Tulipa Cv Research Articles

Hydrocarbons Associated with the Developing Microspores in the Anther Loculus of Tulipa

Abstract: Hydrocarbons associated with the developing microspores in the anther loculus of Tulipa cv. Apeldoorn were investigated. They occurred mainly at chain lengths between C19 and C29. At the lower chain lengths the n‐alkanes, at higher chain lengths the alkenes dominated. During microspore development the ratio of unsaturated to saturated hydrocarbons in the extracts changed. While the content of all main hydrocarbons increased, the content of the alkanes C19, C21 and C23 increased to 3.8, 3.4 and 3.3 times the initial value compared to 2.1, 2.0 and 2.2 times in case of the alkenes C25, C27 and C29. Surface lipids from petals, leaves and ripe pollen were analyzed in comparison and these analyses confirmed that a high amount of alkenes was unique for microspores. Iso‐heptacosane was the main hydrocarbon from ripe pollen, heptacosene was a main component when microspore extracts were analyzed.

Molecular cloning of two different mannose-binding lectins from tulip bulbs.

Two lectins were isolated from the bulbs of Tulipa cv. Apeldoorn and their corresponding cDNA clones analyzed. The first, called TxLMII (second mannose-binding Tulipa hybrid lectin), is a novel mannose-binding tulip lectin. Based on its molecular structure, carbohydrate-binding specificity and amino acid sequence, TxLMII belongs to the superfamily of mannose-binding monocot lectins which are also found in representatives of the plant families Amaryllidaceae, Alliaceae, Orchidaceae and Araceae. Molecular cloning of the second lectin, called TxLCI (first Tulipa hybrid lectin with complex specificity), allowed determination unambiguously of the molecular structure of this previously described protein. In addition, evidence is presented that each TxLCI subunit possesses a mannose-binding site and an N-acetylgalactosamine-binding site, which act independently of each other. Both binding sites are located in a separate domain of the lectin polypeptide. Since the first domain of TxLCI shows sequence similarity to TxLMII, it is suggested that their genes evolved from a common ancestor.

Comparative FTIR and 13C CP/MAS NMR Spectroscopic Investigations on Sporopollenin of Different Systematic Origins

Abstract Comparative phytochemical investigations were carried out on sporopollenins of the follow­ ing plant species: Bryophyta -Reboulia hemisphaerica and Polytrichum strictum; Pteridophyta -Equisetum arvense, Selaginella selaginoides micro-and megaspores; Gymnospermae -Cycas circinalis; Angiospermae -Typha angustifolia and Tulipa cv. “Apeldoorn”. Although the range of the analyses included species from the Bryophyta to the Angiospermae, fundamen tal correspondences could be detected in the 13C NMR and FTIR spectra concerning the occur­rence of aliphatics, aromatics, ether and carbonyl/carboxylic functions in varying degrees. While the amount of aliphatics strongly dominated in the molecular structure of all other sporopollenins investigated here, it was found in sporopollenin of Equisetum arvense in a much lower extent. Despite the different morphology of Selaginella selaginoides micro-and mega­ spores, their sporopollenin spectra were nearly identical

Studies on Sporopollenin Biosynthesis in Cucurbita maxima I: The Substantial Labeling of Sporopollenin from Cucurbita maxima after Application of [14C]Phenylalanine

The results of tracer experiments performed with anthers of Tulipa cv. Apeldoorn have already shown that a high level of incorporation into the sporopollenin fraction was achieved, when [14C]phenylalanine was applicated as a precursor. In order to investigate whether the substantial incorporation of [14C]phenylalanine is a unique phenomenon restricted to Tulipa, tracer experiments were carried out on anthers of Cucurbita maxima. The sporopollenin fraction was isolated and purified by a gentle method including extractions with various solvents, incubations with hydrolysing enzymes and fractionated saponifications. The remaining, as well as the released radioactivity, was determined after each purification step. After the application of [U-14C]phenylalanine, a substantial incorporation into the sporopollenin fraction was determined. The values were clearly higher than those obtained with [1-14C]glucose or those from corresponding experiments on Tulipa anthers. After potash fusion of sporopollenin fractions labeled via [U-14C]phenylalanine, p-hydroxybenzoic acid was shown to be the main com ponent among the ether soluble acids; moreover it showed the highest level of radioactivity. No radioactivity was detected in the degradation products oxalic acid, benzoic acid, phthalic acid and terephthalic acid or octanedioic acid- and decanedioic acid-dimethylester.

