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Flavonoid and carotenoid pigments in flower tissue of Sandersonia aurantiaca (Hook.)
Scientia Horticulturae 72 Ž1998. 179–192
Flavonoid and carotenoid pigments in flower tissue of Sandersonia aurantiaca žHook. / David H. Lewis
a,)
, Stephen J. Bloor b, Kathy E. Schwinn
a
a
New Zealand Institute for Crop and Food Research, LeÕin Research Centre, PriÕate Bag 4005, LeÕin, New Zealand b New Zealand Institute for Industrial Research, Gracefield Research Centre, PO Box 31-310, Lower Hutt, New Zealand Accepted 15 September 1997
Abstract Luteolin, cryptoxanthin and zeaxanthin were detected as the major pigments present in mature flowers of Sandersonia aurantiaca ŽHook... Both the flavonoids and carotenoid pigments were examined at four stages of flower development from green flower bud to senescent flower prior to tepal necrosis. The major component of the orange colour of the Sandersonia flowers is due to the presence of the carotenoids zeaxanthin and cryptoxanthin, both predominantly present in the esterified form. The main change during development is the loss of chlorophyll, b-carotene and lutein, and the increase in zeaxanthin and cryptoxanthin as the flower matures from a green bud to an open flower. The major flavonoids present were luteolin and luteolin 7-O-glucoside. Flavonoid concentration decreased over the course of development to a mature flower but there was an increase in content per flower. The flavonoids were present in greater quantities than the carotenoids. However, as they absorb mostly in the UV region, they are not believed to contribute significantly to the observed colour. q 1998 Elsevier Science B.V. Keywords: Carotenoids; Flavonoids; Flower pigments; Sandersonia
1. Introduction Sandersonia aurantiaca ŽHook.. is native to South Africa but is grown as a commercial flower crop in New Zealand, where it is currently the third ranked cut flower crop in terms of export earnings ŽStatistics New Zealand.. It is a tuberous plant, )
Corresponding author. Tel.: q64 6 368 7059; fax: q64 6 367 5656; e-mail: [email protected]
0304-4238r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 2 3 8 Ž 9 7 . 0 0 1 2 4 - 6
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with each tuber having two growing points from which the flowering shoots develop. Each shoot is 400–700 mm long and bears a number of bright yellow–orange bell-shaped flowers in the leaf axils ŽClark, 1994.. The distinctive shape of the flowers is due to the fused sepals and petals Žtepals. that form a single complete structure. Sandersonia is a monotypic species ŽBrundell and Reyngoud, 1985. and no new cultivars with altered morphological features have been released. The range of flower colours for this species is therefore limited to yellow–orange. Flower colour and the aesthetic value associated with it, has prompted research into the compounds that are responsible for the colour of plant organs and into the selection and breeding of cultivars with different colours ŽForkmann, 1991.. Besides chlorophyll, there are three main groups of plant pigments: flavonoids which are responsible primarily for the red and blue colours, carotenoids that give orange and yellow colours and the betalains that may give violets or yellows, although their presence is restricted to certain plant families ŽHaslam, 1993.. Research on the genes controlling the synthesis of plant pigments, and in particular the flavonoids, has resulted in the production of transgenic plants with modified flower colours Žsee reviews by Forkmann, 1991; Davies and Scwhinn, 1997.. The same technology could be utilised to develop novel colours for Sandersonia through genetic manipulation of the pigment biosynthetic pathways. However, it is important to know which pigments are normally present so that specific steps in the synthesis of the pigments can be targeted. This study details the pigment composition of Sandersonia flowers at four stages of development.
2. Materials and methods 2.1. Plant material Sandersonia tubers were obtained from Lilies International ŽLevin, New Zealand.. The tubers were dipped in fungicide, 1 g ly1 Benlate ŽDupont, Auckland, New Zealand., prior to planting in polythene bags in a sterilised bark:pumice Ž1:1. mix. The plants were grown in a glasshouse Žheated at 158C, vented at 258C. under ambient light conditions. Four stages of flower development were chosen for examination: stage 1—an immature green bud, stage 4—flower open but still has green tips, stage 7—mature flower completely orange and stage 10—tepals wilting and orange colour fading ŽEason and Webster, 1995.. Flowers were harvested randomly from 40 plants over the period of a month to collect sufficient tissue Žapproximately 10 g fresh weight ŽFW. per flower stage. for analysis. Flowers were harvested, the pedicel, anthers and ovary removed and the remaining tepal tissue frozen in liquid nitrogen, crushed to a fine powder, freeze dried and stored at y208C. The whole flower and tepal tissue weight and flower colour were recorded for five flowers from each development stage, prior to the tissue being frozen. Two extra samples of stage 7 tepal tissue were collected and extracted as fresh tissue for the initial qualitative flavonoid analysis. Colour parameters Žlightness, chroma and hue angle; L, C, H8. were measured with a Minolta CR-200 chroma meter ŽMinolta Camera Co., Osaka, Japan., standardised on a
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white tile supplied with the instrument. The chroma meter has an 8 mm reflectance port size, a dr0 illuminating system and a D65 light source for illumination. Two readings were taken per flower on separate axes Ž908 apart. through a cross section of the centre of the flower. Lightness represents the proportion of total incident light that is reflected, chroma is a measure of colour intensity Žin relative intensity units. and hue angle relates colour to a position on a colour circle or wheel, with red purple at 08, yellow at 908, bluergreen at 1808, and blue at 2708 2.2. FlaÕonoid analysis Fresh tepal tissue was frozen in liquid nitrogen, homogenised using a mortar and pestle and extracted at room temperature in 90% methanol Ž10 ml gy1 FW. for 72 h. The sample was centrifuged at 1200 = g for 10 min, the supernatant decanted and the pellet re-extracted in 50% methanol Ž5 ml gy1 FW. for 24 h. The sample was centrifuged again and the supernatants combined. Final extract volume was reduced to 10 ml using a Savant SC210 Speedvac. Eight aliquots, each equivalent to 600 mg FW of tepal tissue, were partitioned with chloroform Ž1:1. to remove any carotenoids and other low polarity molecules before being loaded onto separate paper chromatograms. Individual flavonoids were separated by 2D paper chromatography ŽPC. on Whatmann 3MM paper in TBA Ž t-butanol:acetic acid:water 3:1:1. and 15% acetic acid. Dried chromatograms were viewed in ultraviolet light alone and after exposure to ammonia fumes to detect spots, whose colour and Rf values were noted. The