Meeting Reports
(Editorial comment)
Dr. Yasunari Ogihara summarized the meeting report of "the Triticeae Meeting of Japan" held on November 11 and 12 in Kyoto Japan. We circulate the abstracts of oral presentations and the titles of poster presentations as edited by Dr. Ogihara.
The Triticeae Meeting of Japan
Yasunari Ogihara
Laboratory of Genetic Engineering, Graduate School of Agriculture, Kyoto Prefectural University and Kyoto Prefectural Institute of Agricultural Biotechnology, Hangi-cho 1-5, Shimogamo, Kyoto 606-8522, Japan
e-mail: yogihara@kab.seika.kyoto.jp
The Wheat Genetics Symposium of Japan (WGSJ) has been restarted as The Triticeae Meeting of Japan (TMJ). A joint meeting between WGSJ and The Molecular Biology Meeting of Triticeae was held in Tottori University, 2004. In that meeting, it made a decision that two meetings should be united and held annually in several places in Japan. The new meeting covers wide range of fields such as molecular biology, genomics, molecular cytogenetics, physiology, breeding and genecology and evolution.
The first joint meeting named “The Triticeae Meeting of Japan” was held on November 11 and 12, 2005 in Kyoto Prefectural Institute of Agricultural Biotechnology, Seika-cho Kyoto. 94 researchers including students participated to the TMJ meeting (Fig. 1). We had three special lectures, seven oral presentations and 28 poster presentations. The meeting was active and impressive, mainly because many young scientists participated to the meeting and made valuable discussions.
The abstracts and titles are presented below. I hope that many scientists are going to attend to the next meeting and give nice data, even if those are not complete.
Thank you again, indeed.
ABSTRACTS & TITLES
Special Lecture
S1. Regulation of plastid gene transcription in higher plants
Takashi Shiina
Faculty of Human Environment, Kyoto Prefectural University, Shimogamo, Kyoto, 606-8522 Japan
The plastid genome is transcribed by nuclear-encoded (NEP) and plastid-encoded (PEP) RNA polymerases. Interestingly, PEP is responsible for transcription of a subset of plastid genes, including photosystem I and II genes, whereas many housekeeping genes are transcribed by both PEP and NEP. Analogous to the eubacterial enzyme, PEP is a multi-subunit enzyme composed of a catalytic core complex (α2ββ’β”) and a sigma factor that confers transcription specificity. Arabidopsis contains six nuclear-encoded sigma factors (AtSIG1 – AtSIG6), although the core subunits are encode by plastid genome. Recent molecular studies have shown that each plastid sigma factor likely has a specific function. Like eubacterial sigma factors, plastid sigma factors can also be grouped into general factors responsible for transcription of standard PEP-dependent genes and specialized factors involved in the recognition of unique PEP promoters. AtSIG6 likely acts as a general sigma factor and supports early chloroplast development in young cotyledons. By contrast, AtSIG5 is structurally distinct among plastid sigma factors and is probably involved in transcription of photosystem reaction center genes, including psbD, psbA and psaAB. Furthermore, it has been demonstrated that AtSIG2 is responsible for transcription of several tRNA genes. It is presumed that nuclear-encoded multiple sigma factors may play a crucial role in transcriptional regulation of plastid-encoded photosystem and tRNA genes in higher plants.
S2. Current status and future prospect of chloroplast transformation in higher plants
Toru Terachi
Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Motoyama, Kamigamao, Kita-ku, Kyoto 603-8555, JAPAN
Chloroplast transformation is an attractive technology for obtaining the plants with new useful characteristics for human being. Transplastomic plants generally have a number of advantages over conventional nuclear transformants, e.g. 1) strict containment of the transgene due to maternal inheritance of chloroplasts and to the lack of gene flow through pollen, 2) ability to accumulate a large amount of transgene's product in the chloroplast, 3) multigene engineering through the appropriate use of an "operon", 4) absence of gene silencing and position effects on the transgene, and so on.