Tracer Experiments on Sporopollenin Biosynthesis

Abstract Tracer experiments were carried out with anthers of Tulipa cv. Apeldoorn and Cucurbita maxima. Labelled sporopollenin fractions were enriched either by a conventional harsh or by a gentle method. In each case phenylalanine was incorporated at higher rates into the sporopollenin fraction of Tulipa than any of the other precursors used. The high incorporation of phenylalanine was observed at several stages of the postmeiotic development of Tulipa anthers. Even higher incorporation rates were obtained using Cucurbita anthers. The incorporation of [U-l4C]-phenylalanine, [ring U-14C]-phenylalanine and [l-14C]-phenylaIanine into the sporopollenin of Tulipa was compared. The results indicate, that the whole carbon skeleton of phenylalanine is incorporated, pointing to an involvement of the phenylpropanoid metabolism. After potash fusion of a sporopollenin fraction labelled via [ring U-14C]-phenylalanine, p-hydroxybenzoic acid was the main labelled product among the ether soluble acids. Incubation of who...

Metabolism of Ferulic Acid Sucrose Esters in Anthers of Tulipa cv. Apeldoorn: I. The Accumulation of Esters and Free Sugars

Abstract The authors of Tulipa cv. Apeldoorn accumulate a large amount of ferulic acid sucrose esters. In addition to the well-known diferuloyl-(DFS) and triferuloyl sucrose esters (TFS), a new phenylpropanoid characterized as a monoester of ferulic acid and sucrose is described. Like T F S and D F S , this ester exhibits an accumulation m a x i m u m in the early stages of pollen development. Numerous free sugars have been extracted from Tulipa anthers. Three of them were identified as sucrose, glucose and fructose. Sucrose as the main component is accumulated in extremely high amounts during specific developmental stages (= 20 % of the dry weight). After separation of the anthers into an anther wall fraction (AWF) and pollentapetum (PTF), each fraction shows a different accumulation kinetic of the free sugars and the ferulic acid esters. A correlation could be observed between the occurrence of the esters and sucrose in the pollentapetum fraction during the anther development.

Metabolism of Ferulic Acid Sucrose Esters in Anthers of Tulipa cv. Apeldoorn: II. Highly Specific Degradation of the Esters by Different Esterase Activities

Abstract Protein extracts from anthers of Tulipa cv. Apeldoorn catalyze the degradation of ferulic acid sucrose esters. Different products are generated when triferuloyl sucrose (TFS) and diferuloyl sucrose (DFS) were applied as substrates. By the aid of reversed-phase HPLC , TLC and spectroscopy the products could be identified as free ferulic acid, monoferuloyl sucrose ester [feruloylsucrose(mono)] and two different diesters of ferulic acid and sucrose [feruloylsucrose(di) and the endogenously occurring diferuloylsucrose (DFS)]. By means of protein fractionation (chromatofocusing, anion exchange HPLC and molecular sieving HPLC) , four different enzyme activities involved in the degradation process could be separated. According to their catalytic properties, they were characterized as esterases (= EA). The partially purified esterase activity I (EAI) obtained after fractionation by chromatofocusing catalyzes the formation of feruloylsucrose(di) and ferulic acid when TFS is used as substrate. Incubations with EA la or EA Ib isolated in smaller portions lead to the same product pattern. The esterase activity II (EA II) degrades TFS to ferulic acid and DFS . DFS as substrate is only accepted by the EA I activities, in all three cases ferulic acid and feruloyl sucrose(mono) are formed as products. The kinds of different degradation reactions clearly indicate that one enzyme (= the EA II activity) catalyzes exclusively the formation of DFS from TFS. Both enzymes, EA I and EA II, exhibit a high specificity towards ferulic acid sucrose esters. Hydroxycinnamic sucrose esters with only sinapic acid moieties do not function as substrates. When enzymatically formed sucrose esters like feruloylsucrose(di), feruloylsucrose(mono) and monosinapoylsucrose were used as substrates, no product formation could be observed. Applying SFS as substrate, only the ferulic acid moiety was released by EA I. Further, naturally occurring esters (glucose- and CoA-esters of p-coumaric, caffeic, ferulic and sinapic acid; chlorogenic acid; BGM ) tested so far were not degraded by EA I and EA II. It is assumed that these esterase activities play a specific role in the ferulic acid metabolism in Tulipa anthers.

