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Oriental hybrid lilies, known for their vibrant colors, diverse flower shapes, and long blooming seasons, require annual bulb propagation in horticultural production. This necessity can lead to higher production costs and limit their use in landscaping. The LA hybrid lily 'Aladdin' has shown strong self-reproduction capabilities in optimal cultivation environments, producing numerous high-quality underground stem bulblets. This makes it a valuable model for studying bulblet formation in lilies under natural conditions. Through transcriptome data analysis of different developmental stages of 'Aladdin' bulblets, the LaLBD37 gene, linked to bulblet formation, was identified. Bioinformatics analysis, subcellular localization studies, and transcriptional activation activity tests were conducted to understand the characteristics of LaLBD37 . By introducing the LaLBD37 gene into 'Sorbonne' aseptic seedlings via Agrobacterium-mediated transformation, resistant plants were obtained. Positive plants were identified through various methods such as GUS activity detection, PCR, and fluorescence quantitative PCR. Phenotypic changes in positive plants were observed, and various physiological indicators were measured to confirm the role of LaLBD37 in bulblet formation, including soluble sugar content, starch content, sucrose synthase activity, and endogenous hormone levels. The findings suggest that the LaLBD37 gene plays a significant role in promoting the development of lily bulblets, offering insights for enhancing the reproductive capacity of Oriental hybrid lilies and exploring the molecular mechanisms involved in lily bulb regeneration.

Lilium is one of the most widely cultivated ornamental bulbous plants in the world. Although research has shown that variable temperature treatments can accelerate the development process from vegetative to reproductive growth in Lilium , the molecular regulation mechanisms of this development are not clear. In this study, Lbr -miR171b and its target gene, LbrSCL6 , were selected and validated using transgenic functional verification, subcellular localization, and transcriptional activation. This study also investigated the differential expression of Lbr -miR171b and LbrSCL6 in two temperature treatment groups (25 °C and 15 °C). Lbr -miR171b expression significantly increased after the temperature change, whereas that of LbrSCL6 exhibited the opposite trend. Through in situ hybridization experiments facilitated by the design of hybridization probes targeting LbrSCL6 , a reduction in LbrSCL6 expression was detected following variable temperature treatment at 15 °C. The transgenic overexpression of Lbr -miR171b in plants promoted the phase transition, while LbrSCL6 overexpression induced a delay in the phase transition. In addition, LbrWOX4 interacted with LbrSCL6 in yeast two-hybrid and bimolecular fluorescence complementation assays. In conclusion, these results explain the molecular regulatory mechanisms governing the phase transition in Lilium .

As the main forms of carbohydrates, starch and sucrose play a vital role in the balance and coordination of various carbohydrates. Lanzhou lily is the most popular edible lily in China, mainly distributed in the central region of Gansu. To clarify the relationship between carbohydrate metabolism and bulb development of Lanzhou lily, so as to provide a basis for the promotion of the growth and development in Lanzhou lily and its important economic value, we studied lily bulbs in the squaring stage, flowering stage, half withering stage and withering stage. The plant height, fresh weight of mother and daughter bulbs continued to increase during the whole growth period and fresh weight of stem and leaf began to decrease in the half withering stage. The content of starch, sucrose and total soluble sugar in the lily mother bulb accumulated mostly in the flowering, withering and half withering stages, respectively. Starch, sucrose and total soluble sugar accumulated in the daughter bulb with the highest concentration during the withering stage. In the transcription level, sucrose synthase (SuSy1) and sucrose invertase (INV2) expressed the highest in squaring stage, and the expression was significantly higher in the mother bulb than in the daughter bulb. In flowering stage, the expression levels of soluble starch synthase (SSS1), starch-branching enzyme (SBE) and adenosine diphosphate-glucose pyrophosphorylase (AGP1) genes were higher in the mother bulb than in the daughter bulb. Altogether, our results indicate that starch and sucrose are important for the bulb growth and development of Lanzhou lily., Competing Interests: The authors have declared that no competing interests exist.

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.

