Genetic analysis of floral anthocyanin pigmentation traits in Asiatic hybrid lily using molecular linkage maps.

To understand the genetic background of two floral anthocyanin pigmentation traits, anthocyanin pigmentation in the flower tepals and spot formation, in the Asiatic hybrid lily (2n = 24), segregation of the two traits among 96 F(1) plants derived from a cross between commercial cultivars 'Montreux' and 'Connecticut King' were investigated. 'Montreux' has anthocyanin pigmentation in the tepals with many spots, and 'Connecticut King' has flowers with carotenoid pigmentation without spots. The F(1) plants with or without anthocyanin pigment in the tepals segregated with a 1:1 segregation ratio, indicating that a single gene controls anthocyanin pigmentation in the tepals. The number of spots per square centimeter of all tepals showed continuous distribution in the F(1) plants. To map the loci for the two anthocyanin pigmentation traits, molecular linkage maps in the Asiatic hybrid lily were constructed using a double pseudo-testcross strategy, with the same F(1) plants used for phenotypic evaluation, and 212 PCR-based DNA markers. The trait for anthocyanin pigmentation in tepals was used as a trait marker. The map of 'Montreux' comprised 95 markers in 26 linkage groups, and the map of 'Connecticut King' used 119 markers in 24 linkage groups. The total map lengths were 867.5 and 1,114.8 cM, respectively. The trait locus for anthocyanin pigmentation in the tepals was between markers ASR35-180 and P506-40 in linkage group 1 of the 'Montreux' map with a map distance of 1.2 cM and 2.6 cM, respectively. A single-point analysis of quantitative trait loci (QTLs) for tepal spot number identified two putative QTLs in linkage groups 1 and 19 of the 'Connecticut King' map. One putative QTL in linkage group 19 explained 64% of the total phenotypic variation. Because both putative QTLs were mapped on the linkage map of 'Connecticut King' that has no spots, dominant alleles of them might suppress spot formation.

Production of dwarf lettuce by overexpressing a pumpkin gibberellin 20-oxidase gene.

We investigated the effect of overexpressing a pumpkin gibberellin (GA) 20-oxidase gene encoding an enzyme that forms predominantly biologically inactive products on GA biosynthesis and plant morphology in transgenic lettuce (Lactuca sativa cv Vanguard) plants. Lettuce was transformed with the pumpkin GA 20-oxidase gene downstream of a strong constitutive promoter cassette (El2-35S-Omega). The transgenic plants in which the pumpkin gene was detected by polymerase chain reaction were dwarfed in the T(2) generation, whereas transformants with a normal growth phenotype did not contain the transgene. The result of Southern-blot analysis showed that the transgene was integrated as a single copy; the plants segregated three dwarfs to one normal in the T(2) generation, indicating that the transgene was stable and dominant. The endogenous levels of GA(1) and GA(4) were reduced in the dwarfs, whereas large amounts of GA(17) and GA(25), which are inactive products of the pumpkin GA 20-oxidase, accumulated in these lines. These results indicate that a functional pumpkin GA 20-oxidase is expressed in the transgenic lettuce, resulting in a diversion of the normal pathway of GA biosynthesis to inactive products. Furthermore, this technique may be useful for controlling plant stature in other agricultural and horticultural species.

Endogenous gibberellins in immature seeds of Prunus persica L.: identification of GA(118), GA(119), GA(120), GA(121), GA(122) and GA(126).

The endogenous gibberellins in immature seeds of Prunus persica were analyzed by gas chromatography-mass spectrometry. Eleven known gibberellins, GA(3), GA(9), GA(17), GA(19), GA(30), GA(44), GA(61), GA(63), GA(87), GA(95) and GA(97) were identified. Additionally, several hitherto unknown gibberellins were detected and their putative structures were verified by synthesis of the authentic gibberellins. These gibberellins were then assigned trivial numbers, e.g. 1alpha-hydroxy GA(20) (GA(118)), 1alpha-hydroxy GA(9) (GA(119)), 1,2-didehydro GA(9) (GA(120)), 1,2-didehydro GA(70) (GA(121)), 1,2-didehydro GA(69) (GA(122)) and 1,2-didehydro GA(77) (GA(126)). GA(118) and GA(119) were the first 1alpha-hydroxy gibberellins identified from higher plants. The above profile of 1,2-didehydro gibberellins suggests that 1,2-dehydrogenation might occur prior to 3beta-hydroxylation in biosynthesis of GA(3), GA(30) and GA(87) in immature seeds of P. persica.

slender rice, a constitutive gibberellin response mutant, is caused by a null mutation of the SLR1 gene, an ortholog of the height-regulating gene GAI/RGA/RHT/D8.

