A Modified Method for Transient Transformation via Pollen Magnetofection in Lilium Germplasm.

Lily (Lilium spp.) is a popular ornamental plant. Traditional genetic transformation methods have low efficiency in lily, thus development of a high-efficiency genetic transformation system is important. In this study, a novel transient transformation method involving pollen magnetofection was established and optimized pollen viability, and exogenous gene expression in magnetofected pollen and that of different germplasm were assessed. The highest germination percentage of Lilium regale pollen was 85.73% in medium containing 100 g/L sucrose, 61.5 mg/L H3BO3, and 91.5 mg/L CaCl2. A 1:4 ratio of nanomagnetic beads to DNA plasmid and transformation time of 0.5 h realized the highest transformation efficiency (88.32%). The GFP activity in transformed pollen averaged 69.66%, while that of the control pollen was 0.00%. In contrast to the control, transgenic seedlings obtained by pollination with magnetofected pollen showed strong positive GUS activity with 56.34% transformation efficiency. Among the lily germplasm tested, 'Sweet Surrender' and L. leucanthum had the highest transformation efficiency (85.80% and 54.47%), whereas L. davidii var. willmottiae was not successfully transformed. Transformation efficiency was positively correlated with pollen equatorial diameter and negatively correlated with polar axis/equatorial diameter ratio. The results suggest that pollen magnetofection-mediated transformation can be applied in Lilium but might have species or cultivar specificity.

Comprehensive 16S rDNA Sequencing and LC-MS/MS-Based Metabolomics to Investigate Intestinal Flora and Metabolic Profiles of the Serum, Hypothalamus and Hippocampus in p­Chlorophenylalanine-Induced Insomnia Rats Treated with Lilium brownie.

Gut microbiota homeostasis in the organism and insomnia have been reported to influence each other. In the study, a method of 16S rRNA gene sequencing combined with ultra-high performance liquid chromatography-mass/mass spectrometry was adopted to evaluate the effects of Lilium brownie (LB) on intestinal flora and metabolic profiles of serum, hypothalamus and hippocampus in insomnia rat induced by p­chlorophenylalanine (PCPA). It was observed that the imbalance in the diversity and abundance of gut microbiota induced by PCPA was restored after LB intervention. Among these, the Porphyromonadaceae, Lactobacillus and Escherichia were significantly adjusted at the genus level by PCPA and LB, respectively. It was also found that the most of metabolic phenotypes in serum, hypothalamus and hippocampus perturbed by PCPA were regulated towards normal after LB intervention, especially 5-hydroxy-L-tryptophan of the hypothalamus involving in 5-HT metabolism. Moreover, the arachidonic acid metabolism in serum, hypothalamus and hippocampus, and the serotonergic synapse in hypothalamus and hippocampus were the most fundamentally and significantly affected pathways after LB intervention. The results of correlation analysis showed that several floras including Pseudoruegeria have an outstanding contribution to the change of differential metabolites. In brief, the results confirm that gut microbiota is significantly returned to normal and may interact with the corresponding metabolites to relieve insomnia under LB intervention.

Transcriptome and Metabolome Analyses Provide Insights into the Stomium Degeneration Mechanism in Lily.

Lily (Lilium spp.) is a widely cultivated horticultural crop that has high ornamental and commercial value but also the serious problem of pollen pollution. However, mechanisms of anther dehiscence in lily remain largely unknown. In this study, the morphological characteristics of the stomium zone (SZ) from different developmental stages of 'Siberia' lily anthers were investigated. In addition, transcriptomic and metabolomic data were analyzed to identify the differentially expressed genes (DEGs) and secondary metabolites involved in stomium degeneration. According to morphological observations, SZ lysis occurred when flower buds were 6-8 cm in length and was completed in 9 cm. Transcriptomic analysis identified the genes involved in SZ degeneration, including those associated with hormone signal transduction, cell structure, reactive oxygen species (ROS), and transcription factors. A weighted co-expression network showed strong correlations between transcription factors. In addition, TUNEL (TdT-mediated dUTP nick-end labeling) assays showed that programmed cell death was important during anther SZ degeneration. Jasmonates might also have key roles in anther dehiscence by affecting the expression of the genes involved in pectin lysis, water transport, and cysteine protease. Collectively, the results of this study improve our understanding of anther dehiscence in lily and provide a data platform from which the molecular mechanisms of SZ degeneration can be revealed.