Lily ( Lilium spp.) is an important cut flower around the world. Flower senescence in lilies is characterized by the wilting and abscission of tepals, which results in a decrease in flower quality and huge economic loss. However, the mechanism underlying flower senescence in lilies is largely unknown. In this study, single-molecule, real-time (SMRT) and Illumina sequencing were carried out in L . oriental 'Siberia'. Sequencing yielded 73,218 non-redundant transcripts, with an N50 of 3792 bp. These data were further integrated with three published transcriptomes through cogent analysis, which yielded 62,960 transcripts, with an increase in N50 of 3935 bp. Analysis of differentially expressed genes showed that 319 transcription factors were highly upregulated during flower senescence. The expression of twelve NAC genes and eleven senescence-associated genes ( SAGs ) showed that LoNAC29 and LoSAG39 were highly expressed in senescent flowers. Transient overexpression of LoNAC29 and LoSAG39 in tepals of lily notably accelerated flower senescence, and the promoter activity of LoSAG39 was strongly induced by LoNAC29 . This work supported new evidence for the molecular mechanism of flower senescence and provided better sequence data for further study in lilies.

The bulbil is an important vegetative reproductive organ in triploid Lilium lancifolium whose development is promoted by cytokinins. Type-B response regulators (RRs) are critical regulators that mediate primary cytokinin responses and promote cytokinin-induced gene expression. However, the function of cytokinin type-B Arabidopsis RRs (ARRs) in regulating bulbil formation is unclear. In this study, we identified five type-B LlRR s, LlRR1 , LlRR2 , LlRR10 , LlRR11 and LlRR12 , in L. lancifolium for the first time. The five LlRR s encode proteins of 715, 675, 573, 582 and 647 amino acids. All of the regulators belong to the B-I subfamily, whose members typically contain a conserved CheY-homologous receiver (REC) domain and an Myb DNA-binding (MYB) domain at the N-terminus. As transcription factors, all five type-B LlRRs localize at the nucleus and are widely expressed in plant tissues, especially during axillary meristem (AM) formation. Functional analysis showed that type-B LlRR s are involved in bulbil formation in a functionally redundant manner and can activate LlRR9 expression. In summary, our study elucidates the process by which cytokinins regulate bulbil initiation in L. lancifolium through type-B LlRRs and lays a foundation for research on the molecular mechanism of bulbil formation in the lily.

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 Arabidopsis DTX35 gene demonstrated that LhDTX35 could restore silique-infertility and the anthocyaninless phenotype of an Arabidopsis DTX35 mutant. These results indicated that LhDTX35 might be involved in anthocyanin accumulation in Lilium.

The Formosa lily ( Lilium formosanum ) is one of the most common horticultural species in Taiwan. To explore gene regulation involved in this species, we used transcriptome analysis to generate PH-FB (mixed floral buds) and PH-LF (mature leaves) datasets. Combination of the PH-FB and PH-LF constructed a de novo assembly of the ALL dataset, including 18,041 contigs and 23,807 unigenes by Nr, GO, COG, and KEGG databases. The differential gene expression (DGE) analysis revealed 9937 genes were upregulated while 10,383 genes were downregulated in the developing floral buds compared to mature leaves. Seven putative genes ( LFMADS1 to 7 ) encoding floral organ identity proteins were selected for further analysis. LFMADS1-6 genes were specifically expressed in the floral organ, while LFMADS7 in the floral buds and mature leaves. Phylogenetic analysis revealed that LFMADS1-3 is classified into B-class, LFMADS4 into C-class, LFMADS5 into D-class, and LFMADS6-7 into E-class, respectively. LFMADS-GFP fusion proteins appeared to localize in the nucleus, supporting their roles as transcription factors (TFs). Overexpression of the LFMADS2 , LFMADS4 , and LFMADS6 genes in Arabidopsis resulted in early flowering and floral defect, however, only early flowering in transgenic tobacco was observed. Highly expressed floral integrator genes, including AtFT , AtLFY , and AtFUL in transgenic Arabidopsis and NtFUL and NtSOC1 in transgenic tobacco, resulted in early flowering phenotype through qRT-PCR analysis. Yeast two-hybrid analysis suggested that LFMADSs may form higher order complexes with the B-, C-, D, and/or E-class proteins to determine the floral organ identity. Furthermore, E-class LFMADS proteins may function as a glue to mediate and strengthen the protein-protein interactions. Therefore, our de novo datasets would provide information for investigating other differentially expressed candidate transcripts. In addition, functional conservation of LFMADSs appears to be vital in floral transition and floral organ identity.

