specific and/or a low abundance mitochondrial protein(s), whose synthesis eluded detection under our experimental conditions. The synthesis of the mitochondria-encoded polypeptides by isolated rice mitochondria was inhibited by chloramphenicol and incompletely inhibited by erythromycin. A minor chloramphenicol-insensitive, cycloheximide-sensitive translation activity was found consistently to copurify with the mitochondria. This activity generated a reproducible electrophoretic profile of a poorly resolved, weakly labelled population of polypeptides and of a few conspicuous polypeptides, including a 42 kDa species.

GFP‐Targeted Mitochondria Show Heterogeneity of Size, Morphology, and Dynamics in Transgenic Nicotiana tabacum L. Plants In Vivo

This study reports on the heterogeneity and dynamics of green fluorescent protein (GFP)–tagged mitochondria in transgenic tobacco (Nicotiana tabacum) plants in vivo. We investigated the size, shape, and dynamics of mitochondria in different plant cells using confocal microscopy. The mitochondria exhibit different mobility and shapes according to their location, developmental stages, and physiological conditions. Because mitochondria in the shoot apical meristem cells vary greatly in size—from 100‐nm specks to 2‐μm spherical structures—an alternative mitochondrial propagation pathway other than fission may exist in fast‐growing eudicots. Mitochondria as small as 100 nm may undergo a progressive development in situ. Real‐time analysis demonstrated highly dynamic movement of mitochondria in vivo. We present evidence of fusion and fission of mitochondria in eudicots for the first time. We also found that some mitochondria were interconnected to each other by tubular structures and formed networks in the cell.

Characterization of the Structure and DNA Complexity of Mung Bean Mitochondrial Nucleoids

Electron microscopic images of mitochondrial nucleoids isolated from mung bean seedlings revealed a relatively homogeneous population of particles, each consisting of a chromatin-like structure associated with a membrane component. Association of F-actin with mitochondrial nucleoids was also observed. The mitochondrial nucleoid structure identified in situ showed heterogeneous genomic organization. After pulsed-field gel electrophoresis (PFGE), a large proportion of the mitochondrial nucleoid DNA remained in the well, whereas the rest migrated as a 50–200 kb smear zone. This PFGE migration pattern was not affected by high salt, topoisomerase I or latrunculin B treatments; however, the mobility of a fraction of the fastmoving DNA decreased conspicuously following an in-gel ethidium-enhanced UV-irradiation treatment, suggesting that molecules with intricately compact structures were present in the 50-200 kb region. Approximately 70% of the mitochondrial nucleoid DNA molecules examined via electron microscopy were open circles, supercoils, complex forms, and linear molecules with interspersed sigma-shaped structures and/or loops. Increased sensitivity of mtDNA to DNase I was found after mitochondrial nucleoids were pretreated with high salt. This result indicates that some loosely bound or peripheral DNA binding proteins protected the mtDNA from DNase I degradation.

The presence of actin-like protein filaments in higher plant mitochondria

In this report we demonstrate that a 42 kDa actin-like protein filament may exist in mung bean seedling mitochondria. The results of a fine structure study. immunogold localization. immunoblot, fluorescent microscopy, and flow cytometric analyses all point to the existence of this actin-like protein inside mung bean mitochondria.

ABSENCE OF HEAT-SHOCK PROTEIN SYNTHESIS IN ISOLATED RICE (ORYZA SATIVA L.) MITOCHONDRIA

We investigated whether isolated mitochondria of rice (Oryza sativa L.) respond to elevated temperatures by synthesizing a set of unique polypeptides in organello. Mitochondrial samples were prepared by conventional procedures that included surface sterilization and sucrose-gradient purification. The heat-shock treatment adopted for isolated mitochondria was based on the most effective temperatures to induce in vitro synthesis of heat-shock proteins by cytosolic polysomes isolated from etiolated rice seedlings. Heat-induced unique polypeptides were detected in mitochondrial samples prepared from growth-chamber-grown seedlings. In contrast, no heat-shock protein was detected in mitochondrial samples isolated from aseptically grown rice seedlings. Three different rice varieties, including a cytoplasmic male-sterile line, Zhen Shen 97A, gave the same results. We found that the extent of bacterial contamination in mitochondrial samples could be readily determined by plating. Mitochondrial preparations of growth-chamber-grown rice seedlings were found to contain a significant number of bacteria, whereas very few or no bacteria were detected in mitochondrial samples isolated from aseptically grown seedlings. We conclude that isolated rice mitochondria do not respond to elevated temperatures by synthesizing a unique set of proteins and that bacteria are responsible for the synthesis of heat-shock proteins in contaminated rice mitochondrial samples.

Variation in protein and RNA synthesis activity in isolated mitochondria of the developing rice (Oryza sativa L.) panicle

We have studied variation in mitochondrial protein and RNA synthesis during the development of a specialized rice (Oryza sativa L.) reproductive organ in a bacteria-free environment. Mitochondria were prepared from the maturing panicle during microsporogenesis when meiosis occurred and from etiolated seedlings at two growth stages. We found (1) that there was no discernible qualitative difference among the polypeptides synthesized by these three mitochondrial samples; (2) that the quantity of proteins synthesized by panicle mitochondria was approximately 3 times that of the seedling mitochondria, while the two seedling samples exhibited only a minor quantitative difference; (3) that panicle and seedling mitochondria samples synthesized qualitatively the same RNA but at distinctly different rates and that more RNA products were synthesized by panicle than by seedling mitochondria. These results, taken together, suggest that either the regulation of mitochondrial transcription and translation or the copy number of mitochondrial DNA per mitochondrion change discretely in the developing panicle and consequently that the level of mitochondrial gene expression increases considerably during the development of the reproductive structure in rice.