The rice slender mutant (slr1-1) is caused by a single recessive mutation and results in a constitutive gibberellin (GA) response phenotype. The mutant elongates as if saturated with GAs. In this mutant, (1) elongation was unaffected by an inhibitor of GA biosynthesis, (2) GA-inducible alpha-amylase was produced by the aleurone layers without gibberellic acid application, and (3) endogenous GA content was lower than in the wild-type plant. These results indicate that the product of the SLR1 gene is an intermediate of the GA signal transduction pathway. SLR1 maps to OsGAI in rice and has significant homology with height-regulating genes, such as RHT-1Da in wheat, D8 in maize, and GAI and RGA in Arabidopsis. The GAI gene family is likely to encode transcriptional factors belonging to the GRAS gene superfamily. DNA sequence analysis revealed that the slr1-1 mutation is a single basepair deletion of the nuclear localization signal domain, resulting in a frameshift mutation that abolishes protein production. Furthermore, introduction of a 6-kb genomic DNA fragment containing the wild-type SLR1 gene into the slr1-1 mutant restored GA sensitivity to normal. These results indicate that the slr1-1 mutant is caused by a loss-of-function mutation of the SLR1 gene, which is an ortholog of GAI, RGA, RHT, and D8. We also succeeded in producing GA-insensitive dwarf rice by transforming wild-type rice with a modified SLR1 gene construct that has a 17-amino acid deletion affecting the DELLA region. Thus, we demonstrate opposite GA response phenotypes depending on the type of mutations in SLR1.

Malvidin 3-rutinoside as the pigment responsible for bract color in Curcuma alismatifolia.

Malvidin 3-rutinoside was the only anthocyanin identified from pink bracts of Curcuma alismatifolia cultivars. The concentration of malvidin 3-rutinoside in three cultivars increased as the intensity of the pink color in the bracts increased.

Cyclic malyl anthocyanins in Dianthus caryophyllus.

3,5-Di-O-(beta-glucopyranosyl) pelargonidin 6''-O-4,6'''-O-1-cyclic malate and a previously reported cyanidin equivalent, 3,5-di-O-(beta-glucopyranosyl) cyanidin 6''-O-4,6'''-O-1-cyclic malate were identified from petals of deep pink and red-purple flower cultivars of Dianthus caryophyllus, respectively.

Phenolic antioxidants from the leaves of Corchorus olitorius L.

Six phenolic antioxidative compounds [5-caffeoylquinic acid (chlorogenic acid), 3,5-dicaffeoylquinic acid, quercetin 3-galactoside, quercetin 3-glucoside, quercetin 3-(6-malonylglucoside), and quercetin 3-(6-malonylgalactoside) (tentative)] were identified from the leaves of Corchorus olitorius L. (moroheiya) by NMR and FAB-MS. The contents of these phenolic compounds, ascorbic acid, and alpha-tocopherol in C. olitorius leaves were determined, and their antioxidative activities were measured using the radical generator-initiated peroxidation of linoleic acid. The results obtained showed that 5-caffeoylquinic acid was a predominant phenolic antioxidant in C. olitorius leaves.

Anthocyanins in The Dark Purple Anthers of Tulipa gesneriana: Identification of Two Novel Delphinidin 3-O-(6-O-(Acetyl-α-Rhamnopyranosyl)-ß-Glucopyranosides).

Two novel anthocyanins, delphinidin 3-O-(6-O-(2-O-acetyl-α-rhamnopyranosyl)-ß-glucopyranoside) and delphinidin 3-O-(6-O-(3-O-acetyl-α-rhamnopyranosyl)-ß-glucopyranoside), were identified from the anthers of Tulipa gesneriana. These and delphinidin 3-O-(6-O-(α-rhamnopyranosyl)-ß-glucopyranoside) made up over 80% of the anthocyanin content in the dark purple anthers and could be responsible for the intense color of the anthers.

Identification of Endogenous Gibberellins in the Leaves and Xylem Sap of Tea Plants.

Endogenous gibberellins (GAs) in the young leaves and xylem sap of tea plants (Camellia sinensis L.) were analyzed by GC-MS. The following GAs were identified by comparing their mass spectra and KRIs with those of authentic specimens: GA9 and GA20 in the leaves; GA9, GA12, GA15, GA20, GA44, GA51 and GA53 in the xylem sap.

