Euphytica 130: 25–34, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
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Characterization of carotenoid pigments and their biosynthesis in two yellow flowered lines of Sandersonia aurantiaca (Hook) Karen M. Nielsen∗ , David H. Lewis & Ed R. Morgan New Zealand Institute for Crop & Food Research, Fitzherbert Science Centre, Batchelor Rd Private Bag 11 600, Palmerston North, New Zealand; (∗ author for correspondence; e-mail:
[email protected]) Received 10 April 2002; accepted 10 September 2002
Key words: β-carotene hydroxylase, carotenoid, flower color, pigments, phytoene desaturase, Sandersonia
Summary The basis of the novel cream/yellow flower color found in two Sandersonia aurantiaca lines was examined as part of a project to develop new colors for this cut flower crop in New Zealand. The original color, bright orange, is due to the accumulation of the carotenoid pigments zeaxanthin and β-cryptoxanthin. The cream/yellow lines have much lower levels of total carotenoid pigments (17% and 21%) in their tepal tissue compared to the wild type progenitor. Microscopic analysis of epidermal cells showed alteration in the pigment cluster bodies of tepal tissue of the cream/yellow lines compared to the orange wild type. HPLC analysis of the pigments showed that one cream/yellow line (Y-H) produced the same pigment profile as the wild type (zeaxanthin and β-cryptoxanthin). In comparison, the other cream/yellow line (Y-S) produced the carotenoid profile normally found in green vegetative tissue (β-carotene and lutein). Analysis of carotenoid biosynthetic gene expression in Sandersonia indicated that the cream/yellow Y-H line showed expression patterns similar to the wild type, and gene expression in the Y-S line is decreased relative to the wild type and the Y-H line.
Introduction Sandersonia aurantiaca is a tuberous plant native to South Africa and grown in New Zealand as a cut flower crop. The flowers are 2–2.5 cm long and 1.3 cm in width, are golden orange, lantern shaped and hang down from curved, wiry flower-stalks originating at the leaf axils. Mature plants are produced from forkshaped tubers where the stems arise from the single growing points on each tip. During the growing season, the original tuber withers and is lost but is replaced by daughter tubers that develop at the base of each stem (Brundell & Reyngoud, 1986). Sandersonia is a monospecific genus in which little morphological variation has been observed. Variants have been developed for the New Zealand horticulture industry by hybridization with species in related genera, eg. Littonia modesta (Morgan et al., 2001). Sandersonia: Littonia hybrids differed from the parent species in morphological characteristics of the leaves and flowers, although the flower color remained un-
changed. The pigments responsible for the golden orange flower color of Sandersonia have been characterized (Lewis et al., 1998), and the major components giving rise to the orange color are the carotenoid derived pigments zeaxanthin and β-cryptoxanthin. These pigments are synthesized as an extension of the well characterized carotenoid pathway (Figure 1) by hydroxylation of the cyclic rings of β-carotene. Synthesis and accumulation of carotenoids is localized to subcellular organelles called chromoplasts and cDNA clones have been isolated for all the catalytic enzymes involved in the carotenoid pathway (Cunningham & Gantt, 1998). The enzyme phytoene desaturase (PDS) catalyzes an intermediate step in carotenoid biosynthesis and expression of the pds gene is correlated with levels of carotenoid accumulation during flower and fruit development of capsicum (Hugueney et al., 1996), tomato (Pecker et al., 1992; Fraser et al., 1994), daffodil (Al-Babili et al., 1996) and in green tissues of tomato (Corona et al., 1996). β-cryptoxanthin and zeaxanthin are derived from β-carotene by hy-
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Figure 1. Diagrammatic representation of a section of the carotenoid biosynthetic pathway.
