Nobuhiro Tsutsumi

Characterization of the gene family for alternative oxidase from Arabidopsis thaliana

We investigated the copy number of the gene for alternative oxidase (AOX) of Arabidopsis thaliana by amplification by PCR and Southern hybridization. These studies indicated that there are at least four copies of the AOX gene in Arabidopsis. We isolated genomic clones containing individual copies (designated as AOX1a, AOX1b, AOX1c and AOX2) of the AOX genes. Interestingly, two of the AOX genes (AOX1a and AOX1b) were located in tandem in a ca. 5 kb region on one of the chromosomes of Arabidopsis. Comparison between genomic and cDNA sequences of the four AOX genes showed that all AOX genes are divided by three introns and the positions of the introns in AOX1a, AOX1b, AOX1c and AOX2 are the same. We examined whether expression of Arabidopsis AOX genes, like the tobacco AOX1a gene, is enhanced by treatment with antimycin A, an inhibitor of complex III in the mitochondrial respiratory chain. We found that, in young plants, the amount of Arabidopsis AOX1a mRNA was dramatically increased by addition of antimycin A, while the transcription of the other three genes (AOX1b, AOX1c and AOX2) did not respond to antimycin A. Amplification by RT-PCR showed that AOX1a and AOX1c were expressed in all organs examined (flowers and buds, stems, rosette, and roots of 8-week old plants). In contrast, transcripts of AOX1b were detected only in the flowers and buds, and transcripts of AOX2 were detected mainly in stems, rosette and roots. These results suggested that transcriptions of the four genes for alternative oxidase of Arabidopsis are differentially regulated.

A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division

Recently, the FtsZ protein, which is known as a key component in bacterial cell division, was reported to be involved in mitochondrial division in algae. In yeast and animals, however, mitochondrial fission depends on the dynamin-like proteins Dnm1p and Drp1, respectively, whereas in green plants, no potential mitochondrial division genes have been identified. BLAST searches of the nuclear and mitochondrial genome sequences of Arabidopsis thaliana did not find any obvious homologue of the α-proteobacterial-type ftsZ genes. To determine whether mitochondrial division of higher plants depends on a dynamin-like protein, we cloned a cDNA for ADL2b, an Arabidopsis homologue of Dnm1p, and tested its subcellular localization and its dominant-negative effect on mitochondrial division. The fusion protein of green fluorescent protein and ADL2b was observed as punctate structures localized at the tips and at the constriction sites of mitochondria in live plant cells. Cells expressing dominant-negative mutant ADL2b proteins (K56A and T77F) showed a significant fusion, aggregation, and/or tubulation of mitochondria. We propose that mitochondrial division in higher plants is conducted by dynamin-like proteins similar to ADL2b in Arabidopsis. The evolutional points of loss of mitochondrial FtsZ and the functional acquisition of dynamin-like proteins in mitochondrial division are discussed.

Transcript levels of tandem-arranged alternative oxidase genes in rice are increased by low temperature

We identified two genes for alternative oxidase (AOX) from rice. One AOX gene (designated AOX1a) is located approx. 1.9 kb downstream of another AOX gene (designated AOX1b). Comparison of the genomic and cDNA sequences of the two AOX genes showed that the AOX1a gene is interrupted by three introns, as are AOX genes of other plants. On the other hand, two introns are inserted in the AOX1b gene. The predicted AOX1a and AOX1b precursor proteins consist of 332 and 335 amino acid residues, respectively. A genomic Southern hybridization analysis indicated that rice has several AOX genes other than the two tandem-arranged AOX genes. Steady-state mRNA levels of both of the genes for AOX1a and AOX1b were increased under low temperature (4 degrees C). However, no difference in the pattern of induction of transcription between the genes for AOX1a and AOX1b was observed.

