Molecular identification of a laccase that catalyzes the oxidative coupling of a hydroxycinnamic acid amide for hordatine biosynthesis in barley.

Plants produce dimerized phenolic compounds as secondary metabolites. Hordatine A (HA), a dehydrodimer of p-coumaroylagmatine (pCA), is an antifungal compound accumulated at high levels in young barley (Hordeum vulgare) seedlings. The enzyme responsible for the oxidative dimerization of pCA, which is the final step of the hordatine biosynthetic pathway, has not been identified. In this study, we first verified the presence of this enzyme activity in the crude extract of barley seedlings. Because the enzyme activity was not dependent on H2 O2 , the responsible enzyme was not peroxidase, which was previously implicated in HA biosynthesis. The analysis of the dissection lines of wheat (Triticum aestivum) carrying aberrant barley 2H chromosomes detected HA in the wheat lines carrying the distal part of the 2H short arm. This chromosomal region contains two laccase genes (HvLAC1 and HvLAC2) that are highly expressed at the seedling stage and may encode enzymes that oxidize pCA during the formation of HA. Changes in the HvLAC transcript levels coincided with the changes in the HA biosynthesis-related enzyme activities in the crude extract and the HA content in barley seedlings. Moreover, HvLAC genes were heterologously expressed in Nicotiana benthamiana leaves and in bamboo (Phyllostachys nigra) suspension cells and HA biosynthetic activities were detected in the crude extract of transformed N. benthamiana leaves and bamboo suspension cells. The HA formed by the enzymatic reaction had the same stereo-configuration as the naturally occurring HA. These results demonstrate that HvLAC enzymes mediate the oxidative coupling of pCA during HA biosynthesis.

Regulation of the plastochron by three many-noded dwarf genes in barley.

The plastochron, the time interval between the formation of two successive leaves, is an important determinant of plant architecture. We genetically and phenotypically investigated many-noded dwarf (mnd) mutants in barley. The mnd mutants exhibited a shortened plastochron and a decreased leaf blade length, and resembled previously reported plastochron1 (pla1), pla2, and pla3 mutants in rice. In addition, the maturation of mnd leaves was accelerated, similar to pla mutants in rice. Several barley mnd alleles were derived from three genes-MND1, MND4, and MND8. Although MND4 coincided with a cytochrome P450 family gene that is a homolog of rice PLA1, we clarified that MND1 and MND8 encode an N-acetyltransferase-like protein and a MATE transporter-family protein, which are respectively orthologs of rice GW6a and maize BIGE1 and unrelated to PLA2 or PLA3. Expression analyses of the three MND genes revealed that MND1 and MND4 were expressed in limited regions of the shoot apical meristem and leaf primordia, but MND8 did not exhibit a specific expression pattern around the shoot apex. In addition, the expression levels of the three genes were interdependent among the various mutant backgrounds. Genetic analyses using the double mutants mnd4mnd8 and mnd1mnd8 indicated that MND1 and MND4 regulate the plastochron independently of MND8, suggesting that the plastochron in barley is controlled by multiple genetic pathways involving MND1, MND4, and MND8. Correlation analysis between leaf number and leaf blade length indicated that both traits exhibited a strong negative association among different genetic backgrounds but not in the same genetic background. We propose that MND genes function in the regulation of the plastochron and leaf growth and revealed conserved and diverse aspects of plastochron regulation via comparative analysis of barley and rice.

Genomic traces of Japanese malting barley breeding in two modern high-quality cultivars, 'Sukai Golden' and 'Sachiho Golden'.

