Optics & Laser Technology 43 (2011) 282–294

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Optics & Laser Technology

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journal homepage: www.elsevier.com/locate/optlastec

Plants and colour: Flowers and pollination

Renee Miller, Simon J. Owens �, Bjørn Rørslett

Directorate, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, United Kingdom

a r t i c l e i n f o

Available online 10 March 2009

Keywords:

Flowers

Pigments

Pollination

92/$ - see front matter Crown Copyright & 2

016/j.optlastec.2008.12.018

esponding author.

ail address: s.owens@kew.org (S.J. Owens).

: http://www.naturfotograf.com/UV_flowers_

a b s t r a c t

While there is a range of colours found in plants the predominant colour is green. Pigments in plants

have several roles e.g. photosynthesis and signalling. If colour is to be used as a signal then it must stand

out from green. However, one should be aware that there are also coloured compounds where we have

not yet fully investigated the role of colour in their functions—they may have roles in, for example,

defence or heat exchange.

In this paper, we will describe the basic chemistry of the major pigments found in plants and

especially floral pigments. We will then discuss their locations in parts of the flower (such as sepals,

petals, pollen and nectar), the cells in which they are found and their sub-cellular locations.

Floral pigments have a large role to play in pollination of flowers by animals. They can and are

modified in many ways during the development of flowers in nature, for example, at emergence and

post-pollination. There are a range of biochemical mechanisms of colour change both within flowers

and in isolated pigments. Some of the factors influencing colour are temperature, co-pigments, pH,

metals, sugars, anthocyanin stacking and cell shape.

There is a renewed interest in analysing floral pigments and how they are modified partly because of

advances in recombinant DNA technologies, but also because of pollinators and their significance to

biodiversity and for evolutionary studies. There is continued strong interest from the horticultural

industry for the introduction of new colours e.g. the blue rose and for the exploitation of natural dyes.

Funding in this area may impact future research in a potentially beneficial way but it must not deflect us

from science-based conservation.

Crown Copyright & 2009 Published by Elsevier Ltd. All rights reserved.

1. Introduction

The aerial parts of higher plants are mainly concerned withmaking food (photosynthesis), and are thus, for the most part, thecolour green. In order to stand out from the predominant greencolours of leaves and stems (Fig. 1), plants have flowers (andfruits) of many and sometimes multiple colours (Figs. 2 and 3).Some of the molecules produced are pigments creating visualsignals and in flowers these coloured signals are often used toattract animal pollinators [1]. Thus, colour is more often observedin animal rather than wind-pollinated species. What a pollinator‘‘sees’’ will depend upon its visual organs, their acuity and coloursensitivities and the same flower may appear quite different invisible, ultraviolet or infra-red light.

2. The flower

Flowers are composed of several parts—sepals, petals, stamenswith anthers containing pollen, an ovary with a stigma and style

009 Published by Elsevier Ltd. All

list.html (S.J. Owens).

and nectaries. The presence or absence of any one of these floralorgans may again vary considerably. All or any of these parts maybe coloured although not all might be the same colour even in thesame flower.

It is only in flowering plants where, in reproductive organs,photosynthetic pigments are degraded, there is a sacrifice of foodproduction and other molecules are developed as colour signals.These signals are mainly in the flowers and fruits, thus ensuringthat these organs are different from the green background, andconsequently attractive to other organisms.

We have come to expect colour in petals in particular but againthere is variation. In the kangaroo paw flower (Anigozanthos

flavidus D.C.), it is the stamens and particularly the anthers thatstand out (Fig. 4). In tulips (Tulipa armena Boiss.), one can easilydistinguish the black anthers and the green stigma and ovary fromthe background petal colours (Fig. 5). Pollen itself is oftencoloured, for example, that of Achillea millefolium L. is white.Pollen colour may be expressed in the external walls of pollengrains, the exine, which is composed of a complex of compoundscalled sporopollenin (Fig. 6). The exine is built up from materialsdelivered to it during pollen grain development (Fig. 7) andproducts from the nurse tissue, the tapetum, may becomeattached to the surface of the mature pollen grain adding colour.Mature pollen may be white, yellow, blue, red or black but is often

rights reserved.