Floral induction in Tulipa gesneriana and Lilium longiflorum is triggered by contrasting temperature conditions, high and low temperature, respectively. In Arabidopsis, the floral integrator FLOWERING LOCUS T (FT), a member of the PEBP (phosphatidyl ethanolamine-binding protein) gene family, is a key player in flowering time control. In this study, one PEBP gene was identified and characterized in lily (LlFT) and three PEBP genes were isolated from tulip (TgFT1, TgFT2 and TgFT3). Overexpression of these genes in Arabidopsis thaliana resulted in an early flowering phenotype for LlFT and TgFT2, but a late flowering phenotype for TgFT1 and TgFT3. Overexpression of LlFT in L. longiflorum also resulted in an early flowering phenotype, confirming its proposed role as a flowering time-controlling gene. The tulip PEBP genes TgFT2 and TgFT3 have a similar expression pattern in tulip, but show opposite effects on the timing of flowering in Arabidopsis. Therefore, the difference between these two proteins was further investigated by interchanging amino acids thought to be important for the FT function. This resulted in the conversion of phenotypes in Arabidopsis upon overexpressing the substituted TgFT2 and TgFT3 genes, revealing the importance of these interchanged amino acid residues. Based on all obtained results, we hypothesize that LlFT is involved in creating meristem competence to flowering-related cues in lily, and TgFT2 is considered to act as a florigen involved in the floral induction in tulip. The function of TgFT3 remains unclear, but, based on our observations and phylogenetic analysis, we propose a bulb-specific function for this gene., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Pollen tube tip growth is an extreme form of polarized cell growth, which requires polarized exocytosis based on a dynamic actin cytoskeleton. However, the molecular basis for the connection between actin filaments and exocytic vesicles is unclear. Here, we identified a Lilium longiflorum pollen-specific formin (LlFH1) and found that it localized at the apical vesicles and plasma membrane (PM). Overexpression of LlFH1 induced excessive actin cables in the tube tip region, and downregulation of LlFH1 eliminated the actin fringe. Fluorescence recovery after photobleaching (FRAP) analysis revealed that LlFH1-labeled exocytic vesicles exhibited an initial accumulation at the shoulder of the apex and coincided with the leading edge of the actin fringe. Time-lapse analysis suggested that nascent actin filaments followed the emergence of the apical vesicles, implying that LlFH1 at apical vesicles could initiate actin polymerization. Biochemical assays showed that LlFH1 FH1FH2 could nucleate actin polymerization, but then capped the actin filament at the barbed end and inhibited its elongation. However, in the presence of lily profilins, LlFH1 FH1FH2 could accelerate barbed-end actin elongation. In addition, LlFH1 FH1FH2 was able to bundle actin filaments. Thus, we propose that LlFH1 and profilin coordinate the interaction between the actin fringe and exocytic vesicle trafficking during pollen tube growth of lily., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Petal wilting and/or abscission terminates the life of the flower. However, how wilting and abscission are coordinated is not fully understood. There is wide variation in the extent to which petals wilt before abscission, even between cultivars of the same species. For example, tepals of Lilium longiflorum wilt substantially, while those of the closely related Lilium longiflorum×Asiatic hybrid (L.A.) abscise turgid. Furthermore, close comparison of petal death in these two Lilium genotypes shows that there is a dramatic fall in fresh weight/dry weight accompanied by a sharp increase in ion leakage in late senescent L. longiflorum tepals, neither of which occur in Lilium L.A. Despite these differences, a putative abscission zone was identified in both lilies, but while the detachment force was reduced to zero in Lilium L.A., wilting of the fused tepals in L. longiflorum occurred before abscission was complete. Abscission is often negatively regulated by auxin, and the possible role of auxin in regulating tepal abscission relative to wilting was tested in the two lilies. There was a dramatic increase in auxin levels with senescence in L. longiflorum but not in Lilium L.A. Fifty auxin-related genes were expressed in early senescent L. longiflorum tepals including 12 ARF-related genes. In Arabidopsis, several ARF genes are involved in the regulation of abscission. Expression of a homologous transcript to Arabidopsis ARF7/19 was 8-fold higher during senescence in L. longiflorum compared with abscising Lilium L.A., suggesting a conserved role for auxin-regulated abscission in monocotyledonous ethylene-insensitive flowers., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background: The formation and development of bulblets are crucial to the Lilium genus since these processes are closely related to carbohydrate metabolism, especially to starch and sucrose metabolism. However, little is known about the transcriptional regulation of both processes. To gain insight into carbohydrate-related genes involved in bulblet formation and development, we conducted comparative transcriptome profiling of Lilium davidii var. unicolor bulblets at 0 d, 15 d (bulblets emerged) and 35 d (bulblets formed a basic shape with three or four scales) after scale propagation., Results: Analysis of the transcriptome revealed that a total of 52,901 unigenes with an average sequence size of 630 bp were generated. Based on Clusters of Orthologous Groups (COG) analysis, 8% of the sequences were attributed to carbohydrate transport and metabolism. The results of KEGG pathway enrichment analysis showed that starch and sucrose metabolism constituted the predominant pathway among the three library pairs. The starch content in mother scales and bulblets decreased and increased, respectively, with almost the same trend as sucrose content. Gene expression analysis of the key enzymes in starch and sucrose metabolism suggested that sucrose synthase (SuSy) and invertase (INV), mainly hydrolyzing sucrose, presented higher gene expression in mother scales and bulblets at stages of bulblet appearance and enlargement, while sucrose phosphate synthase (SPS) showed higher expression in bulblets at morphogenesis. The enzymes involved in the starch synthetic direction such as ADPG pyrophosphorylase (AGPase), soluble starch synthase (SSS), starch branching enzyme (SBE) and granule-bound starch synthase (GBSS) showed a decreasing trend in mother scales and higher gene expression in bulblets at bulblet appearance and enlargement stages while the enzyme in the cleavage direction, starch de-branching enzyme (SDBE), showed higher gene expression in mother scales than in bulblets., Conclusions: An extensive transcriptome analysis of three bulblet development stages contributes considerable novel information to our understanding of carbohydrate metabolism-related genes in Lilium at the transcriptional level, and demonstrates the fundamentality of carbohydrate metabolism in bulblet emergence and development at the molecular level. This could facilitate further investigation into the molecular mechanisms underlying these processes in lily and other related species.

