Putative regulatory role of hexokinase and fructokinase in terpenoid aroma biosynthesis in Lilium 'Siberia'.

Lily is one of the most economically important flowers worldwide due to its elegant appearance and appealing scent, which is mainly composed of monoterpene ocimene, linalool and benzenoids. Sugars are the primary products of plants, with fructose and hexose sugars being the substrate material for most organic compounds and metabolic pathways in plants. Herein, we isolated and functionally characterized hexokinase (LoHXK) and fructokinase (LoFRK) from Lilium 'Siberia' flower, which indicated their potential roles in floral aroma production. Real-time PCR analysis showed that LoHXK and LoFRK were highly expressed in the flower filament. Overexpression and virus-induced gene silencing (VIGS) assays revealed that LoHXK and LoFRK significantly modified the emission of ß-ocimene and linalool contents via regulation of expression of key structural volatile synthesis genes (LoTPS1 and LoTPS3). Under exogenous glucose and fructose application, the volatile contents of ß-ocimene and linalool were increased and the expression levels of key structural genes were upregulated. The emission of ß-ocimene and linalool followed a diurnal circadian rhythm. Determination of carbon fluxes via 13C-labeled glucose and 13C-labeled fructose experiments showed that the mass spectra of ocimene and linalool significantly increased, however, the m/z ratio of ethyl benzoate did not change. Furthermore, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that LoFRK interacted with LoMYB1 and LoMYB2 proteins. Together, these results suggest that hexokinase and fructokinase may play significant roles in the regulation of ocimene and linalool biosynthesis in Lilium 'Siberia'.

Isolation and functional identification of a Botrytis cinerea-responsive caffeoyl-CoA O-methyltransferase gene from Lilium regale wilson.

In plants, genes involved in the Phenylpropanoid/monolignol pathway play important roles in lignin biosynthesis and plant immunity. However, their biological function in Lilium remains poorly characterized. Comparative RNA sequencing of the expression profiles of the monolignol pathway genes from fungi-resistant species Lilium regale after inoculation with Botrytis cinerea was performed. One upregulated caffeoyl-CoA O-methyltransferase gene, LrCCoAOMT, was cloned for functional characterization by reverse genetic methods. LrCCoAOMT encodes a putative protein of 246 amino acids and is highly expressed in stem tissues and responsive to salicylic acid (SA) signaling and B. cinerea infection. LrCCoAOMT was largely directed to the cytoplasm. LrCCoAOMT overexpression in Arabidopsis resulted in an increased lignin deposition in vascular tissues and conferred resistance to B. cinerea infection in transgenic plants. Transient transformation of LrCCoAOMT in nonresistant Lilium sargentiae leaves also identified the defense function to B. cinerea. In addition, transcript levels of genes involved in the monolignol and SA-dependent signaling pathways were altered in transgenic Arabidopsis, suggesting that LrCCoAOMT might play vital roles in the resistance of L. regale to B. cinerea related to the levels of lignin and the regulation of SA signaling. This is the first report to functionally characterize a CCoAOMT gene in Lilium, a potential molecular target for lily molecular improvement.

Cloning, functional characterization and expression analysis of LoTPS5 from Lilium 'Siberia'.

Lilium 'Siberia' is a perennial herbaceous plant that is commercially significant because of its snowy white floral color and appealing scent which is mainly due to the presence of monoterpenes and benzoids compounds in floral volatile profile. In the current study, LoTPS5 was cloned and functionally characterized. Results revealed that LoTPS5 specifically generates squalene from FPP, whereas no product was produced when it was incubated with GPP or GGPP. The subcellular localization experiment showed that LoTPS5 was located in plastids. Furthermore, LoTPS5 showed its high expression in the leaf followed by petals and sepals of the flower. Moreover, the expression of LoTPS5 gradually increased from the bud stage and peak at the full-bloom stage. Besides, LoTPS5 showed a diurnal circadian rhythmic pattern with a peak in the afternoon (16:00) followed by deep night (24:00) and morning (8:00), respectively. LoTPS5 is highly responsive to mechanical wounding by rapidly elevating its mRNA transcript level. The current study will provide significant information for future studies of terpenoid and squalene biosynthesis in Lilium 'Siberia'.

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.

De novo transcriptome characterization of Lilium 'Sorbonne' and key enzymes related to the flavonoid biosynthesis.

