Masumi Yamagishi

Sugar signalling mediates cluster root formation and phosphorus starvation-induced gene expression in white lupin

Cluster root (CR) formation contributes much to the adaptation to phosphorus (P) deficiency. CR formation by white lupin (Lupinus albus L.) is affected by the P-limiting level in shoots, but not in roots. Thus, shoot-derived signals have been expected to transmit the message of P-deficiency to stimulate CR formation. In this study, it is shown that sugars are required for a response to P starvation including CR formation and the expression of P starvation-induced genes. White lupin plants were grown in vitro on P-sufficient or P-deficient media supplemented with sucrose for 4 weeks. Sucrose supply stimulated CR formation in plants on both P-sufficient and P-deficient media, but no CR appeared on the P-sufficient medium without sucrose. Glucose and fructose also stimulated CR formation on the P-sufficient medium. On the medium with sucrose, a high concentration of inorganic phosphate in leaves did not suppress CR formation. Because sorbitol or organic acid in the media did not stimulate CR formation, the sucrose effect was not due to increased osmotic pressure or enriched energy source, that is, sucrose acted as a signal. Gene transcription induced by P starvation, LaPT1 and LaPEPC3, was magnified by the combination of P limitation and sucrose feeding, and that of LaSAP was stimulated by sucrose supply independently of P supply. These results suggest that at least two sugar-signalling mediating systems control P starvation responses in white lupin roots. One system regulates CR formation and LaSAP expression, which acts even when P is sufficient if roots receive sugar as a signal. The other system controls LaPT1 and LaPEPC3 expression, which acts when P is insufficient.

Spatial and temporal expression of chalcone synthase and dihydroflavonol 4-reductase genes in the Asiatic hybrid lily

Abstract cDNA clones of three chalcone synthase (CHS) genes (LhCHSA, LhCHSB and LhCHSC) and one dihydroflavonol 4-reductase (DFR) gene (LhDFR) were isolated, and spatial and temporal expression of these genes was analyzed for two Asiatic hybrid lily cultivars, ‘Montreux’ (pink tepals with spots) and ‘Connecticut King’ (yellow tepals without spots). Pink tepals and tepal spots contain the same kind of anthocyanin, and yellow tepals have no anthocyanin. In both cultivars, the expression of all four genes was not detected in unpigmented leaves, stems and white bulbscales, but was detected in anthocyanin pigmentation-induced bulbscales, indicating that these genes are transcriptionally active in both cultivars. In flower organs, three CHS genes were expressed in anthocyanin-pigmented tepals, filaments and pistils of ‘Montreux’, and in unpigmented filaments and pistils of ‘Connecticut King’. Pigmented anthers accumulated only LhCHSC mRNA in both cultivars. LhDFR was abundantly expressed in pigmented tepals, anthers, filaments and pistils of ‘Montreux’, and in pigmented anthers of ‘Connecticut King’. These results indicate that different expression of these genes, especially of the LhDFR gene, controls the distinct pigmentation pattern in flower organs in both cultivars. In three F1 plants with many spots on yellow or white tepals, derived from a cross between ‘Montreux’ and ‘Connecticut King’, anthocyanin accumulation in tepal spots was accompanied by the LhCHSA and LhDFR expression, indicating that the expression of CHS and DFR genes is independently controlled between tepals and tepal spots.

PCR-based molecular markers in Asiatic hybrid lily

PCR-based DNA markers in Asiatic hybrid lily were examined. Random primers with various lengths (10-, 12-, 15- and 20-base) were tested in randomly amplified polymorphic DNA (RAPD) reactions with total DNA from two cultivars, ‘Montreux’ and ‘Connecticut King’. Thirty-three out of 61 (54%) 15-base primers and 14 out of 21 (67%) 20-base primers generated polymorphic fragments whereas 17 out of 145 (12%) and 4 out of 24 (17%) of the 10- and 12-base primers, respectively, amplified them. The efficiency of the RAPD reaction increased with increasing primer length. We examined inter-simple sequence repeat (ISSR) reactions using 3 0 -anchored simple sequence repeat (ASSR) primers, and found 33 out of 63 (52%) primers that amplified polymorphic bands between the two cultivars. We repeated all RAPD and ISSR reactions at least two times and obtained the same DNA fingerprints, indicating high reproducibility obtained with these reactions. All polymorphic fragments were inherited into their F1 hybrids, indicating that these DNA markers are genetically stable. # 2002 Elsevier Science B.V. All rights reserved.