Two modern high-quality Japanese malting barley cultivars, 'Sukai Golden' and 'Sachiho Golden', were subjected to RNA-sequencing of transcripts extracted from 20-day-old immature seeds. Despite their close relation, 2,419 Sukai Golden-specific and 3,058 Sachiho Golden-specific SNPs were detected in comparison to the genome sequences of two reference cultivars: 'Morex' and 'Haruna Nijo'. Two single nucleotide polymorphism (SNP) clusters respectively showing the incorporation of (1) the barley yellow mosaic virus (BaYMV) resistance gene rym5 from six-row non-malting Chinese landrace Mokusekko 3 on the long arm of 3H, and (2) the anthocyanin-less ant2 gene from a two-row Dutch cultivar on the long arm of 2H were detected specifically in 'Sukai Golden'. Using 221 recombinant inbred lines of a cross between 'Ishukushirazu' and 'Nishinochikara', another BaYMV resistance rym3 gene derived from six-row non-malting Japanese cultivar 'Haganemugi' was mapped to a 0.4-cM interval on the proximal region of 5H. Haplotype analysis of progenitor accessions of the two modern malting cultivars revealed that rym3 of 'Haganemugi' was independently introduced into 'Sukai Golden' and 'Sachiho Golden'. Residual chromosome 5H segments of 'Haganemugi' surrounding rym3 were larger in 'Sukai Golden'. Available results suggest possibilities for malting quality improvement by minimizing residual segments surrounding rym3.

Mutations in a�Golden2-Like�Gene Cause Reduced Seed Weight in�Barley�albino lemma 1�Mutants.

The albino lemma 1 (alm1) mutants of barley (Hordeum vulgare L.) exhibit obvious chlorophyll-deficient hulls. Hulls are seed-enclosing tissues on the spike, consisting of the lemma and palea. The alm1 phenotype is also expressed in the pericarp, culm nodes and basal leaf sheaths, but leaf blades and awns are normal green. A single recessive nuclear gene controls tissue-specific alm1 phenotypic expression. Positional cloning revealed that the ALM1 gene encodes a Golden 2-like (GLK) transcription factor, HvGLK2, belonging to the GARP subfamily of Myb transcription factors. This finding was validated by genetic evidence indicating that all 10 alm1 mutants studied had a lesion in functionally important regions of HvGLK2, including the three alpha-helix domains, an AREAEAA motif and the GCT box. Transmission electron microscopy revealed that, in lemmas of the alm1.g mutant, the chloroplasts lacked thylakoid membranes, instead of stacked thylakoid grana in wild-type chloroplasts. Compared with wild type, alm1.g plants showed similar levels of leaf photosynthesis but reduced spike photosynthesis by 34%. The alm1.g mutant and the alm1.a mutant showed a reduction in 100-grain weight by 15.8% and 23.1%, respectively. As in other plants, barley has HvGLK2 and a paralog, HvGLK1. In flag leaves and awns, HvGLK2 and HvGLK1 are expressed at moderate levels, but in hulls, HvGLK1 expression was barely detectable compared with HvGLK2. Barley alm1/Hvglk2 mutants exhibit more severe phenotypes than glk2 mutants of other plant species reported to date. The severe alm1 phenotypic expression in multiple tissues indicates that HvGLK2 plays some roles that are nonredundant with HvGLK1.

Biosynthesis of Phenylamide Phytoalexins in Pathogen-Infected Barley.

Phytoalexins are inducible antimicrobial metabolites in plants, and have been indicated to be important for the rejection of microbial infection. HPLC analysis detected the induced accumulation of three compounds 1-3 in barley (Hordeum vulgare) roots infected by Fusarium culmorum, the causal agent of Fusarium root rot. Compounds 1-3 were identified as cinnamic acid amides of 9-hydroxy-8-oxotryptamine, 8-oxotryptamine, and (1H-indol-3-yl)methylamine, respectively, by spectroscopic analysis. Compounds 1 and 2 had been previously reported from wheat, whereas 3 was an undescribed compound. We named 1-3 as triticamides A-C, respectively, because they were isolated from barley and wheat, which belong to the Triticeae tribe. These compounds showed antimicrobial activities, indicating that triticamides function as phytoalexins in barley. The administration of deuterium-labeled N-cinnamoyl tryptamine (CinTry) to barley roots resulted in the effective incorporation of CinTry into 1 and 2, which suggested that they were synthesized through the oxidation of CinTry. Nine putative tryptamine hydroxycinnamoyl transferase (THT)-encoding genes (HvTHT1-HvTHT9) were identified by database search on the basis of homology to known THT gene sequences from rice. Since HvTHT7 and HvTHT8 had the same sequences except one base, we measured their expression levels in total by RT-qPCR. HvTHT7/8 were markedly upregulated in response to infection by F. culmorum. The HvTHT7 and HvTHT8 enzymes preferred cinnamoyl- and feruloyl-CoAs as acyl donors and tryptamine as an acyl acceptor, and (1H-indol-3-yl)methylamine was also accepted as an acyl acceptor. These findings suggested that HvTHT7/8 are responsible for the induced accumulation of triticamides in barley.