Lily is an important cut-flower and bulb crop in the commercial market. Here, transcriptome profiling of Lilium 'Sorbonne' was conducted through de novo sequencing based on Illumina platform. This research aims at revealing basic information and data that can be used for applied purposes especially the molecular regulatory information on flower color formation in lily. In total, 36,920,680 short reads which corresponded to 3.32 GB of total nucleotides, were produced through transcriptome sequencing. These reads were assembled into 39,636 Unigenes, of which 30,986 were annotated in Nr, Nt, Swiss-Prot, KEGG, COG, GO databases. Based on the three public protein databases, a total of 32,601 coding sequences were obtained. Meanwhile, 19,242 Unigenes were assigned to 128 KEGG pathways. Those with the greatest representation by unique sequences were for ''metabolic pathways'' (5,406 counts, 28.09 %). Our transcriptome revealed 156 Unigenes that encode key enzymes in the flavonoid biosynthesis pathway including CHS, CHI, F3H, FLS, DFR, etc. MISA software identified 2,762 simple sequence repeats, from which 1,975 primers pairs were designed. Over 2,762 motifs were identified, of which the most frequent was AG/CT (659, 23.86 %), followed by A/T (615, 22.27 %) and CCG/CGG (416, 15.06 %). Based on the results, we believe that the color formation of the Lilium 'Sorbonne' flower was mainly controlled by the flavonoid biosynthesis pathway. Additionally, this research provides initial genetic resources that will be valuable to the lily community for other molecular biology research, and the SSRs will facilitate marker-assisted selection in lily breeding.

Transcriptome analysis of carbohydrate metabolism during bulblet formation and development in Lilium davidii var. unicolor.

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.

Expression and localisation of a senescence-associated KDEL-cysteine protease from Lilium longiflorum tepals.

Senescence is a tightly regulated process and both compartmentalisation and regulated activation of degradative enzymes is critical to avoid premature cellular destruction. Proteolysis is a key process in senescent tissues, linked to disassembly of cellular contents and nutrient remobilisation. Cysteine proteases are responsible for most proteolytic activity in senescent petals, encoded by a gene family comprising both senescence-specific and senescence up-regulated genes. KDEL cysteine proteases are present in senescent petals of several species. Isoforms from endosperm tissue localise to ricinosomes: cytosol acidification following vacuole rupture results in ricinosome rupture and activation of the KDEL proteases from an inactive proform. Here data show that a Lilium longiflorum KDEL protease gene (LlCYP) is transcriptionally up-regulated, and a KDEL cysteine protease antibody reveals post-translational processing in senescent petals. Plants over-expressing LlCYP lacking the KDEL sequence show reduced growth and early senescence. Immunogold staining and confocal analyses indicate that in young tissues the protein is retained in the ER, while during floral senescence it is localised to the vacuole. Our data therefore suggest that the vacuole may be the site of action for at least this KDEL cysteine protease during tepal senescence.

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.

Enhancement of alkaline phytase production in Pichia pastoris: influence of gene dosage, sequence optimization and expression temperature.

Supplementation of animal feed with phytases has proven to be an effective strategy to alleviate phosphorous contamination of soil and water bodies. The inability of non-ruminant animals to digest phytates in corn and soybeans contributes to environmental contamination. Alkaline phytase from lily pollen (LlALP) exhibits unique catalytic and thermal stability properties that could be useful as a feed supplement. rLlALP2 was successfully expressed in Pichia pastoris; however, enzyme yields were modest (8-10 mg/L). In this paper, we describe our efforts to enhance rLlALP2 yield by investigating the influence of the following potential limiting factors: transgene copy number, codon bias, sequence optimization, and temperature during expression. Data presented indicate that increasing rLlAlp2 copy number was detrimental to heterologous expression, clones with one copy of wt-rLlAlp2 produced the highest activity, clones with two, four and seven or more copies produced 70%, 25% and 10% respectively, of enzyme activity implying that gene dosage is not limiting rLlALP2 yield. Use of a sequence-optimized rLlAlp2 increased the yield of the active enzyme by 25-50% in one/two copy clones, suggesting that translational efficiency is not a major bottleneck for rLlALP2 expression. Reducing the temperature during heterologous expression led to increases of 1.2-20-fold suggesting that protein folding and post-translational processes may be the dominant factors limiting rLlALP2 expression. Early knowledge of the transgene copy number allowed us to develop a more rational strategy for yield enhancement. Cumulatively, sequence optimization and temperature reduction led to the doubling of rLlALP2 enzyme activity in P. pastoris.