corresponding spots from each chromatogram were combined and eluted in 80% methanol. Spectral data for each compound were measured over the 200–500 nm range in both methanol and with the addition of shift reagents ŽMarkham, 1982. and compared with reported results for reference compounds ŽMabry et al., 1970; Markham, 1982.. Each putative flavonoid was run on a separate Sep-pak C 18 preparative cartridge ŽWaters Associates, Milford, MA, USA. and eluted in methanol before being hydrolysed with 2 N HCL at 958C for 1 h. The hydrolysed flavonoids were then taken to dryness under reduced pressure, the sugars dissolved in water and the aglycones in methanol. The sugars were separated by 1D PC and analysed by treatment with aniline hydrogen pthalate ŽMarkham, 1982.. The aglycones were analysed on cellulose thin layer chromatography plates ŽSchleicher and Schull, Dassel, Germany. against known standards in TBA, 50% acetic acid or Forestal ŽMarkham, 1982. and on silica TLC plates ŽSchleicher and Schull, Dassel, Germany. in chloroform:acetone:formic acid Ž9:2:1.. Freeze dried tissue was used for the high performance liquid chromatography ŽHPLC. analysis of flavonoid content in the different stages of flower tissue and to confirm the identity of the flavonoids that were isolated. Three samples of approximately 60 mg of ground freeze-dried tepal tissue from each stage was extracted at room temperature overnight with 5 ml 95% ethanol:formic acid:water Ž75:5:20.. The extracts were centrifuged and analysed by HPLC, using a Waters 600 solvent delivery system with a Merck Lichrosphere Ž5 m m, 119 = 4 mm. 100 RP-18 endcapped column Žcolumn temperature 308C. and a Waters 996 PDA detector with integration at 352 nm. Elution Ž0.8 ml miny1 . was performed using a solvent system comprising solvent A
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wHOAc:CH 3 CN:H 3 PO4 :H 2 O Ž20:24:1.5:54.5.x and 1.5% H 3 PO4 Žsolvent B. and a linear gradient starting with 80% A, decreasing to 33% A at 30 min, 10% A at 33 min and 0% A at 39.3 min. As only luteolin and apigenin derivatives were present in these samples, the amounts of flavones were determined as apigenin-7-neohesperidoside equivalents, since a suitably pure crystalline sample of this compound was available. Results are reported as the mean of the three replicates taken from each development stage. A portion of the extract from each flower stage was hydrolysed by adding an equal volume of 2 N HCl and heating in a sealed vial on a boiling water bath for 40 min. The aglycones were extracted using n-BuOH, dried and analysed by HPLC. The extract remaining after HPLC analysis was pooled, reduced in volume to approximately 10 ml and chromatographed on a polyamide column Ž35 = 350 mm Machery-Nagel SC-6., using water with an increasing proportion of methanol. Fractions containing pure luteolin 7-O-neohesperidoside and luteolin 7,4X-di-O-glucoside were eluted with 1:1 and 4:6 water:methanol respectively. NMR spectra were obtained in dimethylsulphoxide-d6 on a Bruker AC-300 instrument. NMR data from the flavonoid diglycosides isolated from Sandersonia are listed below. Luteolin 7,4X-di-O-glucoside. 1 H NMR data Ž d Žppm., multiplicity, coupling constant ŽHz..; 7.55 dd 8,2 ŽH-6X ., 7.53 d 2 ŽH-2X ., 7.26 d 8 ŽH-5X ., 6.90 s ŽH-3., 6.84 d 2 ŽH-8., 6.45 d 2 ŽH-6., 5.10 d 7 ŽH-1 of 7-glc., 4.89 d 7 ŽH-1 of 4-glc.. 13 C NMR data Ž d Žppm..; 182.6ŽC-4., 163.6ŽC-2., 161.7ŽC-5., 157.6ŽC-9., 149.3ŽC-4X ., 147.5ŽC-3X ., 125.2ŽC-1X ., 119.2ŽC-6X ., 116.6ŽC-5X ., 114.3ŽC-2X ., 106.0ŽC-10., 104.8ŽC-3., 101.9ŽC-1 of 4X glc., 100.4ŽC-6., 100.2ŽC-1 of 7 glc., 95.4ŽC-8., 77.9, 77.7Žglc C-5., 77.0, 76.4Žglc C-3., 73.8, 73.7Žglc C-2., 70.3, 70.1Žglc C-4., 61.2Ž2C, glc C-6.. Luteolin 7-Oneohesperidoside. 1 H NMR data Ž d Žppm., multiplicity, coupling constant ŽHz..; 7.41 s ŽH-2X ., 7.4 dd 8, 1 ŽH-6X ., 6.91 d 8 ŽH-5X .. 6.75 s ŽH-3., 6.74 d 2 ŽH-8., 6.38 d 2 ŽH-6., 5.23 d 7 ŽH-1X of glc., 5.14 s ŽH-1 of rha., 1.20 d 6.2 ŽH-6 of rha.. 2.3. Carotenoid analysis Three 50 mg dry weight ŽDW. samples of powdered freeze-dried tepal material from each flower development stage were extracted by soaking twice in 3 ml of acetone Ž4 h then overnight. and once in diethyl ether Ž2 h.. The combined acetone extracts were dried and added to the ether extract which was then washed once with water, and dried. The extract, made up to 2 ml with ethyl acetate, was then used for saponification and further analysis. Total carotenoid and chlorophyll content of Sandersonia tepal tissue was estimated using the methods of Wellburn Ž1994.. An aliquot of the ethyl acetate extract was dried under a stream of O 2-free N2 and then redissolved in chloroform for spectrophotometric analysis. Saponification of one of the carotenoid extracts was carried out to identify the parent carotenoids. A sample of the extract was taken and evaporated to dryness. The dried sample was dissolved completely in 500 m l ethanol and 100 m l of 60% KOH in water Žwrv. added. The solution was shaken and left at room temperature for 2 h. The carotenoids were then extracted by adding ethyl acetate Ž0.5 ml. and water to the saponification mixture. The ethyl acetate layer was taken, re-extracted with water, and used directly for HPLC.
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Table 1 Flower bud size and colour characteristics of Sandersonia flowers at four stages of development ŽMean"SE, ns 5. Flower development stage
Flower weight Žg FW.
Tepal tissue per flower 1 Žg FW.
Žg DW.
1 4 7 10
0.13"0.01 0.34"0.02 0.60"0.03 0.35"0.04
0.05 0.19 0.45 0.21
0.008 0.026 0.054 0.027
1
Lightness Ž% refl..
Chroma Žintensity.
Hue angle Ždegrees.
54.7"0.4 58.7"0.6 53.9"0.5 55.0"0.3
46.1"0.5 46.3"0.7 46.7"1.2 42.4"0.3
109.9"1.2 85.3"0.9 73.9"0.3 76.4"1.4
Mean tepal weight was determined from the total amount of tissuertotal flower number.
HPLC analyses were performed on the same instrument and column type used for flavonoid analysis. Elution Ž1 ml miny1 . was performed using a solvent system comprising solvent A wCH 3 CN:H 2 O:triethylamine Ž80:20:0.1.x and ethyl acetate Žsolvent
Fig. 1. HPLC chromatogram showing separation of the flavonoids extracted from Sandersonia tepals at stage 7.
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B. and a linear gradient starting with 100% A, decreasing to 0% A at 25 min and held at 0% A for a further 5 min. A modified gradient was employed for analysis of the saponified samples to separate lutein and zeaxanthin. Elution Ž0.9 ml miny1 . was performed using a solvent system comprising solvent C wCH 3 CN:H 2 O:isopropanol Ž86:10:4.x and ethyl acetate Žsolvent B. and a linear gradient starting with 100% C, decreasing to 50% C at 20 min, 0% C at 25 min and held at 0% A for a further 5 min.
Fig. 2. Total flavonoid concentration ŽA. or content per flower ŽB. for Sandersonia flowers at four stages of development ŽMean"SE.. Concentration and content were calculated as apigenin 7-neohesperidoside equivalents. Where error bar is not visible, standard error was too small to distinguish from the mean.
