FERONIA controls pectin- and nitric oxide-mediated male-female interaction.

Species that propagate by sexual reproduction actively guard against the fertilization of an egg by multiple sperm (polyspermy). Flowering plants rely on pollen tubes to transport their immotile sperm to fertilize the female gametophytes inside ovules. In Arabidopsis, pollen tubes are guided by cysteine-rich chemoattractants to target the female gametophyte1,2. The FERONIA receptor kinase has a dual role in ensuring sperm delivery and blocking polyspermy3. It has previously been reported that FERONIA generates a female gametophyte environment that is required for sperm release4. Here we show that FERONIA controls several functionally linked conditions to prevent the penetration of female gametophytes by multiple pollen tubes in Arabidopsis. We demonstrate that FERONIA is crucial for maintaining de-esterified pectin at the filiform apparatus, a region of the cell wall at the entrance to the female gametophyte. Pollen tube arrival at the ovule triggers the accumulation of nitric oxide at the filiform apparatus in a process that is dependent on FERONIA and mediated by de-esterified pectin. Nitric oxide nitrosates both precursor and mature forms of the chemoattractant LURE11, respectively blocking its secretion and interaction with its receptor, to suppress pollen tube attraction. Our results elucidate a mechanism controlled by FERONIA in which the arrival of the first pollen tube alters ovular conditions to disengage pollen tube attraction and prevent the approach and penetration of the female gametophyte by late-arriving pollen tubes, thus averting polyspermy.

Characterization of a Cis-Prenyltransferase from Lilium longiflorum Anther.

A group of prenyltransferases catalyze chain elongation of farnesyl diphosphate (FPP) to designated lengths via consecutive condensation reactions with specific numbers of isopentenyl diphosphate (IPP). cis-Prenyltransferases, which catalyze cis-double bond formation during IPP condensation, usually synthesize long-chain products as lipid carriers to mediate peptidoglycan biosynthesis in prokaryotes and protein glycosylation in eukaryotes. Unlike only one or two cis-prenyltransferases in bacteria, yeast, and animals, plants have several cis-prenyltransferases and their functions are less understood. As reported here, a cis-prenyltransferase from Lilium longiflorum anther, named LLA66, was expressed in Saccharomyces cerevisiae and characterized to produce C40/C45 products without the capability to restore the growth defect from Rer2-deletion, although it was phylogenetically categorized as a long-chain enzyme. Our studies suggest that evolutional mutations may occur in the plant cis-prenyltransferase to convert it into a shorter-chain enzyme.

A WD40 protein, AtGHS40, negatively modulates abscisic acid degrading and signaling genes during seedling growth under high glucose conditions.

The Arabidopsis thaliana T-DNA insertion mutant glucose hypersensitive (ghs) 40-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The ghs40-1 mutant displayed severely impaired cotyledon greening and expansion and showed enhanced reduction in hypocotyl elongation of dark-grown seedlings when grown in Glc concentrations higher than 3 %. The Glc-hypersensitivity of ghs40-1 was correlated with the hyposensitive phenotype of 35S::AtGHS40 seedlings. The phenotypes of ghs40-1 were recovered by complementation with 35S::AtGHS40. The AtGHS40 (At5g11240) gene encodes a WD40 protein localized primarily in the nucleus and nucleolus using transient expression of AtGHS40-mRFP in onion cells and of AtGHS40-EGFP and EGFP-AtGHS40 in Arabidopsis protoplasts. The ABA biosynthesis inhibitor fluridone extensively rescued Glc-mediated growth arrest. Quantitative real time-PCR analysis showed that AtGHS40 was involved in the control of Glc-responsive genes. AtGHS40 acts downstream of HXK1 and is activated by ABI4 while ABI4 expression is negatively modulated by AtGHS40 in the Glc signaling network. However, AtGHS40 may not affect ABI1 and SnRK2.6 gene expression. Given that AtGHS40 inhibited ABA degrading and signaling gene expression levels under high Glc conditions, a new circuit of fine-tuning modulation by which ABA and ABA signaling gene expression are modulated in balance, occurred in plants. Thus, AtGHS40 may play a role in ABA-mediated Glc signaling during early seedling development. The biochemical function of AtGHS40 is also discussed.

Characterization of a lily anther-specific gene encoding cytoskeleton-binding glycoproteins and overexpression of the gene causes severe inhibition of pollen tube growth.