A Homolog of Blade-On-Petiole 1 and 2 (BOP1/2) Controls Internode Length and Homeotic Changes of the Barley Inflorescence.

Inflorescence architecture in small-grain cereals has a direct effect on yield and is an important selection target in breeding for yield improvement. We analyzed the recessive mutation laxatum-a (lax-a) in barley (Hordeum vulgare), which causes pleiotropic changes in spike development, resulting in (1) extended rachis internodes conferring a more relaxed inflorescence, (2) broadened base of the lemma awns, (3) thinner grains that are largely exposed due to reduced marginal growth of the palea and lemma, and (4) and homeotic conversion of lodicules into two stamenoid structures. Map-based cloning enforced by mapping-by-sequencing of the mutant lax-a locus enabled the identification of a homolog of BLADE-ON-PETIOLE1 (BOP1) and BOP2 as the causal gene. Interestingly, the recently identified barley uniculme4 gene also is a BOP1/2 homolog and has been shown to regulate tillering and leaf sheath development. While the Arabidopsis (Arabidopsis thaliana) BOP1 and BOP2 genes act redundantly, the barley genes contribute independent effects in specifying the developmental growth of vegetative and reproductive organs, respectively. Analysis of natural genetic diversity revealed strikingly different haplotype diversity for the two paralogous barley genes, likely affected by the respective genomic environments, since no indication for an active selection process was detected.

Distribution of the tryptophan pathway-derived defensive secondary metabolites gramine and benzoxazinones in Poaceae.

The Poaceae is a large taxonomic group consisting of approximately 12,000 species and is classified into 12 subfamilies. Gramine and benzoxazinones (Bxs), which are biosynthesized from the tryptophan pathway, are well-known defensive secondary metabolites in the Poaceae. We analyzed the presence or absence of garamine and Bxs in 64 species in the Poaceae by LC-MS/MS. We found that Hordeum brachyantherum and Hakonechloa macra accumulated gramine, but the presence of gramine was limited to small groups of species. We also detected Bxs in four species in the Pooideae and six species in the Panicoideae. In particular, four species in the Paniceae tribe in Panicoideae accumulaed Bxs, indicating that this tribe is a center of the Bx distribution. Bxs were absent in the subfamilies other than Pooideae and Panicoideae. These findings provide an overview of biased distribution of gramine and Bxs in Poaceae species.

Barley NARROW LEAFED DWARF1 encoding a WUSCHEL-RELATED HOMEOBOX 3 (WOX3) regulates the marginal development of lateral organs.