ABA-mediated heterophylly is regulated by differential expression of 9-cis-epoxycarotenoid dioxygenase 3 in lilies.

Although exogenous ABA-regulated heterophylly has been well documented in multiple plant species, the effect of endogenous ABA and its molecular mechanism remain uncharacterized. In the present study, the effects of endogenous ABA on heterophyllous switching were investigated in two different lily varieties, Lilium formosanum and Lilium oriental hybrid 'Casa Blanca'. Seedlings of L. formosanum, which have scale-leaf-type growth, displayed low levels of both 9-cis-epoxycarotenoid dioxygenase 3 (LfNCED3) transcripts and ABA, whereas seedlings of L. oriental hybrid 'Casa Blanca', which have scale-type growth, displayed high levels of both LoNCED3 transcripts and ABA. Sucrose induced endogenous ABA production in cultured lilies; low ABA induction shows scale-leaf-type growth, whereas scale-type growth becomes predominant when ABA levels are high. Heterologous expression of either LfNCED3 or LoNCED3 was found to complement the Arabidopsis Atnced3 mutant. Interestingly, the expression patterns of LfNCED3 and LoNCED3 in transgenic Arabidopsis plants are distinguishable. Further promoter analysis revealed that a putative E2F-like element in the LfNCED3 promoter, but not in the LoNCED3 promoter, plays a negative role in controlling its activity. Collectively, our results demonstrate that NCED3 plays a key role in ABA-mediated heterophylly in lilies.

Characterization of a chalcone synthase (CHS) flower-specific promoter from Lilium orential 'Sorbonne'.

The first enzyme in the flavonoid pathway, chalcone synthase, is encoded by a gene (CHS) whose expression is normally under developmental control. In our previous studies, an 896-bp promoter region of a flower-specific CHS gene was isolated from Lilium orential 'Sorbonne', and designated as PLoCHS. Here, the PLoCHS promoter was fused to the ß-glucuronidase (GUS) gene to characterize its spatial and temporal expression in Petunia hybrida 'Dreams Midnight' using an Agrobacterium-mediated leaf disc transformation method. Our results demonstrated that GUS expression was present in flowers, but reduced or absent in the other tissues (leaf and stem) examined. In petals, GUS activity reached its peak at flower developmental stage 4, and decreased at later stages. Deletion analysis indicated that even a 307-bp fragment of the PLoCHS promoter could still direct flower-specific expression. Further deletion of the region from -261 to -72 bp resulted in weak expression in different organs, including flowers, leaves and stems. This evidence combined with prediction of cis-acting elements in the PLoCHS promoter suggests that the TACPyAT box located in this promoter plays a key role in the regulation of organ-specific expression.

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.

Changes in ultrastructure, protease and caspase-like activities during flower senescence in Lilium longiflorum.

The last phase of flower development is senescence during which nutrients are recycled to developing tissues. The ultimate fate of petal cells is cell death. In this study we used the ethylene-insensitive Lilium longiflorum as a model system to characterize Lily flower senescence from the physiological, biochemical and ultrastructural point of view. Lily flower senescence is highly predictable: it starts three days after flower opening, before visible signs of wilting, and ends with the complete wilting of the corolla within 10 days. The earliest events in L. longiflorum senescence include a fall in fresh and dry weight, fragmentation of nuclear DNA and cellular disruption. Mesophyll cell degradation is associated with vacuole permeabilization and rupture. Protein degradation starts later, coincident with the first visible signs of tepal senescence. A fall in total protein is accompanied by a rise in total proteases, and also by a rise of three classes of caspase-like activity with activities against YVAD, DEVD and VEID. The timing of the appearance of these caspase-like activities argues against their involvement in the regulation of the early stages of senescence, but their possible role in the regulation of the final stages of senescence and cell death is discussed.

Osmoregulation in Lilium pollen grains occurs via modulation of the plasma membrane H+ ATPase activity by 14-3-3 proteins.