27 droxylation of the cyclic rings, a reaction catalyzed by the enzyme β-carotene hydroxylase (β-OH) (Sun et al., 1996). Antisense inhibition of production of this enzyme has been shown to alter the accumulation of βcarotene derived xanthophylls in Arabidopsis (Rissler & Pogson, 2001). Therefore, activity of either or both the pds and β-oh genes could be crucial in determining pigmentation and flower color in Sandersonia. In New Zealand, Sandersonia is grown for export as both cut flowers and as tubers. Cream/yellow flowered lines have recently been identified and one line has been released commercially. We have investigated the pigment changes responsible for this atypical flower color and examined expression of the carotenoid biosynthetic genes pds and β-oh. This has been done to achieve an understanding of the mechanism underlying this flower phenotype and to assist in the development of further novel lines.
Materials and methods Plant material Sandersonia tubers were obtained from Lilies International (Levin, New Zealand) and the yellow flowered lines (spontaneous mutations identified in seed grown crops) were made available to us by Sanza New Zealand. The plants were grown in a greenhouse (heated at 15 ◦ C, vented at 25 ◦ C) under ambient light conditions. The stages of flower development referred to in this work are those described by Eason & Webster (1995), where stage 1 corresponds to immature green buds and stage 7 to fully mature, pre-senescent flowers. Pigment analysis Comparisons of pigment profiles of the standard orange line with the novel cream/yellow lines were made in tissues from young leaf, stage 3 bud tepal tissue and pooled stage 6 and 7 tepal tissue. Fresh tissue was frozen in liquid nitrogen, homogenized using a mortar and pestle and tissue for pigment analysis was freeze dried. Two 30 mg dry weight (DW) samples of powdered freeze-dried tepal or leaf material were analyzed from each selection or development stage. Samples were moistened with water (50 µl) and then extracted, initially overnight, in 10ml of Acetone:Methanol (7:3) with 200 mg ml−1 CaCO3 . Extracts were kept at room
temperature and covered with aluminium foil to exclude light. The extract was centrifuged 3000 rpm × 5 min, the supernatant removed and re-extracted in a further 10 ml of Acetone:Methanol (7:3). This process was repeated at 1 hour intervals until tissue was colorless. The combined supernatants for each sample were partitioned with equal volumes of diethyl ether and water, and the diethyl ether fraction removed. This process was repeated until the acetone aqueous phase was colorless. The combined diethyl ether fractions were dried under O2 -free N2 and the carotenoids dissolved in 2 ml of ethyl acetate. The extract was then used for saponification and further analysis. Saponification of the carotenoid extracts was carried out to identify the parent carotenoids. A 300 µl sample of the extract was taken and evaporated to dryness. The dried sample was dissolved completely in 25 µl ethyl acetate, 275 µl solvent A (see below) and 300 µl of 20% KOH in methanol (w/v) added. The solution was shaken and left at room temperature overnight. The carotenoids were then extracted (3x) by adding diethyl ether (0.6 ml) and water to the saponification mixture. The diethyl ether layer was taken, dried under N2 , and redissolved in 300 µl of ethyl acetate. High performance liquid chromatography (HPLC) analyses were performed on a Waters 600 solvent delivery system with a Beckman Ultrasphere (5 µm, 250 × 4.6 mm) ODS endcapped column (column temperature 25 ◦ C) and a Waters 996 PDA detector. Elution (0.9 ml min−1 ) was performed using a solvent system comprising solvent A [CH3 CN:H2 O:isoPrOH:triethylamine (86:10:4:0.1)] and ethyl acetate (solvent B) and a linear gradient starting with 90% A, decreasing to 75% A at 10 min, 60% A at 13 min, 0% A at 21 min and held at 0% A for a further 9 min. Carotenoids were detected at 450 nm and the levels of carotenoids