Rice Expression Atlas In Reproductive Development

Gene expression throughout the reproductive process in rice (Oryza sativa) beginning with primordia development through pollination/fertilization to zygote formation was analyzed. We analyzed 25 stages/organs of rice reproductive development including early microsporogenesis stages with 57,381 probe sets, and identified around 26,000 expressed probe sets in each stage. Fine dissection of 25 reproductive stages/organs combined with detailed microarray profiling revealed dramatic, coordinated and finely tuned changes in gene expression. A decrease in expressed genes in the pollen maturation process was observed in a similar way with Arabidopsis and maize. An almost equal number of ab initio predicted genes and cloned genes which appeared or disappeared coordinated with developmental stage progression. A large number of organ-/stage-specific genes were identified; notably 2,593 probe sets for developing anther, including 932 probe sets corresponding to ab initio predicted genes. Analysis of cell cycle-related genes revealed that several cyclin-dependent kinases (CDKs), cyclins and components of SCF E3 ubiquitin ligase complexes were expressed specifically in reproductive organs. Cell wall biosynthesis or degradation protein genes and transcription factor genes expressed specifically in reproductive stages were also newly identified. Rice genes homologous to reproduction-related genes in other plants showed expression profiles both consistent and inconsistent with their predicted functions. The rice reproductive expression atlas is likely to be the most extensive and most comprehensive data set available, indispensable for unraveling functions of many specific genes in plant reproductive processes that have not yet been thoroughly analyzed.

A unified nomenclature for Arabidopsis dynamin-related large GTPases based on homology and possible functions

Z. Hong1, S.Y. Bednarek2, E. Blumwald3, I. Hwang4, G. Jurgens5, D. Menzel6, K.W. Osteryoung7, N.V. Raikhel8, K. Shinozaki9, N. Tsutsumi10 and D.P.S. Verma1,∗ 1Department of Molecular Genetics and Plant Biotechnology Center, Ohio State University, Columbus, OH 43210, USA (∗author for correspondence; e-mail verma.1@osu.edu; 2Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706, USA; 3Department of Pomology, University of California, One Shields Ave., Davis, CA 95616, USA; 4Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, 790-784, Korea; 5Lehrstuhl fur Entwicklungsgenetik, Universitat Tubingen, 72076 Tubingen, Germany; 6Zellbiologie der Pflanzen, Botanisches Institut, Universitat Bonn, Kirschallee 1, Bonn, 53115, Germany; 7Department of Plant Biology, 166 Plant Biology Building, Michigan State University, East Lansing, MI 48824, USA; 8Department of Botany and Plant Sciences and Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA; 9Laboratory of Plant Molecular Biology, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba 305-0074, Japan; 10Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Various spatiotemporal expression profiles of anther-expressed genes in rice.

The male gametophyte and tapetum play different roles during anther development although they are differentiated from the same cell lineage, the L2 layer. Until now, it has not been possible to delineate their transcriptomes due to technical difficulties in separating the two cell types. In the present study, we characterized the separated transcriptomes of the rice microspore/pollen and tapetum using laser microdissection (LM)-mediated microarray. Spatiotemporal expression patterns of 28,141 anther-expressed genes were classified into 20 clusters, which contained 3,468 (12.3%) anther-enriched genes. In some clusters, synchronous gene expression in the microspore and tapetum at the same developmental stage was observed as a novel characteristic of the anther transcriptome. Noteworthy expression patterns are discussed in connection with gene ontology (GO) categories and gene annotations, which are related to important biological events in anther development, such as pollen maturation, pollen germination, pollen tube elongation and pollen wall formation.

Identification of genes expressed in maize root cortical cells during lysigenous aerenchyma formation using laser microdissection and microarray analyses

• To adapt to waterlogging in soil, some gramineous plants, such as maize (Zea mays), form lysigenous aerenchyma in the root cortex. Ethylene, which is accumulated during waterlogging, promotes aerenchyma formation. However, the molecular mechanism of aerenchyma formation is not understood. • The aim of this study was to identify aerenchyma formation-associated genes expressed in maize roots as a basis for understanding the molecular mechanism of aerenchyma formation. Maize plants were grown under waterlogged conditions, with or without pretreatment with an ethylene perception inhibitor 1-methylcyclopropene (1-MCP), or under aerobic conditions. Cortical cells were isolated by laser microdissection and their mRNA levels were examined with a microarray. • The microarray analysis revealed 575 genes in the cortical cells, whose expression was either up-regulated or down-regulated under waterlogged conditions and whose induction or repression was suppressed by pretreatment with 1-MCP. • The differentially expressed genes included genes related to the generation or scavenging of reactive oxygen species, Ca(2+) signaling, and cell wall loosening and degradation. The results of this study should lead to a better understanding of the mechanism of root lysigenous aerenchyma formation.