Fig. 1. Green frog on green leaf.

Fig. 2. Mauve primula flowers with yellow centres.

Fig. 3. Orange Caesalpinia pulcherrima (L.) Sw. (Leguminosae) flowers with yellow

anthers.

Fig. 4. Anigozanthos flavidus DC. (Haemodoraceae; Kangaroo paw) dull flowers

with prominent stamens and anthers.

Fig. 5. Tulipa armena Boiss. flowers with black anthers and light green stigma/

ovary.

R. Miller et al. / Optics & Laser Technology 43 (2011) 282–294 283

species or even genus specific. Colours of the stigma, style andovary appear to be recorded rarely and can vary between species.For example, in Fuchsia denticulata Riuz & Pav., the ovary, style andstigmas are coloured red in similar fashion to the petals while inFuchsia microphylla H.B. & K. all these organs are white while thepetals are red (Fig. 8). In Turraea nilotica Kotschy & Peyr., there is abright green stigma which stands proud of the flower (Fig. 9).

Fig. 6. Scanning electron micrograph of Achillea millefolium L. pollen. Fig. 7. Scanning electron micrograph of the exine layer of Achillea pollen.

R. Miller et al. / Optics & Laser Technology 43 (2011) 282–294284

Hansen et al. (2007) [2] have recently discussed, there are manyplants (67 taxa) known to have coloured nectar (Fig. 10).

Flowers which are wind-pollinated are almost never colourful;fruits where the seeds are dispersed by wind are almost neverbrightly coloured. Pollinating animals use colour as a clue to maketheir choices about flowers to visit. Colour can help with plantspecies selection, flower location and ripeness, and nectar andpollen location within the flower. Thus pigmentation in flowersseems to play a major role in pollination success. In fruit, colourschange as the seeds reach maturity and the fruit ripens, thusensuring that animals will be attracted to mature fruit and sodisperse mature seed.

Fig. 8. Fuchsia denticulata Ruiz & Pav. with red petals and white stamens, ovary,

style and stigma, and F. microphylla H.B. & K., where all floral parts are red

(Oenotheraceae).

3. Pigments

3.1. Pigment chemistry

Pigments in plants are organic molecules that absorb lightenergy and retain it for a few nano/microseconds. They can retainthe light energy due to the presence of multiple carbon atoms thatshare the light energy across alternating double bonds. In this waythey produce colour (see Grotewold 2006 for review [3]).

There are many roles played by pigments in plants:

�

Several are involved in the conversion of light energy tochemical energy. The chlorophylls are the main players here.

�

Signalling, either for the plant to detect aspects of itsenvironment, or enabling other organisms such as animals tobe aware of plants. These pigments may be present in small orlarge quantities. � Molecules that may show colour (e.g., some of the mitochon-

drial cytochromes, in this case due to the presence of iron) butfor which we have no clear idea of how their interaction withlight energy can affect plant function.

Fig. 9. Turraea nilotica Kotschy & Peyr. (Meliaceae) with bright green stigma

standing proud of the flowers.Fig. 10. Plant orders containing species with coloured nectar (reproduced by kind

permission from Hansen et al. [2]).

R. Miller et al. / Optics & Laser Technology 43 (2011) 282–294 285

Plant pigments fall into four major categories (see also Table 1)

a.

Tetrapyrroles such as chlorophylls (which contain magne-sium). The chlorophylls are all green and are required forphotosynthesis. They are the most abundant pigments in theworld. Other tetrapyrroles include all cytochromes (includingthe light-sensing pigment cryptochrome), phytochrome (an-other light-sensing pigment), and phycobilins (accessoryphotosynthetic pigments in algae). These pigments are oftenmembrane-bound but some, for example phycobilins, arefound in complexes in cells.

b.