were determined by peak integration as β-carotene equivalents per gram dry weight of tissue. β-carotene, lutein, zeaxanthin and β-cryptoxanthin were identified in the sample extracts by comparison of retention times and on-line spectral data with standard samples. Trans-β-carotene was purchased from Sigma Chemicals (St Louis, Missouri, USA). Samples of lutein, β-cryptoxanthin and zeaxanthin were supplied by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Total carotenoid and individual carotenoid levels were quantified for each of the extracts from each selection and developmental stage and the results reported as a mean from the two replicates. Total carotenoid content was also estimated
28 spectrophotometrically using the methods of Wellburn (1994). Color parameters were measured with a Minolta CR-200 chroma meter (Minolta Camera, Osaka, Japan), standardized on a white tile supplied with the instrument. The chroma meter has an 8 mm reflectance port size, a d/0◦ illuminating viewing geometry, and the ‘C’ light source was used for illumination. Readings were taken from at least 4 separate flowers. PCR amplification of a β-oh partial cDNA A β-oh partial cDNA was isolated by RT-PCR using primers designed according to conserved regions identified by comparison of published sequences in the GENBANK database. Primers specific for β-oh were: PBOH-5: 5’-TGGARTTYTGGGCDARRTGGG-3’ and PBOH-3: 5’-TCYTCMARYTCCTTCRGGWCC3’. First strand cDNA was synthesized from 2 µg of total RNA isolated from stage 4 tepals annealed to 5 pmol of oligo dT using Superscript II according to the manufacturer’s protocol (BRL, Gaithersburg, MD). 2 µl of the first strand cDNA was subjected to PCR amplification using AmpliTaq (BRL, Gaithersburg, MD) according to the manufacturer’s protocol, in 50 µl total volume containing 50 pmol of each primer. Annealing temperature was 50 ◦ C. PCR amplified products were cloned into pGEMT (Promega, Madison WI, USA). Library screening A pds cDNA was isolated from a Sandersonia tepal cDNA library (Eason, et al., 2000). A total of 104 plaque forming units were transferred to Hybond N+ discs (Amersham Pharmacia Biotech, UK Ltd), prehybridized and hybridized at 55 ◦ C in 0.5M NaPhosphate (pH 7.4); 7%SDS (Church & Gilbert 1984) and washed at 55 ◦ C in 2XSSC;0.1%SDS. A tomato pds cDNA homologue (K.M. Nielsen, unpublished data) was used to screen the library for a pds homologue. All Sandersonia clones isolated were sequenced at the University of Waikato DNA Sequencing Facility to confirm identity. Both Sandersonia sequences are available from Genbank, Accession numbers AY077686 (pds) and AY077687 (β-oh). Northern RNA analysis Northern RNA analysis was carried out for tepal tissue from mixed stages 1 and 2, 3, 4, 5 and mixed stages 6 and 7 of flower development for the original Sandersonia line and the two cream/yellow lines. For a descrip-
tion of Sandersonia flower development, see Eason & Webster (1995). Total RNA was isolated as per the protocol of Prescott & Martin (1987). Fifteen micrograms of total RNA were separated in a horizontal formaldehyde agarose gel, blotted to Hybond N+ membrane (Amersham Pharmacia Biotech, UK Ltd). DNA fragments to be used as probes were purified by electrophoresis in 1% agarose and recovered using a Qiagen Minelute kit (Qiagen GmbH, Germany). Radioactive labeling probes were produced using a Rediprime II random prime labeling kit (Amersham Pharmacia Biotech, UK Ltd) and α[32 P]dCTP (3000 Ci/mmol) according to the manufacturer’s instructions. Membranes were hybridized at 65 ◦ C in 0.5 M NaPhosphate (pH 7.4); 7%SDS (Church & Gilbert 1984). Membranes were washed at 65 ◦ C in 2XSSC; 0.1%SDS. The size of mRNA transcripts was estimated by comparison of the position of the hybridization signal with a 9.5 kb to 0.24 kb RNA molecular weight ladder (BRL, Gaithersburg, MD). Total RNA loadings were determined by hybridization to a cDNA probe encoding a 25/26S ribosomal RNA from Asparagus (King & Davies, 1992) at 55 ◦ C.