Developing an efficient DNA barcoding system to differentiate between Lilium species.

BACKGROUND: Lilium is an important ornamental bulb, possesses medicinal properties, and is also edible. Species within the Lilium genus share very similar morphology and macroscopic characteristics, thus they cannot be easily and clearly distinguished from one another. To date, no efficient species-specific markers have been developed for classifying wild lily species, which poses an issue with further characterizing its medicinal properties. RESULTS: To develop a simple and reliable identification system for Lilium, 45 representative species from 6 sections were used to develop a DNA barcoding system, which was based on DNA sequence polymorphisms. In this study, we assessed five commonly used DNA barcode candidates (ITS, rbcL, ycf1b, matK and psbA-trnH) and five novel barcode candidates obtained from highly variable chloroplast genomic regions (trnL-trnF, trnS-trnG, trnF-ndhJ, trnP-psaJ-rpI33 and psbB-psbH). We showed that a set of three novel DNA barcodes (ITS + trnP-psaJ-rpI33 + psbB-psbH) could be efficiently used as a genetic marker to distinguish between lily species, as assessed by methods including DNAsp, BI and ML tree, and Pair Wise Group (PWG). CONCLUSIONS: A rapid and reliable DNA barcoding method was developed for all 45 wild Lilium species by using ITS, trnP-psaJ-rpI33, and psbB-psbH as DNA barcoding markers. The method can be used in the classification of wild Lilium species, especially endangered species, and also provides an effective method for selective lily breeding.

Cloning and Functional Characterization of a Flavonoid Transport-Related MATE Gene in Asiatic Hybrid Lilies (Lilium spp.).

Previous studies have suggested that multidrug and toxic compound extrusion (MATE) proteins might be involved in flavonoid transportation. However, whether MATE proteins are involved in anthocyanin accumulation in Lilium is unclear. Here, a flavonoid transport-related MATE candidate gene, LhDTX35, was cloned from the Asiatic hybrid lily cultivar 'Tiny Padhye' by rapid amplification of 5' and 3' cDNA ends (RACE) and found to encode 507 amino acids. BLASTx results indicated that LhDTX35 showed high homology to the DTX35 genes of other species. Bioinformatics analysis predicted that the protein encoded by LhDTX35 possessed 12 typical transmembrane segments and had functional domains typical of the MATE-like superfamily. Phylogenetic analysis grouped LhDTX35 in the same clade as the DTX35 of other species. Notably, the expression pattern of LhDTX35 was positively correlated with floral anthocyanin accumulation in 'Tiny Padhye'. A subcellular localization assay showed that the protein encoded by LhDTX35 was plasmalemma localized but not nuclear, indicating that the LhDTX35 gene may function as a carrier protein to transport anthocyanins in Lilium. Functional complementation of the ArabidopsisDTX35 gene demonstrated that LhDTX35 could restore silique-infertility and the anthocyaninless phenotype of an ArabidopsisDTX35 mutant. These results indicated that LhDTX35 might be involved in anthocyanin accumulation in Lilium.

Physiological and transcriptomic responses of Lanzhou Lily (Lilium davidii, var. unicolor) to cold stress.

