Daisuke Saisho

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.

Barley grain with adhering hulls is controlled by an ERF family transcription factor gene regulating a lipid biosynthesis pathway

In contrast to other cereals, typical barley cultivars have caryopses with adhering hulls at maturity, known as covered (hulled) barley. However, a few barley cultivars are a free-threshing variant called naked (hulless) barley. The covered/naked caryopsis is controlled by a single locus (nud) on chromosome arm 7HL. On the basis of positional cloning, we concluded that an ethylene response factor (ERF) family transcription factor gene controls the covered/naked caryopsis phenotype. This conclusion was validated by (i) fixation of the 17-kb deletion harboring the ERF gene among all 100 naked cultivars studied; (ii) two x-ray-induced nud alleles with a DNA lesion at a different site, each affecting the putative functional motif; and (iii) gene expression strictly localized to the testa. Available results indicate the monophyletic origin of naked barley. The Nud gene has homology to the Arabidopsis WIN1/SHN1 transcription factor gene, whose deduced function is control of a lipid biosynthesis pathway. Staining with a lipophilic dye (Sudan black B) detected a lipid layer on the pericarp epidermis only in covered barley. We infer that, in covered barley, the contact of the caryopsis surface, overlaid with lipids to the inner side of the hull, generates organ adhesion.

A Semidwarf Phenotype of Barley uzu Results from a Nucleotide Substitution in the Gene Encoding a Putative Brassinosteroid Receptor

Brassinosteroids (BRs) play important roles throughout plant growth and development. Despite the importance of clarifying the mechanism of BR-related growth regulation in cereal crops, BR-related cereal mutants have been identified only in rice (Oryza sativa). We previously found that semidwarf barley (Hordeum vulgare) accessions carrying the “uzu” gene, called “uzu” barley in Japan, are non-responding for brassinolide (BL). We then performed chemical and molecular analyses to clarify the mechanisms of uzu dwarfism using isogenic line pairs of uzu gene. The response of the uzu line to BL was significantly lower than that of its corresponding normal line. Measurement of BRs showed that the uzu line accumulates BRs, similar to known BR-insensitive mutants. The marker synteny of rice and barley chromosomes suggests that the uzu gene may be homologous to rice D61, a rice homolog of Arabidopsis BR-insensitive 1 (BRI1), encoding a BR-receptor protein. A barley homolog of BRI1, HvBRI1, was isolated by using degenerate primers. A comparison of HvBRI1 sequences in uzu and normal barley varieties showed that the uzu phenotype is correlated with a single nucleotide substitution. This substitution results in an amino acid change at a highly conserved residue in the kinase domain of the BR-receptor protein. These results may indicate that uzu dwarfism is caused by the missense mutation in HvBRI1. The uzu gene is being introduced into all hull-less barley cultivars in Japan as an effective dwarf gene for practical use, and this is the first report about an agronomically important mutation related to BRs.

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.

Widespread Endogenization of Genome Sequences of Non-Retroviral RNA Viruses into Plant Genomes

Non-retroviral RNA virus sequences (NRVSs) have been found in the chromosomes of vertebrates and fungi, but not plants. Here we report similarly endogenized NRVSs derived from plus-, negative-, and double-stranded RNA viruses in plant chromosomes. These sequences were found by searching public genomic sequence databases, and, importantly, most NRVSs were subsequently detected by direct molecular analyses of plant DNAs. The most widespread NRVSs were related to the coat protein (CP) genes of the family Partitiviridae which have bisegmented dsRNA genomes, and included plant- and fungus-infecting members. The CP of a novel fungal virus (Rosellinia necatrix partitivirus 2, RnPV2) had the greatest sequence similarity to Arabidopsis thaliana ILR2, which is thought to regulate the activities of the phytohormone auxin, indole-3-acetic acid (IAA). Furthermore, partitivirus CP-like sequences much more closely related to plant partitiviruses than to RnPV2 were identified in a wide range of plant species. In addition, the nucleocapsid protein genes of cytorhabdoviruses and varicosaviruses were found in species of over 9 plant families, including Brassicaceae and Solanaceae. A replicase-like sequence of a betaflexivirus was identified in the cucumber genome. The pattern of occurrence of NRVSs and the phylogenetic analyses of NRVSs and related viruses indicate that multiple independent integrations into many plant lineages may have occurred. For example, one of the NRVSs was retained in Ar. thaliana but not in Ar. lyrata or other related Camelina species, whereas another NRVS displayed the reverse pattern. Our study has shown that single- and double-stranded RNA viral sequences are widespread in plant genomes, and shows the potential of genome integrated NRVSs to contribute to resolve unclear phylogenetic relationships of plant species.

