Faculty Profiles - SAKAMOTO Wataru

Papers - SAKAMOTO Wataru

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A Conserved, Mg2+-Dependent Exonuclease Degrades Organelle DNA during Arabidopsis Pollen Development International journal

Matsushima, Ryo, Tang, Lay Yin, Zhang, Lingang, Yamada, Hiroshi, Twell, David, Sakamoto, Wataru

Plant Cell 23 ( 4 ) 1608 - 24 2011

Language：English Publishing type：Research paper (scientific journal)

In plant cells, mitochondria and plastids contain their own genomes derived from the ancestral bacteria endosymbiont. Despite their limited genetic capacity, these multicopy organelle genomes account for a substantial fraction of total cellular DNA, raising the question of whether organelle DNA quantity is controlled spatially or temporally. In this study, we genetically dissected the organelle DNA decrease in pollen, a phenomenon that appears to be common in most angiosperm species. By staining mature pollen grains with fluorescent DNA dye, we screened Arabidopsis thaliana for mutants in which extrachromosomal DNAs had accumulated. Such a recessive mutant, termed defective in pollen organelle DNA degradation1 (dpd1), showing elevated levels of DNAs in both plastids and mitochondria, was isolated and characterized. DPD1 encodes a protein belonging to the exonuclease family, whose homologs appear to be found in angiosperms. Indeed, DPD1 has Mg²⁺-dependent exonuclease activity when expressed as a fusion protein and when assayed in vitro and is highly active in developing pollen. Consistent with the dpd phenotype, DPD1 is dual-targeted to plastids and mitochondria. Therefore, we provide evidence of active organelle DNA degradation in the angiosperm male gametophyte, primarily independent of maternal inheritance; the biological function of organellar DNA degradation in pollen is currently unclear.

DOI： 10.1105/tpc.111.084012

Highly efficient visual selection of transgenic rice plants using green fluorescent protein or anthocyanin synthetic genes

Saika, Hiroaki, Sakamoto, Wataru, Maekawa, Masahiko, Toki, Seiichi

Plant Biotechnology 28 ( 1 ) 2011

Reactive oxygen species derived from impaired quality control of photosystem II are irrelevant to plasma-membrane NADPH oxidases. International journal

Eiko Miura, Yusuke Kato, Wataru Sakamoto

Plant signaling & behavior 5 ( 3 ) 264 - 6 2010.3

Language：English Publishing type：Research paper (scientific journal)

Protein quality control plays an important role in the photosynthetic apparatus because its components receive excess light energy and are susceptible to photooxidative damage. In chloroplasts, photodamage is targeted to the D1 protein of Photosystem II (PSII). The coordinated PSII repair cycle (PSII disassembly, D1 degradation and synthesis, and PSII reassembly) is necessary to mitigate photoinhibition. A thylakoid protease FtsH, which is formed predominantly as a heteromeric complex with two isoforms of FtsH2 and FtsH5 in Arabidopsis, is the major protease involved in PSII repair. A mutant lacking FtsH2 (termed var2) shows compromised D1 degradation. Furthermore, var2 accumulates high levels of chloroplastic reactive oxygen species (cpROS), reflecting photooxidative stress without functional PSII repair. To examine if the cpROS produced in var2 are connected to a ROS signaling pathway mediated by plasma membrane NADPH oxidase (encoded by AtRbohD or AtRbohF), we generated mutants in which either Rboh gene was inactivated under var2 background. Lack of NADPH oxidases had little or no impact on cpROS accumulation. It seems unlikely that cpROS in var2 activate plasma membrane NADPH oxidases to enhance ROS production and the signaling pathway. Mutants that are defective in PSII repair might be valuable for investigating cpROS and their physiological roles.