The Separation of Two Different Enzymes Catalyzing the Formation of Hydroxycinnamic A dd Glucosides and Esters

Protein extracts from anthers of Tulipa cv. Apcldoorn catalyze the formation of glucosidcs and glucose esters of hydroxycinnamic acid with UDP-glucose as the glucosyl donor. By chromatofocusing and by HPLC (anion exchange chromatography) it could be demonstrated for the first time that two different enzymes are involved in these reactions. By using the molecular sieving (HPLC) a molecular weight of about 45000 D was determined for the GT (E) (= catalyz­ing the formation of esters) and of about 25000 D for the GT (G) (= catalyzing the formation of glucosidcs). Both enzymes exhibit a high spezificity for free hydroxycinnamic acids. It is assumed that these transferases arc involved in the formation of feruloylglucose and ferulic acid glucoside, the latter of which can be isolated from anthers at early developmental stages. Furthermore, it is hypothesized that feruloylglucose functions as a starter molecule for the transacylation reaction by which di- and triferulovlsucrose are formed.

Conversion of Phloroglucinyl-type Chalcones by Purified Chalcone Isomerase from Tulip Anthers and from Cosmos Petals

Summary Pollen of various plant species have the ability to accumulate chalcone aglycones of the phloroglucinyl- type (2’ ,4’ ,6’-O H). These compounds could represent remnants of a chalcone pool built up in previous stages of anther development. Chalcone isomerase (EC 5.5.1.6) catalyzes the formation of flavanones from correspondingly substituted chalcones. This enzyme was isolated and purified from anthers of Tulipa cv. “Apeldoorn” as well as from petals of Cosmos sulphureus with the aim at looking at the affinity of the enzyme for its naturally occurring substrates. The isomerase from tulip anthers and the enzyme from Cosmos petals both have molecular weights of 31,000 ± 2,000 daltons, pI (pH 5.0), and a broad pH optimum ranging between pH 7.5 and pH 8.2. As calculated from the Km/V max ratios, both the enzymes had a high affinity for 3,4,2’,4’ ,6’-pentahydroxychalcone and a lower affinity for 4,2’, 4’, 6’-tetrahydroxychalcone. The tulip enzyme did not convert 6’-deoxychalcones at all whereas the Cosmos isomerase showed slow conversion rates with isoliquiritigenin and butein as substrates. The isomerase from Tulipa was further characterized by its sensitivity towards distinct SH reagents and bivalent cations. The presence of isoenzymic forms was not observed and the possibility of a peroxidase catalyzed reaction was excluded.

Formation of Flavonol 3-O-Diglycosides and Flavonol 3-O-Triglycosides by Enzyme Extracts from Anthers of Tulipa cv. Apeldoorn / Characterization and Activity of Three Different O -Glycosyltransferases during Anther Development