Oriental hybrid lily Sorbonne homologue of LhMYB12 regulates anthocyanin biosyntheses in flower tepals and tepal spots

Anthocyanins are the predominant visible pigments in flowers of the Oriental hybrid lily (Lilium spp.). To understand the regulatory mechanisms of flower coloration in this hybrid lily, full-length cDNA of the R2R3-MYB gene, LhSorMYB12, was isolated from anthocyanin-accumulating tepals of the cultivar Sorbonne. The deduced amino acid sequence of LhSorMYB12 showed 85% (209/246) identity, including a gap of seven amino acids, with an amino acid sequence in LhMonMYB12 that regulates anthocyanin biosynthesis in the Asiatic hybrid lily. Transcripts of LhSorMYB12 accumulated in anthocyanin-accumulating tepals of three cultivars but not in white tepals of two other cultivars. The white-tepal cultivar Rialto showed LhMYB12 transcription in tepals, but the Rialto sequence contained amino acid replacements in the R2 repeat, resulting in reduced transcription of anthocyanin biosynthesis genes and in no pigmentation. Spatial and temporal transcription of LhSorMYB12 showed that transcription of LhMYB12 corresponded well with transcription of anthocyanin biosynthesis genes in tepal regions where anthocyanins accumulated and in anthocyanin spots on tepals. These results indicate that LhSorMYB12 definitely regulates anthocyanin biosynthesis and is responsible for the tepal color difference between Oriental hybrid lily cultivars.

QTL analysis for panicle characteristics in temperate japonica rice

To understand the genetic background of panicle characteristics in temperate japonica rice (Oryza sativa L.), we genetically analyzed DH lines derived from a cross between two temperate japonica rice cultivars, ‘Akihikari’ and ‘Koshihikari’,in 1996 and 1997. Four traits of panicle characteristics, number of primary branches per panicle (NPB), number of secondary branches per panicle (NSB), average number of spikelets on one primary branch (NSP)and average number of spikelets on one secondary branch (NSS), in 212 DH lines were measured, and the interval mapping of QTLs for these traits was carried out using169 DNA markers with an LOD threshold of2.5. Five, three and one putative QTLs for NPB, NSB and NSS were identified,respectively, and no QTLs relating to NSP appeared. The percentages in total phenotypic variation explained by all putative QTLs for NPB were 35.5%: and43.8% in 1996 and 1997, respectively. All putative QTLs for NSB accounted for 35.5%and 27.5% of total phenotypic variation in1996 and 1997, respectively. The QTLs identified in this study will be useful intemperate japonica rice breeding for improved spikelet yield.

A review of target gene specificity of flavonoid R2R3-MYB transcription factors and a discussion of factors contributing to the target gene selectivity

Flavonoid biosynthetic genes are often coordinately regulated in a temporal manner during flower or fruit development, resulting in specific accumulation profiles of flavonoid compounds. R2R3-MYB-type transcription factors (TFs) “recruit” a set of biosynthetic genes to produce flavonoids, and, therefore, R2R3-MYBs are responsible for the coordinated expression of structural genes. Although a wealth of information regarding the identified and functionally characterized R2R3-MYBs that are involved in flavonoid accumulation is available to date, this is the first review on the global regulation of MYB factors in the flavonoid pathway. The data presented in this review demonstrate that anthocyanin, flavone/flavonol/3-deoxyflavonoid (FFD), proanthocyanidin (PA), and isoflavonoid are independently regulated by different subgroups of R2R3-MYBs. Furthermore, FFD-specific R2R3-MYBs have a preference for early biosynthetic genes (EBGs) as their target genes; anthocyanin-specific R2R3-MYBs from dicot species essentially regulate late biosynthetic genes (LBGs); the remaining R2R3-MYBs have a wider range of target gene specificity. To elucidate the nature of the differential target gene specificity between R2R3-MYBs, we analyzed the DNA binding domain (also termed the MYB-domain) of R2R3-MYBs and the distribution of the recognition cis-elements. We identified four conserved amino acid residues located in or just before helix-3 of dicot anthocyanin R2R3-MYBs that might account for the different recognition DNA sequence and subsequently the different target gene specificity to the remaining R2R3-MYB TFs.

Characterization of segregation distortion on chromosome 3 induced in wide hybridization between indica and japonica type rice varieties