In our laboratory, being supported by the Foundation for Bio-venture Research Center from the Ministry of Education, Culture, Sports, Science and Technology, Japan, the attempts to obtain useful recombinant plants by chloroplast transformation technology have been done, and several transplastomic tobacco plants have already been produced. Our transplastomic plants belong to either categories A) the plants with improved characteristics, or B) the plants producing pharmaceutical products, or C) the plants which can be used for phytoremediation process. In this meeting, transplantomic tobacco with Arabidopsis fatty acids desaturase gene (fad8) was presented as an example of the plant in the category A. PCR and Southern analyses showed that fad8 gene is integrated into the chloroplast genome as expected, and preliminary data on fatty acid composition indicated that the ratio of triunsaturated-/diunsaturated-fatty acids was slightly increased in the transplastomic tobacco. Another example of the category A is transplastomic tobacco plants with soybean's ferritin gene. PCR and Southern analyses confirmed that the gene is integrated into the chloroplast genome, and quantitative measurement of iron contents showed that the amounts of iron are increased three times in the transplastomic plants compared to the wild-type tobacco. A single successful example in the category B is a transplastomic tobacco with hrd gene encoding thrombin inhibitor hirudin. PCR, Southern, Northern and Western analyses showed the hrd gene is integrated into the chloroplast genome correctly, and mRNA and protein are accumulated in the chloroplast, although biological activity of chloroplast hirudin have yet to be elucidated. Our laboratory presently is involved with the construction of transplastomic plants in the category C. Radish genes involving phytochelatin synthesis, such as glutathione synthetase (gs), γ-glutamylcysteine synthetase (gsh1) and phytochelatin synthase (pcs), are in our hand, and they will be delivered to the tobacco chloroplast genome, to test the possibility of obtaining a hyperaccumlator for heavy metals.
S3. Diversity of Lolium temulentum, an associated weed of wheat and barley, in Malo, Ethiopia
Tohru Tominaga
Graduate School of Agriculture, Kyoto University
Lolium temulentum, darnel, is an associated weed of wheat and barley. In Malo, south-western Ethiopia, where people maintain traditional ways of subsistence by growing various kinds of crops and livestock by conducting sustainable shifting cultivation, man's impacts, especially cereal cultivation, on the diversity of darnel was surveyed. The grains of darnel are either awned or awnless, and the awnless is dominant over the awned. Awned form was found in emmer wheat, and awnless one was generally associated with bread, macaroni and rivet wheat. In Malo, grain cleaning is done by winnowing and subsequent hand removal of contaminants. Emmer wheat has non-free threshing grains, and the other three crops have free threshing grains. Awned darnel's grain morphology is similar to that of emmer wheat grain, and the awnless darnel grain resembles the free threshing grains of bread, macaroni and rivet wheat. Separating awned darnel grains from emmer wheat grains is difficult, as is separating awnless grains from free threshing wheat grains. The free threshing wheat grains contaminated with darnel grains are sown in the emmer wheat field because the boundaries between the two fields are unclear. Crop seed exchange and contamination of crop grains with darnel grains during storage or seeding of crops lead to unintended artificial gene flow of darnel and consequently conserve the genetic diversity of darnel.
Oral Presentation
O1. Various functions of aquaporins in MIP gene family
Maki Katsuhara
Research Institute for Bioresources, Okayama University
Aquaporins are membrane proteins that facilitate water movement across bio-membranes. Aquaporins are suggested to mediate not only water but also other molecules transports. Aquaporin genes are known as Major Intrinsic Protein (MIP) genes.
In barley EST database, putative 24 MIPs (contigs) were identified and 11 genes of plasma membrane type aquaporin (PIPs) were detected by PCR. Expression of these 11 PIPs were investigated under salt (NaCl), osmotic (manitol), heavy metals (CuCl2 and CdCl2), and oxidative (H2O2) stresses. One of them, HvPIP2;1, was most abundant and its protein expression was also analyzed. It was confirmed that HvPIP2;1, encoded water channel activity in X. laevis oocytes injected with HvPIP2;1 cRNA. Transcripts and proteins of HvPIP2;1 were reduced in barely roots under salt stress. Over-expression of HvPIP2;1 increased the shoot/root ratio and raised salt sensitivity in transgenic rice plants, indicating HvPIP2;1 is involved in the cellular mechanism of salt tolerance. Over-expression of the HvPIP2;1 also increased internal CO2 conductance and CO2 assimilation in the leaves of transgenic rice plants, suggesting that HvPIP2;1 permeates CO2 in addition to H2O. Recent reports from other researchers suggested that aquaporins were involved in the flood-induced reduction of root water uptake, chilling-induced decrease of root water permeability, and other many physiological functions in plants.
