Katsuyuki Kakeda

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 residues in a hydrophilic loop of the Papaver rhoeas S protein that play a crucial role in recognition of incompatible pollen.

The self-incompatibility response involves S allele–specific recognition between stigmatic S proteins and incompatible pollen. This response results in pollen inhibition. Defining the amino acid residues within the stigmatic S proteins that participate in S allele–specific inhibition of incompatible pollen is essential for the elucidation of the molecular basis of the self-incompatibility response. We have constructed mutant derivatives of the S1 protein from Papaver rhoeas by using site-directed mutagenesis and have tested their biological activity. This has enabled us to identify amino acid residues in the stigmatic S proteins of P. rhoeas that are required for S-specific inhibition of incompatible pollen. We report here the identification of several amino acid residues in the predicted hydrophilic loop 6 of the P. rhoeas stigmatic S1 protein that are involved in the inhibition of S1 pollen. Mutation of the only hypervariable amino acid, which is situated in this loop, resulted in the complete loss of ability of the S protein to inhibit S1 pollen. This clearly demonstrates that this residue plays a crucial role in pollen recognition and may also participate in defining allelic specificity. We have also established the importance of highly conserved amino acids adjacent to this hypervariable site. Our studies demonstrate that both variable and conserved amino acids in the region of the S protein corresponding to surface loop 6 are key elements that play a role in the recognition and inhibition of incompatible pollen in the pollen–pistil self-incompatibility reaction.

Molecular and genetic characterization of the S locus in Hordeum bulbosum L., a wild self-incompatible species related to cultivated barley.

Gametophytic self-incompatibility (GSI) in the grasses is controlled by a distinct two-locus genetic system governed by the multiallelic loci S and Z. We have employed diploid Hordeum bulbosum as a model species for identifying the self-incompatibility (SI) genes and for elucidating the molecular mechanisms of the two-locus SI system in the grasses. In this study, we attempted to identify S haplotype-specific cDNAs expressed in pistils and anthers at the flowering stage in H. bulbosum, using the AFLP-based mRNA fingerprinting (AMF, also called cDNA-AFLP) technique. We used the AMF-derived DNA clones as markers for fine mapping of the S locus, and found that the locus resided in a chromosomal region displaying remarkable suppression of recombination, encompassing a large physical region. Furthermore, we identified three AMF-derived markers displaying complete linkage to the S locus, although they showed no significant homology with genes of known functions. Two of these markers showed expression patterns that were specific to the reproductive organs (pistil or anther), suggesting that they could be potential candidates for the S gene.

SLG/SRK-like genes are expressed in the reproductive tissues of Ipomoea trifida

Self-incompatibility of Ipomoea trifida is under the control of a single multi-allelic locus, called the S-locus, and is genetically the same as the sporophytic system found in some species of Brassica. We investigated whether the S-locus-specific glycoprotein (SLG) and S receptor kinase (SRK) genes associated with the self-incompatibility of Brassica are expressed in the reproductive tissues of I. trifida. We amplified mRNAs from reproductive tissues of I. trifida by reverse transcriptase PCR using primers corresponding to conserved regions of the Brassica SLGs. Four kinds of PCR fragments were amplified in this experiment. These fragments were designated IPG1 to IPG4 based on dot-blot cross-hybridization. Nucleotide sequencing of the clones revealed 40–46% similarity to the Brassica SLGs and SRKs at the protein level. Northern analysis using IPG1 as a probe revealed a major transcript of 2.8 kb that would correspond in size to a fully spliced SRK transcript of Brassica. The transcript for IPG1 was detected in both mature stigma and anther tissue and was developmentally regulated. The experimental results reported here indicate that at least four kinds of SLG/SRK-like genes are expressed in the reproductive tissues of Ipomoea.

Molecular Genetics of Sporophytic Self-Incompatibility in Ipomoea, aMember of the Convolvulaceae

Diploid Ipomoea trifida in the Convolvulaceae is a close relative of the cultivated hexaploid species, the sweet potato, and has sporophytic self-incompatibility controlled by a single multi-allelic S-locus. Genetic analyses of I. trifida plants collected from native populations in Central America have identified a number of different S-haplotypes, which show a linear dominance hierarchy with some codominance relationships. A linkage map of DNA markers showed that the S-locus is delimited to a 0.23 cM region and is located in the S-haplotype-specific divergent region (SDR) that has a physical size of 35–95 kb. Of the six genes located within the SDR, three stigma-specific novel genes, SE1, SE2 and SEA, and an antherspecific gene, AB2, are candidates for encoding pistil and pollen determinants of self-incompatibility, respectively, suggesting that a unique recognition mechanism is involved in the self-incompatibility system of Ipomoea.

