Two populations of solanum elaeagnifolium Cav. F. albiflorum Cockll. (Solanaceae) are blocked at distinct sites in the anthocyanin biosynthetic pathway.
Solanum elaeagnifolium Cav. (silverleaf nightshade, horsenettle) is a rhizomatous perennial occurring in dry, sterile soils, open woods, and prairies from March to October. The natural range of S. elaeagnifolium extends from Missouri and Kansas south to Louisiana, Texas, Arizona and adjacent Mexico. The species has been introduced and become a troublesome weed of crop lands in many subarid regions of the world by distribution of propagules mixed with agricultural exports (Boyd et al. 1984).
The corollas of S. elaeagnifolium range from pale to deep violet in color presumably due to anthocyanin pigments as is true for other members of the Solanaceae (Harborne 1967). White-flowered populations of the species, designated as S. elaeagnifolium Cav. f. albiflorum Cockll. by Correll & Johnston (1970), Stanford (1976) and others, occur rarely. Biosynthesis of corolla pigments has not previously been examined in this species. However, anthocyanin biosynthetic pathways have been characterized in maize (Poaceae), snapdragon (Scrophulariaceae) and petunia (Solanaceae). A composite biosynthetic pathway is presented in Figure 1 (Holton & Cornish 1995). The branch points and consequent biosynthetic product(s) of the pathway are species-specific. White (colorless) phenotypes result from blocks in the biosynthetic pathway prior to the production of colored products (Durbin et al. 1995; Levin & Black 1994).
[FIGURE 1 OMITTED]
Thin-layer chromatography (TLC) was employed to identify the anthocyanin pigments in colored corollas of S. elaeagnifolium and to locate the sites of biosynthetic blocks resulting in white corollas in two distinct populations of S. elaeagnifolium f. albiflorum. Precursor feeding experiments confirmed the TLC results.
MATERIALS AND METHODS
Collection sites. -- Colored corollas of Solanum elaeagnifolium were collected from plants growing adjacent to Sikes Lake (Wichita Falls, Wichita County, Texas). White corollas were collected from two distinct populations growing in the same locality. Specimen plants from each collection site were transplanted to pots and grown in the greenhouse where subsequent corolla sampling occurred.
Chemicals. -- Pelargonidin, taxifolin (+/-dihydroquercetin), kaempferol, myricetin, quercetin, D-(+)-glucose, D-(+)-galactose, D-(-)-arabinose, D-(+)-xylose, L-(+)-rhamnose and N-methyldioctylamine, purchased from Sigma Chemical Company, were of reagent grade. Cyanidin was extracted from corollas of Centaurea cyanus. Delphinidin was extracted from Delphinium hybrida (Harborne 1967). All other chemicals were of reagent grade.
Extraction and purification of pigments and precursors. -- Corollas were excised, weighed and extracted in 10 mL acidifed methanol [1% (w/v) HCl in methanol] per gram tissue. Extractions were performed overnight in 15 mL polypropylene tubes at room temperature in the dark. Plant tissues were removed by extraction with 2.4 volumes chloroform: water (5:1). The pigmented aqueous phase was placed in a 1.5 mL microcentrifuge tube and dried under vacuum overnight.
Deglycosylation of anthocyanins. -- The dried extract from one corolla was dissolved in 1 mL methanol, combined with 1 mL 4 N HCl in a 15 mL conical polypropylene tube and placed in a boiling water bath for 40 minutes (Strack & Wray 1989). Cooled hydrolyzate was extracted with 0.5 mL Iso-amyl alcohol. The pigmented organic layer was dried under vacuum overnight.
Anthocyanidin chromatography. -- The dried pigment extract was reconstituted in 40 mL 0.1% HCl/methanol (w/v) and spotted onto cellulose plates. Plates were developed in the dark with Forestal (glacial acetic acid: concentrated HCl:deionized water, 30:3:10) (Francis 1982), or Iso-PrOH (isopropanol:5% aqueous HCl, 55:45) (Mullick & Brink 1967) mobile phases. Developed plates were dried at room temperature and observed under visible and ultraviolet illumination. Spots were marked, their colors were noted and [R.sub.f] values were calculated.
Sugar analysis. -- Following deglycosylation, the aqueous phase was washed three times with 10% (v/v) N-methyldioctylamine in chloroform to remove acid residue. Samples were washed once with chloroform and dried overnight under vacuum. Dried sugars were dissolved in 40 mL water and spotted on cellulose plates with glucose, galactose, and rhamnose as reference solutions. TLC was developed in BBPW (n-butanol-benzene-pyridine-water, 5:1:3:3, upper layer) or Phenol (phenol:water, 4:1) and dried before spraying with aniline-hydrogen phthalate sugar reagent (1.6 g sodium hydrogen phthalate, 9.1 mL aniline, 48 mL n-butanol, 48 mL ethyl ether, 4.0 mL water) (Francis 1982). The sprayed plates were heated at 93[degrees]C for 10 minutes to visualize sugars. Spots were marked and [R.sub.f] values were calculated.
