Masahiko Ichii

A Rice Mutant Defective in Si Uptake

Rice (Oryza sativa) accumulates silicon (Si) in the tops to levels up to 10.0% of shoot dry weight, but the mechanism responsible for high Si uptake by rice roots is not understood. We isolated a rice mutant (GR1) that is defective in active Si uptake by screening M2 seeds (64,000) of rice cv Oochikara that were treated with 10−3 m sodium azide for 6 h at 25oC. There were no phenotypic differences between wild type (WT) and GR1 except that the leaf blade of GR1 remained droopy when Si was supplied. Uptake experiments showed that Si uptake by GR1 was significantly lower than that by WT at both low and high Si concentrations. However, there was no difference in the uptake of other nutrients such as phosphorus and potassium. Si concentration in the xylem sap of WT was 33-fold that of the external solution, but that of GR1 was 3-fold higher than the external solution at 0.15 mm Si. Si uptake by WT was inhibited by metabolic inhibitors including NaCN and 2,4-dinitrophenol and by low temperature, whereas Si uptake by GR1 was not inhibited by these agents. These results suggest that an active transport system for Si uptake is disrupted in GR1. Analysis of F2 populations between GR1 and WT showed that roots with high Si uptake and roots with low Si uptake segregated at a 3:1 ratio, suggesting that GR1 is a recessive mutant of Si uptake.

Monophyletic origin of naked barley inferred from molecular analyses of a marker closely linked to the naked caryopsis gene (nud )

To elucidate the origin of naked barley, molecular variation of the marker sKT7 tightly linked to the nud locus was examined. A total of 259 (53 wild, 106 hulled domesticated, and 100 naked domesticated) barley accessions were studied. Restriction analysis of the sKT7 PCR-amplified product revealed the alleles I, II, III, and IV. All four alleles were found in wild barley, but allele IV was found only in a single accession from southwestern Iran. Hulled domesticated accessions showed alleles I, II, or III, but all naked domesticated accessions had allele IV. The distribution of allele IV in wild barley and its pervasive presence in naked domesticated lines support the conclusion that naked barley has a monophyletic origin, probably in southwestern Iran. The available results suggest two scenarios for the origin of naked barley: either directly from a wild barley with allele IV or from a hulled domesticated line with allele IV that later became extinct. Naked domesticated accessions from different regions of the world have extremely homogeneous DNA sequences at the sKT7 locus, supporting the monophyletic origin of naked barley. For allele IV, four haplotypes (IVb to IVe) were found in 30 naked accessions: IVb was predominant (66.7%) and widely distributed, while the other three haplotypes, differing by only one nucleotide at different positions relative to IVb, showed a localized distribution. The geographical distribution of the haplotypes of sKT7 allele IV suggests migration routes of naked domesticated barley in central and eastern Asia.

Efficient fine mapping of the naked caryopsis gene (nud) by HEGS (High Efficiency Genome Scanning)/AFLP in barley

The hulled or naked caryopsis character of barley (Hordeum vulgare L.) is an important trait for edibility and to follow its domestication process. A single recessive gene, nud, controls the naked caryopsis character, and is located on the long arm of chromosome 7H. To develop a fine map around the nud locus efficiently, the HEGS (High Efficiency Genome Scanning) electrophoresis system was combined with amplified fragment length polymorphism (AFLP). From bulked segregant analysis of 1,894 primer combinations, 12 AFLP fragments were selected as linked markers. For mapping, an F2 population of 151 individuals derived from a cross between Kobinkatagi (naked type) and Triumph (hulled type) was used. Seven AFLP markers were localized near the nud region. A fine map was developed with one-order higher resolution than before, along with the seven anchor markers. Among the seven linked AFLP markers (KT1–7), KT1, KT2 and KT6 were co-dominant, and the former two were detected for their single-nucleotide polymorphisms (SNPs) in the same length of fragments after electrophoresis with the non-denaturing gels of HEGS. The nud locus has co-segregated with KT3 and KT7, and was flanked by KT2 and KT4, at the 0.3-cM proximal and the 1.2-cM distal side, respectively. Four of these AFLP markers were converted into sequence-characterized amplified region (SCAR) markers, one of which was a dominant marker co-segregating with the nud gene.

The effects of auxin on lateral root initiation and root gravitropism in a lateral rootless mutant Lrt1 of rice (Oryza sativa L.)