A Rapid, Direct Observation Method to Isolate Mutants with Defects in Starch Grain Morphology in Rice

Matsushima, Ryo, Maekawa, Masahiko, Fujita, Naoko, Sakamoto, Wataru

Plant and Cell Physiology 51 ( 5 ) 728 - 41 2010

Language：English Publishing type：Research paper (scientific journal)

Starch forms transparent grains, referred to as starch grains (SGs), in amyloplasts. Despite the simple glucose polymer composition of starch, SGs exhibit different morphologies depending on plant species, especially in the endosperm of the Poaceae family. This study reports a novel method for preparing thin sections of endosperm without chemical fixation or resin embedding that allowed us to visualize subcellular SGs clearly. Using this method, we observed the SG morphologies of >5,000 mutagenized rice seeds and were able to isolate mutants in which SGs were morphologically altered. In five mutants, named ssg (substandard starch grain), increased numbers of small SGs (ssg1-ssg3), enlarged SGs (ssg4) and abnormal interior structures of SGs (ssg5) were observed. Amylopectin chain length distribution analysis and identification of the mutated gene suggested a possible allelic relationship between ssg1, ssg2, ssg3 and the previously isolated amylose-extender (ae) mutants, while ssg4 and ssg5 seemed to be novel mutants. Compared with conventional observation methods, the methods developed here are more effective for obtaining fine images of subcellular SGs and are suitable for the observation of a large number of samples.

DOI： 10.1093/pcp/pcq040

NEW INSIGHTS INTO THE TYPES AND FUNCTION OF PROTEASES IN PLASTIDS International journal

Kato, Yusuke, Sakamoto, Wataru, Jeon, KW

International Review of Cell and Molecular Biology, Vol 280 280 185 - 218 2010

Language：English Publishing type：Research paper (scientific journal)

Plastids change their morphology dynamically in response to environmental conditions and developmental status. Related to these plastid dynamics, the quality and quantity controls of proteins are necessary. Therefore, proteases are important as key regulators in almost all processes during the conversion of plastid types and the maintenance of plastid homeostasis. Recent progress in this field has revealed that various proteases and peptidases act on plastids. Results of the studies indicate that the vast majority of plastid proteases are homologous to prokaryotic ones because plastids are thought to originate from endosymbiosis of ancestral cyanobacteria. Moreover, the diversification of subunits of several plastid proteases has been revealed along with new insights into the functions of these homologues. This review provides basic information related to plastid proteases and the current view of their physiological roles in plastid homeostasis and biogenesis.

DOI： 10.1016/S1937-6448(10)80004-8

The FtsH Protease Heterocomplex in Arabidopsis: Dispensability of Type-B Protease Activity for Proper Chloroplast Development International journal

Zhang, Di, Kato, Yusuke, Zhang, Lingang, Fujimoto, Masaru, Tsutsumi, Nobuhiro, Sodmergen, Sakamoto, Wataru

Plant Cell 22 ( 11 ) 3710 - 25 2010

Language：English Publishing type：Research paper (scientific journal)

FtsH is an ATP-dependent metalloprotease present as a hexameric heterocomplex in thylakoid membranes. Encoded in the Arabidopsis thaliana YELLOW VARIEGATED2 (VAR2) locus, FtsH2 is one isoform among major Type A (FtsH1/5) and Type B (FtsH2/8) isomers. Mutants lacking FtsH2 (var2) and FtsH5 (var1) are characterized by a typical leaf-variegated phenotype. The functional importance of the catalytic center (comprised by the zinc binding domain) in FtsH2 was assessed in this study by generating transgenic plants that ectopically expressed FtsH2(488), a proteolytically inactive version of FtsH2. The resulting amino acid substitution inhibited FtsH protease activity in vivo when introduced into Escherichia coli FtsH. By contrast, expression of FtsH2(488) rescued not only leaf variegation in var2 but also seedling lethality in var2 ftsh8, suggesting that the protease activity of Type B isomers is completely dispensable, which implies that the chloroplastic FtsH complex has protease sites in excess and that they act redundantly rather than coordinately. However, expression of FtsH2(488) did not fully rescue leaf variegation in var1 var2 because the overall FtsH levels were reduced under this background. Applying an inducible promoter to our complementation analysis revealed that rescue of leaf variegation indeed depends on the overall amount of FtsH. Our results elucidate protein activity and its amount as important factors for the function of FtsH heterocomplexes that are composed of multiple isoforms in the thylakoid membrane.