Barley (Hordeum vulgare L.) is the fourth most-produced cereal in the world and is mainly utilized as animal feed and malts. Recently barley attracts considerable attentions as healthy food rich in dietary fiber. However, limited knowledge is available about developmental aspects of barley leaves. In the present study, we investigated barley narrow leafed dwarf1 (nld1) mutants, which exhibit thin leaves accompanied by short stature. Detailed histological analysis revealed that leaf marginal tissues, such as sawtooth hairs and sclerenchymatous cells, were lacked in nld1, suggesting that narrowed leaf of nld1 was attributable to the defective development of the marginal regions in the leaves. The defective marginal developments were also appeared in internodes and glumes in spikelets. Map-based cloning revealed that NLD1 encodes a WUSCHEL-RELATED HOMEOBOX 3 (WOX3), an ortholog of the maize NARROW SHEATH genes. In situ hybridization showed that NLD1 transcripts were localized in the marginal edges of leaf primordia from the initiating stage. From these results, we concluded that NLD1 plays pivotal role in the increase of organ width and in the development of marginal tissues in lateral organs in barley.

Isolation of candidate genes for the barley Ant1 and wheat Rc genes controlling anthocyanin pigmentation in different vegetative tissues.

MYB transcription factors exist in a large copy number and control various plant phenotypes. We cloned R2R3 MYB-type transcription factors that determine the coloration of basal sheaths in barley and wheat coleoptiles. These genes are highly homologous to maize C1 and rice OsC1, regulators for anthocyanin biosynthesis, but they control seed pigmentation in maize and rice. On the basis of high homology, barley and wheat counterparts are designated HvC1 and TaC1, respectively. HvC1 gene is located on the short arm of chromosome 7H, and TaC1 genes are located on the short arms of chromosomes 7A, 7B, and 7D (TaC1-A1, B1, and D1, respectively). HvC1 is a strong candidate for Ant1 because of (1) complete co-segregation of anthocyanin pigmentation phenotype of the basal sheath with the HvC1 genotype in genetic mapping, and (2) complete deletion of the HvCl gene in two anthocyanin-decreased allelic mutants (ant1.1 and ant1.2) that were induced by irradiation. In contrast, colorless coleoptile wheat lines had lesions in all three genomes consisting of a single-nucleotide substitution or a 1-bp deletion of TaC1-A1, a 1.7-kb insertion of TaC1-B1, and a 2.0-kb insertion of TaC1-D1. At least one normal TaC1 gene appears to be sufficient to produce anthocyanin pigments in wheat coleoptiles. Previous crossing experiments localized Rc (red coleoptile) genes to homoeologous group 7 chromosomes and deduced Rc genotypes of several wheat lines. Their TaC1 gene sequence variation coincided with deduced Rc genotypes; therefore, the present molecular genetic study demonstrates that TaC1 is a strong candidate for Rc in wheat.

Multiple origins of the phenol reaction negative phenotype in foxtail millet, Setaria italica (L.) P. Beauv., were caused by independent loss-of-function mutations of the polyphenol oxidase (Si7PPO) gene during domestication.

Foxtail millet shows variation in positive phenol color reaction (Phr) and negative Phr in grains, but predominant accessions of this crop are negative reaction type, and the molecular genetic basis of the Phr reaction remains unresolved. In this article, we isolated polyphenol oxidase (PPO) gene responsible for Phr using genome sequence information and investigated molecular genetic basis of negative Phr and crop evolution of foxtail millet. First of all, we searched for PPO gene homologs in a foxtail millet genome database using a rice PPO gene as a query and successfully found three copies of the PPO gene. One of the PPO gene homologs on chromosome 7 showed the highest similarity with PPO genes expressed in hulls (grains) of other cereal species including rice, wheat, and barley and was designated as Si7PPO. Phr phenotypes and Si7PPO genotypes completely co-segregated in a segregating population. We also analyzed the genetic variation conferring negative Phr reaction. Of 480 accessions of the landraces investigated, 87 (18.1 %) showed positive Phr and 393 (81.9 %) showed negative Phr. In the 393 Phr negative accessions, three types of loss-of-function Si7PPO gene were predominant and independently found in various locations. One of them has an SNP in exon 1 resulting in a premature stop codon and was designated as stop codon type, another has an insertion of a transposon (Si7PPO-TE1) in intron 2 and was designated as TE1-insertion type, and the other has a 6-bp duplication in exon 3 resulting in the duplication of 2 amino acids and was designated as 6-bp duplication type. As a rare variant of the stop codon type, one accession additionally has an insertion of a transposon, Si7PPO-TE2, in intron 2 and was designated as "stop codon +TE2 insertion type". The geographical distribution of accessions with positive Phr and those with three major types of negative Phr was also investigated. Accessions with positive Phr were found in subtropical and tropical regions at frequencies of ca. 25-67 % and those with negative Phr were broadly found in Europe and Asia. The stop codon type was found in 285 accessions and was broadly distributed in Europe and Asia, whereas the TE-1 insertion type was found in 99 accessions from Europe and Asia but was not found in India. The 6-bp duplication type was found in only 8 accessions from Nansei Islands (Okinawa Prefecture) of Japan. We also analyzed Phr in the wild ancestor and concluded that the negative Phr type was likely to have originated after domestication of foxtail millet. It was also implied that negative Phr of foxtail millet arose by multiple independent loss of function of PPO gene through dispersal because of some advantages under some environmental conditions and human selection as in rice and barley.