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.

The growing pollen tube is central to plant reproduction and is a long-standing model for cellular tip growth in biology. Rapid osmotically driven growth is maintained under variable conditions, which requires osmosensing and regulation. This study explores the mechanism of water entry and the potential role of osmosensory regulation in maintaining pollen growth. The osmotic permeability of the plasmalemma of Lilium pollen tubes was measured from plasmolysis rates to be 1.32±0.31×10(-3) cm s(-1). Mercuric ions reduce this permeability by 65%. Simulations using an osmotic model of pollen tube growth predict that an osmosensor at the cell membrane controls pectin deposition at the cell tip; inhibiting the sensor is predicted to cause tip bursting due to cell wall thinning. It was found that adding mercury to growing pollen tubes caused such a bursting of the tips. The model indicates that lowering the osmotic permeability per se does not lead to bursting but rather to thickening of the tip. The time course of induced bursting showed no time lag and was independent of mercury concentration, compatible with a surface site of action. The submaximal bursting response to intermediate mercuric ion concentration was independent of the concentration of calcium ions, showing that bursting is not due to a competitive inhibition of calcium binding or entry. Bursting with the same time course was also shown by cells growing on potassium-free media, indicating that potassium channels (implicated in mechanosensing) are not involved in the bursting response. The possible involvement of mercury-sensitive water channels as osmosensors and current knowledge of these in pollen cells are discussed.