Carotenoids are long-chain lipid-soluble molecules present inall eukaryotic cells. In plants, some carotenoids are involved inphotosynthesis either as accessory photosynthetic pigmentsor as anti-oxidants. These carotenoids are membrane-boundoften within the photosynthetic complexes in the chloroplasts.Other carotenoids form droplets in cells, giving colour mainlyto flowers and fruits.The carotenoids, or tetraterpenoids, are the most abundantpigments after the chlorophylls. They are usually yellow,orange and red in colour. There are 2 groups: the carotenes,for example lycopene, are hydrocarbons, the xanthophylls, forexample lutein, contain oxygen. There are approximately 600known carotenoids but only 10% of these are found in plants.Their colours are influenced by the number of double bondsin the central hydrocarbon chain and the nature of the endgroups. In flower cells they are mostly found in chromoplasts.Daffodils, marigolds, and dandelions are good examples ofcarotenoid-containing flowers.

Unlike anthocyanins and betalains, the carotenoids are essen-tial for plant life. But they also make up much of the pigment inyellow and orange flowers. They can co-exist with the otherpigment groups. The general precursor, lycopene, is colourless,and this is converted to phytoene (pink) and carotene (yellow).Many of the biochemical pathways have been worked out.In tomato plants, one of the initial enzymes exists in 2forms—one in green tissue and the other in the fruit. Inmarigolds, the carotenoid lutein (a xanthophyll) is the mainpigment. In light yellow marigolds, there is reduced expressionof genes for enzymes in the carotenoid biosynthetic pathway.However, the leaves have normal carotenoid levels showingthat the photosynthetic roles of carotenoids are independentlyregulated from those involved in flower colour.Carotenoids are being used as natural colouring agents forfood, generally for yellow and/or orange. Another, a ketocar-otenoid astaxanthin from the blood-red Adonis aestivalis L., iscurrently being considered as a source of anti-oxidants/redcolouring agents from plants (thus avoiding bacterial genesand genetically modified organisms).

c.

Flavonoids are complex, phenolic, water-soluble moleculesthat occur in almost all vascular plants. More than 9000 areknown and included in this group are the flavones, flavonols,anthocyanins and isoflavonoids. Flavonoids are present in mostaerial plant parts and are even found in roots. They can absorbultraviolet light, thus conferring some protection for landplants.Anthocyanins are a group of flavonoids that are major sourcesof colour in flowers and fruits. Despite the range of colours

Table 1Overview of plant pigments.

Colours Category Associations/Modifiers Locations in cells/plants Functions

Green, blue-green, red Tetrapyrroles (e.g.,

chlorophylls)

Mg, Fe, Cu Membrane-bound mainly in plastids, but

others found in other cell parts/chlorophyll

found in green tissue, but others found

throughout plant

Photosynthesis (chlorophylls) or

signalling

Yellow, orange & red Carotenoids Some have molecular

oxygen

Either membrane-bound or in droplets/in

all cells; high levels in pollen, petals and

fruit

Accessory pigment for

photosynthesis, protection of

chlorophyll from photo-oxidation,

signalling, storage

Colourless, white, pale

yellow; red, purple,

blue

Flavonoids (flavones

and flavonols;

anthocyanins)

Affected by age and

temperature, also pH,

metals, sugars, organic

acids, co-pigments, shape

of cells, stacking

Accumulate in cell vacuoles; in all cells,

but accumulate in pollen, petals and fruit

Protection from UV damage to

photosynthetic apparatus,

signalling, defence

Red, purple and blue Betalains Unlike flavonoids, they all

contain N

Accumulate in cell vacuoles in

Caryophyllales (no anthocyanins) and

fungi; mainly found in flowers and fruit

Same as anthocyanins? Signalling,

defence?