This work characterizes an anther/pollen-specific gene that encodes potential intermediate filament (IF)-binding glycoproteins in lily (Lilium longiflorum Thunb. cv. Snow Queen) anthers during the development and pollen germination. LLP13 is a single gene that encodes a polypeptide of 807 amino acids, and a calculated molecular mass of 91 kDa. The protein contains a predicted transmembrane domain at the N-terminus and a conserved domain of unknown function (DUF)593 at the C-terminal half of the polypeptide. Sequence analysis revealed that LLP13 shares significant identity (37-41 %) with two intermediate filament antigen-binding proteins, representing a unique subgroup of DUF593 domain proteins from known rice and Arabidopsis species. The expression of LLP13 gene is anther-specific, and the transcript accumulates only at the stage of pollen maturation. Both premature drying and abscisic acid (ABA) treatment of developing pollen indicated that LLP13 was not induced by desiccation and ABA, but by other developmental cues. Antiserum was raised against the overexpressed LLP13C fragment of the protein in Escherichia coli and affinity-purified antibodies were prepared. Immunoblot analyses revealed that the LLP13 protein was a heterogeneous, anther-specific glycoprotein that accumulated only at the stage of pollen maturation. The protein is not heat-soluble. The level of LLP13 protein remained for 24 h during germination in vitro. Overexpression of LLP13-GFP or GFP-LLP13 in lily pollen tubes caused severe inhibition of tube elongation. The LLP13 protein codistributed with mTalin in growing tubes, suggesting that it apparently decorates actin cytoskeleton and is likely a cytoskeleton-binding protein that binds with IFs that potentially exist in pollen tubes.

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.

AtRH57, a DEAD-box RNA helicase, is involved in feedback inhibition of glucose-mediated abscisic acid accumulation during seedling development and additively affects pre-ribosomal RNA processing with high glucose.

The Arabidopsis thaliana T-DNA insertion mutant rh57-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The other two rh57 mutants also showed Glc hypersensitivity similar to rh57-1, strongly suggesting that the Glc-hypersensitive feature of these mutants results from mutation of AtRH57. rh57-1 and rh57-3 displayed severely impaired seedling growth when grown in Glc concentrations higher than 3%. The gene, AtRH57 (At3g09720), was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc and salt. The new AtRH57 belongs to class II DEAD-box RNA helicase gene family. Transient expression of AtRH57-EGFP (enhanced green fluorescent protein) in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. Purified AtRH57-His protein was shown to unwind double-stranded RNA independent of ATP in vitro. The ABA biosynthesis inhibitor fluridone profoundly redeemed seedling growth arrest mediated by sugar. rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. A feedback inhibition of ABA accumulation mediated by AtRH57 exists within the sugar-mediated ABA signaling. AtRH57 mutation and high Glc conditions additively caused a severe defect in small ribosomal subunit formation. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type Col-0 under high Glc conditions. These results suggested that AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth.

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.

Lily ASR protein-conferred cold and freezing resistance in Arabidopsis.

The lily LLA23 protein is a member of the abscisic acid, stress and ripening-induced (ASR) protein family. Constitutive overexpression of LLA23 under the cauliflower mosaic virus 35S promoter confers cold and freezing tolerance in Arabidopsis. The phenotypical growth and survival percentage of the two transgenic 35S::LLA23 plants showed higher resistance to cold and freezing conditions than those of wild-type (WT) plants. The electrolyte leakage in WT leaves increased by approximately fourfold at -2 °C relative to that at 22 °C whereas both transgenic leaves showed little ion leakage under the same conditions. A microarray analysis of LLA23-overexpressing transgenic line, 35S::LLA23E, under normal growing conditions was previously conducted by Yang et al. (Protoplasma, 2008, 233:241-254). Microarray analysis showed that 12 cold-responsive genes are upregulated and 25 cold-responsive genes are downregulated by lily ASR. Many ASR-regulated genes encode proteins involved in the classes of defense/stress-related, transcription, and metabolism. Quantitative polymerase chain reaction analysis confirms the changes in mRNA levels observed in the microarray analysis. Thus, our results provide in vivo evidence implying that LLA23 mediates cold/freezing stress-responsive signaling. To gain further insight into the functions of LLA23 protein, an in vitro enzyme protection assay was used in which lactate dehydrogenase and malate dehydrogenase were subjected to unfavorable conditions. The assay revealed that both enzyme activities were significantly retained with the addition of LLA23, which was superior to either trehalose or BSA, suggesting that the LLA23 protein can protect enzymatic activities against freeze-thaw cycles. The 35S::LLA23 seedlings also exhibited enzyme activity superior to WT at -4 °C. These results suggest that LLA23 may act as an osmoprotectant as well as a transcription factor to confer 35S::LLA23 plants enhanced cold and freezing resistance.