Two lily (Lilium longiflorum) PISTILLATA (PI) genes, Lily MADS Box Gene 8 and 9 (LMADS8/9), were characterized. LMADS9 lacked 29 C-terminal amino acids including the PI motif that was present in LMADS8. Both LMADS8/9 mRNAs were prevalent in the first and second whorl tepals during all stages of development and were expressed in the stamen only in young flower buds. LMADS8/9 could both form homodimers, but the ability of LMADS8 homodimers to bind to CArG1 was relatively stronger than that of LMADS9 homodimers. 35S:LMADS8 completely, and 35S:LMADS9 only partially, rescued the second whorl petal formation and partially converted the first whorl sepal into a petal-like structure in Arabidopsis pi-1 mutants. Ectopic expression of LMADS8-C (with deletion of the 29 amino acids of the C-terminal sequence) or LMADS8-PI (with only the PI motif deleted) only partially rescued petal formation in pi mutants, which was similar to what was observed in 35S:LMADS9/pi plants. In contrast, 35:LMADS9+L8C (with the addition of the 29 amino acids of the LMADS8 C-terminal sequence) or 35S:LMADS9+L8PI (with the addition of the LMADS8 PI motif) demonstrated an increased ability to rescue petal formation in pi mutants, which was similar to what was observed in 35S:LMADS8/pi plants. Furthermore, ectopic expression of LMADS8-M (with the MADS domain truncated) generated more severe dominant negative phenotypes than those seen in 35S:LMADS9-M flowers. These results revealed that the 29 amino acids including the PI motif in the C-terminal region of the lily PI orthologue are valuable for its function in regulating perianth organ formation.

Background: Pollination is a crucial step in angiosperm (flowering plant) reproduction. Highly orchestrated pollen-pistil interactions and signalling events enable plant species to avoid inbreeding and outcrossing as a species-specific barrier. In compatible pollination, pollen tubes carrying two sperm cells grow through the pistil transmitting tract and are precisely guided to the ovules, discharging the sperm cells to the embryo sac for fertilization., Scope: In Lilium longiflorum pollination, growing pollen tubes utilize two critical mechanisms, adhesion and chemotropism, for directional growth to the ovules. Among several molecular factors discovered in the past decade, two small, secreted cysteine-rich proteins have been shown to play major roles in pollen tube adhesion and reorientation bioassays: stigma/style cysteine-rich adhesin (SCA, approx. 9·3 kDa) and chemocyanin (approx. 9·8 kDa). SCA, a lipid transfer protein (LTP) secreted from the stylar transmitting tract epidermis, functions in lily pollen tube tip growth as well as in forming the adhesive pectin matrix at the growing pollen tube wall back from the tip. Lily chemocyanin is a plantacyanin family member and acts as a directional cue for reorienting pollen tubes. Recent consecutive studies revealed that Arabidopsis thaliana homologues for SCA and chemocyanin play pivotal roles in tip polarity and directionality of pollen tube growth, respectively. This review outlines the biological roles of various secreted proteins in angiosperm pollination, focusing on plant LTPs and chemocyanin.

The ubiquitin-proteasome pathway mediates protein degradation and is involved in diverse aspects of plant development and differentiation, including pollen tube elongation and self-incompatibility. We characterized three lily (Lilium longiflorum) SKP1-like genes, LSK1-LSK3, that are specifically expressed in late pollen developmental stages and the elongating pollen tube. The encoded peptide sequences reveal that LSK1-LSK3 share high identity with Arabidopsis ASK1 and contain a putative N-terminal CUL1- and a C-terminal F-box-interacting domain. Yeast two-hybrid and in vitro affinity binding assays revealed that the LSKs associate with lily CULLIN1. In addition, the LSK genes can functionally complement the yeast skp1 deletion mutant YDR328C. To investigate their biological functions in pollen tube elongation, an in vivo approach for green fluorescent protein (GFP)-tagged dominant-negative LSK1-LSK3 was developed. Microprojectile bombardment with N-terminally truncated LSK1-LSK3 (LSK1-LSK3Delta-GFP) significantly retarded pollen tube elongation in both in vitro germination and in vivo self- and cross-pollination after >12 h incubation. Interestingly, elongation of pollen tubes harboring overexpressed LSK2Delta-GFP and LSK3Delta-GFP was substantially inhibited within the self-pollinated styles. The elongation of most LSK2Delta-GFP-transformed pollen tubes could germinate only on the stigmatic surface of self style and showed statistically significant growth arrest as compared with control pollen tubes. Lily exhibits typical gametophytic self-incompatibility via an unknown mechanism, but LSK2 and LSK3 may be involved in this complex machinery. These results suggest critical roles for LSK1-LSK3 in regulating fundamental pollen tube elongation in vitro and in vivo and that the 26S proteasome-mediated protein pathway plays an important role in pollen tube elongation.