To allow successful germination and growth of a pollen tube, mature and dehydrated pollen grains (PGs) take up water and have to adjust their turgor pressure according to the water potential of the surrounding stigma surface. The turgor pressure of PGs of lily (Lilium longiflorum) was measured with a modified pressure probe for simultaneous recordings of turgor pressure and membrane potential to investigate the relation between water and electrogenic ion transport in osmoregulation. Upon hyperosmolar shock, the turgor pressure decreased, and the plasma membrane (PM) hyperpolarizes in parallel, whereas depolarization of the PM was observed with hypoosmolar treatment. An acidification and alkalinization of the external medium was monitored after hyper- and hypoosmotic treatments, respectively, and pH changes were blocked by vanadate, indicating a putative role of the PM H(+) ATPase. Indeed, an increase in PM-associated 14-3-3 proteins and an increase in PM H(+) ATPase activity were detected in PGs challenged by hyperosmolar medium. We therefore suggest that in PGs the PM H(+) ATPase via modulation of its activity by 14-3-3 proteins is involved in the regulation of turgor pressure.

Characterization of bacterial-type phosphoenolpyruvate carboxylase expressed in male gametophyte of higher plants.

BACKGROUND: Phosphoenolpyruvate carboxylase (PEPC) is a critical enzyme catalyzing the ß-carboxylation of phosphoenolpyruvate (PEP) to oxaloacetate, a tricarboxylic acid (TCA) cycle intermediate. PEPC typically exists as a Class-1 PEPC homotetramer composed of plant-type PEPC (PTPC) polypeptides, and two of the subunits were reported to be monoubiquitinated in germinating castor oil seeds. By the large-scale purification of ubiquitin (Ub)-related proteins from lily anther, two types of PEPCs, bacterial-type PEPC (BTPC) and plant-type PEPC (PTPC), were identified in our study as candidate Ub-related proteins. Until now, there has been no information about the properties of the PEPCs expressed in male reproductive tissues of higher plants. RESULTS: Expression analyses showed that lily BTPC (LlBTPC) and Arabidopsis BTPC (AtBTPC) were significantly expressed in pollen. The fusion protein AtBTPC-Venus localized in the cytoplasm of the vegetative cell (VC). Both LlBTPC and AtBTPC expression initiated after the last mitosis before pollen germination. Lily PTPC (LlPTPC) and monoubiquitinated LlPTPC (Ub-LlPTPC) remained at constant levels during pollen development. In late bicellular pollen of lily, LlBTPC forms a hetero-octameric Class-2 PEPC complex with LlPTPC to express PEPC activity. CONCLUSION: Our results suggest that an LlBTPC:Ub-LlPTPC:LlPTPC complex is formed in the VC cytoplasm during late pollen development. Both LlBTPC and AtBTPC expression patterns are similar to the patterns of the appearance of storage organelles during pollen development in lily and Arabidopsis, respectively. Therefore, BTPC is thought to accelerate the metabolic flow for the synthesis of storage substances during pollen maturation. Our study provides the first characterization of BTPC in pollen, the male gametophyte of higher plants.

Heterologous expression and functional characterization of a plant alkaline phytase in Pichia pastoris.

Phytases catalyze the sequential hydrolysis of phytic acid (myo-insositol hexakisphosphate), the most abundant inositol phosphate in cells. Phytic acid constitutes 3-5% of the dry weight of cereal grains and legumes such as corn and soybean. The high concentration of phytates in animal feed and the inability of non-ruminant animals such as swine and poultry to digest phytates leads to phosphate contamination of soil and water bodies. The supplementation of animal feed with phytases results in increased bioavailability to animals and decreased environmental contamination. Therefore, phytases are of great commercial importance. Phytases with a range of properties are needed to address the specific digestive needs of different animals. Alkaline phytase (LlALP1 and LlALP2) which possess unique catalytic properties that have the potential to be useful as feed and food supplement has been identified in lily pollen. Substantial quantities of alkaline phytase are needed for animal feed studies. In this paper, we report the heterologous expression of LlALP2 from lily pollen in Pichia pastoris. The expression of recombinant LlALP2 (rLlALP2) was optimized by varying the cDNA coding for LlALP2, host strain and growth conditions. The catalytic properties of recombinant LlALP2 were investigated extensively (substrate specificity, pH- and temperature dependence, and the effect of Ca(2+), EDTA and inhibitors) and found to be very similar to that of the native LlALP2 indicating that rLlALP2 from P. pastoris can serve as a potential source for structural and animal feed studies.