O2. Why is wheat so unique? - Understanding a mechanism of gluten formation based on a comparison of seed storage proteins among cereals.
Tatsuya M. Ikeda
National Agricultural Research Station for Western Region, 6-12-1 Nishifukatsu, Fukuyama, 721-8514, JAPAN
Wheat seed proteins consist of glutenin and gliadin, which belongs to prolamin superfamily. The prolamin superfamily also includes rice prolamins, rye secalins, maize zeins and barley hordeins. Among these prolamin proteins, only wheat prolamins can form a gluten macropolymer. Based on a sequence comparison, it was clear that glutenin, gliadin, hordein and secalin but rice prolamins contained glutamine-rich tandem repetitive sequences. This structure might be involved in protein elasticity. The number of cysteine residues in these proteins involved in inter- and intra- disulfide bonds was varied even within a same species. Fractionation of seed proteins by aqueous alcohol with DTT (insoluble polymeric proteins) or without DTT (soluble polymeric and monomeric proteins) showed that barley hordeins did not form insoluble polymers, probably due to a very low amount of D-hordein, which shared a similar structure to wheat high-molecular-weight glutenin subunits (HMW-GSs). Since HMW-GSs is known to be important to form a structural framework of gluten polymers, the amount of D-hordein might be critical for hordein polymerization.
O3. Wheat production and target of wheat breeding program in Japan
Kanenori Takata
Department of Crop Breeding, National Agricultural Research Center for Western Region
Japan consume about 5.7million ton of wheat a year. Domestic wheat production is 860 thousand ton. Improvement of Japanese wheat quality has been demanded by food industry. Wheat breeders are trying the improvement of quality and resistance to disease or severe environment. Pre-harvest sprouting (PHS) causes severe damage to wheat quality. Zenkouji-komugi is the most resistant to PHS in Japanese wheat cultivars. Our objective is to raise all cultivars to the resistance. Fusarium head bright (FHB) resistance is the most important breeding target. The disease causes significant economic losses, especially production of toxin. We are trying to introduce the resistant genes from Sumai 3. However the resistance for FHB may be inadequate for actual wheat cultivation.
The amylose content is associated with elasticity of white salt noodle. The wx-B1b is responsible for a good noodle quality. High molecular weight glutenin subunits relate bread making quality. Low molecular weight glutenin are not fully understood the relations of noodle and bread making quality. We are trying to introduce the optimum glutenin subunits compositions for each food products. Grain hardness of Japanese soft wheat cultivars is softer than that of foreign wheat cultivars. The soft grain make small flour particle by milling. Arabinoxylan content of flour is associated with flour yield. We believe that both low arabinxylan content and a large flour particle achieve a good milling property.
O4. Expression profile of gliadin and glutenin gene families in hexaploid wheat by large-scale EST analysis
Kanako Kawaura1, Keiichi Mochida2 and Yasunari Ogihara1
1: Kyoto Prefectural University
2: RIKEN Plant Science Center
For discerning the expression patterns of individual members of storage protein gene families in hexaploid wheat, we analyzed comprehensive ESTs by the bioinformatics method. The genes for α/β-gliadins and low-molecular-weight (LMW)-glutenin subunits (GS) were selected from the EST database. The SNP sites among each member of both genes were traced by the alignment. The combinations of the SNPs allowed us to assign those haplotypes into their homoeologous chromosomes by allele-specific PCR. The phylogenetic analysis showed that both of the genes rapidly diverged after differentiation of three genomes, namely A, B and D. Expression patterns of these genes were estimated based on the frequencies of ESTs. These storage protein genes were expressed only in the seed developing stages. The α/β-gliadin genes revealed at least two distinct expression patterns during the course of seed maturation. While early expressing genes of α/β-gliadin and LMW-GS genes showed similar expression patterns and were preferentially expressed from D genome, late expressing genes of α/β-gliadin were drastically expressed from the A genome. Phylogenetic relationships and their expression patterns were not correlated. These evidences suggest that expressions of the two gene families are independently regulated among multi-gene members, and α/β-gliadin genes should possess novel regulation system(s) in addition to the prolamin box.