Two stage-specific genes have been isolated from a subtractive cDNA library constructed from developing anthers of lily (Lilium longiflorum). The proteins encoded by the two genes have a strong hydrophobic region at the N-terminus, indicating the presence of a signal peptide. The deduced LLA-67 is a new type of small cysteine-rich protein whose sequence exhibits four consecutive CX(3)CX(6-10) repeats that could form signal-receiving finger motifs, while the deduced LLA-115 protein shows significant similarities to a rice unknown protein, and putative cell wall proteins of Medicago truncatula and Arabidopsis. The transcripts of LLA-67 and LLA-115 were anther specific and differentially detected at the phase of microspore development. In situ hybridization with antisense riboprobes of the two genes in the anther showed strong signals localized to the tapetal layer of the anther wall. The LLA-67 mRNA was also detected in the microspore at the phase of microspore development but the LLA-115 mRNA was not. The LLA-115 gene can be exogenously induced by gibberellin (GA), whereas the LLA-67 gene cannot be induced. Studies with the GA biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that the two genes were negatively regulated by ethylene and a cross-talk between GA and ethylene was involved in the regulation of the two genes occurring in young anthers. The treatment of NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development to a status close to that of control. DNA blots of lily genomic DNA indicated that the two genes were encoded by a small gene family. The different actions of hormones on gene expression and the possible function of the gene products in young anthers are discussed.

Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca(2+): LlLIM1 showed a preference for F-actin binding under low pH and low Ca(2+) concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed.

Exhibiting rapid polarized growth, the pollen tube delivers the male gametes into the ovule for fertilization in higher plants. To get an overall picture of gene expression during pollen germination and pollen tube growth, we profiled the transcription patterns of 1,536 pollen cDNAs from lily (Lilium longiflorum) by microarray. Among those that exhibited significant differential expression, a cDNA named lily ankyrin repeat-containing protein (LlANK) was thoroughly studied. The full-length LlANK cDNA sequence predicts a protein containing five tandem ankyrin repeats and a RING zinc-finger domain. The LlANK protein possesses ubiquitin ligase activity in vitro. RNA blots demonstrated that LlANK transcript is present in mature pollen and its level, interestingly contrary to most pollen mRNAs, up-regulated significantly during pollen germination and pollen tube growth. When fused with green fluorescent protein and transiently expressed in pollen, LlANK was found dominantly associated with membrane-enclosed organelles as well as the generative cell. Overexpression of LlANK, however, led to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. Taken together, these results showed that LlANK is a ubiquitin ligase associated with membrane-enclosed organelles and required for polarized pollen tube growth.

Two AGL2-like MADS-box genes, Lily MADS Box Gene (LMADS) 3 and LMADS4, with extensive homology of LMADS3 to the Arabidopsis SEPALLATA3 were characterized from the lily (Lilium longiflorum). Both LMADS3 and LMADS4 mRNA were detected in the inflorescence meristem, in floral buds of different developmental stages, and in all four whorls of the flower organ. LMADS4 mRNA is also expressed in vegetative leaf and in the inflorescence stem where LMADS3 expression is absent. Transgenic Arabidopsis, which ectopically expresses LMADS3, showed novel phenotypes by significantly reducing plant size, flowering extremely early, and loss of floral determinacy. By contrast, 35S::LMADS4 transgenic plants were morphologically indistinguishable from wild-type plants. The early-flowering phenotype in 35S::LMADS3 transgenic Arabidopsis plants was correlated with the up-regulation of flowering time genes FT, SUPPRESSOR OF OVEREXPRESSION OF CO 1, LUMINIDEPENDENS, and flower meristem identity genes LEAFY and APETALA1. This result was further supported by the ability of 35S::LMADS3 to rescue the late-flowering phenotype in gigantea-1 (gi-1), constans-3 (co-3), and luminidependens-1 but not for ft-1 or fwa-1 mutants. The activation of these flowering time genes is, however, indirect because their expression was unaffected in plants transformed with LMADS3 fused with rat glucocorticoid receptor in the presence of both dexamethasone and cycloheximide.