Low temperature induces changes in plants at physiological and molecular levels, thus affecting growth and development. The Lanzhou lily (Lilium davidii, var. unicolor) is an important medicinal plant with high economic value. However, the molecular mechanisms underlying its photosynthetic and antioxidation responses to low temperature still remain poorly understood. This study subjected the Lanzhou lily to the two temperatures of 20°C (control) and 4°C (low temperature) for 24 h. Physiological parameters related to membrane integrity, photosynthesis, antioxidant system, and differentially expressed genes were investigated. Compared with control, low temperature increased the relative electrical conductivity by 43.2%, while it decreased net photosynthesis rate, ratio of variable to maximal fluorescence, and catalase activity by 47.3%, 10.1%, and 11.1%, respectively. In addition, low temperature significantly increased the content of soluble protein, soluble sugar, and proline, as well as the activity of superoxide dismutase and peroxidase. Comparative transcriptome profiling showed that a total of 238,109 differentially expressed genes were detected. Among these, 3,566 were significantly upregulated while 2,982 were significantly downregulated in response to low temperature. Gene Ontology enrichment analysis indicated that in response to low temperature, the mostly significantly enriched differentially expressed genes were mainly involved in phosphorylation, membrane and protein kinase activity, as well as photosynthesis, light harvesting, light reaction, and alpha,alpha-trehalose-phosphate synthase activity. Kyoto Encyclopedia of Genes and Genomes enrichment analysis also indicated that the most significantly enriched pathways involved ribosome biogenesis in eukaryotes, phenylalanine metabolism, circadian rhythm, porphyrin and chlorophyll metabolism, photosynthesis of antenna proteins, photosynthesis, and carbon fixation in photosynthetic organisms. Moreover, the expression patterns of 10 randomly selected differentially expressed genes confirmed the RNA-Seq results. These results expand the understanding of the physiological and molecular mechanisms underlying the response of the Lanzhou lily to low temperature stress.

Analysis of Pollen Allergens in Lily by Transcriptome and Proteome Data.

The lily (Lilium spp.) anther contains a lot of pollen. It is not known if lily pollen contains allergens, and therefore screening pollen allergy-related proteins and genes is necessary. The pollen development period of lily 'Siberia' was determined by microscope observation. Early mononuclear microspores and mature pollens were used as sequencing materials. The analysis of the pollen transcriptome identified differentially expressed genes (DEGs), e.g., Profilin, Phl p 7 (Polcalcin), Ole e 1, and Phl p 11, which are associated with pollen allergens. The proteome analysis positively verified a significant increase in pollen allergenic protein content. The expression levels of LoProfiilin and LoPolcalcin, annotated as allergen proteins, gradually increased in mature pollen. LoProfiilin and LoPolcalcin were cloned and their open reading frame lengths were 396 bp and 246 bp, which encoded 131 and 81 amino acids, respectively. Amino acid sequence and structure alignment indicated that the protein sequences of LoProfilin and LoPolcalcin were highly conserved. Subcellular localization analysis showed that LoProfilin protein was localized in the cell cytoplasm and nucleus. LoProfilin and LoPolcalcin were highly expressed in mature pollen at the transcriptional and protein levels. A tertiary structure prediction analysis identified LoProfilin and LoPolcalcin as potential allergens in lily pollen.

Meiotic chromosome behavior of the male-fertile allotriploid lily cultivar 'Cocossa'.

KEY MESSAGE: Cytological observations of microsporogenesis in the allotriploid lily cultivar 'Cocossa' showed that viable pollen production could be attributed mainly to disoriented spindles, abnormal cytokinesis, and cytomixis during male meiosis. To identify the reasons why the allotriploid lily cultivar 'Cocossa' can produce aneuploid and euploid functional male gametes and can be used as the paternal parent in lily introgression breeding, we performed a detailed investigation of microsporogenesis using the conventional cytological methods. The allotriploid not only produced single pollen grains with variable sizes but also produced adherent pollen grains. Pollen viability was estimated at 50.1% based on staining and 30.8% based on germination. Based on the chromosomal analysis of BC2 plants derived from Oriental cultivars (♀) crossed with the OOT cultivar 'Cocossa' (♂), it was concluded that the objective allotriploid contributed haploid (x), diploid (2x), and aneuploid chromosome complements. Common meiotic abnormalities were observed, indicating the high genetic imbalance of this allotriploid. In addition to normally oriented metaphase II spindles (linear and perpendicular), abnormal spindles, such as parallel, tripolar, fused, and multiple spindles, accounted for 6.21, 6.41, 14.27, and 1.17%, respectively. Tripolar and fused spindles resulted in the production of triads and dyads, which contributed to unreduced pollen production. Some microsporocytes exhibited complete or partial absence of cytokinesis, which led to relatively high frequencies of monads, dyads, and triads. Furthermore, the phenomenon of cytomixis during microsporogenesis occurred mainly in the first meiotic prophase and early development of pollen grains, which we assume is a possible cause of unreduced gamete generation. Our study offers a new resource for lily introgression breeding.