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Non-carotenoid peaks in the HPLC chromatogram at 430 nm and 480 nm were identified as such from on-line spectral analysis. The levels of carotenoids were determined by summing the carotenoid peak areas and calculating the m g b-carotene equivalentsrg DW of tepal. Lutein, zeaxanthin and cryptoxanthin were identified in the flower extracts by comparison of retention times and on-line spectral data with standard samples. The identity of the cryptoxanthin peak in the saponified flower extract was also verified by co-chromatography with a b-cryptoxanthin standard. Total carotenoid and individual carotenoid levels were quantified for each of the three extracts from each development stage and the results reported as a mean from the three replicates. 2.4. FlaÕonoid and carotenoid standards Flavonoid standards for luteolin, apigenin and apigenin 7-neohesperidoside were purchased from Apin Chemicals ŽAbingdon, Oxon, UK.. Luteolin 7-O-glucoside, luteolin 7,3X-di-O-glucoside, luteolin 7-O-rutinoside and apigenin 7-O-glucoside were from a collection of standard samples held in the Gracefield laboratory. Trans-b-carotene was purchased from Aldrich Chemicals ŽMilwaukee, WI, USA.. The concentration of a solution of b-carotene in hexane was determined by measuring the absorbance at 449 nm and using a A11%cm value of 2592 ŽBritton, 1985.. This standard solution was then dried and redissolved in ethyl acetate and used as an external standard for the HPLC carotenoid quantification. Samples of lutein and zeaxanthin were kindly donated by Dr. D. Della Penna, University of Nevada Reno. b-Cryptoxanthin was isolated from a saponified extract of peach. 3. Results 3.1. Flower deÕelopment Whole flowers and tepals of Sandersonia increased in weight as the flower developed from an immature bud to an open flower, stages 1–7 ŽTable 1.. A decrease in flower Table 2 Mean concentration and content per flower for individual flavonoids in flowers of Sandersonia at different stages of development ŽMean"SE, ns 3.. Flavonoids were quantified as apigenin-7-neohesperidoside equivalents Flower development stage
Luteolin
conc. Ž m mol gy1 DW. 1 15.6"0.5 4 50.2"0.2 7 36.3"0.5 10 45.0"0.5 content Ž m mol flowery1 . 1 0.12"0.02 4 1.30"0.02 7 1.94"0.03 10 1.19"0.02
Luteolin 7-O-glucoside
Luteolin 7-O-neo-hesperidiside
Luteolin X 7,4 -di-O glucoside
Apigenin 7-O-glucoside
257.8"0.2 99.7"1.2 50.2"0.2 73.9"1.2
10.3"0.2 5.2"0.2 3.5"0.2 5.2"0.2
58.8"0.2 24.7"0.5 12.1"0.2 15.6"0.2
14.4"0.5 6.9"0.2 3.5"0.2 5.2"0.2
0.09"0.02 0.14"0.02 0.19"0.02 0.14"0.02
0.47"0.02 0.64"0.02 0.66"0.02 0.42"0.02
0.12"0.02 0.17"0.02 0.19"0.02 0.14"0.02
2.06"0.02 2.60"0.03 2.72"0.02 1.99"0.03
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weight was observed for flowers at stage 10 as the tissue wilted and senescence proceeded. The change in flower size was accompanied by a marked change in colour ŽTable 1.. Buds at stage 1 were dark green while the mature flowers were bright orange. This is shown by the change in hue angle from 110 to 74. The colour did revert somewhat to a more yellow rather than red colour as the flowers reached stage 10.
Fig. 3. Chlorophyll and total carotenoid concentration ŽA. or content per flower ŽB. for Sandersonia flowers at four stages of development ŽMean"SE.. Concentration and content were calculated as b-carotene equivalents. Where error bar is not visible, standard error was too small to distinguish from the mean. ŽB s chlorophyll, s total carotenoids..
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Major changes in lightness or intensity of colour were not observed although a decrease in colour intensity was detected at stage 10. 3.2. FlaÕonoids Five significant flavonoids were evident in the HPLC chromatograms from extracts of Sandersonia flower tissue ŽFig. 1.. All were flavones, as hydrolysis of the extracts gave only luteolin and apigenin, with luteolin predominant. The least polar compound, 5, was identified as luteolin by co-chromatography ŽHPLC. with authentic samples, combined with the results from the 2D-PCs and shift reagents. Similarly, the two monoglycosides, 2 and 4, were shown to be luteolin-7-O-glucoside and apigenin 7-O-glucoside respectively. The precise structures of the two diglycosides, 1 and 3, were more difficult to determine and NMR spectroscopy was used for their identification. The most polar glycoside, 1, had 13 C and 1 H NMR spectra consistent with a luteolin 7,4X or 7,3X-diglucoside, however, a reference sample of luteolin 7,3X-diglucoside had a shorter HPLC retention time and the reported NMR shifts of the 7,3X compound are different from those observed for 1. No literature data or reference compound was available for the 7,4X-diglucoside but the 13 C NMR shift values for the flavonoid nucleus of 1 are consistent with this compound and with values for the similarly substituted luteolin-7neohesperidoside-4X-glucoside ŽAgrawal, 1989.. Hence 1 is luteolin 7,4X-di-O-glucoside. The other diglycoside, 3, had 1 H NMR signals for both rhamnose and glucose and substitution only at the C-7 position. The 1 H NMR chemical shifts of the anomeric protons were consistent with a neohesperidoside rather than a rutinoside and this was confirmed by comparison with reported 1 H NMR shifts for the neohesperidoside ŽStein and Zinsmeister, 1990.. A reference sample of luteolin 7-O-rutinoside eluted earlier from the HPLC than 3. Thus 3 is luteolin 7-O-neohesperidoside Žs a-L-rhamnosyl-Ž1 ™ 2.-b-D-glucoside.. The total flavonoid concentration decreased as the flower buds developed from stage 1 to 7 but then increased at stage 10 ŽFig. 2.. The flavonoid content per flower, however, showed the opposite pattern ŽFig. 2B.. All four flower stages showed similar flavonoid profiles although the concentration and content of the individual flavonoids did change during development ŽTable 2.. Luteolin 7-O-glucoside and luteolin 7,4X-di-O-glucoside Table 3 Mean concentration and content per flower for individual carotenoids in flowers of Sandersonia at different stages of development. Carotenoids were quantified as b-carotene equivalents. nd s not detected Flower development stage b-carotene
Lutein
Zeaxanthin Crypto-xanthin Esterified Xanthophylls
0.27"0.01 0.19"0.01 nd nd
0.32"0.01 0.01"0.01 nd nd
0.26"0.01 nd 0.19"0.01 0.07"0.01 0.16"0.01 0.25"0.01 0.07"0.01 0.13"0.01
nd 1.39"0.3 3.82"0.05 2.33"0.04
2.14"0.02 4.97"0.06 nd nd
2.59"0.09 0.09"0.02 nd nd
2.12"0.07 nd 5.00"0.19 1.73"0.06 8.60"0.19 13.48"0.43 1.90"0.04 3.52"0.06
nd 36.11"0.84 206.41"2.72 62.79"0.95
y1
conc. Ž m mol g DW. 1 4 7 10 content Žnmol flowery1 . 1 4 7 10
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were the major flavonoids at stage 1 but by stage 4 luteolin is present in greater quantities than 7,4X-di-O-glucoside. 3.3. Carotenoids Distinct changes occurred in both carotenoid concentration ŽFig. 3. and the individual carotenoids detected at different stages during flower development ŽTable 3.. Flowers at stage 1 had the highest levels of chlorophyll and the carotenoids lutein, zeaxanthin and b-carotene were also present. At stage 4 the chlorophyll concentration had decreased by
Fig. 4. HPLC chromatogram of the carotenoids extracted from Sandersonia tepals at stage 7.