AbstractWe previously surveyed chromosomal regions showing segregation distortion of RFLP markers in the F2 population from the cross between a japonica type variety ‘Nipponbare’ and an indica type variety ‘Milyang23’, and showed that the most skewed segregation appeared on the short arm of chromosome 3. By comparison with the marker loci where distortion factors were previously identified, this region was assumed to be a gametophytic selection-2 (ga2) gene region. To evaluate this region, two near isogenic lines (NILs) were developed. One NIL had the ‘Nipponbare’ segment of this region on the genetic background of ‘Milyang23’ (NIL9-23), and the other NIL had the ‘Milyang23’ segment on the genetic background of ‘Nipponbare’ (NIL33-18). NIL9-23 and ‘Milyang23’, NIL33-18 and ‘Nipponbare’, and ‘Nipponbare’ and ‘Milyang23’ were respectively crossed to produce F1 and F2 populations. The F1 plants of NIL9-23 × ‘Milyang23’ and NIL33-18 × ‘Nipponbare’ showed high seed fertility and the same pollen fertility as their parental cultivars, indicating that ga2 does not reduce seed and pollen fertility. Segregation ratio of a molecular marker on the ga2 region in the three F2 populations was investigated to clarify whether segregation distortion occurred on the different genetic backgrounds. Segregation distortion of the ga2 region appeared in the both F2 populations from the NIL9-23 and ‘Milyang23’ cross (background was ‘Milyang23’ homozygote) and the ‘Nipponbare’ and ‘Milyang23’ cross (background was heterozygote), but did notin the F2 population from the NIL33-18 and ‘Nipponbare’ cross (background was ‘Nipponbare’ homozygote). This result indicates that ga2 interacts with a ‘Milyang23’ allele(s) on the different chromosomal region(s) to cause skewed segregation of the ga2 region. In addition, segregation ratio was the same between the F2 populations from NIL9-23 × ‘Milyang23’ and ‘Nipponbare’ × ‘Milyang23’ crosses, suggesting that the both genotypes, ‘Milyang23’ homozygote and heterozygote, of gene(s) located on the different chromosomal region(s) have the same effect on the segregation distortion.

Chromosomal regions controlling anther culturability in rice (Oryza sativa L.)

Diallel analysis has revealed that anther culturability in rice (Oryza sativa L.) is a quantitative trait controlled by the nuclear genome. Mapping of anther culturability is important to increase the efficiency for green plant regeneration from microspores. In the previous study, we detected distorted segregation of RFLP markers in rice populations derived from the anther culture of an F1 hybrid between a japonica cultivar ‘Nipponbare’ and an indica cultivar ‘Milyang 23’. To clarify the association between chromosomal regions showing distorted segregation and anther culturability, the anther culturability of doubled haploid lines derived from the same cross combination was examined, and the association between alleles of the RFLP markers, which exhibiting the most distorted segregation on chromosomes 1, 3, 7, 10 and 11, and the anther culturability was evaluated. One region on chromosome 1 was found to control callus formation from microspores, and one region on chromosome 10 appeared to control the ratio of green to albino regenerated plants. In both regions, the Nipponbare allele had positive effects. Three regions on chromosomes 3, 7 and 11, however, showed no significant effect on anther culturability.

Pigment accumulation and transcription of LhMYB12 and anthocyanin biosynthesis genes during flower development in the Asiatic hybrid lily (Lilium spp.).

Anthocyanin biosynthesis is often regulated by MYB transcription factors that are classified into AN2 and C1 subgroups. The AN2 subgroup regulates the late genes in the anthocyanin biosynthesis pathway of eudicots, whereas the C1 subgroup controls both early and late genes in monocots. Anthocyanin is a major pigment in Asiatic hybrid lilies (Lilium spp.), with LhMYB12 being the first AN2 subgroup in monocots. In this study, the accumulation of pigments and gene transcripts during flower bud development was evaluated to determine the genes regulated by LhMYB12. LhMYB12 and anthocyanin biosynthesis genes showed the same transcription profiles, with LhMYB12 directly activating the promoters of chalcone synthase and dihydroflavonol 4-reductase. This indicates that LhMYB12 regulates both early and late genes, despite belonging to the AN2 subgroup. The cultivar Landini accumulated anthocyanin and flavonol. The contents of these pigments increased during the late stages of flower bud development; this might result from the coordinated expression of early and late genes. During the early stages of flower bud development, the tepals contained no flavonoids but accumulated cinnamic acid derivatives. These results indicate that the profiles of pigment accumulation and gene transcription in lily tepals are unique among angiosperm flowers.

Effects of culture temperature on the enlargement, sugar uptake, starch accumulation, and respiration of in vitro bulblets of Lilium japonicum Thunb.

The effects of culture temperature on the enlargement, sugar uptake, starch accumulation and respiration of in vitro bulblets of Lilium japonicum were investigated. Enlargement of bulblets was higher at a culture temperature of 20°C than at 15°C or 26°C. Sugar uptake from the medium was at a maximum at 20°C. Respiration of the bulblets increased with increasing temperature. Starch concentrations were similar in the bulblets cultured at 15°C, 20°C and 26°C. These findings indicate that the suppression of bulblet enlargement at a high culture temperature was accompanied by both reduced sugar uptake from the medium and the increased consumption of accumulated carbon compounds by respiration.