DOI： 10.1105/tpc.110.079202

Comparative transcriptome analysis of green/white variegated sectors in Arabidopsis yellow variegated2: responses to oxidative and other stresses in white sectors International journal

Miura, Eiko, Kato, Yusuke, Sakamoto, Wataru

Journal of Experimental Botany 61 ( 9 ) 2433 - 45 2010

Language：English Publishing type：Research paper (scientific journal)

The yellow variegated2 (var2) mutant in Arabidopsis thaliana has been studied as a typical leaf-variegated mutant whose defect results from the lack of FtsH2 metalloprotease in chloroplasts. The var2 green sectors suffer from photo-oxidative stress and accumulate high levels of reactive oxygen species (ROS) because of compromised Photosystem II repair. This study investigated and compared microarray-based expression profiles of green and white sectors of var2 leaves. Results suggest that ROS that accumulate in chloroplasts of var2 green sectors do not cause much significant change in the transcriptional profile related to ROS signalling and scavenging. By contrast, transcriptome in the white sectors apparently differs from those in the green sectors and wild type. Numerous genes related to photosynthesis and chloroplast functions were repressed in the white sectors. Furthermore, many genes related to oxidative stress were up-regulated. Among them, ROS scavenging genes were specifically examined, such as Cu/Zn superoxide dismutase 2 (CSD2), that were apparently up-regulated in white but not in the green sectors. Up-regulation of CSD2 appears to be partly attributable to the lack of a microRNA (miR398) in the white sectors. It was concluded that the white sectors exhibit a response to oxidative and other stresses, including CSD2 up-regulation, which might be commonly found in tissues with abnormal chloroplast differentiation.

DOI： 10.1093/jxb/erq075

Identification and Characterization of High Molecular Weight Complexes Formed by Matrix AAA Proteases and Prohibitins in Mitochondria of Arabidopsis thaliana International journal

Piechota, Janusz, Kolodziejczak, Marta, Juszczak, Ilona, Sakamoto, Wataru, Janska, Hanna

Journal of Biological Chemistry 285 ( 17 ) 12512 - 21 2010

Language：English Publishing type：Research paper (scientific journal)

We identify and characterize two matrix (m)-AAA proteases (AtFtsH3 and AtFtsH10) present in the mitochondria of Arabidopsis thaliana. AtFtsH3 is the predominant protease in leaves of wild type plants. Both proteases assemble with prohibitins (PHBs) into high molecular weight complexes (approximately 2 MDa), similarly to their yeast counterparts. A smaller PHB complex (approximately 1 MDa), without the m-AAA proteases, was also detected. Unlike in yeast, stable prohibitin-independent high molecular weight assemblies of m-AAA proteases could not be identified in A. thaliana. AtFtsH3 and AtFtsH10 form at least two types of m-AAA-PHB complexes in wild type plants. The one type contains PHBs and AtFtsH3, and the second one is composed of PHBs and both AtFtsH3 and AtFtsH10. Complexes composed of PHBs and AtFtsH10 were found in an Arabidopsis mutant lacking AtFtsH3 (ftsh3). Thus, both AtFtsH3 and AtFtsH10 may form hetero- and homo-oligomeric complexes with prohibitins. The increased level of AtFtsH10 observed in ftsh3 suggests that functions of the homo- and hetero-oligomeric complexes containing AtFtsH3 can be at least partially substituted by AtFtsH10 homo-oligomers. The steady-state level of the AtFtsH10 transcripts did not change in ftsh3 compared with wild type plants, but we found that almost twice more of the AtFtsH10 transcripts were associated with polysomes in ftsh3. Based on this result, we assume that the AtFtsH10 protein is synthesized at a higher rate in the ftsh3 mutant. Our results provide the first data on the composition of m-AAA and PHB complexes in plant mitochondria and suggest that the abundance of m-AAA proteases is regulated not only at the transcriptional but also at the translational level.