O5. Molecular mechanisms of the accumulation process of seed storage proteins in rice and other cereals
Takehiro Masumura1,2
1: Laboratory of Genetic Engineering, Faculty of Agriculture, Kyoto Prefecture University, Shimogamo, Sakyo, Kyoto 606-8522, Japan
2: Kyoto Prefecture Institute of Agricultural Biotechnology, Seika-cho, Kyoto 619-0244, Japan
The endosperm tissues of cereal seeds are the principal storage organs for protein, oil, and carbohydrate. In other words, cereal endosperm cells are extremely important cells that provide basic food for humankind. An understanding of the formation mechanisms of endosperm tissues of cereals is extremely important in stabilizing world crop production. The rice endosperm is an ideal tissue for basic studies of cereal endosperm development, because rice has been selected as a model plant for studying the genome science.
In the ripening period, rice seed stored many kinds of substances, i.e. starch, protein, mineral, and oil. These substances are rapidly biosynthesized mainly in the endosperm cells, leading the formation of the cellular organelles as a specific deposition site for each storage substance. The physiological role of storage proteins is to provide nutrients such as nitrogen and sulfur for germination. Up to 95% of the endosperm protein is deposited in protein granules called protein body (PB). Most of the stored rice protein is glutelin, globulin and prolamin, although these proteins are accumulated in different PB. The type-I protein body (PB-I) is spherical and contains prolamin polypeptide. On the other hand, the type-II protein body (PB-II) is rich in glutelin and globulin. These protein bodies develop in an endosperm cell basically at the same time, and different types of storage proteins are correctly sorted into the proper deposition sites. In this study, I consider the molecular mechanisms of seed storage protein synthesis and accumulation in rice compared with other cereals.
O6. Cytogenetic analysis of wheat-barley hybrids
Shin Taketa
Faculty of Agriculture, Kagawa University
In wheat-barley intergeneric hybridization, many cytogenetically interesting phenomena are observed. In this presentation, I summarize our work on the production and genetic analysis of wheat-barley hybrids. Genetic variation in crossability with wheat was detected among barley germplasms. QTL analysis detected four genes controlling the crossability of barley with wheat. In a certain cross combination with an enhanced crossability, barley chromosome 4H was preferentially eliminated in hybrid plants, while another cross combination showed preferential elimination of barley chromosome 1H. In wheat-barley hybrids, not all barley dominant genes controlling morphological traits are expressed, indicating that complicated genetic interaction is operating between the genomes of wheat and barley. A barley gene on the long arm of chromosome 1H causes sterility in hybrids with wheat (Shw). This gene was mapped both genetically and physically by introducing a series of aberrant barley 1H chromosomes into wheat followed by molecular marker analysis. Finally, homoeologous chromosome pairing between barley chromosome 5H and wheat homoeologues was successfully induced by nullisomy 5B. However, all recombinant chromosomes recovered had a cross-over point in the distal region of the group 5 chromosomes. This may hamper transfer of proximally located barley genes into the wheat genetic background with a minimum amount of barley chromatin.
O7. Chromosomal localization pattern of the heterochromatin protein 1 (HP1) in wheat
Michiaki Okuda
Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University
In eukaryote nuclei, genomic DNA builds up the macrostructure with histones and other non-histone proteins, which is called chromatin. The highly condensed region of the chromatin structure is referred as heterochromatin that functionally involves in nuclear organization, chromosomal segregation, and gene regulation. Most of genes in heterochromatic regions are transcriptionally inactive, and this state of chromatin is epigenetically inherited through mitotic and meiotic cell divisions. Heterochromatin Protein 1 (HP1) is one of the non-histone proteins associated with heterochromatins. Recent studies in yeasts revealed that HP1 plays a key role in formation and maintenance of heterochromatin by intermediating heterochromatin-specifically modified histone H3 (methylated at lysine 9) and other heterochromatin components.
