Novel QTLs for photoperiodic flowering revealed by using reciprocal backcross inbred lines from crosses between japonica rice cultivars.

The rice japonica cultivars Nipponbare and Koshihikari differ in heading date and response of heading to photoperiod (photoperiod sensitivity). Using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers, we conducted quantitative trait locus (QTL) analyses for heading date in a set of reciprocal backcross inbred lines (BILs) from crosses between Nipponbare and Koshihikari. Under natural-day conditions, transgressive segregation in days to heading (DTH) toward both early and late heading was observed in both BIL populations. QTL analyses revealed that two QTLs--on chromosomes 3 and 6--were involved in the difference in heading date between the parental cultivars. The Nipponbare allele at the QTLs on chromosomes 3 and 6 showed, respectively, increasing and decreasing effects on DTH in both BIL populations. The transgressive segregation observed in the BILs could be accounted for mainly by the complementary action of a set of alleles with opposing effects. Both QTLs were finely mapped as single Mendelian factors in secondary mapping populations (BC2F2 plants/BC2F3 lines). The QTL on chromosome 3 was mapped in the 1,140-kb interval between 94O03-4 (SSR) and OJ21G19-4 (SNP) and was designated Hd16. The QTL on chromosome 6 was mapped in the 328-kb interval between P548D347 (SSR) and 0007O20 (SSR) and was designated Hd17. Both Hd16 and Hd17 were involved in photoperiod sensitivity, as revealed by observation of the DTH of nearly isogenic lines of Nipponbare under short- and long-day conditions, suggesting that allelic differences in both Hd16 and Hd17 account for most of the difference in photoperiod sensitivity between the parental cultivars.

Detection of quantitative trait loci controlling extremely early heading in rice.

To clarify the genetic basis of extremely early heading in rice, we conducted quantitative trait locus (QTL) analyses using F2 populations from two genetically wide cross combinations, Hayamasari/Kasalath (HaF2) and Hoshinoyume/Kasalath (HoF2). Hayamasari and Hoshinoyume are extremely early-heading japonica cultivars. Photoperiod sensitivity is completely lost in Hayamasari and weak in Hoshinoyume. Three QTLs, QTL(chr6), QTL(chr7), and QTL(chr8), for days-to-heading (DTH) in HaF2 were detected on chromosomes 6, 7, and 8, respectively, and QTL(chr6) and QTL(chr7) were detected in HoF2. On the basis of the chromosomal locations, QTL(chr6), QTL(chr7), and QTL(chr8) may be likely to be Hd1, Hd4, and Hd5, respectively, which had been detected previously as QTLs for DTH in an F2 population of NipponbarexKasalath. Alleles of QTL(chr7) decreased DTH dramatically in both Hayamasari and Hoshinoyume, suggesting that QTL(chr7) has a major role in determining extremely early heading. In addition, allele-specific interactions were detected between QTL(chr6), QTL(chr7) and QTL(chr8). This result suggests that not only allelic differences but also epistatic interactions contribute to extremely early heading. QTL(chr8) was detected in HaF2, but not in HoF2, suggesting that it determines the difference in DTH between Hayamasari and Hoshinoyume. A major QTL was also detected in the region of QTL(chr8) in QTL analysis using an F2 population of HayamasarixHoshinoyume. This result supports the idea that QTL(chr8) is a major factor that determines the difference in DTH between Hayamasari and Hoshinoyume, and is involved in photoperiod sensitivity.

Accumulation of additive effects generates a strong photoperiod sensitivity in the extremely late-heading rice cultivar 'Nona Bokra'.