DOI： 10.1074/jbc.M109.063644

Arrested Differentiation of Proplastids into Chloroplasts in Variegated Leaves Characterized by Plastid Ultrastructure and Nucleoid Morphology

Sakamoto, Wataru, Uno, Yasuyuki, Zhang, Quan, Miura, Eiko, Kato, Yusuke, Sodmergen

Plant and Cell Physiology 50 ( 12 ) 2069 - 83 2009

Language：English Publishing type：Research paper (scientific journal)

Leaf variegation is seen in many ornamental plants and is often caused by a cell-lineage type formation of white sectors lacking functional chloroplasts. A mutant showing such leaf variegation is viable and is therefore suitable for studying chloroplast development. In this study, the formation of white sectors was temporally investigated in the Arabidopsis leaf-variegated mutant var2. Green sectors were found to emerge from white sectors after the formation of the first true leaf. Transmission electron microscopic examination of plastid ultrastructures confirmed that the peripheral zone in the var2 shoot meristem contained proplastids but lacked developing chloroplasts that were normally detected in wild type. These data suggest that chloroplast development proceeds very slowly in var2 variegated leaves. A notable feature in var2 is that the plastids in white sectors contain remarkable globular vacuolated membranes and prolamellar body-like structures. Although defective plastids were hardly observed in shoot meristems, they began to accumulate during early leaf development. Consistent with these observations, large plastid nucleoids detected in white sectors by DNA-specific fluorescent dyes were characteristic of those found in proplastids and were clearly distinguished from those in chloroplasts. These results strongly imply that in white sectors, differentiation of plastids into chloroplasts is arrested at the early stage of thylakoid development. Interestingly, large plastid nucleoids were detected in variegated sectors from species other than Arabidopsis. Thus, plastids in variegated leaves appear to share a common feature and represent a novel plastid type.

DOI： 10.1093/pcp/pcp127

Activation of the heterotrimeric G protein alpha-subunit GPA1 suppresses the ftsh-mediated inhibition of chloroplast development in Arabidopsis International journal

Zhang, Lingang, Wei, Qing, Wu, Wenjuan, Cheng, Yuxiang, Hu, Guangzhen, Hu, Fenhong, Sun, Yi, Zhu, Ying, Sakamoto, Wataru, Huang, Jirong

Plant Journal 58 ( 6 ) 1041 - 53 2009

Language：English Publishing type：Research paper (scientific journal)

Heterotrimeric G protein knock-out mutants have no phenotypic defect in chloroplast development, and the connection between the G protein signaling pathway and chloroplast development has only been inferred from pharmaceutical evidence. Thus, whether G protein signaling plays a role in chloroplast development remains an open question. Here, we present genetic evidence, using the leaf-variegated mutant thylakoid formation 1 (thf1), indicating that inactivation or activation of the endogenous G protein alpha-subunit (GPA1) affects chloroplast development, as does the ectopic expression of the constitutively active Galpha-subunit (cGPA1). Molecular biological and genetic analyses showed that FtsH complexes, which are composed of type-A (FtsH1/FtsH5) and type-B (FtsH2/FtsH8) subunits, are required for cGPA1-promoted chloroplast development in thf1. Furthermore, the ectopic expression of cGPA1 rescues the leaf variegation of ftsh2. Consistent with this finding, microarray analysis shows that ectopic expression of cGPA1 partially corrects mis-regulated gene expression in thf1. This overlooked function of G proteins provides new insight into our understanding of the integrative signaling network, which dynamically regulates chloroplast development and function in response to both intracellular and extracellular signals.