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Table 4 Concentration and content per flower for individual carotenoids in a saponified extract from flowers of Sandersonia at different stages of development. Carotenoids were quantified as b-carotene equivalents. nd s not detected Flower development stage conc. Ž m mol gy1 DW. 1 4 7 10 content Žnmol flowery1 . 1 4 7 10
b-carotene
Lutein
0.25 0.39 nd nd
0.41 0.02 nd nd
0.34 0.79 1.69 1.01
nd 0.52 2.54 1.52
2.05 10.24 nd nd
3.35 0.37 nd nd
2.79 20.48 91.43 27.37
nd 13.59 137.06 40.97
Zeaxanthin
Cryptoxanthin
70% but the carotenoid concentration had doubled. b-carotene and zeaxanthin were the major carotenoids, but the lutein concentration had decreased and the monohydroxy xanthophyll, b-cryptoxanthin, was now present. Carotenoid concentration and content per flower were greatest at stage 7. The carotenoid concentration had doubled again between stages 4 and 7 while the content per flower had increased by a factor of four. Carotenoid levels, however, decreased at stage 10. The xanthophylls, zeaxanthin and cryptoxanthin, were the major carotenoids present in flowers at stages 7 and 10 but they were present mainly in their esterified forms ŽFig. 4.. Saponification of the carotenoid extract revealed no other parent carotenoids ŽTable 4.. Lutein, b-carotene and the chlorophylls were not detected in flowers at these development stages. 4. Discussion Both flavonoids and carotenoids were detected in the tepal tissue of Sandersonia flowers. The changes in these compounds, especially with respect to the carotenoids, were linked with the changes in colour of the flower as it developed from a green bud to an orange flower. The green colour of the buds at stage 1 was due to the presence of chlorophyll. Carotenoids and flavonoids were also present but these were masked by the presence of the chlorophyll. Carotenoids are responsible for the colour of the mature flower. The orange colour of the flower developed in the period between stage 1 and 7 and this correlated with the increase in both the carotenoid concentration and content per flower. Zeaxanthin and cryptoxanthin, the major carotenoids detected, have absorbance maxima between 430–490 nm ŽBritton, 1995. and are orangeryellow in colour. The flavonoid concentration did not increase with flower development and luteolin and luteolin 7-O-glucoside, the most abundant flavonoids Žwith absorbance maxima at 349 nm, Mabry et al., 1970., would provide a background creamy yellow colour to the flower. No strongly coloured flavonoids, such as anthocyanins, were detected. The carotenoids present in the flower changed during development, with lutein and b-carotene being replaced by zeaxanthin and cryptoxanthin. This is consistent with the degradation of chloroplasts and the formation of chromoplasts and the associated
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changes in carotenoid metabolism resulting in the accumulation of apolar carotenoids and xanthophylls and their acylated and esterified forms ŽBritton, 1982; Marano et al., 1993; Bartley and Scolnik, 1995.. Approximately 90% of the zeaxanthin and cryptoxanthin in stage 7 Sandersonia flowers was present in the esterified form ŽTables 3 and 4.. Esterification of the carotenoids appears to be important for the formation of the chromoplasts, the accumulation and longevity of the carotenoid pigments and ultimately the colour of the tissue. The esterified form is considered more stable and more liposoluble, so is more easily incorporated into membranes ŽMinguez-Mosquera and Hornero-Mendez, 1994.. The accumulation of carotene bodies in the chromoplast and the different forms of the carotene bodies, influence the structure and properties of that chromoplast ŽMarano et al., 1993; Bartley and Scolnik, 1995.. Carotenoids are the major pigment in many redryellow flowers including daffodils ŽLiedvogel et al., 1976., marigolds ŽQuackenbush and Miller, 1972. and in yellow varieties of Viola tricolor ŽHansmann and Kleinig, 1982.. Their main role appears to be pollinator attraction, although carotenoids have also been linked with photosynthesis, photoprotection and plastid structure ŽBartley and Scolnik, 1995.. The total carotenoid level measured in Sandersonia was within the range recorded for the flowers listed above, although the predominant carotenoid varied with the different species. Lutein was the most abundant carotenoid detected in marigold flowers ŽTagetes erecta., varying between 76 and 93% of the total carotenoids depending on the variety ŽQuackenbush and Miller, 1972.. The difference in colour between the varieties examined Žred, gold or yellow. appeared to be due to the variation in the total carotenoid concentration and to smaller changes in the concentration of the more minor carotenoids. Thus subtle changes in the carotenoid content can result in more obvious visual changes in flower colour. The flavonoids detected in Sandersonia flower tissue were all flavones or their glycosides, the most abundant being luteolin and luteolin 7-O-glucoside. The major change during development was the increase in luteolin, possibly as an external flavonoid on the surface of the tepals. The lipophillic nature of aglycones such as luteolin often results in their accumulation on external plant surfaces ŽWollenweber and Jay, 1988.. Colourless flavonoids, including the flavones, play various roles in plant tissue. These include acting as co-pigments, providing background colour, producing patterns on flower petals that are attractive to pollinators, acting as UV protectants and they have also been linked with antibacterial and antifungal activity and with deterring insect herbivory ŽHarborne, 1988; Forkmann, 1991; Shirley, 1996.. The flavonoids were present in Sandersonia flower tissue in much greater quantities than the carotenoids ŽFigs. 2 and 3., despite their smaller contribution to the overall colour of the flower. The presence of both flavonoids and carotenoids is not uncommon. Flavonol glycosides were also the major flavonoids detected in Lathyrus aphaca petals and were estimated to make up 12.7% of the dry weight ŽMarkham and Hammett, 1994., although the colour of the Lathyrus flowers was attributed to the carotenoids. The total flavonoid concentration in Sandersonia flowers decreased as the flower developed although there was an increase in content per flower, indicating continued flavonoid synthesis. The increase in concentration at stage 10 is probably due to the loss of fresh weight either from passive water loss during senescence or the transport of soluble metabolites out of the flower.
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The pigments in the flower tissue of Sandersonia were examined as the first step in a study to determine whether flower colour in this crop could be modified. The presence of both the flavonoid and carotenoid biosynthetic pathways means that several strategies could be pursued. Inhibition of the carotenoid pathway may result in paler flower colour, while redirection of the pathway may allow production of new novel colours. Manipulation of the carotenoid biosynthetic pathway in tomato altered both flower and fruit colour ŽBramley et al., 1992.. Alternatively the introduction of the appropriate regulatory genes may allow the whole flavonoid pathway to be activated, resulting in anthocyanin production ŽLloyd et al., 1992..
Acknowledgements The authors thank Mr. Ray Rains for cultivating the plant material and the New Zealand Lottery Grants Board for a grant towards the purchase of the Savant Speedvac. This research was supported by funding from the New Zealand Foundation for Research, Science and Technology.
References Agrawal, P.K., 1989. Carbon-13 NMR of flavonoids. Elsevier, Amsterdam. Bartley, G.E., Scolnik, P.A., 1995. Plant carotenoids: pigments for photoprotection, visual attraction and human health. Plant Cell 7, 1027–1038. Bramley, P., Teulieres, C., Blain, I., Bird, C., Schuch, W., 1992. Biochemical characterisation of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5. Plant J. 2, 343–349. Britton, G., 1982. Carotenoid biosynthesis in higher plants. Physiol. Veg. 20, 735–755. Britton, G., 1985. General carotenoid methods. Methods Enzymol. 111, 113–149. Britton, G., 1995. UVrVisible Spectroscopy. In: Britton, G., Liaaen-Jensen, S. ŽEds.., Carotenoids. Vol. 1b Spectroscopy. Birkhauser Verlag, Basel, pp. 13–62. Brundell, D.J., Reyngoud, J.L., 1985. Observations on the development and culture of Sandersonia. Acta Hortic. 177, 439–447. Clark, G.E., 1994. Assessment of tuber storage and sprouting treatments for Sandersonia aurantiaca. N. Z. J. Crop Hortic. Sci. 22, 431–437. Davies, K.M., Scwhinn, K.E., 1997. Flower colour. In: Geneve, R.L., Preece, J.E., Merkle, S.A. ŽEds.., Biotechnology of Ornamental Plants. CAB International, Wallingford, UK, Chap. 15, pp. 259–294. Eason, J.R., Webster, D., 1995. Development and senescence of Sandersonia aurantiaca ŽHook.. flowers. Sci. Hortic. 63, 113–121. Forkmann, G., 1991. Flavonoids as flower pigments: The formation of the natural spectrum and its extension by genetic engineering. Plant Breeding 106, 1–26. Hansmann, P., Kleinig, H., 1982. Violaxanthin esters from Viola tricolor flowers. Phytochemistry 21, 238–239. Harborne, J.B., 1988. The flavonoids: recent advances. In: Goodwin, T.W. ŽEd.., Plant Pigments. Academic Press, London, pp. 299–343. Haslam, E., 1993. Natures Palette. Chem. Bri. October, pp. 875–878. Liedvogel, B., Sitte, P., Falk, H., 1976. Chromoplasts in the daffodil: fine structure and chemistry. Cytobiologie 12, 155–174.