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.

Morphogenesis of complex plant cell shapes: the mechanical role of crystalline cellulose in growing pollen tubes.

Cellulose is the principal component of the load-bearing system in primary plant cell walls. The great resistance to tensile forces of this polysaccharide and its embedding in matrix components make the cell wall a material similar to a fiber composite. In the rapidly growing pollen tube, the amount of cellulose in the cell wall is untypically low. Therefore, we want to investigate whether the load-bearing function of cellulose is nevertheless important for the architecture of this cell. Enzymatic digestion with cellulase and inhibition of cellulose crystal formation with CGA (1-cyclohexyl-5-(2,3,4,5,6-pentafluorophenoxy)-1lambda4,2,4,6-thiatriazin-3-amine) resulted in the formation of tubes with increased diameter in Solanum chacoense and Lilium orientalis when present during germination. In pre-germinated tubes, application of both agents resulted in the transient arrest of growth accompanied by the formation of an apical swelling indicating a role in the mechanical stabilization of this cellular region. Once growth resumed in the presence of cellulase, however, the cell wall in the newly formed tube showed increased amounts of pectins, possibly to compensate for the reduced amount of cellulose. Scanning electron microscopy of pollen tubes subjected to digestion of matrix polysaccharides revealed the mechanical anisotropy of the cell wall. In both Lilium and Solanum, the angle of highest stability revealed by crack formation was significantly below 45 degrees , an indication that in the mature part of the cell cellulose may not the main stress-bearing component against turgor pressure induced tensile stress in circumferential direction.

Cloning of Glucuronokinase from Arabidopsis thaliana, the last missing enzyme of the myo-inositol oxygenase pathway to nucleotide sugars.

Nucleotide sugars are building blocks for carbohydrate polymers in plant cell walls. They are synthesized from sugar-1-phosphates or epimerized as nucleotide sugars. The main precursor for primary cell walls is UDP-glucuronic acid, which can be synthesized via two independent pathways. One starts with the ring cleavage of myo-inositol into glucuronic acid, which requires a glucuronokinase and a pyrophosphorylase for activation into UDP-glucuronate. Here we report on the purification of glucuronokinase from Lilium pollen. A 40-kDa protein was purified combining six chromatographic steps and peptides were de novo sequenced. This allowed the cloning of the gene from Arabidopsis thaliana and the expression of the recombinant protein in Escherichia coli for biochemical characterization. Glucuronokinase is a novel member of the GHMP-kinase superfamily having an unique substrate specificity for d-glucuronic acid with a K(m) of 0.7 mm. It requires ATP as phosphate donor (K(m) 0.56 mm). In Arabidopsis, the gene is expressed in all plant tissues with a preference for pollen. Genes for glucuronokinase are present in (all) plants, some algae, and a few bacteria as well as in some lower animals.

Nonradioactive enzyme measurement by high-performance liquid chromatography of partially purified sugar-1-kinase (glucuronokinase) from pollen of Lilium longiflorum.

Here we present a highly sensitive and simple high-performance liquid chromatography (HPLC) method that enables specific quantification of glucuronokinase activity in partially purified extracts from pollen of Lilium longiflorum without radioactive labeled substrates. This assay uses a recombinant UDP-sugar pyrophosphorylase with broad substrate specificity from Pisum sativum (PsUSP) or Arabidopsis thaliana (AtUSP) as a coupling enzyme. Glucuronokinase was partially purified on a DEAE-sepharose column. Kinase activity was measured by a nonradioactive coupled enzyme assay in which glucuronic acid-1-phosphate, produced in this reaction, is used by UDP-sugar pyrophosphorylase and further converted to UDP-glucuronic acid. This UDP-sugar, as well as different by-products, is detected by HPLC with either a strong anion exchange column or a reversed phase C18 column at a wavelength of 260 nm. This assay is adaptive to different kinases and sugars because of the broad substrate specificity of USP. The HPLC method is highly sensitive and allows measurement of kinase activity in the range of pmol min(-1). Furthermore, it can be used for determination of pure kinases as well as crude or partially purified enzyme solutions without any interfering background from ATPases or NADH oxidizing enzymes, known to cause trouble in different photometric assays.