Feeding corollas anthocyanin precursors. -- Corollas were excised and placed in 1.5 mL microcentrifuge tubes filled with feeding solution. Feeding solutions consisted of 2 mg of commercially prepared dihydroquercetin (DHQ), quercetin, myricetin, or kaempferol dissolved in 10 mL water or synthesized dihydrokaempferol (DHK) or dihydromyricetin (DHM) (Pew 1948) dissolved in about one mL of water. Control corollas received water. Feeding occurred under ambient light at room temperature (Forkmann 1977).
Anthocyanidin identification. -- Deglycosylation of anthocyanin extracts yielded two violet-colored bands with different mobilities following thin-layer chromatography (TLC) with Forestal (Table 1). The [R.sub.f] value of the lower mobility band corresponded with delphinidin. The [R.sub.f] of the higher mobility band was comparable to that of cyanidin, but its violet color differed from the magenta color of cyanidin. TLC with Iso-PrOH yielded distinct [R.sub.f] values for cyanidin and the S. elaeagnifolium pigment (Table 2). Furthermore, the absorbance maximum of the unknown pigment was 556 nm while that of cyanidin was 546 nm. Based on these data, the higher mobility aglycone of S. elaeagnifolium was identified as petunidin. The intensity of pigmentation of S. elaeagnifolium corollas varies dramatically from population to population, ranging from pale to deeply colored. Extracts of corollas from several different populations contained pigments of identical color and TLC mobilities.
Sugar analysis. -- The sugar component of S. elaeagnifolium anthocyanin was analyzed by TLC of extracts following acid hydrolysis. TLC with glucose, galactose and rhamnose standards and BBPW as mobile phase identified the moiety as glucose (Table 3). This result was confirmed by TLC with galactose and glucose standards and Phenol mobile phase for increased resolution (Table 4).
TLC analysis of white corollas. -- Extracts of white corollas were compared by TLC with flavonol standards and Forestal mobile phase. White corollas from site A contained the same non-pigment compounds as colored corollas. Kaempferol accumulated in site A corollas, although the quantity was not significantly increased compared with colored corollas. In contrast, white corollas from site B did not accumulate kaempferol or any other compounds that corresponded to flavonol standards on TLC. Based on the presence of kampferol in site A corollas, a biochemical block subsequent to the conversion of DHK to DHM was hypothesized. The absence of flavonol precursors in site B corollas suggested that a block occurs prior to the conversion of naringenin to DHK.
Precursor feeding of white corollas. -- Cut white corollas were fed kaempferol, quercetin and myricetin as well as the corresponding dihydroflavonols. Corollas fed flavonols failed to produce colored pigments. Corollas from site A did not produce colored product when fed DHK, but converted both DHQ and DHM to colored pigments. The color of the product was determined by the precursor that was fed to the corollas. DHQ was converted to a magenta pigment whose aglycone exhibited mobility in Forestal and Iso-PrOH mobile phase similar to cyanidin. Corollas from the same site converted DHM to a violet product resembling delphinidin. Site B corollas produced colored pigmentation when fed any of the three dihydroflavonol precursors. In each case, the pigment exhibited a violet color. Extracts of DHQ-fed site B corollas exhibited mobility similar to delphinidin in Iso-PrOH (Table 2). Naringenin-fed corollas from each site failed to accumulate pigmentation.
Timberlake & Bridle (1982) identify petunidin as a component pigment of nearly all members of the genus Solanum. The pigments of S. elaeagnifolium, which have not previously been examined, are based on the aglycones delphinidin and petunidin (3'-methyldelphinidin). Rare white-flowered populations of S. elaeagnifolium fail to synthesize the colored pigments, presumably as a consequence of the loss of a specific enzymatic activity.
White corollas from site A accumulated kaempferol but not quercetin or myricetin. This suggested that a block occurred after the conversion of naringenin to DHK but before the synthesis of either DHQ or DHM (Figure 1). Two enzymes are potentially involved in the conversion of DHK to either DHQ or DHM: flavonoid 3'-hydroxylase (F3' Hase) and flavonoid 3',5'-hydroxylase (F3'5'Hase), respectively. Feeding experiments indicated that the corollas could produce cyanidin when fed DHQ, delphinidin when fed DHM, but no colored product when fed DHK. The failure to produce colored product when fed DHK confirms the absence of F3'Hase and F3'5'Hase activities in the corollas. The production of only cyanidin when fed DHQ in this population indicates that F3'5'Hase is not present to convert DHQ to DHM. However, the enzymes necessary to convert DHQ or DHM into the corresponding colored products are present. The natural violet pigment composition of S. elaeagnifolium colored corollas coupled with the production of cyanidin when white corollas were fed DHQ indicates that these organs do not naturally exhibit F3'Hase activity. If this activity were present, DHK would presumably be partitioned between pools of DHQ and DHM and corolla pigment composition should include a mixture of cyanidin and delphinidin or their derivatives. The absence of cyanidin from colored corollas indicates that DHQ is not naturally formed. Therefore, site A white corollas are colorless due to a failure to produce DHM resulting from a deficiency in F3'5'Hase activity.