Barley Ant17, encoding flavanone 3-hydroxylase (F3H), is a promising target locus for attaining anthocyanin/proanthocyanidin-free plants without pleiotropic reduction of grain dormancy.

Preharvest sprouting is a serious problem in grain crop production because it causes quality deterioration and economic losses. It is well known that grain colour is closely associated with grain dormancy in wheat; white-grained lines without accumulating proanthocyanidins in testa tend to be more susceptible to preharvest sprouting than red ones. All available white-grained wheat lines are restricted to triple recessive mutations at the R loci (R-A1, R-B1, and R-D1), but barley is known to have 11 independent loci conferring the proanthocyanidin-free grain phenotype. In this study, we evaluated the dormancy levels of anthocyanin/proanthocyanidin-free ant17 mutants. Three ant17 mutants showed the same levels of dormancy as their respective wild types. Sequencing of three independent ant17 alleles detected a point mutation within the coding regions of flavanone-3-hydroxylase (F3H), which are predicted to cause a premature stop codon at different sites. The F3H locus completely cosegregated with the Ant17 position on the chromosome arm 2HL. Expression of the barley F3H gene was observed in pigmented tissues, but not in nonpigmented roots and stems. This result indicates that wheat F3H may be a promising new target locus for breeding white-grained lines with a practical level of preharvest sprouting resistance.

Functional characterization of barley betaglucanless mutants demonstrates a unique role for CslF6 in (1,3;1,4)-ß-D-glucan biosynthesis.

(1,3;1,4)-ß-D-glucans (mixed-linkage glucans) are found in tissues of members of the Poaceae (grasses), and are particularly high in barley (Hordeum vulgare) grains. The present study describes the isolation of three independent (1,3;1,4)-ß-D-glucanless (betaglucanless; bgl) mutants of barley which completely lack (1,3;1,4)-ß-D-glucan in all the tissues tested. The bgl phenotype cosegregates with the cellulose synthase like HvCslF6 gene on chromosome arm 7HL. Each of the bgl mutants has a single nucleotide substitution in the coding region of the HvCslF6 gene resulting in a change of a highly conserved amino acid residue of the HvCslF6 protein. Microsomal membranes isolated from developing endosperm of the bgl mutants lack detectable (1,3;1,4)-ß-D-glucan synthase activity indicating that the HvCslF6 protein is inactive. This was confirmed by transient expression of the HvCslF6 cDNAs in Nicotiana benthamiana leaves. The wild-type HvCslF6 gene directed the synthesis of high levels of (1,3;1,4)-ß-D-glucans, whereas the mutant HvCslF6 proteins completely lack the ability to synthesize (1,3;1,4)-ß-D-glucans. The fine structure of the (1,3;1,4)-ß-D-glucan produced in the tobacco leaf was also very different from that found in cereals having an extremely low DP3/DP4 ratio. These results demonstrate that, among the seven CslF and one CslH genes present in the barley genome, HvCslF6 has a unique role and is the key determinant controlling the biosynthesis of (1,3;1,4)-ß-D-glucans. Natural allelic variation in the HvCslF6 gene was found predominantly within introns among 29 barley accessions studied. Genetic manipulation of the HvCslF6 gene could enable control of (1,3;1,4)-ß-D-glucans in accordance with the purposes of use.