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Lloyd, A.M., Walbot, V., Davis, R.W., 1992. Arabidopsis and Nicotiana anthocyanin production activated by Maize regulators R and C1. Science 258, 1773–1775. Mabry, T.J., Markham, K.E., Thomas, M.B., 1970. The systematic identification of the flavonoids. Springer Verlag, Berlin. Marano, M.R., Serra, E.C., Orellano, E.G., Carillo, N., 1993. The path of chromoplast development in fruits and flowers. Plant Sci. 94, 1–17. Markham, K.R., 1982. Techniques of flavonoid identification. Academic Press, London. Markham, K.R., Hammett, K.R.W., 1994. The basis of yellow colouration in Lathyrus aphaca flowers. Phytochemistry 37, 163–165. Minguez-Mosquera, M.I., Hornero-Mendez, D., 1994. Changes in carotenoid esterification during the fruit ripening of Capsicum anuum cv. Bola. J. Agric. Food Chem. 42, 640–644. Shirley, B.W., 1996. Flavonoid biosynthesis: new functions for an old pathway. Trends Plant Sci. 1, 377–382. Stein, W., Zinsmeister, H.D., 1990. New flavonoids from the moss Bryum pseudotriquetrum. Z. Naturforsch. 45c, 25–31. Quackenbush, F.W., Miller, S.L., 1972. Composition and analysis of the carotenoids in marigold petals. J. Assoc. Off. Anal. Chem. 55, 617–621. Wellburn, A.R., 1994. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various spectrophotometers of different resolution. J. Plant Physiol. 144, 307–313. Wollenweber, E., Jay, M., 1988. Flavones and flavonols. In: Harborne, J.B. ŽEd.., The Flavonoids. Chapman and Hall, London, Chap. 7, pp. 233–302.
Flavonoid and carotenoid pigments in flower tissue of Sandersonia aurantiaca žHook. / David H. Lewis
a,)
, Stephen J. Bloor b, Kathy E. Schwinn
a
a
New Zealand Institute for Crop and Food Research, LeÕin Research Centre, PriÕate Bag 4005, LeÕin, New Zealand b New Zealand Institute for Industrial Research, Gracefield Research Centre, PO Box 31-310, Lower Hutt, New Zealand Accepted 15 September 1997
Abstract Luteolin, cryptoxanthin and zeaxanthin were detected as the major pigments present in mature flowers of Sandersonia aurantiaca ŽHook... Both the flavonoids and carotenoid pigments were examined at four stages of flower development from green flower bud to senescent flower prior to tepal necrosis. The major component of the orange colour of the Sandersonia flowers is due to the presence of the carotenoids zeaxanthin and cryptoxanthin, both predominantly present in the esterified form. The main change during development is the loss of chlorophyll, b-carotene and lutein, and the increase in zeaxanthin and cryptoxanthin as the flower matures from a green bud to an open flower. The major flavonoids present were luteolin and luteolin 7-O-glucoside. Flavonoid concentration decreased over the course of development to a mature flower but there was an increase in content per flower. The flavonoids were present in greater quantities than the carotenoids. However, as they absorb mostly in the UV region, they are not believed to contribute significantly to the observed colour. q 1998 Elsevier Science B.V. Keywords: Carotenoids; Flavonoids; Flower pigments; Sandersonia
1. Introduction Sandersonia aurantiaca ŽHook.. is native to South Africa but is grown as a commercial flower crop in New Zealand, where it is currently the third ranked cut flower crop in terms of export earnings ŽStatistics New Zealand.. It is a tuberous plant, )
Corresponding author. Tel.: q64 6 368 7059; fax: q64 6 367 5656; e-mail: [email protected]
0304-4238r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 2 3 8 Ž 9 7 . 0 0 1 2 4 - 6
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with each tuber having two growing points from which the flowering shoots develop. Each shoot is 400–700 mm long and bears a number of bright yellow–orange bell-shaped flowers in the leaf axils ŽClark, 1994.. The distinctive shape of the flowers is due to the fused sepals and petals Žtepals. that form a single complete structure. Sandersonia is a monotypic species ŽBrundell and Reyngoud, 1985. and no new cultivars with altered morphological features have been released. The range of flower colours for this species is therefore limited to yellow–orange. Flower colour and the aesthetic value associated with it, has prompted research into the compounds that are responsible for the colour of plant organs and into the selection and breeding of cultivars with different colours ŽForkmann, 1991.. Besides chlorophyll, there are three main groups of plant pigments: flavonoids which are responsible primarily for the red and blue colours, carotenoids that give orange and yellow colours and the betalains that may give violets or yellows, although their presence is restricted to certain plant families ŽHaslam, 1993.. Research on the genes controlling the synthesis of plant pigments, and in particular the flavonoids, has resulted in the production of transgenic plants with modified flower colours Žsee reviews by Forkmann, 1991; Davies and Scwhinn, 1997.. The same technology could be utilised to develop novel colours for Sandersonia through genetic manipulation of the pigment biosynthetic pathways. However, it is important to know which pigments are normally present so that specific steps in the synthesis of the pigments can be targeted. This study details the pigment composition of Sandersonia flowers at four stages of development.