Many rice cultivars that originated from lower-latitude regions exhibit a strong photoperiod sensitivity (PS) and show extremely late heading under long-day conditions. Under natural day-length conditions during the cropping season in Japan, the indica rice cultivar 'Nona Bokra' from India showed extremely late heading (202 days to heading) compared to the japonica cultivar 'Koshihikari' (105 days), from Japan. To elucidate the genetic factors associated with such extremely late heading, we performed quantitative trait locus (QTL) analyses of heading date using an F(2) population and seven advanced backcross progeny (one BC(1)F(2) and six BC(2)F(2)) derived from a cross between 'Nona Bokra' and 'Koshihikari'. The analyses revealed 12 QTLs on seven chromosomes. The 'Nona Bokra' alleles of all QTLs contributed to an increase in heading date. Digenic interactions were rarely observed between QTLs. Based on the genetic parameters of the QTLs, such as additive effects and percentage of phenotypic variance explained, these 12 QTLs are likely generate a large proportion of the phenotypic variation observed in the heading dates between 'Nona Bokra' and 'Koshihikari'. Comparison of chromosomal locations between heading date QTLs detected in this study and QTLs previously identified in 'Nipponbare' x 'Kasalath' populations revealed that eight of the heading date QTLs were recognized nearby the Hd1, Hd2, Hd3a, Hd4, Hd5, Hd6, Hd9, and Hd13. These results suggest that the strong PS in 'Nona Bokra' was generated mainly by the accumulation of additive effects of particular alleles at previously identified QTLs.

Expression of the Pib rice-blast-resistance gene family is up-regulated by environmental conditions favouring infection and by chemical signals that trigger secondary plant defences.

The rice blast resistance gene Pib is a member of the nucleotide binding site (NBS) and leucine-rich repeat (LRR) class of plant disease resistance (R) genes and belongs to a small gene family. We describe here the isolation and characterization of a Pib homologue (PibH8), and extensive investigation of the expression of the Pib gene family (Pib, PibH8, HPibH8-1, HPibH8-2) under various environmental and chemical treatments. PibH8 shows 42% identity and 60% similarity to Pib and, like Pib, has a duplication of the kinase 1a, 2, and 3a motifs of the NBS region in the N-terminal half of the protein. Interestingly, genes of the Pib family exhibit a diurnal rhythm of expression. RNA gel blot analysis revealed that their expression was regulated dramatically by environmental signals. such as temperature, light and water availability. Their expression was also induced by chemical treatments, such as jasmonic acid, salicylic acid, ethylene and probenazole. Our findings suggest that expression of the Pib gene family is up-regulated by environmental conditions that would favour pathogen infection. This may reflect the evolution of anticipatory control of R gene expression.

Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS.

A major quantitative trait locus (QTL) controlling response to photoperiod, Hd1, was identified by means of a map-based cloning strategy. High-resolution mapping using 1505 segregants enabled us to define a genomic region of approximately 12 kb as a candidate for Hd1. Further analysis revealed that the Hd1 QTL corresponds to a gene that is a homolog of CONSTANS in Arabidopsis. Sequencing analysis revealed a 43-bp deletion in the first exon of the photoperiod sensitivity 1 (se1) mutant HS66 and a 433-bp insertion in the intron in mutant HS110. Se1 is allelic to the Hd1 QTL, as determined by analysis of two se1 mutants, HS66 and HS110. Genetic complementation analysis proved the function of the candidate gene. The amount of Hd1 mRNA was not greatly affected by a change in length of the photoperiod. We suggest that Hd1 functions in the promotion of heading under short-day conditions and in inhibition under long-day conditions.

Identification of cis-regulatory elements required for endosperm expression of the rice storage protein glutelin gene GluB-1.