Genetic variability and diversity of the main resources of lily assessed via phenotypic characters, pollen morphology, and ISSR markers.

Lily (Lilium spp), which belongs to Lilium, is one kind of monocotyledon. As a perennial ornamental plant with extremely high esthetic, edible, and medicinal value, lily has gained much favor due to its mostly showy flowers of various colors and elegant shape. In this research, we studied experimental materials in a sample of 49 individuals including 40 cultivars, nine species of wild lily, and their variants. The collection of 40 cultivars covered all six hybrids in the genus, i.e., Asiatic hybrids, Oriental hybrids, Longiflorum hybrids, LA hybrids, LO hybrids, and OT hybrids. Genetic diversity and inter-relationships were assessed through analysis of phenotypic characteristics, pollen morphology, and ISSR molecular markers. Quantitative characters were selected to analyze phenotypic variation, with results indicating greater variability in petiole length as compared to other characters. Pollen morphological observations suggested that the largest variation coefficient between all hybrids and wild species was the lumina. ISSR makers demonstrated that both cultivars and wild species possess a high level of genetic diversity. Specifically, the genetic diversity of wild lily was higher than cultivars.

Proteomic analysis reveals the differential histone programs between male germline cells and vegetative cells in Lilium davidii.

In flowering plants, male germline fate is determined after asymmetric division of the haploid microspore. Daughter cells have distinct fates: the generative cell (GC) undergoes further mitosis to generate sperm cells (SCs), and the vegetative cell (VC) terminally differentiates. However, our understanding of the mechanisms underlying germline development remains limited. Histone variants and modifications define chromatin states, and contribute to establishing and maintaining cell identities by affecting gene expression. Here, we constructed a lily protein database, then extracted and detailed histone entries into a comprehensive lily histone database. We isolated large amounts of nuclei from VCs, GCs and SCs from lily, and profiled histone variants of all five histone families in all three cell types using proteomics approaches. We revealed 92 identities representing 32 histone variants: six for H1, 11 for H2A, eight for H2B, five for H3 and two for H4. Nine variants, including five H1, two H2B, one H3 and one H4 variant, specifically accumulated in GCs and SCs. We also detected H3 modification patterns in the three cell types. GCs and SCs had almost identical histone profiles and similar H3 modification patterns, which were significantly different from those of VCs. Our study also revealed the presence of multiple isoforms, and differential expression patterns between isoforms of a variant. The results suggest that differential histone programs between the germline and companion VCs may be established following the asymmetric division, and are important for identity establishment and differentiation of the male germline as well as the VC.

Study on the homology of the genomes of tetraploid Asiatic lilies (Lilium) using FISH.

Asiatic lily cultivars, bred by hybridization and (or) chromosome doubling of species of section Sinomartagon of Lilium, are diploid, triploid, or tetraploid, but the homology of the genomes among species of section Sinomartagon and Asiatic lilies remains unclear. In the present research, two tetraploid Asiatic cultivars were analyzed, using 45S rDNA as probe, for their FISH karyotypes and their chromosomal association, anaphase I, telophase II, and pollen viability were surveyed to assess the multivalent segregation. Chromosomal assortment of six progenies of the two tetraploid cultivars were also investigated. The results showed that the tetraploid cultivars had similar FISH karyotypes, they predominantly formed multivalents, and these were equally separated because their anaphase I, telophase II, and pollen viability were similar to those of diploid species. Apart from minor variations, FISH karyotypes of progenies were similar to each other and to their parents. Based on these results and considering the high crossability among species of section Sinomartagon and (or) Asiatic lilies, we concluded that species of section Sinomartagon and their resulting cultivars share a common genome; thus, polyploidy Asiatic lilies are autopolyploid.

Morphology and Physicochemical Properties of 3 Lilium Bulb Starches.