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.

A bifurcated palea mutant infers functional differentiation of WOX3 genes in flower and leaf morphogenesis of barley.

Barley (Hordeum vulgare) is the fourth most highly produced cereal in the world after wheat, rice and maize and is mainly utilized as malts and for animal feed. Barley, a model crop of the tribe Triticeae, is important in comparative analyses of Poaceae. However, molecular understanding about the developmental processes is limited in barley. Our previous work characterized one of two WUSCHEL-RELATED HOMEOBOX 3 (WOX3) genes present in the barley genome: NARROW LEAFED DWARF1 (NLD1). We demonstrated that NLD1 plays a pivotal role in the development of lateral organs. In the present study, we describe a bifurcated palea (bip) mutant of barley focusing on flower and leaf phenotypes. The palea in the bip mutant was split into two and develop towards inside the lemma surrounding the carpels and anthers. The bip mutant is devoid of lodicules, which develop in a pair at the base of the stamen within the lemma in normal barley. bip also exhibited malformations in leaves, such as narrow leaf due to underdeveloped leaf-blade width, and reduced trichome density. Map-based cloning and expression analysis indicated that BIP is identical to another barley WOX3 gene, named HvWOX3. The bip nld1 double mutant presented a more severe reduction in leaf-blade width and number of trichomes. By comparing the phenotypes and gene expression patterns of various WOX3 mutants, we concluded that leaf bilateral outgrowth and trichome development are promoted by both NLD1 and HvWOX3, but that HvWOX3 serves unique and pivotal functions in barley development that differ from those of NLD1.

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.

Identification of methoxylchalcones produced in response to CuCl2 treatment and pathogen infection in barley.

Changes in specialized metabolites were analyzed in barley (Hordeum vulgare) leaves treated with CuCl2 solution as an elicitor. LC-MS analysis of the CuCl2-treated leaves showed the induced accumulation of three compounds. Among them, two were purified by silica gel and ODS column chromatography and preparative HPLC and were identified as 2',3,4,4',6'-pentamethoxychalcone and 2'-hydroxy-3,4,4',6'-tetramethoxychalcone by spectroscopic analyses. The remaining compound was determined as 12-oxo-phytodienoic acid (OPDA), a major oxylipin in plants, by comparing its spectrum and retention time from LC-MS/MS analysis with those of the authentic compound. The accumulation of these compounds was reproduced in leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of the Poaceae species. This inoculation increased the amounts of other oxylipins, including jasmonic acid (JA), JA-Ile, 9-oxooctadeca-10,12-dienoic acid (9-KODE), and 13-oxooctadeca-9,11-dienoic acid (13-KODE). The treatments of the barley leaves with JA and OPDA induced the accumulation of methoxylchalcones, but treatment with 9-KODE did not. These methoxylchalcones inhibited conidial germination of B. sorokiniana and Fusarium graminearum, thereby indicating that these compounds possessed antifungal activity. Consequently, they are considered to be involved in the chemical defense processes as phytoalexins in barley. Accumulation of methoxylchalcones in response to JA treatment was observed in all seven barley cultivars tested, but was not detected in other wild Hordeum species, wheat, and rice, thus indicating that their production was specific to cultivated barley.

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.

Duplicate polyphenol oxidase genes on barley chromosome 2H and their functional differentiation in the phenol reaction of spikes and grains.