Auxins control growth and development in plants, including lateral rootinitiation and root gravity response. However, how endogenous auxin regulatesthese processes is poorly understood. In this study, the effects of auxins onlateral root initiation and root gravity response in rice were investigatedusing a lateral rootless mutant Lrt1, which fails to formlateral roots and shows a reduced root gravity response. Exogenous applicationof IBA to the Lrt1 mutant restored both lateral rootinitiation and root gravitropism. However, application of IAA, a major form ofnatural auxin, restored only root gravitropic response but not lateral rootinitiation. These results suggest that IBA is more effective than IAA in lateralroot formation and that IBA also plays an important role in root gravitropicresponse in rice. The application of NAA restored lateral root initiation, butdid not completely restore root gravitropism. Root elongation assays ofLrt1 displayed resistance to 2,4-D, NAA, IBA, and IAA.This result suggests that the reduced sensitivity to exogenous auxins may be due tothe altered auxin activity in the root, thereby affecting root morphology inLrt1.

A SHORT INTERNODES (SHI) family transcription factor gene regulates awn elongation and pistil morphology in barley

The awn, an apical extension from the lemma of the spikelet, plays important roles in seed dispersal, burial, and photosynthesis. Barley typically has long awns, but short-awn variants exist. The short awn 2 (lks2) gene, which produces awns about 50% shorter than normal, is a natural variant that is restricted to Eastern Asia. Positional cloning revealed that Lks2 encodes a SHI-family transcription factor. Allelism tests showed that lks2 is allelic to unbranched style 4 (ubs4) and breviaristatum-d (ari-d), for which the phenotypes are very short awn and sparse stigma hairs. The gene identity was validated by 25 mutant alleles with lesions in the Lks2 gene. Of these, 17 affected either or both conserved regions: the zinc-binding RING-finger motif and the IGGH domain. Lks2 is highly expressed in awns and pistils. Histological observations of longitudinal awn sections showed that the lks2 short-awn phenotype resulted from reduced cell number. Natural variants of lks2 were classified into three types, but all shared a single-nucleotide polymorphism (SNP) that causes a proline-to-leucine change at position 245 in the IGGH domain. All three lks2 natural variants were regarded as weak alleles because their awn and pistil phenotypes are mild compared with those of the 25 mutant alleles. Natural variants of lks2 found in the east of China and the Himalayas had considerably different sequences in the regions flanking the critical SNP, suggesting independent origins. The available results suggest that the lks2 allele might have a selective advantage in the adaptation of barley to high-precipitation areas of Eastern Asia.

Studies on sodium bypass flow in lateral rootless mutants lrt1 and lrt2, and crown rootless mutant crl1 of rice (Oryza sativa L.)

An apoplastic pathway, the so-called bypass flow, is important for Na+ uptake in rice (Oryza sativa L.) under saline conditions; however, the precise site of entry is not yet known. We report the results of our test of the hypothesis that bypass flow of Na+ in rice occurs at the site where lateral roots emerge from the main roots. We investigated Na+ uptake and bypass flow in lateral rootless mutants (lrt1, lrt2), a crown rootless mutant (crl1), their wild types (Oochikara, Nipponbare and Taichung 65, respectively) and in seedlings of rice cv. IR36. The results showed that shoot Na+ concentration in lrt1, lrt2 and crl1 was lower (by 20-23%) than that of their wild types. In contrast, the bypass flow quantified using trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS) was significantly increased in the mutants, from an average of 1.1% in the wild types to 3.2% in the mutants. Similarly, bypass flow in shoots of IR36 where the number of lateral and crown roots had been reduced through physical and hormonal manipulations was dramatically increased (from 5.6 to 12.5%) as compared to the controls. The results suggest that the path of bypass flow in rice is not at the sites of lateral root emergence.

Morphological and physiological characteristics of a root-hairless mutant in rice (Oryza sativa L.)

This paper reports morphological and physiological characteristics of a first root-hairless mutant (RH2) of rice (Oryza sativa L.), which can be useful in advancing knowledge on the role of root hairs in water and nutrient uptake, and genetics of root hairs. The mutant was selected among NaN3 mutagenized progeny of the rice cultivar Oochikara. Microscopic observations showed absence of root hairs in RH2. At the seedling stage, RH2 showed shorter seedling height and shorter roots compared to the wild type variety Oochikara. Because of the differences in seedling growth, all comparisons between Oochikara and RH2 in uptake-related characters were made on the basis of values adjusted by the dry weight of either the shoot or the root. When grown at low water potential in soil, Oochikara and RH2 were similar in shoot water content and transpiration per unit shoot dry weight, and similarly, at low water potential in solution culture, there was no significant difference between Oochikara and RH2 in transpiration per unit shoot dry weight. These results suggest that at the seedling stage, root hairs do not significantly contribute to uptake of water. In solution culture, Oochikara and RH2 did not significantly differ in phosphate uptake per unit root dry weight. This result supports the previous work that root hairs do not contribute to phosphate uptake in solution culture. Regarding to response to plant hormones, RH2 showed a higher level of resistance to two synthetic auxins, 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthaleneacetic acid (NAA) than Oochikara. NAA treatment induced very short root hairs in RH2, suggesting that the absence of root hairs in RH2 may be due to a shortage of endogenous auxin. Genetic analysis showed that the root hairless character in RH2 is inherited as a single recessive gene.