Lilium (Liliaceae) is an important wild plant and is used as food and traditional medicine worldwide. One Lilium cultivar (Lilium lancifolium) and 2 wild types (Lilium leucanthum and Lilium rosthornii) that are commonly distributed in Western China were investigated to completely utilize Lilium resources. The morphology of the flowers, bulbs, and scales and soluble sugar, total starch and amylose contents was remarkably different among the 3 Lilium species. Starches from the 3 Lilium species presented different granule size and shape. The starch of L. lancifolium exhibited higher swelling power and solubility than that of L. leucanthum and L. rosthornii. The starches from the 3 Lilium bulbs presented similar X-ray diffraction patterns and Fourier transform infrared spectroscopy. Among the 3 Lilium species, L. lancifolium showed the lowest crystallinity and the largest proportion of ordered structures in granule external region. Gelatinization temperatures and retrogradation percentage were significantly lower, but gelatinization enthalpy was significantly higher in L. lancifolium than those in L. leucanthum and L. rosthornii. Pasting properties of starch were different among the 3 Lilium species. Starch from L. lancifolium showed the highest degree of amylopectin branching, followed by L. leucanthum and L. rosthornii. Starches from L. leucanthum and L. rosthornii showed higher resistance to porcine pancreatic α-amylase hydrolysis compared to that of L. lancifolium. These results indicated that 3 Lilium bulbs exhibited remarkable differences in morphological, crystal, thermal, pasting, and hydrolysis properties of starches.

De novo sequencing and analysis of the lily pollen transcriptome: an open access data source for an orphan plant species.

Pollen grains of Lilium longiflorum are a long-established model system for pollen germination and tube tip growth. Due to their size, protein content and almost synchronous germination in synthetic media, they provide a simple system for physiological measurements as well as sufficient material for biochemical studies like protein purifications, enzyme assays, organelle isolation or determination of metabolites during germination and pollen tube elongation. Despite recent progresses in molecular biology techniques, sequence information of expressed proteins or transcripts in lily pollen is still scarce. Using a next generation sequencing strategy (RNAseq), the lily pollen transcriptome was investigated resulting in more than 50 million high quality reads with a length of 90 base pairs. Sequenced transcripts were assembled and annotated, and finally visualized with MAPMAN software tools and compared with other RNAseq or genome data including Arabidopsis pollen, Lilium vegetative tissues and the Amborella trichopoda genome. All lily pollen sequence data are provided as open access files with suitable tools to search sequences of interest.

A novel lily anther-specific gene encodes adhesin-like proteins associated with exine formation during anther development.

The anther-specific gene LLA1271 isolated from lily (Lilium longiflorum Thunb.) anthers is novel and exists in two forms. The protein encoded by LLA1271 may represent an adhesin-like protein first found in higher plants. The protein contains a typical N-terminal signal peptide followed by a highly conserved repeat domain. The LLA1271 gene is temporally expressed at the phase of microspore development. RNA blot and RNA in situ hybridization analyses demonstrated that the gene was expressed both in the tapetum and in the microspore. The gene is endo- and exogenously induced by gibberellin. Studies with the gibberellin biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that LLA1271 is negatively regulated by ethylene, and a cross-talk of regulation between gibberellin and ethylene occurs in young anthers. The treatment with NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development in a state close to that of a tapetum without treatment. The LLA1271 protein is heat stable and heterogeneous. An immunoblot of separated protein fractions of the anther revealed that the LLA1271 protein was detected in protein fraction of the microspore released from the cell wall by treatment with either 0.5% or 2% Triton X-100. Ectopic expression of LLA1271 resulted in impaired stamen and low pollen germination. Scanning electron microscopy of TAP::LLA1271 pollen showed distorted exine formation and patterning. The LLA1271 protein once synthesized in both the tapetum and microspore is secreted and deposited on the surface of microspores, moderately affecting exine formation and patterning.

Identification of the tapetum/microspore-specific promoter of the pathogenesis-related 10 gene and its regulation in the anther of Lilium longiflorum.