Rice storage protein glutelin genes are coordinately regulated during seed development. A previous 5' deletion analysis using transgenic tobacco revealed that the minimum 5' region necessary for endosperm specificity was within -245 bp of the transcription start site, and included the AACA and GCN4 motifs that are highly conserved in the 5'-flanking regions of all glutelin genes. In this paper, the sequence elements essential for endosperm-specific expression are characterized in stable transgenic tobacco plants by both loss-of-function and gain-of-function experiments using this minimum promoter. Base substitution analysis shows that the proximal AACA motif between -73 and -61, and the GCN4 motif between -165 and -158 act as critical elements. An ACGT motif between -81 and -75, and Skn-I-like elements between -173 and -169 also play important roles in controlling the seed-specific expression. When the distal region between -245 and -145 containing the AACA and the GCN4 motifs or the proximal region between -113 and -46 containing the ACGT and AACA motifs is fused to a truncated promoter (-90 to +9) of the CaMV 35S gene fused to the beta-glucuronidase (GUS) reporter gene, high levels of seed-specific expression are observed in these fusions, thereby indicating that either pair of motifs is sufficient to confer seed expression in these fusions. However, when substituted for by the CaMV 35S core promoter (-46 to +1), seed expression is abolished, suggesting that the sequence between -90 and -46 of the CaMV 35S promoter containing G-box-like motif (as-1 element) is required for such specific expression in addition to AACA and GCN4 motifs. Therefore, we conclude that at least three cis-regulatory elements, the AACA motif, GCN4 motif and ACGT motif, are necessary to mediate endosperm expression of the GluB-1 glutelin gene.

The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes.

Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most serious diseases of rice. Here we describe the isolation and characterization of Pib, one of the rice blast resistance genes. The Pib gene was isolated by a map-based cloning strategy. The deduced amino acid sequence of the Pib gene product contains a nucleotide binding site (NBS) and leucine-rich repeats (LRRs); thus, Pib is a member of the NBS-LRR class of plant disease resistance genes. Interestingly, a duplication of the kinase 1a, 2 and 3a motifs of the NBS region was found in the N-terminal half of the Pib protein. In addition, eight cysteine residues are clustered in the middle of the LRRs, a feature which has not been reported for other R genes. Pib gene expression was induced upon altered environmental conditions, such as altered temperatures and darkness.

Expression of Xa1, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation.

The Xa1 gene in rice confers resistance to Japanese race 1 of Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight (BB). We isolated the Xa1 gene by a map-based cloning strategy. The deduced amino acid sequence of the Xa1 gene product contains nucleotide binding sites (NBS) and a new type of leucine-rich repeats (LRR); thus, Xa1 is a member of the NBS-LRR class of plant disease-resistance genes, but quite different from Xa21, another BB-resistance gene isolated from rice. Interestingly, Xa1 gene expression was induced on inoculation with a bacterial pathogen and wound, unlike other isolated resistance genes in plants, which show constitutive expression. The induced expression may be involved in enhancement of resistance against the pathogen.

Characterization of common cis-regulatory elements responsible for the endosperm-specific expression of members of the rice glutelin multigene family.

Glutelin is the most abundant storage protein in rice, which is expressed specifically in the endosperm of maturing seed. Glutelin is encoded by about 10 genes per haploid genome, which are clearly divided into two subfamilies (GluA and GluB). Most of them are coordinately expressed during seed maturation in spite of the remarkable divergence in the 5'-flanking regions between members of two subfamilies. In order to identify the common regulatory mechanisms responsible for the endosperm-specific expression, various cis-regulatory elements in the 5'-flanking region of the glutelin GluB-1 gene were characterized by studying the expression of chimeric genes that consisted of the sequentially deleted or mutagenized promoter and a beta-glucuronidase (GUS) reporter gene in transgenic tobacco seeds. The essential cis-regulatory elements governing the spatially and temporally specific expression of the glutelin gene expression were located within the first 245 bp of the promoter region of the GluB-1 gene from the site of initiation of transcription. The AACA motif between positions -73 and -61 common to all the six genes for glutelin sequenced to date and is repeated between positions -212 and -200 is implicated in the seed-specific expression. The GCN4 motif between positions -165 and -158 and between positions -96 and -92 that is conserved at homologous sites in all the members of glutelin gene family is also involved in the seed-specific regulation. However, both are required for the high level of seed-specific expression, because deletion of the region containing one set of both elements or substitution mutation of the AACA or GCN4 motif substantially reduced the activity. As a whole, our results suggest the combinatorial interaction of the elements in regulation of the glutelin gene expression.