White corollas from site B accumulated no kaemferol, suggesting that DHK is not produced in the corollas. Feeding experiments showed that the corollas were capable of producing colored pigments utilizing DHK, DHQ or DHM. It is likely that chalcone synthase (CHSase) and chalcone isomerase (CHIase) activities are present in white corollas from site B and that flavanone 3-hydroxylase (F3Hase) activity is absent or reduced to trace levels for the following reasons. In all plants which have been studied, CHSase is encoded by a multigene family consisting of as many as six members (Holton & Cornish 1995), although all copies may not be expressed in corollas. Isomerization of 4,2',4',6'-tetrahydroxychalcone to naringenin is reported to occur spontaneously at a low rate (Holton & Cornish 1995). Even if CHIase activity were absent, natural pigments would presumably be produced at a low rate and might accumulate. Site B corollas produce a very low level of pigment which was detected only upon extraction of corollas. This would be consistent with the hypothesis that site B corollas are white due to the absence of CHIase activity. However, it is the opinion of the authors that these corollas actually lack F3Hase activity. TLC analysis of site B corollas on Forestal produces a very high mobility compound that is also present in colored corollas. The compound exhibits several characteristics of naringenin. Naringenin accumulation would be consistent with the absence of F3Hase activity rather than CHIase activity. Additionally, feeding of naringenin to corollas of this population failed to produce pigment. The accumulation of a small amount of pigment in the corollas may be due to an incomplete block in the conversion of naringenin to DHK. Regardless of the specific site of blockage in the anthocyanin pathway of site B corollas, it is not the same as the block in site A corollas.
The potential for molecular verification of these results is simplified by the fact that genes encoding both F3Hase and F3'5'Hase have been isolated and their nucleotide sequences are known (Britsch et al. 1992; Britsch et al. 1993; Charrier et al. 1995; Deboo et al. 1995; Holton et al. 1993; Pelletier & Shirley 1995; Tanaka et al. 1996; Weiss et al. 1993). Northern blot analysis of poly (A)+ RNA from colored and white corollas will indicate whether the genes are transcribed at normal levels in the white corollas. The identification of two distinct populations of white flowered S. elaeagnifolium raises the question of whether other white-flowered populations may have arisen from genetic lesions which block other steps in anthocyanin synthesis, such as CHIase or dihydroflavonol reductase (DFRase).
Strict interpretation of the nomenclatural designation of these populations infers descent from a common progenitor and, consequently, derivation from a common molecular event. The results obtained from two distinct white-flowered populations refute this inference.
Table 1. Composition of acid-methanol solutions containing corolla extracts or standards with constituents identified by [R.sub.f] ranges in Forestal mobile phase. [R.sub.f] Ranges of Compounds Resolved by TLC [R.sub.f] = [R.sub.f] = [R.sub.f] = Sample 67-75 51-57 32-36 Solanum elaeagnifolium wild type corollas + + + site A white corollas + - - site B white corollas - trace trace upper leaves - - + Cyanidin - + - Delphinidin - - + Kampferol + - - Table 2. Composition of acid-methanol solutions containing corollas extracts or standards with constituents identified by [R.sub.f] ranges in Iso-PrOH mobile phase. [R.sub.f] Ranges of Compounds Resolved by TLC Sample [R.sub.f] = 67-74 [R.sub.f] = 56-60 Solanum elaeagnifolium wild type corollas - + site A--DHQ fed + - corollas site B--DHQ fed - + corollas Cyanidin + - Delphinidin - + Table 3. Composition of aqueous solutions containing hydrolyzate or sugar standards with constituents identified by [R.sub.f] ranges in BBPW mobile phase. [R.sub.f] Ranges of Compounds Resolved by TLC [R.sub.f] = [R.sub.f] = [R.sub.f] = Sample 22-25 17.2-18.5 16-17 Solanum elaeagnifolium wild type corollas - + - site A white corollas - + - Glucose - + - Galactose - - + Rhamnose + - - Table 4. Composition of aqueous solutions containing hydrolyzate or sugar standards with constituents identified by [R.sub.f] ranges in Phenol mobile phase. [R.sub.f] Ranges of Compounds Resolved by TLC Sample [R.sub.f] = 37.5-39 [R.sub.f] = 31.2-32.1 Solanum elaeagnifolium wild type corollas - + site A white corollas - + Glucose - + Galactose + -
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Tina A. Moore and William B. Cook
Department of Biology, Midwestern State University
Wichita Falls, Texas 76308
WBC at: email@example.com
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|Author:||Moore, Tina A.; Cook, William B.|
|Publication:||The Texas Journal of Science|
|Date:||Nov 1, 1998|
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