2. Materials and methods 2.1. Plant material Sandersonia tubers were obtained from Lilies International ŽLevin, New Zealand.. The tubers were dipped in fungicide, 1 g ly1 Benlate ŽDupont, Auckland, New Zealand., prior to planting in polythene bags in a sterilised bark:pumice Ž1:1. mix. The plants were grown in a glasshouse Žheated at 158C, vented at 258C. under ambient light conditions. Four stages of flower development were chosen for examination: stage 1—an immature green bud, stage 4—flower open but still has green tips, stage 7—mature flower completely orange and stage 10—tepals wilting and orange colour fading ŽEason and Webster, 1995.. Flowers were harvested randomly from 40 plants over the period of a month to collect sufficient tissue Žapproximately 10 g fresh weight ŽFW. per flower stage. for analysis. Flowers were harvested, the pedicel, anthers and ovary removed and the remaining tepal tissue frozen in liquid nitrogen, crushed to a fine powder, freeze dried and stored at y208C. The whole flower and tepal tissue weight and flower colour were recorded for five flowers from each development stage, prior to the tissue being frozen. Two extra samples of stage 7 tepal tissue were collected and extracted as fresh tissue for the initial qualitative flavonoid analysis. Colour parameters Žlightness, chroma and hue angle; L, C, H8. were measured with a Minolta CR-200 chroma meter ŽMinolta Camera Co., Osaka, Japan., standardised on a
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white tile supplied with the instrument. The chroma meter has an 8 mm reflectance port size, a dr0 illuminating system and a D65 light source for illumination. Two readings were taken per flower on separate axes Ž908 apart. through a cross section of the centre of the flower. Lightness represents the proportion of total incident light that is reflected, chroma is a measure of colour intensity Žin relative intensity units. and hue angle relates colour to a position on a colour circle or wheel, with red purple at 08, yellow at 908, bluergreen at 1808, and blue at 2708 2.2. FlaÕonoid analysis Fresh tepal tissue was frozen in liquid nitrogen, homogenised using a mortar and pestle and extracted at room temperature in 90% methanol Ž10 ml gy1 FW. for 72 h. The sample was centrifuged at 1200 = g for 10 min, the supernatant decanted and the pellet re-extracted in 50% methanol Ž5 ml gy1 FW. for 24 h. The sample was centrifuged again and the supernatants combined. Final extract volume was reduced to 10 ml using a Savant SC210 Speedvac. Eight aliquots, each equivalent to 600 mg FW of tepal tissue, were partitioned with chloroform Ž1:1. to remove any carotenoids and other low polarity molecules before being loaded onto separate paper chromatograms. Individual flavonoids were separated by 2D paper chromatography ŽPC. on Whatmann 3MM paper in TBA Ž t-butanol:acetic acid:water 3:1:1. and 15% acetic acid. Dried chromatograms were viewed in ultraviolet light alone and after exposure to ammonia fumes to detect spots, whose colour and Rf values were noted. The corresponding spots from each chromatogram were combined and eluted in 80% methanol. Spectral data for each compound were measured over the 200–500 nm range in both methanol and with the addition of shift reagents ŽMarkham, 1982. and compared with reported results for reference compounds ŽMabry et al., 1970; Markham, 1982.. Each putative flavonoid was run on a separate Sep-pak C 18 preparative cartridge ŽWaters Associates, Milford, MA, USA. and eluted in methanol before being hydrolysed with 2 N HCL at 958C for 1 h. The hydrolysed flavonoids were then taken to dryness under reduced pressure, the sugars dissolved in water and the aglycones in methanol. The sugars were separated by 1D PC and analysed by treatment with aniline hydrogen pthalate ŽMarkham, 1982.. The aglycones were analysed on cellulose thin layer chromatography plates ŽSchleicher and Schull, Dassel, Germany. against known standards in TBA, 50% acetic acid or Forestal ŽMarkham, 1982. and on silica TLC plates ŽSchleicher and Schull, Dassel, Germany. in chloroform:acetone:formic acid Ž9:2:1.. Freeze dried tissue was used for the high performance liquid chromatography ŽHPLC. analysis of flavonoid content in the different stages of flower tissue and to confirm the identity of the flavonoids that were isolated. Three samples of approximately 60 mg of ground freeze-dried tepal tissue from each stage was extracted at room temperature overnight with 5 ml 95% ethanol:formic acid:water Ž75:5:20.. The extracts were centrifuged and analysed by HPLC, using a Waters 600 solvent delivery system with a Merck Lichrosphere Ž5 m m, 119 = 4 mm. 100 RP-18 endcapped column Žcolumn temperature 308C. and a Waters 996 PDA detector with integration at 352 nm. Elution Ž0.8 ml miny1 . was performed using a solvent system comprising solvent A
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wHOAc:CH 3 CN:H 3 PO4 :H 2 O Ž20:24:1.5:54.5.x and 1.5% H 3 PO4 Žsolvent B. and a linear gradient starting with 80% A, decreasing to 33% A at 30 min, 10% A at 33 min and 0% A at 39.3 min. As only luteolin and apigenin derivatives were present in these samples, the amounts of flavones were determined as apigenin-7-neohesperidoside equivalents, since a suitably pure crystalline sample of this compound was available. Results are reported as the mean of the three replicates taken from each development stage. A portion of the extract from each flower stage was hydrolysed by adding an equal volume of 2 N HCl and heating in a sealed vial on a boiling water bath for 40 min. The aglycones were extracted using n-BuOH, dried and analysed by HPLC. The extract remaining after HPLC analysis was pooled, reduced in volume to approximately 10 ml and chromatographed on a polyamide column Ž35 = 350 mm Machery-Nagel SC-6., using water with an increasing proportion of methanol. Fractions containing pure luteolin 7-O-neohesperidoside and luteolin 7,4X-di-O-glucoside were eluted with 1:1 and 4:6 water:methanol respectively. NMR spectra were obtained in dimethylsulphoxide-d6 on a Bruker AC-300 instrument. NMR data from the flavonoid diglycosides isolated from Sandersonia are listed below. Luteolin 7,4X-di-O-glucoside. 1 H NMR data Ž d Žppm., multiplicity, coupling constant ŽHz..; 7.55 dd 8,2 ŽH-6X ., 7.53 d 2 ŽH-2X ., 7.26 d 8 ŽH-5X ., 6.90 s ŽH-3., 6.84 d 2 ŽH-8., 6.45 d 2 ŽH-6., 5.10 d 7 ŽH-1 of 7-glc., 4.89 d 7 ŽH-1 of 4-glc.. 13 C NMR data Ž d Žppm..; 182.6ŽC-4., 163.6ŽC-2., 161.7ŽC-5., 157.6ŽC-9., 149.3ŽC-4X ., 147.5ŽC-3X ., 125.2ŽC-1X ., 119.2ŽC-6X ., 116.6ŽC-5X ., 114.3ŽC-2X ., 106.0ŽC-10., 104.8ŽC-3., 101.9ŽC-1 of 4X glc., 100.4ŽC-6., 100.2ŽC-1 of 7 glc., 95.4ŽC-8., 77.9, 77.7Žglc C-5., 77.0, 76.4Žglc C-3., 73.8, 73.7Žglc C-2., 70.3, 70.1Žglc C-4., 61.2Ž2C, glc C-6.. Luteolin 7-Oneohesperidoside. 1 H NMR data Ž d Žppm., multiplicity, coupling constant ŽHz..; 7.41 s ŽH-2X ., 7.4 dd 8, 1 ŽH-6X ., 6.91 d 8 ŽH-5X .. 6.75 s ŽH-3., 6.74 d 2 ŽH-8., 6.38 d 2 ŽH-6., 5.23 d 7 ŽH-1X of glc., 5.14 s ŽH-1 of rha., 1.20 d 6.2 ŽH-6 of rha.. 2.3. Carotenoid analysis Three 50 mg dry weight ŽDW. samples of powdered freeze-dried tepal material from each flower development stage were extracted by soaking twice in 3 ml of acetone Ž4 h then overnight. and once in diethyl ether Ž2 h.. The combined acetone extracts were dried and added to the ether extract which was then washed once with water, and dried. The extract, made up to 2 ml with ethyl acetate, was then used for saponification and further analysis. Total carotenoid and chlorophyll content of Sandersonia tepal tissue was estimated using the methods of Wellburn Ž1994.. An aliquot of the ethyl acetate extract was dried under a stream of O 2-free N2 and then redissolved in chloroform for spectrophotometric analysis. Saponification of one of the carotenoid extracts was carried out to identify the parent carotenoids. A sample of the extract was taken and evaporated to dryness. The dried sample was dissolved completely in 500 m l ethanol and 100 m l of 60% KOH in water Žwrv. added. The solution was shaken and left at room temperature for 2 h. The carotenoids were then extracted by adding ethyl acetate Ž0.5 ml. and water to the saponification mixture. The ethyl acetate layer was taken, re-extracted with water, and used directly for HPLC.
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Table 1 Flower bud size and colour characteristics of Sandersonia flowers at four stages of development ŽMean"SE, ns 5. Flower development stage
Flower weight Žg FW.
Tepal tissue per flower 1 Žg FW.
Žg DW.
1 4 7 10
0.13"0.01 0.34"0.02 0.60"0.03 0.35"0.04
0.05 0.19 0.45 0.21
0.008 0.026 0.054 0.027
1
Lightness Ž% refl..
Chroma Žintensity.
Hue angle Ždegrees.
54.7"0.4 58.7"0.6 53.9"0.5 55.0"0.3
46.1"0.5 46.3"0.7 46.7"1.2 42.4"0.3
109.9"1.2 85.3"0.9 73.9"0.3 76.4"1.4
Mean tepal weight was determined from the total amount of tissuertotal flower number.
HPLC analyses were performed on the same instrument and column type used for flavonoid analysis. Elution Ž1 ml miny1 . was performed using a solvent system comprising solvent A wCH 3 CN:H 2 O:triethylamine Ž80:20:0.1.x and ethyl acetate Žsolvent
Fig. 1. HPLC chromatogram showing separation of the flavonoids extracted from Sandersonia tepals at stage 7.