Acquisition of aluminium tolerance by modification of a single gene in barley.

Originating from the Fertile Crescent in the Middle East, barley has now been cultivated widely on different soil types including acid soils, where aluminium toxicity is a major limiting factor. Here we show that the adaptation of barley to acid soils is achieved by the modification of a single gene (HvAACT1) encoding a citrate transporter. We find that the primary function of this protein is to release citrate from the root pericycle cells to the xylem to facilitate the translocation of iron from roots to shoots. However, a 1-kb insertion in the upstream of the HvAACT1 coding region occurring only in the Al-tolerant accessions, enhances its expression and alters the location of expression to the root tips. The altered HvAACT1 has an important role in detoxifying aluminium by secreting citrate to the rhizosphere. Thus, the insertion of a 1-kb sequence in the HvAACT1 upstream enables barley to adapt to acidic soils.

Transcript levels of the nuclear-encoded respiratory genes in rice decrease by oxygen deprivation: evidence for involvement of calcium in expression of the alternative oxidase 1a gene

We investigated the effect of oxygen on the expressions of respiratory genes encoded in the nuclear and mitochondrial genomes of rice (Oryza sativa L.). Hypoxic treatment decreased the transcript levels of nuclear‐encoded, but not mitochondrial‐encoded respiratory genes. The effects of ruthenium red (an inhibitor of Ca2+ fluxes from organelles) and/or CaCl2 on plants under hypoxic conditions suggested that Ca2+ is a physiological transducer of a low‐oxygen signaling pathway for expression of the alternative oxidase 1a gene (AOX1a), but not for expressions of genes involved in the cytochrome respiratory pathway, in rice.

ATP synthesis inhibitors as well as respiratory inhibitors increase steady-state level of alternative oxidase mRNA in Arabidopsis thaliana

Summary The oxygen uptake in mitochondria can be uncoupled from ATP synthesis by the product of a single gene, the alternative oxidase ( AOX ) gene, which may be involved in signal transduction from mitochondria to the nucleus. To better understand induction of this gene in higher plants, we investigated the effects of the cytochrome respiratory inhibitors and ATP synthesis inhibitors on the expression of the AOX s of Arabidopsis thaliana . A Northern blot analysis showed the AOX1a transcripts were increased by treatment of antimycin A and myxothiazol, which act on complex III of the respiratory chain, and by NaN 3 , which acts on complex IV. AOX1a mRNA was also strongly induced by oligomycin (F 1 F 0 -ATP synthase inhibitor) and weakly induced by 2,4-DNP (ATP synthesis uncoupler) at the maximum dose. These results indicate that not only inhibition of electron transfer but also inhibition of ATP synthesis in mitochondria induces the transcription of AOX1a