Separated Transcriptomes of Male Gametophyte and Tapetum in Rice: Validity of a Laser Microdissection (LM) Microarray

In flowering plants, the male gametophyte, the pollen, develops in the anther. Complex patterns of gene expression in both the gametophytic and sporophytic tissues of the anther regulate this process. The gene expression profiles of the microspore/pollen and the sporophytic tapetum are of particular interest. In this study, a microarray technique combined with laser microdissection (44K LM-microarray) was developed and used to characterize separately the transcriptomes of the microspore/pollen and tapetum in rice. Expression profiles of 11 known tapetum specific-genes were consistent with previous reports. Based on their spatial and temporal expression patterns, 140 genes which had been previously defined as anther specific were further classified as male gametophyte specific (71 genes, 51%), tapetum-specific (seven genes, 5%) or expressed in both male gametophyte and tapetum (62 genes, 44%). These results indicate that the 44K LM-microarray is a reliable tool to analyze the gene expression profiles of two important cell types in the anther, the microspore/pollen and tapetum.

Arabidopsis dynamin-related proteins DRP2B and DRP1A participate together in clathrin-coated vesicle formation during endocytosis

Endocytosis performs a wide range of functions in animals and plants. Clathrin-coated vesicle (CCV) formation is an initial step of endocytosis, and in animal cells is largely achieved by dynamins. However, little is known of its molecular mechanisms in plant cells. To identify dynamin-related proteins (DRPs) involved in endocytic CCV formation in plant cells, we compared the behaviors of two structurally different Arabidopsis DRPs, DRP2B and DRP1A, with those of the clathrin light chain (CLC), a marker of CCVs, at the plasma membrane by variable incidence angle fluorescent microscopy (VIAFM). DRP2B shares domain organization with animal dynamins whereas DRP1A is plant-specific. We show that green fluorescent protein (GFP)-tagged DRP2B and DRP1A colocalized with CLC tagged with monomeric Kusabira Orange (mKO) in Arabidopsis cultured cells. Time-lapse VIAFM observations suggested that both GFP-DRP2B and GFP-DRP1A appeared and accumulated on the existing mKO-CLC foci and disappeared at the same time as or immediately after the disappearance of mKO-CLC. Moreover, DRP2B and DRP1A colocalized and assembled/disassembled together at the plasma membrane in Arabidopsis cells. A yeast two-hybrid assay showed that DRP2B and DRP1A interacted with each other. An inhibitor of clathrin-mediated endocytosis, tyrphostin A23, disturbed the localization of DRP1A, but had little effect on the localization of DRP2B, indicating that DRP1A and DRP2B have different molecular properties. These results suggest that DRP2B and DRP1A participate together in endocytic CCV formation in Arabidopsis cells despite the difference of their molecular properties.

The rice mitochondrial iron transporter is essential for plant growth

In plants, iron (Fe) is essential for mitochondrial electron transport, heme, and Fe-Sulphur (Fe-S) cluster synthesis; however, plant mitochondrial Fe transporters have not been identified. Here we show, identify and characterize the rice mitochondrial Fe transporter (MIT). Based on a transfer DNA library screen, we identified a rice line showing symptoms of Fe deficiency while accumulating high shoot levels of Fe. Homozygous knockout of MIT in this line resulted in a lethal phenotype. MIT localized to the mitochondria and complemented the growth of Δmrs3Δmrs4 yeast defective in mitochondrial Fe transport. The growth of MIT-knockdown (mit-2) plants was also significantly impaired despite abundant Fe accumulation. Further, the decrease in the activity of the mitochondrial and cytosolic Fe-S enzyme, aconitase, indicated that Fe-S cluster synthesis is affected in mit-2 plants. These results indicate that MIT is a mitochondrial Fe transporter essential for rice growth and development.