Expression, localization and function of a cis-prenyltransferase in the tapetum and microspores of lily anthers.

The cis-prenyltransferase gene LLA66 (Lilium longiflorum anther-66), the first prenyltransferase to be identified in the tapetum and microspores, was selected from a suppression subtractive cDNA library during microspore development in the anther of L. longiflorum. The LLA66 cDNA encodes a polypeptide of 308 amino acids with a calculated molecular mass of 35.7 kDa. Thermal asymmetric interlaced-PCR was employed to obtain the 5'-regulatory region of LLA66. Sequence alignment revealed that the LLA66 protein shares 30-41% identity with cis-prenyltransferases of various broad-spectrum species and is phylogenetically distinct from other monocot cis-prenyltransferases. Based on critical regulatory domains in cis-prenyltransferase, LLA66 was concluded to catalyze the production of long-chain polyprenyl products. RNA blot analysis indicated that the LLA66 gene is anther specific and differentially expressed during microspore development in the anther. In situ hybridization with the digoxigenin-labeled antisense riboprobe of LLA66 showed strong signals at the tapetal layer of the anther wall. The LLA66 mRNA was also coordinately detected in the microspores. Furthermore, gibberellin inhibitor analysis indicated that the LLA66 gene is endogenously induced by gibberellin, but its induction is independent of ethylene regulation. Reverse transcription-PCR analysis indicated that gene expression of LLA66 both in the microspore and in the anther wall increased to the maximum level, at which stage the tapetum became highly active and secretory. The enzyme activity of prenyltransferases in various stages of microspore development correlated with tapetal growth and disintegration. LLA66 was introduced into Saccharomyces cerevisiae, and the His-tagged LLA66 protein was affinity purified using Ni(2+)-nitrilotriacetic acid-agarose. The involvement of cis-prenyltransferase in the anther in the synthesis of dolichols and polyprenols is discussed.

Lily Cdc42/Rac-interactive binding motif-containing protein, a Rop target, involves calcium influx and phosphoproteins during pollen germination and tube growth.

We report unique desiccation-associated ABA signaling transduction through which the Rop (Rho GTPase of plants) and its target LLP12-2 are regulated during the stage of pollen maturation and tube growth. Overexpression of LLP12-2 drastically inhibited pollen germination and tube growth. Studies on the germination inhibitors, Ca (2+) influx blocking agents LaCl 3 and EGTA and an actin-depolymerizing drug, latrunculin B (LatB), revealed that the LLP12-2-induced inhibition of germination and tube growth is significantly suppressed by LaCl 3 and EGTA in the LLP12-2-overexpressing pollen but not by LatB. These results suggested that LLP12-2 is associated with Ca (2+) influx in the cytoplasm and may be not with actin assembly. With the addition of LaCl 3 and EGTA, LLP12-2-overexpressing pollen increased germination and tube growth compared with the one without addition, whereas pollen expressing GFP decreased germination and tube growth. Thus, an optimum level of [Ca (2+) ]cyt influx is crucial for normal germination and tube growth. Studies on the inhibitors, staurosporine and okadaic acid in the LLP12-2-overexpressing pollen, showed no appreciable increase in germination when compared with the one without addition, suggesting that staurosporine-sensitive protein kinases and dephosphorylation of phosphoproteins may be not involved in the LLP12-2 mediated germination. However, the LLP12-2-induced inhibition of tube length was slightly but significantly suppressed by staurosporine, suggesting that staurosporine-sensitive protein kinases involve in the LLP12-2-induced inhibition of tube growth.

Rop GTPase and its target Cdc42/Rac-interactive-binding motif-containing protein genes respond to desiccation during pollen maturation.