Results A comparison of flower color between the wild type (WT) and the variant lines (Y-H and Y-S) is shown in Figure 2A. Flower color for the Y-H and Y-S lines was indistinguishable, and only the Y-S line is shown. The WT plant has a bright orange color and the flowers of both variant lines are a pale cream/yellow. Epidermal cells of mature (stage 7) tepal tissue are shown in Figure 2B. Notable differences are observed in the chromoplast structures. In the WT cells, the pigment containing structures are elongated, tubular structures which give a clear orange color in this line. In comparison, the cream/yellow lines both show small, globular pigment-containing structures. Total carotenoid pigment levels in the Sandersonia lines were determined and results are presented in Figure 3. Total carotenoid levels were similar in leaf tissue, although higher levels were evident in leaves of the Y-S line. For tepal tissue, considerably lower levels of carotenoid pigments were found in the cream/yellow lines as compared to the WT. Over all tissues, only 17% of the wild type levels were detected in Y-H and 21% of the WT level detected in Y-S. A comparison of several color parameters among the three lines is shown in Table 1. Lightness (L) rep-
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Figure 2. A) Flowers of wild type orange (WT) and the cream/yellow line Y-H of Sandersonia. Y-H is indistinguishable in color from Y-S (not shown). B) Epidermal cells of the WT and Y-HSandersonia lines. Cells of Y-H were indistinguishable from Y-S (not shown).
Table 1. Color variables (mean ± standard error of the mean) recorded for the wild type (WT) and cream/yellow Sandersonia lines (Y-H and Y-S) for bud (stage 3) and mature (stage 6/7) tepal tissue. [Y] – yellow, [GY] – green/yellow, [YR] – yellow/red Tissue
Line∗
L (lightness)
C (Chroma)
Stage 6/7
WT (5) Y-H (4) Y-S (4)
58.7 ± 0.8 64.9 ± 1.0 67.1 ± 0.7
59.2 ± 0.9 45.6 ± 0.6 51.7 ± 1.3
71.4 ± 0.8 [YR] 90.7 ± 0.1 [Y] 91.1 ± 0.3 [Y]
WT (4) Y-S (6)
59.3 ± 0.9 62.5 ± 1.3
56.8 ± 0.9 52.3 ± 1.3
102.5 ± 1.8 [GY] 107.5 ± 0.6 [GY]
Stage 3
H◦ (Hue angle)
∗ number in brackets refers to the number of determinations.
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Figure 3. The mean of total carotenoids detected in leaf, bud (stage 3) and mature (stage 6/7) tepal tissue of wild type (WT) and variant cream/yellow (Y-H and Y-S) lines of Sandersonia. Values were calculated as µm per gram dry weight of tissue and are expressed as β-carotene equivalents. Error bars represent the standard error of the mean (n=2).
resents the proportion of total incident light that is reflected, chroma (C) is a measure of color intensity (in relative intensity units) and hue angle (H◦ ) relates color to a position on a color circle or wheel, with red/purple at 0◦ , yellow at 90◦ , blue/green at 180◦, and blue at 270◦ . The Y-H and Y-S lines showed an increase in L and a decrease in C compared to the WT which corresponds to the loss of carotenoid pigments. Hue angle (H◦ ) values also reflect the color change from the orange (yellow/red) of the WT to the more yellow color of the lines Y-H and Y-S. The carotenoid pigments present in the orange (WT) and the cream/yellow lines were identified by HPLC and results are presented in Table 2. For leaf tissue, the pigments present were β-carotene and lutein, and the Y-S line showed a slight increase in lutein accumulation. Differences in carotenoid pigment profiles were observed in tepal tissue for the three lines. In stage 6/7 tepal tissue, β-cryptoxanthin, zeaxanthin with trace levels of β-carotene accumulate in WT and β-cryptoxanthin and zeaxanthin accumulate in Y-H tepal tissue. The ratio of β-cryptoxanthin to zeaxanthin differed between WT and Y-H. In the WT line, the level of β-cryptoxanthin exceeded that of zeaxanthin whereas the reverse was observed in Y-H, where zeaxanthin was the predominant pigment. The pigment profile in Y-S was quite different from WT and Y-H,