Flowering plant male gametic cell-specific gene expression has been reported recently but the regulatory elements controlling specificity of such genes expressed in generative cell and sperm cells have not been identified and studied. Here, we report the 0.8 kb promoter sequence upstream of the start of the transcription site of the generative cell-specific gene, LGC1, sufficient to regulate the expression of reporter genes in a cell-specific manner. In addition, the diphtheria toxin A-chain- (DT-A)-coding region under the control of the LGC1 promoter sequence confirmed unequivocally the lack of LGC1 expression in vegetative tissues. Transgenic tobacco plants carrying the LGC1-DT/A construct showed normal phenotype except for anthers of these plants that contained sterile and aborted pollen. Truncation and internal deletion analysis of the LGC1 promoter identified -242 bp as the minimal sequence necessary for male gametic cell-specific expression. In addition, a regulatory sequence required for determining generative cell-specific expression of LGC1 was identified. Deletion of this regulatory sequence led to loss of the generative cell specificity resulting in activation of this promoter in other tissues where it is normally repressed. Therefore, male gametic cell specificity of the LGC1 gene seems to be regulated by factors that suppress its activation in other plant cells. This is the first report of a male gametic cell-specific promoter, hence can be used as a novel tool in molecular analyses and experimental manipulation of flowering plant spermatogenesis and fertilization.

During pollination the pollen tube grows into the style and toward the ovary via the transmitting tract. In lily the growth of pollen tubes involves tube cell adhesion to transmitting tract cells. We reported two molecules involved in this adhesion event. One is a pectic polysaccharide and the other, a 9 kDa basic protein named SCA for stigma/stylar cysteine-rich adhesin. SCA, which shows some identity with LTP (lipid transfer protein), was localized to the transmitting tract epidermis of the style where pollen tubes adhere. The present studies on the expression of SCA indicate that the protein has a similar expression pattern with LTP1 in Arabidopsis and that the protein is abundant in both the stigma and the style. For further proof of its role in pollen tube adhesion the activity of Escherichia coli-expressed protein has been studied in an in vitro adhesion assay system.

We investigated the cytoskeleton of Lilium longiflorum pollen tubes and examined the effects of the type 2A protein phosphatase (PP2A) inhibitors calyculin A and okadaic acid. An improved method for actin visualization, the simultaneous fixation and staining with rhodamine-labelled phalloidin during microscopical observation, revealed abundant actin filaments of no preferential orientation in the apical clear zone. Microtubules, visualized by indirect immunofluorescence, were mostly absent from the apices of straight-growing pollen tubes but present in those with irregular shape. Double labelling showed that both actin bundles and microtubules had a similar longitudinal or slightly helical orientation in the pollen tube shaft. In the presence of 30 nM calyculin A or okadaic acid, pollen tubes grew very slowly, branched frequently, and contained isolated, randomly oriented, curved actin bundles and microtubules. Treating pollen tubes with calyculin A or okadaic acid after germination arrested growth immediately, reversibly altered the alignment of actin bundles from axial to transverse, and disassembled microtubules. The changes in actin organization caused by the PP2A inhibitors were similar to those observed upon overexpression of AtRop1 (Y. Fu, G. Wu, Z. Yang, Journal of Cell Biology 152: 1019-1032, 2001), suggesting that hyperphosphorylation interferes with the signalling pathway of small GTPases. The effects of the PP2A inhibitors could be ameliorated with nanomolar concentrations of latrunculin B.