Polyphenol oxidases (PPOs) are copper-containing metalloenzymes encoded in the nucleus and transported into the plastids. Reportedly, PPOs cause time-dependent discoloration (browning) of end-products of wheat and barley, which impairs their appearance quality. For this study, two barley PPO homologues were amplified using PCR with a primer pair designed in the copper binding domains of the wheat PPO genes. The full-lengths of the respective PPO genes were cloned using a BAC library, inverse-PCR, and 3'-RACE. Linkage analysis showed that the polymorphisms in PPO1 and PPO2 co-segregated with the phenol reaction phenotype of awns. Subsequent RT-PCR experiments showed that PPO1 was expressed in hulls and awns, and that PPO2 was expressed in the caryopses. Allelic variation of PPO1 and PPO2 was analysed in 51 barley accessions with the negative phenol reaction of awns. In PPO1, amino acid substitutions of five types affecting functionally important motif(s) or C-terminal region(s) were identified in 40 of the 51 accessions tested. In PPO2, only one mutant allele with a precocious stop codon resulting from an 8 bp insertion in the first exon was found in three of the 51 accessions tested. These observations demonstrate that PPO1 is the major determinant controlling the phenol reaction of awns. Comparisons of PPO1 single mutants and the PPO1PPO2 double mutant indicate that PPO2 controls the phenol reaction in the crease on the ventral side of caryopses. An insertion of a hAT-family transposon in the promoter region of PPO2 may be responsible for different expression patterns of the duplicate PPO genes in barley.

Phylogenetic analysis of the Si7PPO gene in foxtail millet, Setaria italica, provides further evidence for multiple origins of the negative phenol color reaction phenotype.

To elucidate the diversity and evolution of the Si7PPO gene that controls phenol color reaction (Phr) in foxtail millet, Setaria italica, we analyzed sequence polymorphisms of the Si7PPO gene in 39 accessions consisting of foxtail millet landraces (32 accessions) and their wild ancestor ssp. viridis (seven accessions) collected from various regions in Europe and Asia. The accessions included wild type (positive Phr) and three different types of loss-of-function phenotype (negative Phr), "stop codon type", "TE1-insertion type" and "6-bp duplication type", found in our previous study. We constructed a phylogenetic tree of the gene and found that accessions with positive Phr showed higher genetic diversity at the nucleotide sequence level. We also found that the three different loss-of-function types formed different clusters, suggesting that landraces with negative Phr have multiple origins from three different lineages including both landrace and ssp. viridis accessions with positive Phr.

Identification of phenylamide phytoalexins and characterization of inducible phenylamide metabolism in wheat.

Changes in specialized metabolites were analyzed in wheat leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of Poaceae species. HPLC analysis detected the accumulation of six compounds in B. sorokiniana-infected leaves. Of these, we purified two compounds by silica gel and ODS column chromatography and preparative HPLC, and identified them as cinnamic acid amides, N-cinnamoyl-9-hydroxy-8-oxotryptamine and N-cinnamoyl-8-oxotryptamine, by spectroscopic analyses. The remaining four compounds were predicted to be p-coumaric acid amides of hydroxyputrescine, hydroxyagmatine, hydroxydehydroagmatine, and agmatine by mass spectrometry. The accumulation of two cinnamic acid amides was also induced by Fusarium graminearum infection, and by treatment with CuCl2, jasmonic acid, and isopentenyladenine. Antifungal activity of these amides was shown by inhibition of conidial germination and germ tube elongation of F. graminearum and Alternaria brassicicola, indicating that they act as phytoalexins. The accumulation of these amides also detected in barley leaves treated with CuCl2. We examined the accumulation of 25 phenylamides in B. sorokiniana-infected wheat leaves using LC-MS/MS. Hydroxycinnamic acid amides of tryptamine, serotonin, putrescine, and agmatine, were induced after infection with B. sorokiniana. Thus, the induced accumulation of two groups of phenylamides, cinnamic acid amides with indole amines, and p-coumaric acid amides with putrescine and agmatine related amines, represents a major metabolic response of wheat to pathogen infection.