DOI： 10.1111/j.1365-313X.2009.03843.x

The Variegated Mutants Lacking Chloroplastic FtsHs Are Defective in D1 Degradation and Accumulate Reactive Oxygen Species International journal

Kato, Yusuke, Miura, Eiko, Ido, Kunio, Ifuku, Kentaro, Sakamoto, Wataru

Plant Physiology 151 ( 4 ) 1790 - 801 2009

Language：English Publishing type：Research paper (scientific journal)

In the photosynthetic apparatus, a major target of photodamage is the D1 reaction center protein of photosystem II (PSII). Photosynthetic organisms have developed a PSII repair cycle in which photodamaged D1 is selectively degraded. A thylakoid membrane-bound metalloprotease, FtsH, was shown to play a critical role in this process. Here, the effect of FtsHs in D1 degradation was investigated in Arabidopsis (Arabidopsis thaliana) mutants lacking FtsH2 (yellow variegated2 [var2]) or FtsH5 (var1). Because these mutants are characterized by variegated leaves that sometimes complicate biochemical studies, we employed another mutation, fu-gaeri1 (fug1), that suppresses leaf variegation in var1 and var2 to examine D1 degradation. Two-dimensional blue native PAGE showed that var2 has less PSII supercomplex and more PSII intermediate lacking CP43, termed RC47, than the wild type under normal growth light. Moreover, our histochemical and quantitative analyses revealed that chloroplasts in var2 accumulate significant levels of reactive oxygen species, such as superoxide radical and hydrogen peroxide. These results indicate that the lack of FtsH2 leads to impaired D1 degradation at the step of RC47 formation in PSII repair and to photooxidative stress even under nonphotoinhibitory conditions. Our in vivo D1 degradation assays, carried out by nonvariegated var2 fug1 and var1 fug1 leaves, demonstrated that D1 degradation was impaired in different light conditions. Taken together, our results suggest the important role of chloroplastic FtsHs, which was not precisely examined in vivo. Attenuated D1 degradation in the nonvariegated mutants also suggests that leaf variegation seems to be independent of the PSII repair.

DOI： 10.1104/pp.109.146589

Dynamic behavior of organellar DNAs during male gametophyte development in higher plants

Sakamoto, Wataru, Matsushita, Ryo

Genes & Genetic Systems 84 ( 6 ) 487 - 487 2009

Leaf-variegated tobacco plants generated by knocking-down FtsH gene

Kouso, Takayoshi, Kato, Yusuke, Sakamoto, Wataru

Genes & Genetic Systems 84 ( 6 ) 471 - 471 2009

Localization and expression of serine racemase in Arabidopsis thaliana International journal

Sugimoto, Manabu, Sakamoto, Wataru, Fujitani, Yoshiyuki

Amino Acids 36 ( 3 ) 587 - 90 2009

Language：English Publishing type：Research paper (scientific journal)

Arabidopsis plants transformed by promoter of A. thaliana serine racemase fused with beta-glucuronidase (GUS) reporter gene showed strong GUS staining in elongating and developing cells such as tip regions of primary and lateral roots, developing leaves, and shoot meristems. RT-PCR and digital northern hybridization showed that expression of the serine racemase gene was not induced by L- and D-serine, light irradiation, biotic and abiotic stresses.

DOI： 10.1007/s00726-008-0112-z

The lack of mitochondrial AtFtsH4 protease alters Arabidopsis leaf morphology at the late stage of rosette development under short-day photoperiod International journal

Gibala, Marta, Kicia, Marta, Sakamoto, Wataru, Gola, Edyta M., Kubrakiewicz, Janusz, Smakowska, Elwira, Janska, Hanna

Plant Journal 59 ( 5 ) 685 - 99 2009

Language：English Publishing type：Research paper (scientific journal)

AtFtsH4 is one of four inner membrane-bound mitochondrial ATP-dependent metalloproteases in Arabidopsis thaliana, called AAA proteases, whose catalytic site is exposed to the intermembrane space. In the present study, we used a reverse-genetic approach to investigate the physiological role of the AtFtsH4 protease. We found that loss of AtFtsH4 did not significantly affect Arabidopsis growth under optimal conditions (long days); however, severe morphological and developmental abnormalities in late rosette development occurred under short-day conditions. The asymmetric shape and irregular serration of expanding leaf blades were the most striking features of the ftsh4 mutant phenotype. The severe abnormal morphology of the leaf blades was accompanied by ultrastructural changes in mitochondria and chloroplasts. These abnormalities correlated with elevated levels of reactive oxygen species and carbonylated mitochondrial proteins. We found that two classes of molecular chaperones, Hsp70 and prohibitins, were over-expressed in ftsh4 mutants during late vegetative growth under both short- and long-day conditions. Taken together, our data indicate that lack of AtFtsH4 results in impairment of organelle development and Arabidopsis leaf morphology under short-day conditions.