R. Miller et al. / Optics & Laser Technology 43 (2011) 282–294286

provided by anthocyanins (red, purple, blue), and the numberof pigments found in plant cells, only 17 anthocyanidins (theproto-pigments lacking any sugars, metals) are involved. Inliving cells most anthocyanidins are linked to one or moresugars, giving different colours.These pigments can adopt multiple forms in solution depend-ing on pH and the solvent. In aqueous solutions with pH 3–6,like the plant vacuole, the coloured pigments are often linkedwith co-pigments, for example, non-coloured flavonols, car-otenoids or metals. The anthocyanin molecules can also bebound to organic acids or aromatic acyl groups. In fact, mostflavonoids, including anthocyanins, are modified by covalentlinks to acyl, methyl or glycosyl groups. These modificationsstabilise, add pigmentation and change hues. For example,adding sugars leads to shifts towards the blue. Acylation isthought to help with the stacking of these pigments.The identity of some of the enzymes for biosynthesis ofanthocyanins, transport to the vacuolar membrane and trans-port into the vacuole has been determined. Anthocyaninbiosynthesis has also been very well studied with respect toregulation. Transcription factors are known, and there aregroups of regulator proteins. Light and hormones can activatethe transcription factors.There are also at least 4000 flavonoids that are not anthocya-nins, many of which are colourless, white or pale yellow. Someof these do add to the white and cream colours of flower petals(flavones, flavonols).The flavonoids are synthesized by a complex pathway, startingwith the removal of an amino group mostly from an aminoacid such as phenylalanine (sometimes tyrosine). Flavonoidsare present in most flowering plants and good examples arehydrangeas and petunias.

d.

Betalains are, like flavonoids, complex water-soluble moleculesbut, unlike flavonoids, they are all indoles, contain nitrogenand are derived from the amino acid tyrosine. They arerestricted to plants in the order Carophyllales which includethe beets, amaranths and cacti and to fungi. There are the redand purple betacyanins and yellow betaxanthins. Thosefamilies that contain betalains do not have anthocyanins.

Betalains are much less studied. However, the first enzyme inthe biosynthetic pathway has now been cloned. Again likeanthocyanins, the pigments are stored in the vacuole, mainly asglycosides. Some of the enzymes that glycosylate these pigmentshave been cloned, and the enzymes will also work with flavonoidanthocyanins. Unike most anthocyanins, betalains have the ability

to react with amino acids. We still do not know how theirsynthesis is controlled and how they are transported to and intothe vacuoles.

We are still not sure why anthocyanins are widely distributedbut betalains are not, and why they do not co-exist. Anthocyaninshave even been able replace betalains in some families within themain betalain order Caryophyllaceae. For example, Dianthus,

carnations, have only anthocyanins. Plants that make betalainsdo in fact make some flavonoids (e.g. flavonols), but they do notmake anthocyanins. Perhaps they have simply lost one enzyme inevolution? Or perhaps betalains could have arisen more recentlyin a small group of angiosperms (and fungi)? Even stranger is thepresence of betalains in fungi (fungal enzymes will function inplants). To date, there is no knowledge of whether either group ofpigments has an advantage over the other.

The pigments found in flowers are mostly those also found inother parts of the plant, where they have different effects. Forexample, carotenoids are found in all cells and can protect againsthigh-energy oxygen species (of molecules). Anthocyanins arefound in all plant cells exposed to light and are part of the defenceagainst ultraviolet light and other harmful influences. Betacyaninsare also thought to be functionally defensive.

3.2. Pigments in petals

In flowers, pigments are thought to be accumulated so as toattract other organisms, either due to visual colour or to the heatthese pigments produce. The final colour of the flower dependsupon the particular pigments synthesized, the concentrations ofthese pigments, and many other physical and chemical cell factorssuch as temperature and pH.

For example, different flowers synthesize different carotenoids,and thus appear red, orange or yellow. The flowers become moreintense–darker—as the amount of pigment increases. Carotenoids,besides having colour in the visible spectrum, are also able toreflect ultraviolet light. This can produce shine—again as pigmentlevels rise, there will be more shine.

Although some flowers have only carotenoids, most contain atleast one other group of pigments. The same flower can containcarotenoids and flavonoids. There are anthocyanin flavonoids thatare red (pelargonidin), violet (cyanidin), or blue (delphinidin), andother flavonoids that are white, ivory or light yellow. Theconcentration of these pigments can increase as the flowerdevelops, and some of the lighter pigments may be converted todarker ones during development. Unlike carotenoids, flavonoidstend to absorb ultraviolet light, producing darker areas.