A tapetum/microspore-specific pathogenesis-related (PR) 10 gene was previously identified in lily (Lilium longiflorum Thunb.) anthers. In situ hybridization and RNA blot analysis indicated that the lily PR10 genes are expressed specifically and differentially in the tapetum of the anther wall and in microspores during anther development. The accumulation of PR10 transcripts was exogenously induced by gibberellic acid (GA) and was suppressed by ethylene. Studies using inhibitors of GA and ethylene revealed that the lily PR10 is modulated by an antagonistic interaction between GA and ethylene. The treatment of norbornadien, an ethylene inhibitor, caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole, an inhibitor of GA biosynthesis, arrested tapetal development to a status close to that of control. The expression of the lily PR10g promoter in transgenic Arabidopsis was determined using the ß-glucuronidase (GUS) reporter gene indicated that the decisive fragment required for anther specificity is located -1183 bp to -880 bp upstream of the transcription start site. The PR10gPro::barnase transgenic lines exhibited complete male sterility because of the disruption of the tapetum and the deformation of microspore/pollen. The anther specificity of lily PR10 highlights the importance of the tapetum/microspore-specific PR10g promoter for future biotechnological and agricultural applications.

Production of polyploids and unreduced gametes in Lilium auratum × L. henryi hybrid.

Intergenomic F1 hybrids between L. auratum x L. henryi and their BC1 progeny were investigated through genomic in situ hybridization technique (GISH) to determine their potential value in lily breeding. We confirmed that F1 intergenomic hybrids possessed a set of chromosomes (x=12) from both parents and that flowers of the F1 auratum × henryi hybrid showed an intermediate morphological phenotype. Pollen size, viability and germination ability were measured through microscopic observations. F1 intergenomic hybrids produced a relevant frequency of 2n-gametes, which were successfully used to perform crosses with Oriental hybrids, resulting in the triploid Oriental Auratum Henryi (OAuH) hybrid. Twenty BC1 plants were generated by crossing between four different Oriental hybrid cultivars and F1 AuH hybrids using an in vitro embryo rescue technique, after which the genome constitution and chromosome composition were analyzed by GISH. All plants were triploid, showing 12 from female parents (diploid Oriental hybrid) and 24 from male parents (diploid F1 AuH hybrid). Overall, 16 out of 20 BC1 progeny possessed recombinant chromosomes with 1-5 crossover sites per plant. Cytological analysis of 20 BC1 plants by GISH verified that the occurrence of 2n pollen formation in all F1 AuH hybrids was derived from the FDR (first division restitution) mechanism, in which the genome composition of all BC1 plants possess 12 Oriental + 12 L. auratum + 12 L. henryi chromosomes. Allotriploids derived from the AuH hybrid were used as female for crossing with the diploid Oriental hybrid cultivar 'Sorbonne' and considerable numbers of plants (0-6.5 plants per ovary) were only obtained when female OAuH (BC1) triploids were used. Taken together, the results of this study indicate that production and analysis of F1 AuH hybrids and their progeny through sexual polyploidization can be useful for efficient creation of important horticultural traits.

New insights into desiccation-associated gene regulation by Lilium longiflorum ASR during pollen maturation and in transgenic Arabidopsis.

LLA23, a member of the abscisic acid-, stress-, and ripening-induced (ASR) protein family, was previously isolated from lily (Lilium longiflorum) pollen. The lily ASR is induced through desiccation-associated ABA signaling transduction in the pollen. ASRs are highly hydrophilic and intrinsically unstructured proteins with molecular masses generally less than 18 kDa. LLA23 is abundant in the cytoplasm and nuclei of both vegetative and generative cells of pollen grains. The protein in the nucleus and in the cytoplasm is partly regulated by dehydration. A dual role is proposed for LLA23, as a regulator and a protective molecule, upon exposure to water deficits. This chapter reviews the current state of literature on Asr genes, protein structure, function, and their responses to various stresses. In a study, a genome-wide microarray was used to monitor the expression of LLA23-regulated genes, focusing on the relationship between ASR-, glucose-, and drought-inducible genes, and outlined the difference and cross talk of gene expression among these signaling networks. A strong association was observed in the expression of stress-responsive genes and found 25 genes that respond to all three treatments. Highly inducible genes were also found in each specific stress treatment. Promoter sequence analysis of LLA23-inducible genes enabled us not only to identify possible known cis-acting elements in the promoter regions but also to expect the existence of novel cis-acting elements involved in ASR-responsive gene expression. ASR can be used to improve crops and economically important plants against various environmental stresses.