DOI： 10.1111/j.1365-313X.2009.03907.x

Visualization of Plastids in Pollen Grains: Involvement of FtsZ1in Pollen Plastid Division

Tang, Lay Yin, Nagata, Noriko, Matsushima, Ryo, Chen, Yuling, Yoshioka, Yasushi, Sakamoto, Wataru

Plant and Cell Physiology 50 ( 4 ) 904 - 8 2009

Language：English Publishing type：Research paper (scientific journal)

Visualizing organelles in living cells is a powerful method to analyze their intrinsic mechanisms. Easy observation of chlorophyll facilitates the study of the underlying mechanisms in chloroplasts, but not in other plastid types. Here, we constructed a transgenic plant enabling visualization of plastids in pollen grains. Combination of a plastid-targeted fluorescent protein with a pollen-specific promoter allowed us to observe the precise number, size and morphology of plastids in pollen grains of the wild type and the ftsZ1 mutant, whose responsible gene plays a central role in chloroplast division. The transgenic material presented in this work is useful for studying the division mechanism of pollen plastids.

DOI： 10.1093/pcp/pcp042

Protein Quality Control in Chloroplasts: A Current Model of D1 Protein Degradation in the Photosystem II Repair Cycle International journal

Kato, Yusuke, Sakamoto, Wataru

Journal of Biochemistry 146 ( 4 ) 463 - 9 2009

Language：English Publishing type：Research paper (scientific journal)

The chloroplast originated from endosymbiosis of photosynthetic bacteria. Thus, mechanisms essential for chloroplast biogenesis/homeostasis (protein synthesis, import from cytosol, assembly, and degradation) are predominantly governed by prokaryotic systems. Among these, the quality control system is crucial, because light energy constantly damages photosynthetic proteins and excessive light often limits plant growth by irreversibly inactivating the photosynthetic apparatuses. Here, we overview prokaryotic proteases (FtsH and Deg) which are two enzymes that play critical roles in this system. We particularly focus on Photosystem II (PSII) in thylakoid membranes, which is composed of more than 20 subunits. Among the subunits is one of the intrinsic reaction centre proteins (D1) which is considered to be the target of photodamage. Its rapid and specific turnover suggests that photodamaged D1 is degraded by these proteases and replaced with a de novo synthesized one in a system which is termed the PSII repair cycle. We discuss a current model of D1 degradation which is executed by a concerted action of particular FtsH and Deg isoforms.

DOI： 10.1093/jb/mvp073

Plant mitochondrial rhomboid, AtRBL12, has different substrate specificity from its yeast counterpart International journal

Kmiec-Wisniewska, Beata, Krumpe, Katrin, Urantowka, Adam, Sakamoto, Wataru, Pratje, Elke, Janska, Hanna

Plant Molecular Biology 68 ( 1-2 ) 159 - 71 2008

Language：English Publishing type：Research paper (scientific journal)

Rhomboid proteins comprise a class of serine proteases that are conserved in all kingdoms of organisms. They contain six or seven transmembrane helices and control a wide range of cellular functions and developmental processes by intramembrane proteolysis. This paper provides experimental evidence for the existence of rhomboid proteases in plant mitochondria and chloroplasts. Among 15 putative rhomboid-like proteins in Arabidopsis thaliana, we selected five predicted as mitochondrially targeted. For these proteins we performed the GFP transient assay, and identified two homologues, AtRBL11 (At5g25752) and AtRBL12 (At1g18600) to be targeted into plastids and mitochondria, respectively. Phylogenetic analysis reveals that AtRBL12 or AtRBL11 have only one clear orthologue in plant species with completely sequenced genomes. Complementation of the yeast lacking a functional copy of mitochondrial rhomboid with AtRBL12 indicates that this plant protease, in contrast to the human orthologue, does not recognize the yeast substrates, cytochrome c peroxidase (Ccp1) or dynamin-like GTPase (Mgm1). In agreement with this, we did not observe processing of Mgm1 when labeled precursor of this protein was incubated in vitro with Arabidopsis mitochondrial extract. Our results imply that plant mitochondrial rhomboids function in a specific manner and thus differ from their yeast and mammal counterparts.