Abstract Three distinct glycosyltransferases have been isolated and partially purified from anthers of Tulipa cv. Apeldoorn. The following designations are proposed; UDP-glucose: flavonol 3-O-glucosyltransferase (GT-I), UDP-rhamnose: flavonol 3-O-glucoside rhamnosyltransferase (GT-II) and UDP-xylose: flavonol 3-glycoside xylosyltransferase (GT-III). The three enzymes exhibited an identical pH optimum within the range of 8.5 -9.0. The estimated molecular weight of GT-I and GT-II was about 40 000, GT-III showed a molecular weight of 30 000. GT-III required ions like NH4 +or Ca2+ whereas these ions have almost no influence on GT-I and GT-II activity. The enzymes have a slight requirement for SH-reagents, particularly DTE. As opposed to GT-II activity of GT-I and GT-III is significantly influenced by SH reagents and PCMB. Sucrose enhanced GT-III activity but only slightly GT-I activity, GT-II activity is not influenced. Flavonol aglycones can function as glycosyl acceptor for the GT-I, whereas flavonol 3-O-glycosides, luteolin, dihydroquercetin, naringenin, cyanidin, p-coumaric acid and some other phenols were inactive as acceptor. The best acceptors were isorhamnetin and quercetin (Km: 0.9 × 10-6 ᴍ). GT-II did not accept aglycones as substrates. For this enzyme flavonol 3-O-glucosides were the most attractive substrates. GT-III did not have any affinity towards aglycones, too. This enzyme exhibited a high specificity for flavonol 3-O-glucosides as well as flavonol 3-O-galactosides. Both enzymes, the GT-II and GT-III, were able to glycosylate flavonol 3-O-diglycosides forming triglycosides. UDP-glucose (Km = 1.0 × 10-4 ᴍ), UDP-rhamnose and UDP-xylose where shown to be the best glycosyl donors for GT-I, GT-II or GT-III respectively. The glycosyl transfer catalysed by the GT-I was shown to be a freely reversible reaction. In the whole anthers, highest specific activities of GT-I and GT-II were found during late stages of anther development. Similar results were obtained using the contents of anthers or the tapetum fraction. In contrary, high GT-III activity can be detected already in young stages of anther devel­opment. The highest activities of the three glycosyltransferases were found in the tapetum fraction, whereas the pollen fraction exhibited only poor activities.

Ferulic Acid-Sucrose Esters from Immature Anthers of Tulipa cv. "Apeldoorn"

Abstract Two ferulic acid conjugates have been isolated from the extract of immature anthers of Tulipa cv. "Apeldoorn" and their structures elucidated using chromatography, electron impact mass spectrometry, and NMR spectroscopy. These two conjugates are 6,3'-diferuloylsucrose and 6,3',4'-triferuloylsucrose. High performance liquid chromatographic analysis of the extract showed that these esters are transient major constituents of the phenylpropanoid pattern of the immature anthers and are present in the ratio of 1:2. Previous investigations showed that these components are accumulated in the loculus.

Purification of Chalcone Synthase from Tulip Anthers and Comparison with the Synthase from Cosmos Petals

Abstract Chalcone synthase was isolated from both anthers of Tulipa cv. “Apeldoorn” and petals of Cosmos sulphureus Cav. After certain prepurification steps, the enzymes were further purified using gel chromatography on Sephadex G-200 followed by repeated hydroxylapatite absorption chromatography. Both the enzymes showed the same chromatographic properties. After gel chromatography as well as after the first hydroxylapatite fractionation, the reaction products appeared as flavanones. However, after the second hydroxylapatite step, production of chalcones was observed. Like the enzyme from tulip anthers, the synthase from Cosmos petals produced the correspondingly substituted chalcones when p-coumaroyl-CoA, caffeoyl-CoA and feruloyl-CoA, respectively, were used as substractes. In both the cases, the ratios of the different chalcones produced were found to be about the same. The appearance of chalcone synthesis in this in vitro assay is caused by the complete elimination of chalcone isomerase in the purification procedure. The importance of the isomerase for flavonoid biosynthesis, particularly in plant systems which are accumulating chalcones, is discussed.