In this study, genes encoding for HP1 homologues were isolated from common wheat Triticum aestivum, and chromosomal locations and expression patterns of the wheat HP1 genes were determined. The indirect fluorescent antibody technique was applied to determine localization patterns of HP1 protein on mitotic chromosomes in wheat and related species. The results showed that the wheat HP1 homologues are encoded by homoeologous genes locating on the group-7 chromosomes and all the genes are expressed constitutively. Wheat HP1 proteins were localized to centromere in diploids and tetraploids, but were distributed to entire length of the chromosomes in hexaploid wheat. The localization patterns of di- or tri-methylated histone H3K9 and other modified histones (monomethylated H3K27, trimethylated histone H4K20, phosphorylated histone H3S10, phosphorylated H3S28 dimethylated H3K4, and acetylated H3K9) did not coincided with those of HP1 in wheat plants at different ploidy levels. In addition, localization of HP1 to chromatin did not change after treament with 5’-azacytidine, an inhibitor of DNA methylation although the localization patterns of some modified histones changed. These findings suggest that localization of HP1 might not be regulated directly by methylation status of either histone H3 at Lysin 9 or DNA.
Poster Presentation
P1. Nishio, Z.1, K. Takata2, T. Tabiki1, M. Ito1, N. Iriki1, T. Ban3 (1. NARCH, 2. NARCW, 3. JIRCAS) The effect of marker assisted selection on Fusarium head blight resistance in winter wheat of Hokkaido.
P2. Tonooka,T., T. Yoshioka (Natl. Inst. Cro. Sci, NARO) Difference of discoloration of boiled grain among proanthocyanidin-free barley NILs.
P3. Konishi, S., T. Sasanuma, T. Sasakuma (Kihara Inst. Biol. Res., Yokohama City U.) Organ-specific expression of Mlo gene family in Triticum aestivum.
P4. Terasawa, Y.1, T. Sasanuma1, T. Kawahara2, T. Sasakuma1 (1. Kihara Inst. Biol. Res., Yokohama City U., 2. Grad.Sch. Agr., Kyoto U.) Diversity of the HMW-glutenin subunits of wheat landraces in Afghanistan, Iran and Pakistan collected in 1955, 1965 and 1979.
P5. Tamura, T., T. Sasanuma, T. Sasakuma (Kihara Inst. Biol. Res., Yokohama City U.) Isolation of vrn1 and 2 homologs in Aegilops umbellulata.
P6. Yamagiwa, H.1, T. Sasanuma1, T. Kawahara2, T. Sasakuma1 (1. Kihara Inst. Biol. Res., Yokohama City U., 2. Grad.Sch. Agr., Kyoto U.) Multiple origins of U genome in US genome tetraploid Aegilops species.
P7. Orihara, K.1,2, T. Sasanuma1, T. Sasakuma1 (1. Kihara Inst. Biol. Res., Yokohama City U., 2. Kanagawa Pref. Livst. Ind. Tech. Ctr.) Breeding for a line with low nitrate concentration of Italian ryegrass (Lolium multiflorum Lam.) by marker assist selection.
P8. Ikari, C., N. Shitsukawa, S. Shimada, K. Murai (Dep. Biosci., Fukui Pref. U.) Einkorn wheat mutant, mvp, which shows maintained vegetative phase is caused by mutation of wheat APETALA1.
P9. Shitsukawa, N.1, A. Takagishi1, C. Tahira1, S. Takumi2, K. Murai1 (1. Dep. Biosci., Fukui Pref. U., 2. Fac. Ari., Kobe U.) The molecular basis of wheat spike formation.
P10. Saraike, T., K. Murai (Dep. Biosci., Fukui Pref. U.) Identification of a protein kinase gene preferentially expressed in young spikes of the alloplasmic wheat lines with pistillody.