DOI： 10.1007/s11103-008-9359-8

Influence of chloroplastic photo-oxidative stress on mitochondrial alternative oxidase capacity and respiratory properties: A case study with arabidopsis yellow variegated 2

Yoshida, Keisuke, Watanabe, Chihiro, Kato, Yusuke, Sakamoto, Wataru, Noguchi, Ko

Plant and Cell Physiology 49 ( 4 ) 592 - 603 2008

Language：English Publishing type：Research paper (scientific journal)

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy-wasteful cyanide (CN)-resistant respiration. Although it has been demonstrated that leaf AOX is up-regulated under high-light (HL) conditions, the in vivo mechanism of AOX up-regulation by light is still unknown. In the present study, we examined whether the photo-oxidative stress in the chloroplast modulates mitochondrial respiratory properties, especially the AOX capacity, using Arabidopsis leaf-variegated mutant yellow variegated 2 (var2) and exposing plants to HL. var2 mutants lack FtsH2 metalloprotease required for the repair of damaged PSII. Indeed, var2-1 suffered from photo-oxidative stress even before the HL treatments. While the activities of tricarboxylic acid cycle enzymes and cytochrome c oxidase in var2-1 were almost identical to those in the wild type, the amount of AOX protein and the CN-resistant respiration rate were higher in var2-1. Real-time PCR analysis revealed that HL treatment induced the expression of some energy-dissipating respiratory genes, including AOX1a, NDB2 and UCP5, more strongly in var2-1. Western blotting using var2-1 leaf extracts specific to green or white sectors, containing functional or non-functional photosynthetic apparatus, respectively, revealed that more AOX protein was induced in the green sectors by the HL treatment. These results indicate that photo-oxidative stress by excess light is involved in the regulation of respiratory gene expression and the modulation of respiratory properties, especially the AOX up-regulation.

DOI： 10.1093/pcp/pcn031

Mitochondrial dynamics in plant male gametophyte visualized by fluorescent live imaging

Matsushima, Ryo, Hamamura, Yuki, Higashiyama, Tetsuya, Arimura, Shin-ichi, Sodmergen, Tsutsumi, Nobuhiro, Sakamoto, Wataru

Plant and Cell Physiology 49 ( 7 ) 1074 - 83 2008

Language：English Publishing type：Research paper (scientific journal)

Visualization of organelles in living cells is a powerful method for studying their dynamic behavior. Here we attempted to visualize mitochondria in angiosperm male gametophyte (pollen grain from Arabidopsis thaliana) that are composed of one vegetative cell (VC) and two sperm cells (SCs). Combination of mitochondria-targeted fluorescent proteins with VC- or SC-specific expression allowed us to observe the precise number and dynamic behavior of mitochondria in the respective cell types. Furthermore, live imaging of SC mitochondria during double fertilization confirmed previous observations, demonstrated by electron microscopy in other species, that sperm mitochondria enter into the egg and central cells. We also attempted to visualize mutant mitochondria that were elongated due to a defect in mitochondrial division. This mutant phenotype was indeed detectable in VC mitochondria of a heterozygous F(1) plant, suggesting active mitochondrial division in male gametophyte. Finally, we performed mutant screening and isolated a putative mitochondrial protein transport mutant whose phenotype was detectable only in haploid cells. The transgenic materials presented in this work are useful not only for live imaging but also for studying mitochondrial functions by mutant analysis.

DOI： 10.1093/pcp/pcn084

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