Die Entwicklung zweier o-Dihydroxyphenol O-Methyltransferasen während der Mikrosporogenese von Tulipa cv. «Apeldoorn»

During the microsporogenesis of Tulipa cv. «Apeldoorn», the activities of the two O-methyltransferases (CMT, SAM: caffeic acid 3-O-methyltransferase and FMT, SAM: 3′, 4′-o-dihydroxyflavonoid 3′-O-methyltransferase) show a different course of development. Using whole anthers as enzyme source, there was a good correlation between the activity of CMT and the accumulation of ferulic acid derivatives during the middle postmeiotic stages. In contrast, the activity of FMT already reached maximum values in the early, flavonoid-free stages of anther development. More distinct results were obtained using the contents of anthers and tapetum fractions of anthers. Whilst the correlation of CMT activity and ferulic acid accumulation was again confirmed, no connexion seemed to exist between the FMT activity and the accumulation of the methylated flavonol glycosides (isorhamnetin) during the stages of pollen ripening. Together with results from earlier analytical and enzymatic investigations, the data support the hypothesis that methylation of quercetin does not occur during flavonol-accumulating stages. Therefore, the substitution pattern of the B-ring of isorhamnetin must occur most probably at the phenylpropanoid stage.

Chalcone synthesis with enzyme extracts from tulip anther tapetum using a biphasic enzyme assay

The in vitro synthesis of chalcones has been demonstrated using a special biphasic enzyme assay. The highly viscous lower phase in this assay stems from a tapetum fraction of anthers of Tulipa cv. “Apeldoorn” which has been used an enzyme source. The upper phase of this system consists of a reaction mixture of the normal “flavanone synthase” assay. It is suggested that chalcone synthesis occurs at the boundary layer between the two phases. To prevent spontaneous as well as enzymatic cyclization of the chalcones formed (phloroglucinyl type), the pH of the upper phase must not be allowed to exceed pH 4.0. Under these pH conditions, chalcone formation by a reverse reaction of chalcone-flavanone isomerase can be excluded. The measured substrate specificity of the “chalcone synthase” corresponds to the conditions of chalcone formation in the natural system. Using p-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA, respectively, as substrates, the enzyme system forms the correspondingly substituted chalcones which are also accumulated in the loculus of tulip anthers. It is suggested that this chalcone synthase is identical to the previously described “flavanone synthase”. The results can be further explained as follows. (i) Not flavanones, but rather chalcones are the first C 15 intermediates of flavonoid biosynthesis in tulip anthers. (ii) In this Tulipa system, the substitution pattern of three different hydroxycinnamic acids can be transferred unchanged into the flavonoid C 15 stage. (iii) The role of chalcone-flavanone isomerase is to cyclize chalcones to flavanones on the direct biosynthetic pathway to the further accumulated flavonol glycosides. (iv) The sensitivity of the reaction with regard to chalcone production points to the localization of chalcone synthase in a most unstable and, up to now, unknown tapetal compartment. Since purification of the enzyme results in exclusive production of flavanones, it is suggested that certain “chalcone stabilizing factors” must occur in the natural system. (v) The phenomenon of chalcone accumulation in tulip anthers, however, must be caused by a complex system, distinguished by cooperation of certain biochemical and physiological conditions, and, finally, by special compartmentation of the enzymes which are responsible for the biosynthesis of flavonoids.

Characterization, Development and Localization of "Flavanone Synthase" in Tulip Anthers

1. “Flavanone synthase” was isolated from anthers of Tulipa cv. “Apeldoorn” and partially purified by (NH4) 2SO4 fractionation, gel chromatography and isoelectric focussing. The enzyme preparation was free of chalcone-flavanone isomerase activity. 2. p-Coumaroyl-CoA, caffeoyl-CoA and feruloyl-CoA were found to be efficient substrates of the synthase. The products formed were naringenin (5,7,4′-trihydroxyflavanone), eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) and homoeriodictyol (5,7,4′-trihydroxy-3′-methoxyflavanone), respectively. Addition of thiol reagents at concentrations exceeding 10-3 м caused inhibition of the enzyme. “ Release products” , however, were not detectable. Although exclusively chalcones accumulate in the tulip anther, only flavanones but no chalcones were detectable in our in vitro system. 3. The apparent Km values for p-coumaroyl-CoA, caffeoyl-CoA and feruloyl-CoA were 1 .7× 10-6 м, 1.6× 10-6 м and 2 .5 ×10-6 м, respectively. Similar data were observed for malonyl- CoA. 4. No cofactors are required for the synthase reaction. The enzyme is strongly inhibited by the reaction products flavanone and coenzyme A . Maximum enzyme activity was found at pH 8.0 and 30 °. The molecular weight was approx. 55,000. 5. Synthase activity develops in early postmeiotic stages of microsporogenesis. Highest specific activities of the enzyme coincide with a maximum in chalcone accumulation within the anthers. 6. The contents of anthers was separated into two fractions, pollen and tapetum. Highest specific activities were observed with tapetum fractions, while pollen fractions exhibited only very low activities. The high enzyme activity in the tapetum fraction points to the important role of the tapetum in the biosynthesis of flavonoids in the loculus of anthers.