A novel short-root gene that affects specifically early root development in rice (Oryza sativa L.)

Abstract We have isolated a novel short-root mutant, srt5 , from an M 2 population of rice ( Oryza sativa L. cv. Oochikara) treated with NaN 3 , and mapped its causal gene to chromosome 2. The mutant showed extreme inhibition of seminal root, crown root and lateral root elongation, and altered root hair formation at the seedling stage. Histological observation of seminal root tips indicated that the short-root phenotype was due to the reduced cell size and cell number. At later stages of plant development, roots of the mutant elongated acceleratingly and recovered normal morphology 45 days after transplantation, and adult plants of the mutant resembled the wild type except for reduced pollen and seed fertilities. Seminal root growth of the mutant was partially rescued by exogenously applied ABA, suggesting that ABA biosynthesis might be affected in srt5 plants. Moreover, the mutant also showed high resistance to 2,4-D, GA 3 and KIN based on seminal root growth assays, but the resistance to these hormones disappeared when ABA was concomitantly applied. Thus, the wild type allele of srt5 seems to be of great importance for early root development by altering plant hormone homeostasis, in which ABA plays a critical role.

Different Behaviour of Indole-3-Acetic Acid and Indole-3-Butyric Acid in Stimulating Lateral Root Development in Rice (Oryza sativa L.)

The plant hormone auxin has been shown to be involved in lateral root development and application of auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), increases the number of lateral roots in several plants. We found that the effects of two auxins on lateral root development in the indica rice (Oryza sativa L. cv. IR8) were totally different from each other depending on the application method. When the roots were incubated with an auxin solution, IAA inhibited lateral root development, while IBA was stimulatory. In contrast, when auxin was applied to the shoot, IAA promoted lateral root formation, while IBA did not. The transport of [3H]IAA from shoot to root occurred efficiently (% transported compared to supplied) but that of [3H]IBA did not, which is consistent with the stimulatory effect of IAA on lateral root production when applied to the shoot. The auxin action of IBA has been suggested to be due to its conversion to IAA. However, in rice IAA competitively inhibited the stimulatory effect of IBA on lateral root formation when they were applied to the incubation solution, suggesting that the stimulatory effect of IBA on lateral root development is not through its conversion to IAA.

Characterization of CMS and maintainer lines in indica rice (Oryza sativa L.) based on RAPD marker analysis

Total DNA from WA type CMS lines: Zhenshan 97 A, Longtepu A and theirmaintainers Zhenshan 97 B, Longtepu B wasextracted by CTAB method. One hundredprimers were used for screening RAPDmarkers to distinguish CMS line (A) andmaintainer (B) plants at seedling stage.Results showed that under the conditions of37 °C annealing temperature and1.5 mM MgCl2 concentration, in Zhenshan97 A, Longtepu A there was a 1600 bp DNAfragment in product amplified by primerOPA12, while in Zhenshan 97 B, and LongtepuB no 1600 bp fragment was found. The 1600 bpfragment was also found in DA type CMS lineXieqingzao A, but was absent in XieqingzaoB. Also in the restorer line, Minghui 63the 1600 bp fragment was absent. In F1and F2 generation of Zhenshan97A/Minghui 63, all plants investigated hadthe 1600 bp fragment. When mitochondrial DNA(mtDNA) was isolated from the three CMS (A)and their B lines and amplified by OPA12,results showed that the 1600 fragment wasfound in all the three A lines and wasabsent in the three B lines. In DA typeXieqingzao A, two other fragments (700 bp,1000 bp) were also found except the 1600 bp.These results indicate that the 1600 bpfragment derived from CMS mitochondrial DNAcan be used as a RAPD marker to distinguishA and B plants at seedling stage, and thefragments (700 bp, 1000 bp) can be used todistinguish WA and DA cytoplasms.