with β-carotene identified as the predominant pigment in conjunction with lower levels of lutein and a trace of β-cryptoxanthin. In stage 3 bud tepal tissue, the WT line accumulates zeaxanthin and β-cryptoxanthin with lower levels of β-carotene present. This was not observed in Y-S. Y-S stage 3 bud tepal pigments were similar to those detected in green leaf tissue (β-carotene and lutein), and in similar proportions. Comparison of carotenoid biosynthetic gene expression (pds and β-oh) in all three lines is shown in Figure 4A. In order to compare transcript levels among the three lines, the band intensities for the autoradiograms were quantified using an AlphaImager 2000 Gel Documentation and Analysis System (Alpha Innotech, USA). The rRNA band intensity values for each sample were normalized relative to the rRNA band for WT stage 1/2. The values recorded for the β-oh and pds transcripts were adjusted accordingly and a graphical representation is presented in Figure 4B. Comparisons cannot be made between expression levels of β-oh and pds. pds transcripts were detectable in stage 1 tepal tissue for all three lines and increased with flower maturity. The transcript steadily increases in abundance, reaching a peak at stage 4 or 5 of floral development and tapering off in stage 6/7 tepal tissue. The levels of pds expression were generally lower in the cream/yellow lines with transcript levels in the Y-S line being considerably lower than either the WT or Y-H line. In Y-S, pds expression (as measured by band intensity) was 52% of the WT levels averaged throughout floral development. WT and Y-H profiles for β-oh expression were comparable although band intensities for Y-H exceeded those for WT from stage 4, and consistently exceeded the levels detected for YS. The extent of the reduction of β-oh transcript levels as the flower matures in Y-S is less marked than observed for the pds transcript in this line. pds and β-oh transcripts were not detectable in leaf tissue (data not shown).
Discussion Two novel lines of Sandersonia have been identified with altered flower color. This color change is due to the accumulation of lower levels of carotenoid pigments. The cream/yellow flowered lines accumulated significantly lower levels of carotenoid pigments than the wild type (Figure 3) and this loss of pigmentation is reflected in the increase in Lightness (L) and decrease in Chroma (C) (Table 1) for both Y-S and
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Figure 4. A) Northern analysis of RNA isolated from tepal tissue of WT, Y-H and Y-S Sandersonia lines. RNA was sampled during floral development: mixed stages 1 and 2, stage 3, 4, 5 and mixed stages 6 and 7. B) Diagrammatic representation of PDS and β-OH band intensities normalized for rRNA band intensity for each developmental tepal stage.
32 Table 2. Identification and quantification of carotenoids in leaf, mature floral petal and young bud tissue of the standard Sandersonia cultivar (WT) and two yellow flowered cultivars (Y-H and Y-S). HPLC traces were run for two replicates of saponified extracts for each tissue sample for each developmental stage. Values were corrected for losses during saponification and calculated as µm per gram dry weight of tissue expressed as β-carotene equivalents (mean ± standard error of the mean) with the percent of total carotenoids given in brackets Tissue
Lutein µM/g DW (%)
Zeaxanthin µM/g DW (%)
β-Cryptoxanthin µM/g DW (%)
β-carotene µM/g DW (%)
Leaf WT Y-H Y-S
3.1 ± 0.12 (60) 3.13 ± 0.03 (62) 4.12 ± 0.44 (64)
– – –
– – –
2.07 ± 0.08 (40) 1.92 ± 0.02 (38) 2.3 ± 0.25 (36)
Stage 6/7 Tepal WT Y-H Y-S
– – 0.24 ± 0.01 (31)
1.44 ± 0.05 (39) 0.6 ± 0.03 (95) –
2.09 ± 0.003 (55) 0.03 ± 0.002 (5) 0.023 ± 0.001 (3)
0.22 ± 0.008 (6) – 0.51 ± 0.015 (66)
Stage 3 Bud Tepal WT Y-S
– 0.39 ± 0.07 (63)
1.17 ± 0.14 (62) –
0.43 ± 0.04 (23) –
0.28 ± 0.18 (15) 0.23 ± 0.05 (37)
– not detected; DW – Dry weight.