The Formation of Flavanones from Hydroxycinnamoyl-Coenzyme A Thiol Esters and Malonyl-Coenzyme A by Enzyme Extracts from Anthers

During microsporogenesis of Tulipa cv. “Apeldoorn” an accumulation of chalcones occurs in specific stages of development. From corresponding anthers an enzyme was isolated catalyzing the formation of the flavonoid skeleton. Partially purified preparations of whole anthers and of the contents of anthers (= pollen + tapetum fractionl were employed for enzyme assay. Naringenin and eriodictyol, respectively, were identified as products of the enzymatic reaction using malonyl-CoA and either p-coumaroyl-CoA or caffeoyl-CoA as substrates.

�ber die Beziehungen zwischen flavonolaufbauenden Enzymen, einem flavonolumwandelnden Enzym und der Akkumulation phenylpropanoider Verbindungen w�hrend der Antherenentwicklung

The difference in activity of enzymes which are involved in the biosynthesis of flavonols (phenylalanineammonium-lyase, chalcone-flavanone isomerase) and of an enzyme which converts flavonols is studied during the development of anthers in Tulipa cv. Apeldoorn. The results are considered in relation to the accumulation of simple phenylpropanes, of an intermediate a chalcone and of different flavonoid compounds.

Aktivit�t der Chalkon-Flavanon Isomerase und Akkumulation von phenylpropanoiden Verbindungen in Antheren

Chalcone-flavanone isomerase from the anthers of Lilium candidum and Tulipa cv. “Apeldoorn” exhibits a distinct substrate specificity. The enzyme catalyses only the isomerization of 2′,4,4′,6′-tetrahydroxychalcone, whereas it is not active with 2′,4,4′-trihydroxychalcone.

Synthesis of phenylpropanes during pollen development

The synthesis and accumulation of several phenylpropanes in the anther content (pollen+tapetum fraction) during microsporogenesis has been investigated by chromatographic techniques in Narcissus pseudonarcissus, Lilium candidum, and in the Darwin tulip “Apeldoorn”. In these species, the pigmentation process is initiated by the synthesis of several cinnamic acid derivates (mainly derivates of ferulic acid) during meiosis II. In Narcissus, and intense synthesis of kaempferol glycosides takes place during the separation of the tetrad which follows immediately upon its formation. In Tulipa and Lilium, however, chalcones are synthesized in an intermediate phase before flavonols and anthocyanins (in Tulipa) are produced in significant amounts. In Tulipa, the investigations revealed the following sequence in the pigmentation process: cinnamic acid derivatives-chalcone-flavonols-anthocyanins. The sequence is discussed in relation to flavonoid biosynthesis. Because of biogenetic considerations a special emphasis is laid on the “chalcone stage”. Chromatographic and spectroscopic data show that the isomerization product of the chalcone is eriodictyol. Accordingly, this chalcone must be 2′,3,4,4′,6′-pentahydroxychalcone. Other chalcones could not be identified. During anthesis the following aglycones are accumulated in the pollen of Tulipa cv. “Apeldoorn”: ferulic acid, p-coumaric acid, kaempferol, quercetin, isorhamnetin, delphinidin, and small traces of the pentahydroxy-chalcone, which is the main pigment in the intermediate stages of microsporogenesis. On the basis of histochemical findings, it is suggested that at least the final steps of synthesis leading to flavonol and anthocyanidin glycosides take place on the pollen wall in the loculus of the anthers, that is, in the extracellular space.