Barley: Emergence as a New Research Material of Crop Science

The completion of genome sequencing, advances in a wide range of analytic technologies, greater research platforms and the emergence of bioinformatics procedures, as well as the development of related resources, have contributed to improvements in the quality of research not only in model species but also in crop plants and livestock. Sustainable agricultural production is an urgent issue in the context of global climate change and food security (Brown and Funk 2008, Turner et al. 2009). In order to address this issue, the integration of a broad spectrum of analytical tools and resources and an understanding of genetic mechanisms for agriculturally important traits is needed (Yamamoto et al. 2009, Mochida and Shinozaki 2010). Several essential features of barley (Hordeum vulgare L.) contribute to the broad utilization of this crop in genetic studies. These features include (i) the crop’s diploid nature with a high degree of inbreeding; (ii) the low chromosome number (2n = 14) with large size; (iii) the ease of cross-breeding; and (iv) the ease of cultivation in a wide range of climatic conditions. Cultivated barley, which ranks fourth among cereals with respect to worldwide production, is also well known as an extensively studied plant species in the field of genetics. Regarding its geographic adaptability, barley is particularly noted for its tolerance to cold, drought, alkalinity and salinity. In recent years, to address its huge genome size ( 5,000 Mb), research resources essential for barley genomic studies have been developed, including a large number of expressed sequence tags (ESTs). These have been widely used for barley genome analyses such as DNA marker generation and the construction of microarrays. Recent innovations in sequencing technology, which allow for working on a massive scale by genotyping thousands of single nucleotide polymorphisms (SNPs) on a genome-wide scale, have enabled us to dissect many genetically and biologically agronomically important complex traits. Advanced mapping populations, including chromosome segment substitution lines (CSSLs), have worked as the engines of genetic dissection of quantitative trait loci (QTLs) (Fukuoka et al. 2010). Resources for barley functional genomics have improved over the last decade, and several high-density genetic maps utilizing various types of molecular markers have been constructed (Close et al. 2009, Schulte et al. 2009). Several CSSLs have also been developed, and these are being used for various QTL discovery and related analyses in barley (e.g. Hori et al. 2005). Furthermore, >370,000 barley germplasms are preserved as ex situ collections in representative genebanks, including Okayama University (http://www .shigen.nig.ac.jp/barley/), and worldwide. In this special issue on barley in Plant and Cell Physiology, we discuss the historic advantages of barley as a genetic research material, as well as crop improvements, and briefly outline recent achievements in the establishment of genomic infrastructure that have enabled us to examine the characteristic traits of barley and/or to compare findings with other plant species such as Arabidopsis, rice, maize and soybean. Sato et al. (pp. 728–737) provided new barley research resources in the form of a doubled haploid population derived from the cross between the malting variety ‘Haruna Nijo’ and the Japanese landrace ‘Akashinriki,’ as well as 35 CSSL introgressions from ‘Akashinriki’ on a ‘Haruna Nijo’ background. Several genes controlling barley flower and inflorescence morphology have been isolated by means of map-based cloning technology, including the genes encoding the six-rowed spike (Komatsuda et al. 2007) and naked caryopsis (Taketa et al. 2008). These morphological characteristics and the nonbrittleness (grain shattering) of barley are well-known characteristic traits of domesticated barley, and a model of the barley domestication process has been developed based on archeological evidence of these characteristics (Zohary and Hopf 2000). Sakuma et al. (pp. 738–749) provides an overview of the disarticulation systems and inflorescence characteristics, along with the genes underlying these traits, occurring in the Triticeae tribe. In spite of its large genome, barley is recognized as a good genomic model of the Triticeae tribe, which includes cultivated wheat (einkorn, durum and bread wheats), rye and their respective wild relatives (Schulte et al. 2009). The evolution of the polyploid wheats is distinctive in that domestication, natural hybridization and allopolyploid speciation have significant impacts on their diversification. In this special issue, Matsuoka (pp. 750–764) outlines the phylogenetic relationship between cultivated wheats and their wild relatives and provides an overview of the recent progress and remaining questions in our

Natural Variation of Barley Vernalization Requirements: Implication of Quantitative Variation of Winter Growth Habit as an Adaptive Trait in East Asia

In many temperate plant species, prolonged cold treatment, known as vernalization, is one of the most critical steps in the transition from the vegetative to the reproductive stage. In contrast to recent advances in understanding the molecular basis of vernalization in Arabidopsis non-vernalization mutants or the spring growth habits of cereal crops such as wheat and barley, natural variations in winter growth habits and their geographic distribution are poorly understood. We analyzed varietal variation and the geographic distribution of the degree of vernalization requirements in germplasms of domesticated barley and wild barley collections. We found a biased geographic distribution of vernalization requirements in domesticated barley: Western regions were strongly associated with a higher degree of spring growth habits, and the extreme winter growth habits were localized to Far Eastern regions including China, Korea and Japan. Both wild accessions and domesticated landraces, the regions of distribution of which overlapped each other, mainly belonged to the moderate class of winter growth habit. As a result of quantitative evaluations performed in this study, we provide evidence that the variation in the degree of winter growth habit in recombinant inbred lines was controlled by quantitative trait loci including three vernalization genes (VRN1, VRN2 and VRN3) that account for 37.9% of the variation in vernalization requirements, with unknown gene(s) explaining the remaining two-thirds of the variation. This evidence implied that the Far Eastern accessions might be a genetically differentiated group derived for an evolutionary reason, resulting in their greater tendency towards a winter growth habit.