Y-H as compared to WT mature tepal tissue. The change in Hue Angle (H◦ ) seen for Y-H and Y-S reflects the change from a red/orange to a cream/yellow flower color and is likely due to the loss of pigment concentration. A number of factors may be influencing the change in pigment accumulation. These include carotenoid biosynthetic gene expression patterns or altered enzyme activity levels, changes in pigment accumulation associated with flower maturity and chromoplast structure. Carotenoid pigments in Stages 1, 4 and 7 flowers of wild type, bright orange Sandersonia have previously been characterized (Lewis et al., 1998). Carotenoid pigment levels steadily increased with flower maturity with peak pigment levels evident in stage 7 flowers. The predominant pigments identified were zeaxanthin and β-cryptoxanthin. These compounds are synthesized by hydroxylation of the cyclic rings of β-carotene by the enzyme β-OH (Figure 1). We examined gene expression levels of β-oh in the cream/yellow lines (Figure 4) and found normal or near-normal transcript levels as compared to the WT. Expression of pds, which catalyzes an earlier biosynthetic step in the pathway (Figure 1), is consistent with the pigment accumulation profile of WT Sandersonia with maximum expression (stages 4 & 5) preceding the floral stage of maximum pigment accumulation (stage 7). pds expression levels have been correlated with levels
of carotenoid accumulation in capsicum (Hugueney et al. 1996), tomato (Pecker et al., 1992; Fraser et al., 1994; Corona et al., 1996), and daffodil (Al-Babili et al., 1996) and may be the rate-limiting step for carotenoid biosynthesis in Sandersonia. HPLC identification of the carotenoid pigments indicated that the cream/yellow line, Y-S, exhibits an altered carotenoid pigment profile from that observed in the WT and the Y-H lines (Table 2). This shift in carotenoid biosynthesis is evident from onset of production of floral specific pigmentation (stage 3 buds). Lutein and β-carotene accumulate rather than β-cryptoxanthin and zeaxanthin and this profile is similar to that observed in green tissue. The change in pigment profile is associated with a decrease in pds expression and slightly decreased levels of β-oh gene expression in this line. The pigment profile of the YH line was similar to WT although overall carotenoid pigment levels were greatly decreased to only 17% of the WT content. This is not associated with a substantial decrease in gene expression. A number of factors are critical for accumulation of floral specific carotenoid pigments. During floral ontogenesis, carotenoid biosynthesis switches from green tissue specific (chloroplastic) to flower specific (chromoplastic) biosynthesis. The transition from chloroplast to chromoplast is concomitant with the degradation of chlorophylls, the upregulation of caroten-
33 oid biosynthetic genes and increased production of floral or fruit specific carotenoids. The transition from chloroplast to chromoplast is influenced by a number of environmental and hormonal factors. Phytochrome expression in tomato fruit regulates lycopene accumulation independently of ethylene production (Alba et al., 2000) and gibberellin and auxin hormones have also been implicated in chromoplastogenesis in both fruits and flowers (Vainstein et al., 1994; Deruere et al., 1994; Vishnevetsky et al., 1999). In the Y-S line, although the loss of chlorophylls is complete, it is possible that the switch from green tissue specific (chloroplastic) to flower specific (chromoplastic) carotenoid biosynthesis is not complete in flowers of this line. Thus, -lycopene cyclase (-Lcy) (Figure 1) activity occurs leading to the presence of lutein in the tepal tissue. However the β-carotene levels increase, while the lutein levels drop as the tepal tissue matures (Table 2) suggesting perhaps both the -Lcy and β-Lcy activity is altered in this line. Further work to characterize -Lcy and β-Lcy expression patterns is necessary to clarify their role in the Y-S pigment profile. Alternatively, the genetic basis for this change may simply be a nonsense mutation in the β-oh gene leading to the production of β-OH enzyme with minimal activity. This would explain the low level