Over-expression of a tobacco homeobox gene, NTH15, decreases the expression of a gibberellin biosynthetic gene encoding GA 20-oxidase.

Ectopic expression of the homeobox gene, NTH15 (Nicotiana tabacum homeobox 15) in transgenic tobacco leads to abnormal leaf and flower morphology, accompanied by a decrease in the content of the active gibberellin, GA1. Quantitative analysis of intermediates in the GA biosynthetic pathway revealed that the step from GA19 to GA20 was blocked in transgenic tobacco plants overexpressing NTH15. To investigate the relationship between the expression of NTH15 and genes involved in GA biosynthesis, we isolated three cDNA clones from tobacco encoding two types of GA 20-oxidase and a 3 beta-hydroxylase. RNA gel blot analysis revealed that the expression of one gene (Ntc12, encoding GA 20-oxidase), which in wild-type tobacco plants was abundantly expressed in leaves, was strongly suppressed in the transformants. The expression level of Ntc12 decreased with increasing severity of phenotype of transgenic tobacco leaves. The abnormal leaf morphology was largely overcome by treatment with GA20 or GA1 but not by GA19. These data strongly suggest that overexpression of NTH15 inhibits the expression of Ntc12, resulting in reduced levels of active GA and abnormal leaf morphology in transgenic tobacco plants. In situ hybridization in wild-type tobacco revealed that expression of Ntc12 occurred mainly in the rib meristem, cells surrounding the procambium and in leaf primordia. Expression was not seen in the tunica, corpus and procambium, tissues in which NTH15 was predominantly expressed. The contrasting expression patterns of these genes may reflect their antagonistic functions in the formation of lateral organs from the shoot apical meristem.

Gibberellin Metabolism in Intact Plants of Raphanus sativus L.

The metabolism of gibberelline (GAs) in intact plants of Raphanus sativus was investigated. With [(2)H]GA feeds, [(2)H]GA1 from [(2)H]GA4, and [(2)H]GA4 and [(2)H]GA20 from [(2)H]GA9 were metabolized. Since [(2)H]GA20 was not converted into [(2)H]GA1, endogenous GA1 may have been biosynthesized from GA9 via GA4 rather than from GA20. The radioactivity of [(3)H]GA9 and [(3)H]GA20 was much more strongly transported among the plant organs than that of [(3)H]GA1 and [(3)H]GA4.

Nondwarf rice seedling bioassay for gibberellins.

The use of nondwarf rice (Oryza sativa L.) cultivars treated with uniconazole as test plants for gibberellin (GA) bioassay instead of Tan-ginbozu dwarf rice variant was investigated. The sensitivity of six nondwarf rice cultivars to GAs was increased substantially by treatment of the seeds with uniconazole. The minimum detectable dose of a GA in the nondwarf cultivars treated with uniconazole was 1- to 1/10-fold of that in the nontreated Tanginbozu and 3- to 10-fold of that in uniconazole-treated Tanginbozu. The relative activity of several GAs on treated nondwarf rice cultivars was not largely different from that to Tan-ginbozu. Considering that seeds of nondwarf rice are available commercially, the nondwarf rice seedling assay would be useful as a simple assay for systematic analysis of GAs, and also as a routine teaching tool in high schools and universities.

Pattern formation and flow control of fine particles by laser-scanning micromanipulation.

A novel micromanipulation technique is proposed for aligning fine particles on micrometer-scale spatial patterns and for moving the particles continuously along the formed patterns. This technique is based on the repetitive scanning of a focused trapping laser beam. The velocity of the particle flow can be controlled by scan speed and laser power. The origin of the driving force is considered theoretically and experimentally.

Metabolism of [H]Gibberellin A(5) by Immature Seeds of Apricot (Prunus armeniaca L.).