[A mathematical model of the regulation mechanism of Ca2+ concentration in lens in vivo].

In order to elucidate the regulation mechanism of homeostasis on intracellular Ca2+ concentration in lens in vivo, the authors proposed a new mathematical model based upon the model of Ca2+ active transport. The mathematical model has new factors: Ca2+ efflux and Ca2+ influx across the cell membrane related to the difference between intracellular and extracellular Ca2+ concentration. Changes in intracellular Ca2+ concentration were investigated by numerical simulation using this model. Ca2+ efflux (active transport) is increased with intracellular Ca2+ concentration and their relationship corresponds to a Michaelis-Menten reaction. Ca2+ influx increases with the Ca2+ permeable coefficient of cell membrane. These results are consistent with the experimental results is a qualitative way and indicate that the model is suitable to elucidate the regulation mechanism of intracellular Ca2+ concentration. From the theoretical point of view, therefore, it is suggested that intracellular Ca2+ concentration may depend on two factors: one is the Ca2+ dependence of the Ca2+ active transport system, the other the Ca2+ permeability of the cell membrane.

[A theoretical study on the regulation mechanism of Ca2+ concentration in lens related to the Ca2+ dependence of Ca2(+)-ATPase activity].

A four-state model for the elementary process of the Ca2+ active transport system in lens was proposed, and based upon this model the Ca2+ dependence of Ca2(+)-ATPase activity was analyzed. The results indicated that the Ca2(+)-ATPase activity reaches a peak at approximately pCa 4-5, and decreases at higher and lower Ca2+ concentrations. In the range of pCa 5-6, ATPase activity increases with Ca2+ concentration. Therefore, the model accounts for the kinetic properties of Ca2(+)-ATPase is a qualitative way. Moreover, it is proved that the Ca2+ dependence of Ca2(+)-ATPase activity is theoretically based upon the changes in Ca2+ affinity of Ca2+ binding sites and the Ca2+ dependence of Ca2(+)-ATPase activity is possibly involved in the regulation of intracellular Ca2+ concentration in vivo as a switching mechanism.

Influence of sulfhydryl agents on cytoskeleton in cultured human trabecular cells.

The effects of three sulfhydryl agents on the cytoskeletal systems of cultured human trabecular cells were examined by the immunofluorescence method. Incubation with iodoacetamide or N-ethyl maleimide (10(-6) M, 10(-5) M, and 10(-4) M) caused a marked alteration on the actin and microtubule organization, but had little effect on the structural integrity of the vimentin intermediate filaments. Incubation with p-chloromercuribenzene sulfonate (10(-6) M, 10(-5) M, and 10(-4) M) caused no remarkable changes in the three major cytoskeletal systems. These findings suggest that the trabecular cell cytoskeleton plays an important role in increasing the aqueous outflow facility after treatment with the sulfhydryl agents, iodoacetamide and N-ethyl maleimide.

Immunofluorescence studies of the cytoskeletal and contractile elements in cultured human trabecular cells.

Characteristics of the distribution of cytoskeletal and contractile elements in cultured human trabecular cells were studied using the immunofluorescence method. Actin microfilaments exhibited stress fiber images which were observed throughout the cell body in a parallel pattern and were concentrated in the cell periphery. Myosin also revealed stress fibers with a distribution corresponding to actin distribution. Vimentin intermediate filaments and microtubules were stained in a filamentous pattern radiating from near the nucleus to the cell periphery. These intermediate filaments and microtubules were more densely and more abundantly observed throughout the cytoplasm in the cultured human trabecular cells than in the cultured human skin fibroblasts. These results suggest that the trabecular cell cytoskeleton plays an important role in maintaining the structural integrity and normal function of the trabecular meshwork.