of β-cryptoxanthin detected in the tepal tissue (Table 2) even though its substrate, β-carotene (Figure 1), accumulated. Chromoplasts accumulate carotenoids in specialized lipoprotein-sequestering structures and elongated structures are characteristic of the association of apolar carotenoids with phospholipids and specific carotenoid associated proteins (Zsila et al., 2001). Carotenoid-associated proteins have been found to play an important role in carotenoid sequestration and accumulation (Vishnevetsky et al., 1999). Treatment of Trapoleum flower buds with cyclohexamide to inhibit cytoplasmic protein synthesis (Emter et al., 1990) prevented the formation of chromoplast tubules resulting in the presence of globules similar to those found in the cream/yellow Sandersonia lines. It is possible that the Y-H line carries a mutation in the carotenoid associated protein, thus preventing the formation of the stable pigment structures normally found in Sandersonia. Pigment that is not stabilized in the lipoprotein structure may be degraded. Alternatively, this line may carry a block in the isoprenoid pathway which provides the precursors for carotenoid biosynthesis. For example, Geranylgeranyl pyrophosphate synthase (GGPPS) catalyzes an important step in the isoprenoid pathway (Cunningham & Gantt, 1998), and
its expression is strongly induced during the development of chromoplasts in ripening pepper fruit (Romer et al., 1992). More recently, activity of the 1-deoxyD-xylulose 5-phosphate synthase (dxs) gene has been implicated as the first potentially regulatory step in carotenoid biosynthesis during tomato fruit ripening (Lois et al., 2000). Loss of activity at either of these points in the pathway could result in reduced levels of carotenoid accumulation in Sandersonia. It is interesting to note that the mutations related to these two novel cream/yellow lines are limited to affecting floral pigmentation. Leaf carotenoid levels were maintained at normal levels. Indeed, greater than normal levels were evident in the Y-S leaf tissue (Table 2). We have examined two novel, although phenotypically indistinguishable, lines of Sandersonia aurantiaca. We found these cream/yellow lines to be deficient in carotenoid biosynthesis and to accumulate distinct pigment combinations. Comparison of pigment profiles, and molecular analysis of carotenoid gene expression, indicates that different mutations give rise to these two lines. The Y-S line showed depressed expression levels of pds, and to a lesser extent β-oh, and may be the result of a mutation in the switch from green tissue (chloroplast) to floral (chromoplast) carotenoid biosynthesis, or in the β-oh structural gene. Gene expression patterns in the Y-H variant did not differ from the wild type progenitor and the loss of carotenoid pigment accumulation may be due to the unavailability of biosynthetic precursors or an inability to form stable pigment lipoprotein structures. Nevertheless, further work is required to accurately delineate the mutations that gave rise to these cream/yellow flowered lines. This study reports on the discovery of a new flower color in Sandersonia aurantiaca, a plant species that was previously considered monotypic. Two variant lines that are phenotypically identical have resulted from different mutations. Both lines show loss of carotenoid pigment concentration although their carotenoid pigment profiles differ, and they also vary in their carotenoid biosynthetic gene expression patterns. The introduction of novel biosynthetic steps or up-regulation of carotenoid biosynthesis in tepal tissue, are strategies with potential for development of further novel flower colors in Sandersonia.
34 Acknowledgements The authors thank Sanza for providing access to the yellow lines, Bruce Dobson for cultivating the plant material, also Helge van Epenhuijsen and Roberta Mayclair for expert library assistance. We thank F. Hoffmann-La Roche Ltd for supplying lutein, zeaxanthin and β-cryptoxanthin standards for use in the analysis of the carotenoid pigments. This research was supported by funding from the New Zealand Foundation for Research Science and Technology.
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