Here, we report unique desiccation-associated ABA signaling transduction through which the Rop (Rho GTPase of plants) gene is regulated during the stage of pollen maturation. A gene encoding Rho GTPase was identified in lily (Lilium longiflorum Thunb.) pollen. Phylogenetic tree analysis of lily LLP-Rop1 revealed that the protein shares greatest similarity with Group 4 Rops. The LLP-Rop1 gene was spatially and temporally regulated in lily plants during anther development. Accumulation of the LLP-Rop1 transcript decreased its level of accumulation while LLP-12-2, a Rop-interactive CRIB motif-containing (RIC) transcript increased either by premature drying of developing anther/pollen or by the exogenous application of various concentrations of abscisic acid (ABA) during pollen maturation and tube growth. Application of norflurazon, an ABA biosynthesis inhibitor, also resulted in the downregulation of the LLP-Rop1 gene while LLP-12-2 was upregulated by ABA. Furthermore, an increase in ABA in the maturing pollen correlated with desiccation that occurred in the anther prior to anthesis. LLP-Rop1 overexpression inhibited tube elongation, and caused tube expansion and the formation of a ballooned tip. CFP-LLP-Rop1 was localized to the cytoplasm having a greater intensity along the tube plasma membrane. Fluorescence resonance energy transfer analysis of lily pollen tubes coexpressing CFP-LLP-Rop1 and YFP-LLP-12-2 demonstrated that LLP-12-2 is a target RIC protein of active LLP-Rop1, but the interaction between LLP-Rop1 and LLP-12-2 proteins is probably irrelevant of dehydration in the dried pollen.

A conserved transcriptional regulator is required for RNA-directed DNA methylation and plant development.

RNA-directed DNA methylation (RdDM) is a conserved mechanism for epigenetic silencing of transposons and other repetitive elements. We report that the rdm4 (RNA-directed DNA Methylation4) mutation not only impairs RdDM, but also causes pleiotropic developmental defects in Arabidopsis. Both RNA polymerase II (Pol II)- and Pol V-dependent transcripts are affected in the rdm4 mutant. RDM4 encodes a novel protein that is conserved from yeast to humans and interacts with Pol II and Pol V in plants. Our results suggest that RDM4 functions in epigenetic regulation and plant development by serving as a transcriptional regulator for RNA Pol V and Pol II, respectively.

An effector of RNA-directed DNA methylation in arabidopsis is an ARGONAUTE 4- and RNA-binding protein.

DNA methylation is a conserved epigenetic mark in plants and mammals. In Arabidopsis, DNA methylation can be triggered by small interfering RNAs (siRNAs) through an RNA-directed DNA methylation (RdDM) pathway. Here, we report the identification of an RdDM effector, KTF1. Loss-of-function mutations in KTF1 reduce DNA methylation and release the silencing of RdDM target loci without abolishing the siRNA triggers. KTF1 has similarity to the transcription elongation factor SPT5 and contains a C-terminal extension rich in GW/WG repeats. KTF1 colocalizes with ARGONAUTE 4 (AGO4) in punctate nuclear foci and binds AGO4 and RNA transcripts. Our results suggest KTF1 as an adaptor protein that binds scaffold transcripts generated by Pol V and recruits AGO4 and AGO4-bound siRNAs to form an RdDM effector complex. The dual interaction of an effector protein with AGO and small RNA target transcripts may be a general feature of RNA-silencing effector complexes.

NRPD4, a protein related to the RPB4 subunit of RNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA-directed DNA methylation.

RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing in plants. The plant-specific RNA polymerases IV and V are required for the generation of 24-nucleotide (nt) siRNAs and for guiding sequence-specific DNA methylation by the siRNAs, respectively. However, unlike the extensively studied multisubunit Pol II, our current knowledge about Pol IV and Pol V is restricted to only the two largest subunits NRPD1a/NRPD1 and NRPD1b/NRPE1 and the one second-largest subunit NRPD2a. It is unclear whether other subunits may be required for the functioning of Pol IV and Pol V in RdDM. From a genetic screen for second-site suppressors of the DNA demethylase mutant ros1, we identified a new component (referred to as RDM2) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway. The differential effects of the mutations on two mechanistically distinct transcriptional silencing reporters suggest that RDM2, NRPD1, NRPE1, NRPD2a, HEN1, and DRD1 function only in the siRNA-dependent pathway of transcriptional silencing, whereas HDA6 and AGO4 have roles in both siRNA-dependent and -independent pathways of transcriptional silencing. In the rdm2 mutants, DNA methylation and siRNA accumulation were reduced substantially at loci previously identified as endogenous targets of Pol IV and Pol V, including 5S rDNA, MEA-ISR, AtSN1, AtGP1, and AtMU1. The amino acid sequence of RDM2 is similar to that of RPB4 subunit of Pol II, but we show evidence that RDM2 has diverged significantly from RPB4 and cannot function in Pol II. An association of RDM2 with both NRPD1 and NRPE1 was observed by coimmunoprecipitation and coimmunolocalization assays. Our results show that RDM2/NRPD4/NRPE4 is a new component of the RdDM pathway in Arabidopsis and that it functions as part of Pol IV and Pol V.