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B. and a linear gradient starting with 100% A, decreasing to 0% A at 25 min and held at 0% A for a further 5 min. A modified gradient was employed for analysis of the saponified samples to separate lutein and zeaxanthin. Elution Ž0.9 ml miny1 . was performed using a solvent system comprising solvent C wCH 3 CN:H 2 O:isopropanol Ž86:10:4.x and ethyl acetate Žsolvent B. and a linear gradient starting with 100% C, decreasing to 50% C at 20 min, 0% C at 25 min and held at 0% A for a further 5 min.
Fig. 2. Total flavonoid concentration ŽA. or content per flower ŽB. for Sandersonia flowers at four stages of development ŽMean"SE.. Concentration and content were calculated as apigenin 7-neohesperidoside equivalents. Where error bar is not visible, standard error was too small to distinguish from the mean.
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Non-carotenoid peaks in the HPLC chromatogram at 430 nm and 480 nm were identified as such from on-line spectral analysis. The levels of carotenoids were determined by summing the carotenoid peak areas and calculating the m g b-carotene equivalentsrg DW of tepal. Lutein, zeaxanthin and cryptoxanthin were identified in the flower extracts by comparison of retention times and on-line spectral data with standard samples. The identity of the cryptoxanthin peak in the saponified flower extract was also verified by co-chromatography with a b-cryptoxanthin standard. Total carotenoid and individual carotenoid levels were quantified for each of the three extracts from each development stage and the results reported as a mean from the three replicates. 2.4. FlaÕonoid and carotenoid standards Flavonoid standards for luteolin, apigenin and apigenin 7-neohesperidoside were purchased from Apin Chemicals ŽAbingdon, Oxon, UK.. Luteolin 7-O-glucoside, luteolin 7,3X-di-O-glucoside, luteolin 7-O-rutinoside and apigenin 7-O-glucoside were from a collection of standard samples held in the Gracefield laboratory. Trans-b-carotene was purchased from Aldrich Chemicals ŽMilwaukee, WI, USA.. The concentration of a solution of b-carotene in hexane was determined by measuring the absorbance at 449 nm and using a A11%cm value of 2592 ŽBritton, 1985.. This standard solution was then dried and redissolved in ethyl acetate and used as an external standard for the HPLC carotenoid quantification. Samples of lutein and zeaxanthin were kindly donated by Dr. D. Della Penna, University of Nevada Reno. b-Cryptoxanthin was isolated from a saponified extract of peach. 3. Results 3.1. Flower deÕelopment Whole flowers and tepals of Sandersonia increased in weight as the flower developed from an immature bud to an open flower, stages 1–7 ŽTable 1.. A decrease in flower Table 2 Mean concentration and content per flower for individual flavonoids in flowers of Sandersonia at different stages of development ŽMean"SE, ns 3.. Flavonoids were quantified as apigenin-7-neohesperidoside equivalents Flower development stage
Luteolin
conc. Ž m mol gy1 DW. 1 15.6"0.5 4 50.2"0.2 7 36.3"0.5 10 45.0"0.5 content Ž m mol flowery1 . 1 0.12"0.02 4 1.30"0.02 7 1.94"0.03 10 1.19"0.02
Luteolin 7-O-glucoside
Luteolin 7-O-neo-hesperidiside
Luteolin X 7,4 -di-O glucoside
Apigenin 7-O-glucoside
257.8"0.2 99.7"1.2 50.2"0.2 73.9"1.2
10.3"0.2 5.2"0.2 3.5"0.2 5.2"0.2
58.8"0.2 24.7"0.5 12.1"0.2 15.6"0.2
14.4"0.5 6.9"0.2 3.5"0.2 5.2"0.2
0.09"0.02 0.14"0.02 0.19"0.02 0.14"0.02
0.47"0.02 0.64"0.02 0.66"0.02 0.42"0.02
0.12"0.02 0.17"0.02 0.19"0.02 0.14"0.02
2.06"0.02 2.60"0.03 2.72"0.02 1.99"0.03
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weight was observed for flowers at stage 10 as the tissue wilted and senescence proceeded. The change in flower size was accompanied by a marked change in colour ŽTable 1.. Buds at stage 1 were dark green while the mature flowers were bright orange. This is shown by the change in hue angle from 110 to 74. The colour did revert somewhat to a more yellow rather than red colour as the flowers reached stage 10.
Fig. 3. Chlorophyll and total carotenoid concentration ŽA. or content per flower ŽB. for Sandersonia flowers at four stages of development ŽMean"SE.. Concentration and content were calculated as b-carotene equivalents. Where error bar is not visible, standard error was too small to distinguish from the mean. ŽB s chlorophyll, s total carotenoids..
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Major changes in lightness or intensity of colour were not observed although a decrease in colour intensity was detected at stage 10. 3.2. FlaÕonoids Five significant flavonoids were evident in the HPLC chromatograms from extracts of Sandersonia flower tissue ŽFig. 1.. All were flavones, as hydrolysis of the extracts gave only luteolin and apigenin, with luteolin predominant. The least polar compound, 5, was identified as luteolin by co-chromatography ŽHPLC. with authentic samples, combined with the results from the 2D-PCs and shift reagents. Similarly, the two monoglycosides, 2 and 4, were shown to be luteolin-7-O-glucoside and apigenin 7-O-glucoside respectively. The precise structures of the two diglycosides, 1 and 3, were more difficult to determine and NMR spectroscopy was used for their identification. The most polar glycoside, 1, had 13 C and 1 H NMR spectra consistent with a luteolin 7,4X or 7,3X-diglucoside, however, a reference sample of luteolin 7,3X-diglucoside had a shorter HPLC retention time and the reported NMR shifts of the 7,3X compound are different from those observed for 1. No literature data or reference compound was available for the 7,4X-diglucoside but the 13 C NMR shift values for the flavonoid nucleus of 1 are consistent with this compound and with values for the similarly substituted luteolin-7neohesperidoside-4X-glucoside ŽAgrawal, 1989.. Hence 1 is luteolin 7,4X-di-O-glucoside. The other diglycoside, 3, had 1 H NMR signals for both rhamnose and glucose and substitution only at the C-7 position. The 1 H NMR chemical shifts of the anomeric protons were consistent with a neohesperidoside rather than a rutinoside and this was confirmed by comparison with reported 1 H NMR shifts for the neohesperidoside ŽStein and Zinsmeister, 1990.. A reference sample of luteolin 7-O-rutinoside eluted earlier from the HPLC than 3. Thus 3 is luteolin 7-O-neohesperidoside Žs a-L-rhamnosyl-Ž1 ™ 2.-b-D-glucoside.. The total flavonoid concentration decreased as the flower buds developed from stage 1 to 7 but then increased at stage 10 ŽFig. 2.. The flavonoid content per flower, however, showed the opposite pattern ŽFig. 2B.. All four flower stages showed similar flavonoid profiles although the concentration and content of the individual flavonoids did change during development ŽTable 2.. Luteolin 7-O-glucoside and luteolin 7,4X-di-O-glucoside Table 3 Mean concentration and content per flower for individual carotenoids in flowers of Sandersonia at different stages of development. Carotenoids were quantified as b-carotene equivalents. nd s not detected Flower development stage b-carotene
Lutein
Zeaxanthin Crypto-xanthin Esterified Xanthophylls
0.27"0.01 0.19"0.01 nd nd
0.32"0.01 0.01"0.01 nd nd
0.26"0.01 nd 0.19"0.01 0.07"0.01 0.16"0.01 0.25"0.01 0.07"0.01 0.13"0.01
nd 1.39"0.3 3.82"0.05 2.33"0.04
2.14"0.02 4.97"0.06 nd nd
2.59"0.09 0.09"0.02 nd nd
2.12"0.07 nd 5.00"0.19 1.73"0.06 8.60"0.19 13.48"0.43 1.90"0.04 3.52"0.06
nd 36.11"0.84 206.41"2.72 62.79"0.95
y1
conc. Ž m mol g DW. 1 4 7 10 content Žnmol flowery1 . 1 4 7 10
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were the major flavonoids at stage 1 but by stage 4 luteolin is present in greater quantities than 7,4X-di-O-glucoside. 3.3. Carotenoids Distinct changes occurred in both carotenoid concentration ŽFig. 3. and the individual carotenoids detected at different stages during flower development ŽTable 3.. Flowers at stage 1 had the highest levels of chlorophyll and the carotenoids lutein, zeaxanthin and b-carotene were also present. At stage 4 the chlorophyll concentration had decreased by
Fig. 4. HPLC chromatogram of the carotenoids extracted from Sandersonia tepals at stage 7.