P11. Shimada, S., C. Ikari, T. Kitagawa, K. Murai (Dep. Biosci., Fukui Pref. U.) Analysis of flowering genes involved in photoperiod pathway in wheat.
P12. Takagishi, A., N. Shitsukawa, K. Murai (Dep. Biosci., Fukui Pref. U.) WFL, a wheat ortholog of FLORICAULA/LEAFY, is associated with the spikelet formation
P13. Zhu, Y., T. Saraike, K. Murai (Dep. Biosci., Fukui Pref. U.) Identification of mitochondrial gene associated with cytoplasmic homeosis in wheat
P14. Ohta, S.1, N. Mori2, H. Ozkan3 (1. Dep. Biosci., Fukui Pref. U., 2. Fac. Agr., Kobe U., 3. Fac. Agr., Cukurova U., Turkey) Geographical distribution of Aegilops neglecta and Ae. columnaris in southern Turkey revealed by the international cooperative field researches from 2003 to 2005.
P15. Iwasaki, R., S. Ohta (Dep. Biosci., Fukui Pref. U.) Intraspecific hybrid sterility observed in Aegilops umbellulata Zhuk.
P16. Yamane, K.1,2, T. Kawahara1 (1. Grad. Sch. of Life. and Envir. Sci., Osaka Pref. U., 2. Grad. Sch. Agr., Kyoto U.) Intra- and interspecific phylogenetic relationships among diploid Triticum-Aegilops species (Poaceae) based on base-pair substitutions, indels, and microsatellites in chloroplast noncoding sequences.
P17. Fukumoto, Y., T. Nakazaki, Y. Okumoto, T. Tanisaka (Grad. Sch. Agr., Kyoto U) Effect of a novel HMW (high-molecular-weight) glutenin subunit on the gluten elasticity in wheat.
P18. Murota, Y., T. Nakazaki, Y. Okumoto, T. Tanisaka (Grad. Sch. Agr.,Kyoto U) Search for LMW (low-molecular-weight) glutenin subunits responsible for the gluten viscoelasticity and Chinese-noodle-making quality.
P19. Ashida, Y., M. Okuda, S. Nasuda, T. R. Endo (Grad. Sch. Agric., Univ. Kyoto) Analysis of gene encoding Shugoshin homolog in common wheat.
P20. Takumi, S., C. Egawa, S. Kume, F. Kobayashi (Fac. Ari., Kobe U.) Trans-activation of Cor/Lea gene expression via wheat CBF/DREB transcription factors.
P21. Kobayashi, F., S. Takumi (Fac. Ari., Kobe U.) Altered gene expression patterns in an ABA less sensitive mutant EH47-1 of common wheat.
P22. Mizuno, N., S. Takumi (Fac. Ari., Kobe U.) Differential expression levels of wheat AOX genes between two cultivars showing distinct freezing tolerance abilities.
P23. Terashima, A., S. Takumi (Fac. Ari., Kobe U.) Genomic structure of a WDREB2 transcription factor gene in Aegilops tauschii.
P24. Ohnishi, N., E. Himi, K. Noda (Res. Inst. Bioresources, Okayama U.) Isolation and expression of ABI5 (ABA insensitive five)-like gene in wheat.
P25. Awayama, T.1, S. Amano1, D. Saisho2, K. Sato2, K. Takeda2, S. Kawasaki3, S. Taketa1 (1. Fac. Agr., Kagawa U., 2. Res. Inst. Bioresour., Okayama U., 3. NIAS) Physical mapping of the nud (naked caryopsis) region in barley.
P26. Saeki, A.1, K. Kawaura1, K. Murai2, Y. Ogihara1 (1 Kyoto Pref. Univ., 2 Fukui Pref. Univ.) Molecular analysis of different types of mitochondrial genes in alloplasmic wheat.
P27. Kamba, C., K. Kawaura, Y. Ogihara (Kyoto Pref. Univ.) Expression analysis of TaDREB1 in response to salt-treatment and screening of salt tolerant wheats.
P28. Kose, A., K. Kawaura, Y. Ogihara (Kyoto Pref. Univ.) Expression analysis of TaGI, a circadian clock-controlled gene, in common wheat.