Expression and regulation of two novel anther-specific genes in Lilium longiflorum.

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.

The LLA23 protein translocates into nuclei shortly before desiccation in developing pollen grains and regulates gene expression in Arabidopsis.

We have isolated the LLA23 gene in the pollen of Lilium longiflorum. The LLA23 gene encodes an ASR (named after abscisic acid, stress and ripening) protein that has a nuclear localization sequence at the C terminus. The gene is interrupted by one single intron and possesses a long 5'-untranslated region. Southern blots of lily genomic DNA indicated that LLA23 is a member of a small gene family. We examined the link between LLA23 location and the desiccation that naturally occurs in developing anthers using immunogold labeling. When pollen reached maturity, a significant increase in LLA23 labeling was observed in the nuclei of both vegetative and generative cells from 10- to 12-cm buds and thereafter. This clearly demonstrates that a marked increase in LLA23 translocation from the cytoplasm to both nuclei of pollen grains occurs in 12-cm buds, a stage shortly before the commencement of desiccation during anther development. In addition, microarray analysis showed that 410 (206 up-regulated and 204 down-regulated) genes have altered expression in LLA23-overexpressing plants. Quantitative PCR analysis confirmed the changes in mRNA levels observed in our microarray analysis. This genome-wide overview of gene expression supports the theory that LLA23 acts as a regulator.

Identification of anther-specific/predominant genes regulated by gibberellin during development of lily anthers.

We successfully identify anther-specific/predominant genes induced by gibberellin (GA) at the microspore stage of lily (Lilium longiflorum) anthers. We used a suppression-subtractive hybridization strategy to identify 22 individual cDNAs followed by a reverse RNA dot plot to determine their specificities at the microspore stage. Of the 22 genes, 12 are clearly anther-specific and three are anther-predominant. Sequence analysis revealed that five anther-specific/predominant genes are novel. The transcripts of anther-specific/predominant genes were differentially detected at the microspore development phase; some began accumulating in level as early as the occurrence of meiosis. When uniconazole, an inhibitor of GA biosynthesis was applied in young lily plants we found that all of the anther-specific/predominant genes, with the exception of LLA-139, were up-regulated by GAs in the anther while only some were responsive to the exogenous addition of 100 microM GA3. In situ hybridization with antisense riboprobes of selected genes in the anther showed a strong signal localized to the tapetal layer. The different actions of GA on gene expression in anthers are discussed.

Gene expression pattern at desiccation in the anther of Lilium longiflorum.

Although gene expression profile of pollen has been described, there is limited information regarding a particular phase during anther/pollen development. This work characterizes gene expression pattern at desiccation in lily (Lilium longiflorum Thunb. cv Snow Queen) anthers. We have applied a suppression-subtractive hybridization (SSH) strategy, through which 90 clones were identified and sequenced. These clones resulted in the identification of 42 individual cDNAs among which 33 genes were specifically expressed at the desiccation phase of anthers of >150-mm buds. Fourteen cDNAs were chosen for further examination. Six genes were both dehydration- and abscisic acid (ABA)-inducible whereas the other eight genes were apparently dehydration-irrelevant. The group of dehydration- and ABA-induced genes was also induced by desiccation that developmentally occurs in the anther. The application of fluridone has a significant effect of inhibition on mRNA accumulation of these genes in maturing anthers during which desiccation occurs. Pollen germination analysis indicated that, of those dehydration-irrelevant genes, three were ABA-responsive and the other five were not. Thus, three separate signal pathways that function in the activation of late genes at desiccation during anther development are established. The first is the ABA-dependent pathway induced by environmental stress of dehydration. The other two pathways of signaling triggered by developmental cues, through which one is ABA-dependent and another is ABA-independent. The 14 gene proteins showed spatial and temporal expression patterns and may participate in membrane/cell wall synthesis, cytoskeletal organization, signaling, RNA binding, ubiquitin-mediated degradation and transportation during germination and tube growth.