Immature seeds of apricot (Prunus armeniaca L.) were fed the native gibberellin A(5) (GA(5)) as 1- and 1,2-[(3)H]GA(5) (5.3 Curies per millimole to 16 milliCuries per millimole) at doses (42 nanograms to 10.6 micrograms per seed) 2 to 530 times the expected endogenous level. After 4 days of incubation, seeds were extracted and free [(3)H]GA-like metabolites were separated from the highly H(2)O-soluble [(3)H]metabolites. For high specific activity feeds the retention times (Rts) of radioactive peaks were compared with Rts of authentic GAs on sequential gradient-eluted --> isocratic eluted reversed-phase C(18) high performance liquid chromatography (HPLC) -radiocounting (RC). From high substrate feeds (530 and 230 x expected endogenous levels) HPLC-RC peak groupings were subjected to capillary gas chromatography-selected ion monitoring (GC-SIM), usually six characteristic ions. The major free GA metabolites of [(3)H] GA(5) were identified as GA(1), GA(3), and GA(6) by GC-SIM. The major highly water soluble metabolite of [(3)H]GA(5) at all levels of substrate GA(5) had chromatographic characteristics similar to authentic GA(1)-glucosyl ester. Expressed as a percentage of recovered radioactivity, low substrate [(3)H]GA(5) feeds (2 x expected endogenous level) yielded a broad spectrum of metabolites eluting at the Rts where GA(1), GA(3), GA(5) methyl ester, GA(6), GA(22), GA(29) (17, 14, 1.6, 7, 1.1, 0.5%, respectively) and GA glucosyl conjugates of GA(1), GA(3), GA(5), and GA(8) (33, 11, 1, 0.1%, respectively) elute. Metabolites were also present at Rts where GA glucosyl conjugates of GA(6) and GA(29) would be expected to elute (8 and 0.1%, respectively). Only 5% of the radioactivity remained as GA(5). Increasing substrate GA(5) levels increased the proportion of metabolites with HPLC Rts similar to GA(1), GA(6), and especially GA(1) glucosyl ester, primarily at the expense of metabolites with HPLC Rts similar to GA(3), GA(3)-glucosyl ester, and a postulated conjugate of GA(6). There was evidence that high doses of substrate GA(5) induced new metabolites which often, but not always, differed from GA(1), GA(3), and GA(6) in HPLC Rt. These same metabolites, when analyzed by GC-SIM yielded m/e ions the same as the M(+) and other characteristic m/e ions of the above GAs, albeit at differing GC Rt and relative intensities.

Identification of Gibberellins A(1), A(5), A(29), and A(32) from Immature Seeds of Apricot (Prunus armeniaca L.).

Gibberellins (GAs) A(1), A(5), and A(29) were identified, and also GA(32) was confirmed, as endogenous GAs of immature seeds (3-4 weeks after anthesis, 0.25-0.5 gram fresh weight) of apricot (Prunus armeniaca L.) based on capillary gas chromatography (GC), retention time (Rt), and selected ion monitoring (SIM), in comparison with authentic standards. Fractions subjected to GC-SIM were purified and separated using sequential solvent partitioning --> paper chromatography --> reverse phase C(18) high performance liquid chromatography (HPLC) --> bioassay on dwarf rice cv Tan-ginbozu. Two other peaks of free GA-like bioactivity (microdrop and immersion dwarf rice assays) were eluted from C(18) HPLC at Rts where GA(4/7) and GA(8) (or other GAs with similar structures) would elute. Also, three unidentified GA glucoside-like compounds (based on bioactivity on the immersion assay, and no bioactivity on the microdrop assay) were noted. There were very high amounts of GA(32) (112 ng of GA(3) equivalents per gram fresh weight), and minor amounts (0.5 ng of GA(3) equivalents) for each of GA(1) and GA(5), respectively, based on the microdrop assay.

Changes in Cytokinins and Gibberellin-Like Substances in Pinus radiata Buds during Lateral Shoot Initiation and the Characterization of Ribosyl Zeatin and a Novel Ribosyl Zeatin Glycoside.

Based on detection and quantitation by bioassay, endogenous gibberellin-like substances (GAs) and cytokinins (CKs) in Pinus radiata D. Don buds during sequential shoot initiation shift from less polar to more polar forms (GAs) and from conjugated to free forms (CKs). As the terminal bud moves from the production of "short shoots" (needle fascicles) to "long shoots" (lateral branches or female conebuds), a more polar GA appears while a glucoside-conjugate of zeatin riboside is reduced, and zeatin riboside levels increase markedly.Permethyl derivatives of the two highly active CK fractions were examined by capillary gas chromatography-mass spectrometry after separation by C(18) reverse phase high performance liquid chromatography. The mass spectra indicated the presence of: 9-beta-d-ribofuranosyl-6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin riboside) and 9-[hexosyl(probably glucosyl)-beta-d-ribofuranosyl]-6-(4-hydroxy-3-methyl-but-2- enylamino)purine (a glycoside of zeatin riboside in which the glycosyl moiety is attached directly to the ribosyl moiety at an unknown position).

Endogenous Gibberellin-Like Substances in Somatic Embryos of Grape (Vitis vinifera x Vitis rupestris) in Relation to Embryogenesis and the Chilling Requirement for Subsequent Development of Mature Embryos.