Cytogenetic studies on meiotic chromosome behaviors in sterile Oriental x Trumpet lily.

In order to determine the reasons for pollen sterility in lily hybrids, four diploid sterile Oriental x Trumpet (OT) lily cultivars ('Nymph', 'Gluhwein', 'Yelloween', and 'Shocking') were used to investigate the meiotic chromosome behaviors in pollen mother cells (PMCs), using genomic in situ hybridization and conventional cytological methods. At metaphase I, chromosome associations were quite variable, not only among different genotypes but also in different PMCs of the same genotype. In addition to bivalents, a certain amount of univalent, trivalents, and quadrivalents were observed in all of the investigated genotypes. In addition, ring octavalents and ring hexavalents were observed in 'Nymph'. Even dodecavalents were observed in 'Nymph'. These abnormal chromosome associations at metaphase I implied the occurrence of chromosome interchanges (translocation) in these intersectional hybrids. At anaphase-telophase, a large number of laggard chromosomes and different kinds of chromosome bridge configurations were observed. At the tetrad stage, micronuclei and polyads were also found in many PMCs. All of these abnormal chromosome behaviors in PMCs were responsible for the pollen sterility in lily hybrids.

Cloning and functional characterization of a gene for capsanthin-capsorubin synthase from tiger lily (Lilium lancifolium Thunb. 'Splendens').

The orange color of tiger lily (Lolium lancifolium 'Splendens') flowers is due, primarily, to the accumulation of two κ-xanthophylls, capsanthin and capsorubin. An enzyme, known as capsanthin-capsorubin synthase (CCS), catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. We cloned the gene for capsanthin-capsorubin synthase (Llccs) from flower tepals of L. lancifolium by the rapid amplification of cDNA ends (RACE) with a heterologous non-degenerate primer that was based on the sequence of a gene for lycopene ß-cyclase (lcyB). The full-length cDNA of Llccs was 1,785 bp long and contained an open reading frame of 1,425 bp that encoded a polypeptide of 474 amino acids with a predicted N-terminal plastid-targeting sequence. Analysis by reverse transcription-PCR (RT-PCR) revealed that expression of Llccs was spatially and temporally regulated, with expression in flower buds and flowers of L. lancifolium but not in vegetative tissues. Stable overexpression of the Llccs gene in callus tissue of Iris germanica, which accumulates several xanthophylls including violaxanthin, the precursor of capsorubin, resulted in transgenic callus whose color had changed from its normal yellow to red-orange. This novel red-orange coloration was due to the accumulation of two non-native κ-xanthophylls, capsanthin and capsorubin, as confirmed by HPLC and ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis with authentic standards. Cloning of the Llccs gene should advance our understanding of the molecular and genetic mechanisms of the biosynthesis of κ-carotenoids in general and in the genus Lilium in particular, and will facilitate transgenic alterations of the colors of flowers and fruits of many plant species.

Generative cell-specific activation of the histone gH2A gene promoter of Lilium longiflorum in tobacco.

The Lilium longiflorum gH2A promoter is active exclusively in the generative cells of mature pollen in transgenic tobacco expressing the gH2A promoter::GUS (ß-glucuronidase) construct as a reporter gene. Temporal and spatial aspects of gH2A promoter activity examined during pollen development in transgenic tobacco reveal that GUS reporter activity was not detected until developing pollen entered the early bicellular developmental stage. Activity was first detected in generative cells at early-mid stages and gradually increased to maximum levels at mid-bicellular stages. The patterns of appearance and longevity of GUS activity in tobacco were very similar to those of gH2A mRNA during pollen development in Lilium. Exogenous treatment with colchicine, a well-known microtubule depolymerize, blocked microspore mitosis and inhibited generative cell differentiation. No GUS signal was detected in the resulting anomalous pollen, which lacked generative cell differentiation. These data strongly suggest that normal generative cell development is essential for activation of the gH2A promoter. Furthermore, these results indicate that common transcriptional activator(s) of the gH2A promoter may be present in both Lilium and Nicotiana, and that such putative factor(s) activates the gH2A promoter only when generative cells undergo normal development.