A self-compatible mutant S allele conferring a dominant negative effect on the functional S allele in Ipomoea trifida

Abstract A spontaneously occurring self-compatible mutant has been identified in Ipomoea trifida, a species possessing sporophytic self-incompatibility controlled by a single multiallelic S locus. Analysis of the segregation of compatibility/incompatibility phenotypes in selfed and crossed progenies of the self-compatible mutant plant indicated that the self-compatibility trait was caused by a mutation at the S locus; the mutated S allele was therefore designated Sc. RFLP analysis of progeny plants segregating for the Sc allele using the SSP gene (a gene linked closely to the S locus of I. trifida) as a probe confirmed that the mutation was present at the S locus. Self-incompatibility responses were examined in F1 progenies obtained from crosses between the self-compatible mutant and self-incompatible plants homozygous for one of three S alleles, S1, S3 and S22, where the dominance relationship is S22>S1>S3. All F1 progeny plants from crosses with S22 and S1 homozygotes were self-incompatible and exhibited the respective phenotypes of each self-incompatible parent (either S22 or S1) in both stigma and pollen. However, of the F1 progeny plants from the cross with the S3 homozygote, those carrying the genotype ScS3 were all self-compatible and cross-compatible as both female and male parents with the S3 homozygote. These results indicate that the dominance relationship between the four S alleles is: S22>S1>Sc>S3 and so reveal the unexpected finding that the mutated Sc allele is dominant over a functional S3 allele. A possible explanation for this observation is that the gene product encoded by the Sc allele confers a dominant negative effect on the S3 gene product.

Sequences of Ipomoea trifida cDNAs related to the Brassica S-locus genes

Self-incompatibility (SI) in the diploid Ipomoea trifida (Convolvulaceae) is sporophytically controlled by a single multiallelic S locus (Kowyama et al. 1980, 1994). In our previous study performed by reverse transcriptase PCR amplification of mRNAs from stigmas and anthers of I. trifida (Kowyama et al. 1995), we detected four kinds of PCR fragment clones (IPG1 to IPG4) with sequence similarity to the S-locus glycoprotein (SLG) and S-receptor kinase (SRK) genes associated with the sporophytic SI of Brassica (Nasrallah and Nasrallah 1993). In subsequent screening of a stigma cDNA library using one of the clones (IPG1) as a probe, we isolated a cDNA clone encoding a putative receptor protein kinase (IRK1) that shares ca. 50% amino acid sequence identity with Brassica SRKs (Kowyama et al. 1996). Restriction fragment length polymorphism (RFLP) analysis, however, showed that neither IRK1 nor the other three genes coding for IPG2 to IPG4 were linked to the S locus of I. trifida (Kowyama et al. 1996). We report here the cloning and sequencing of cDNAs for the latter three genes. A cDNA library (Lambda Zap II) was constructed with poly(A) + RNAs of mature stigmas from a single

Molecular Mechanisms for Covered vs. Naked Caryopsis in Barley

22 homozygous plant. From screening of the stigma cDNA library using IPG2, IPG3 and IPG4 as probes, one, three and one positively hybridizing clones, respectively, were isolated, cDNA inserts of these clones were subcloned into Bluescript plasmids and sequenced. All of the cDNA clones obtained showed sequences with structural similarity to Brassica SLGs but lacked sequences corresponding to either the transmembrane or ki-

Sporophytic Self-incompatibility in Ipomoea trifida, a Close Relative of Sweet Potato

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 vs. naked caryopsis is controlled by a single locus (nud) on chromosome arm 7HL. Positional cloning identified that an ERF (ethylene response factor) family transcription factor gene controls the covered vs. naked caryopsis phenotype. This conclusion was further supported by (1) fixation of the 17-kb deletion, harboring the ERF gene, among all 100 naked cultivars studied; (2) five induced nud alleles with a DNA lesion at a different site, each affecting the putative functional motif; and (3) gene expression strictly localized to the testa. Survey of natural variation at the nud locus indicates that naked barley has monophyletic origin but that covered barley is classified into some clusters, suggesting plural lineages. 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. This observation indicates that in covered barley, lipids on the surface of caryopses act as a glue for their tight adhesion with hulls. Separation of hulls in naked barley is due to the absence of surface lipids on caryopses. Genetic complementation experiment is in progress toward functional validation of the Nud gene.