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Table 4 Concentration and content per flower for individual carotenoids in a saponified extract from flowers of Sandersonia at different stages of development. Carotenoids were quantified as b-carotene equivalents. nd s not detected Flower development stage conc. Ž m mol gy1 DW. 1 4 7 10 content Žnmol flowery1 . 1 4 7 10
b-carotene
Lutein
0.25 0.39 nd nd
0.41 0.02 nd nd
0.34 0.79 1.69 1.01
nd 0.52 2.54 1.52
2.05 10.24 nd nd
3.35 0.37 nd nd
2.79 20.48 91.43 27.37
nd 13.59 137.06 40.97
Zeaxanthin
Cryptoxanthin
70% but the carotenoid concentration had doubled. b-carotene and zeaxanthin were the major carotenoids, but the lutein concentration had decreased and the monohydroxy xanthophyll, b-cryptoxanthin, was now present. Carotenoid concentration and content per flower were greatest at stage 7. The carotenoid concentration had doubled again between stages 4 and 7 while the content per flower had increased by a factor of four. Carotenoid levels, however, decreased at stage 10. The xanthophylls, zeaxanthin and cryptoxanthin, were the major carotenoids present in flowers at stages 7 and 10 but they were present mainly in their esterified forms ŽFig. 4.. Saponification of the carotenoid extract revealed no other parent carotenoids ŽTable 4.. Lutein, b-carotene and the chlorophylls were not detected in flowers at these development stages. 4. Discussion Both flavonoids and carotenoids were detected in the tepal tissue of Sandersonia flowers. The changes in these compounds, especially with respect to the carotenoids, were linked with the changes in colour of the flower as it developed from a green bud to an orange flower. The green colour of the buds at stage 1 was due to the presence of chlorophyll. Carotenoids and flavonoids were also present but these were masked by the presence of the chlorophyll. Carotenoids are responsible for the colour of the mature flower. The orange colour of the flower developed in the period between stage 1 and 7 and this correlated with the increase in both the carotenoid concentration and content per flower. Zeaxanthin and cryptoxanthin, the major carotenoids detected, have absorbance maxima between 430–490 nm ŽBritton, 1995. and are orangeryellow in colour. The flavonoid concentration did not increase with flower development and luteolin and luteolin 7-O-glucoside, the most abundant flavonoids Žwith absorbance maxima at 349 nm, Mabry et al., 1970., would provide a background creamy yellow colour to the flower. No strongly coloured flavonoids, such as anthocyanins, were detected. The carotenoids present in the flower changed during development, with lutein and b-carotene being replaced by zeaxanthin and cryptoxanthin. This is consistent with the degradation of chloroplasts and the formation of chromoplasts and the associated
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changes in carotenoid metabolism resulting in the accumulation of apolar carotenoids and xanthophylls and their acylated and esterified forms ŽBritton, 1982; Marano et al., 1993; Bartley and Scolnik, 1995.. Approximately 90% of the zeaxanthin and cryptoxanthin in stage 7 Sandersonia flowers was present in the esterified form ŽTables 3 and 4.. Esterification of the carotenoids appears to be important for the formation of the chromoplasts, the accumulation and longevity of the carotenoid pigments and ultimately the colour of the tissue. The esterified form is considered more stable and more liposoluble, so is more easily incorporated into membranes ŽMinguez-Mosquera and Hornero-Mendez, 1994.. The accumulation of carotene bodies in the chromoplast and the different forms of the carotene bodies, influence the structure and properties of that chromoplast ŽMarano et al., 1993; Bartley and Scolnik, 1995.. Carotenoids are the major pigment in many redryellow flowers including daffodils ŽLiedvogel et al., 1976., marigolds ŽQuackenbush and Miller, 1972. and in yellow varieties of Viola tricolor ŽHansmann and Kleinig, 1982.. Their main role appears to be pollinator attraction, although carotenoids have also been linked with photosynthesis, photoprotection and plastid structure ŽBartley and Scolnik, 1995.. The total carotenoid level measured in Sandersonia was within the range recorded for the flowers listed above, although the predominant carotenoid varied with the different species. Lutein was the most abundant carotenoid detected in marigold flowers ŽTagetes erecta., varying between 76 and 93% of the total carotenoids depending on the variety ŽQuackenbush and Miller, 1972.. The difference in colour between the varieties examined Žred, gold or yellow. appeared to be due to the variation in the total carotenoid concentration and to smaller changes in the concentration of the more minor carotenoids. Thus subtle changes in the carotenoid content can result in more obvious visual changes in flower colour. The flavonoids detected in Sandersonia flower tissue were all flavones or their glycosides, the most abundant being luteolin and luteolin 7-O-glucoside. The major change during development was the increase in luteolin, possibly as an external flavonoid on the surface of the tepals. The lipophillic nature of aglycones such as luteolin often results in their accumulation on external plant surfaces ŽWollenweber and Jay, 1988.. Colourless flavonoids, including the flavones, play various roles in plant tissue. These include acting as co-pigments, providing background colour, producing patterns on flower petals that are attractive to pollinators, acting as UV protectants and they have also been linked with antibacterial and antifungal activity and with deterring insect herbivory ŽHarborne, 1988; Forkmann, 1991; Shirley, 1996.. The flavonoids were present in Sandersonia flower tissue in much greater quantities than the carotenoids ŽFigs. 2 and 3., despite their smaller contribution to the overall colour of the flower. The presence of both flavonoids and carotenoids is not uncommon. Flavonol glycosides were also the major flavonoids detected in Lathyrus aphaca petals and were estimated to make up 12.7% of the dry weight ŽMarkham and Hammett, 1994., although the colour of the Lathyrus flowers was attributed to the carotenoids. The total flavonoid concentration in Sandersonia flowers decreased as the flower developed although there was an increase in content per flower, indicating continued flavonoid synthesis. The increase in concentration at stage 10 is probably due to the loss of fresh weight either from passive water loss during senescence or the transport of soluble metabolites out of the flower.
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The pigments in the flower tissue of Sandersonia were examined as the first step in a study to determine whether flower colour in this crop could be modified. The presence of both the flavonoid and carotenoid biosynthetic pathways means that several strategies could be pursued. Inhibition of the carotenoid pathway may result in paler flower colour, while redirection of the pathway may allow production of new novel colours. Manipulation of the carotenoid biosynthetic pathway in tomato altered both flower and fruit colour ŽBramley et al., 1992.. Alternatively the introduction of the appropriate regulatory genes may allow the whole flavonoid pathway to be activated, resulting in anthocyanin production ŽLloyd et al., 1992..
Acknowledgements The authors thank Mr. Ray Rains for cultivating the plant material and the New Zealand Lottery Grants Board for a grant towards the purchase of the Savant Speedvac. This research was supported by funding from the New Zealand Foundation for Research, Science and Technology.
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