Endogenous gibberellin (GA)-like substances were examined in suspension cultures of somatic embryos of a hybrid grape (Vitis vinifera x Vitis rupestris) during embryogenesis, and in mature embryos chilled at 4 degrees C, and subsequently incubated at 26 degrees C with and without abscisic acid (ABA). The extract was separated into a nonpolar fraction (would contain GA-precursors); a fraction that would contain free GAs; and a highly H(2)O-soluble fraction (would contain GA glucosyl conjugates and very polar free GAs). Quantitation after SiO(2) partition chromatography was accomplished by microdrop and immersion dwarf rice bioassays. As embryogenesis developed, the free and highly H(2)O-soluble GA-like substances, expressed on a dry weight basis, decreased (however, they increased on a per embryo basis). Chilling at 4 degrees C for 1 week greatly increased activity of free GA-like substances (per g dry weight and per embryo), it then declined over the next three weeks of chilling. Activity (per g dry weight and per embryo) in the H(2)O-soluble fraction declined throughout chilling. Activity in the GA-precursor fraction, however, increased steadily with chilling (per g dry weight and per embryo). Incubation at 26 degrees C after chilling enhanced activity in the free GA and H(2)O-soluble fractions (per g dry weight and per embryo), but activity in the GA-precursor fraction dropped dramatically. Incubation at 26 degrees C with (+/-) ABA after chilling prevented germination and maintained high activity for GA precursors and less polar free GAs and low activity in the polar free GA and H(2)O-soluble fractions.Kaurene and kaurenoic acid were characterized in the GA-precursor fraction of chilled embryos by gas-liquid chromatography-mass spectrometry (GLC-MS). The existence of GA(4) and GA(9) in ABA-treated, chilled embryos was also confirmed by GLC-MS.

Reversible conjugation of gibberellins in situ in maize.

Gibberellins [(3)H]GA(4) (1.33 Curies per millimole) and [(3)H]GA(20) (2.36 Curies per millimole) were injected into the shanks of maize (Zea mays L.) cobs during rapid grain filling and mature seeds were subsequently harvested. Extracts of mature, dry seeds from 1980 feeds yielded only 20 to 30% of the (3)H radioactivity in acidic, ethyl acetate-soluble form, and this was principally associated with the precursor, with lesser amounts of the major metabolite, [(3)H]GA(1) (putative identification based on sequential SiO(2) partition, and gradient-eluted reverse-phase C(18) high performance liquid chromatography [HPLC]). Most of the radioactivity in the dry seeds was associated with compounds having partition characteristics of, and co-chromatographing on, sequential SiO(2) partition and reverse-phase HPLC with glucosyl conjugates of the precursors (GA(4) or GA(20)) and their probable major metabolite (GA(1)). The majority of conjugate associated with the precursor GA(4) eluted coincidental with GA(4) glucoside. Subsequent acid or enzymic hydrolysis (beta-glucosidase or cellulase) yielded the free GAs, putative identification being based on isocratic HPLC of each (3)H-labeled conjugate --> hydrolysis --> isocratic HPLC of the (3)H-labeled hydrolysate. Upon imbibition of the seeds, radioactivity associated with the conjugate fraction decreased; concomitantly, statistically significant increases in levels of free [(3)H]GA-like compounds were observed. Although the specific ratios of GA-like and GA-glucosyl conjugate-like substances varied substantially across years, hybrids, and even, in different plants from the same hybrid, this ;reversible conjugation' (i.e. apparent conjugation during seed maturation followed by release of the GA moiety during germination), was reproducible for [(3)H]GA(20) in seed from two maize hybrids produced over 2 years.

Purification and separation of plant gibberellins from their precursors and glucosyl conjugates.

A procedure using two small preparative columns (in sequence) of C(18) reverse phase Bondapak B material with methanolic extracts of plant tissue (Pisum sativum L., Malus domestica Borkh., Pimpinella anisum L.) yields two fractions: (i) gibberellin (GA) precursors, and (ii) free GA/GA methyl esters (GA-Me)/GA glucosyl conjugates. The discrete separation of (iii) free GA/GA-Me from (iv) GA glucosyl conjugates is then accomplished by a combination of differential solvent solubility and SiO(2) partition chromatography. All fractions are almost pigment free, and appreciable dry weight purification was accomplished for the GA precursor and free GA/GA-Me fractions. Solvent volumes can be kept low, no buffer salts are introduced, and each fraction (i, iii, iv) can be subjected directly to preparative or analytical reverse phase C(18) high performance liquid chromatography without recourse to solvent partitioning, and often without further purification.