R. Miller et al. / Optics & Laser Technology 43 (2011) 282–294 287

The third group of pigments, the betalains, are red/purple.They are not present in most plants, but when they are present,anthocyanins are not. However, they can co-exist with carote-noids.

Kishimoto et al.[4] have provided a good example of howpigment compositions affect flower colour. They looked at thepigment composition of nine species of plants in the samefamily—Compositae (Asteraceae). Cultivars were analysed forcarotenoid and anthocyanin composition. Orange and yellowflowers were seen to contain yellowish carotenoids, but therewere seen to be three possibilities which allow flowers to be moreclearly orange. These included anthocyanins at higher levels thanin the yellow flowers (in Chrysanthemum morifolium Ramat.,Gerbera jamesonii Bolus, and Zinnia elegans Jacq.) or carotenoids athigher levels than in the yellow flowers (in Helianthus annuus L.,Tagetes erecta L. and Tagetes patula L.) or have more of the reddishcarotenoids than in the yellow flowers (in Calendula officinalis L.,Gazania spp and Osteospermum ecklonis (DC.) Norl.).

Colour in petals is definitely seen to be the most importantcolour signal for pollinators. In 1984, Rebelo and Siegfried [5]reported their studies of 341 of the 426 Erica species found in theSouthwest Cape, South Africa. Colour polymorphism was seen inaround 38% of the species. The polymorphs were the species withlarge altitude ranges and long flowering periods, perhaps usingdifferent means of pollination. The shorter pink, purple and whiteflowers may be insect pollinated, the longer red, orange, yellowand green flowers bird pollinated.

Internicola et al. [6] have shown the importance of petal colourto bumblebees, even when there are deceptive plants intermixedwith real flowers. The bumblebees learned to avoid deceptiveplants but this happened more rapidly when the two plant typeswere different colours. Goulson et al. [7] have investigated the roleof colour in decision making by bumblebees and honeybees.When supplied with nasturtium (Tropaeolum majus L.) yellow andorange flowers, the bees choose yellow as this is part of the peaksensitivity of the bee sensitivity to incident light. (It appears to beonly humankind that ensures survival of the orange varieties.) Inreality, there would be an equal award of nectar in the orangeflowers but bees do not take advantage of this. Both bumblebeesand honeybees do not approach these flowers randomly—theyapproach more yellow flowers and also unblemished yellowflowers because blemished flowers might be older and might lacknectar. However, the two types of bees feed differently—thebumblebees can feed on intact flowers while honeybees requireflowers where holes have been made by other insects. Short-rangeacuity assists the honeybees in finding the flowers with holes.

A single gene shift may be all that is needed to alter pollinators.Hoballah et al. [8] have reported on a single change in a gene fortranscription factor anthocyanin2 as the major determinant of thecolour differences between Petunia integrifolia Schinz and Thell.and P. axillaris (Lam.) Britton, Sterns and Poggenb. In the wild,P. axillaris has been found to have had 5 independent instancesof loss of function of this gene. This single gene shift in petalcolour has also caused a major shift in pollination biology, thusshowing that adaptation to pollinators may require only a fewgene changes. Chittka and Raine [9] have provided an excellentreview of how pollinators choose between flowers and how thismight affect plant evolution, and how both visual and olfactoryinformation processing is involved on the part of the pollinator.

However, one must proceed with caution when assumingthat reduced reproduction in a variant flower colour is due todiscrimination between pollinators. Levin and Brack [10] showedthat pollinators did not discriminate between red and white Phlox

and it was just that the white variant had reduced fitness. AlsoTremblay and Ackerman (2007) [11] published results for atropical orchid that exhibits polymorphism with respect to colour

and in which the bicoloured petal form dominates. Colour is notlinked with male or female reproductive success among popula-tions over time (20 months), so it is strange that one formdominates.

The colour palette of flowers varies with the season. We havelong known that there are more blue and purple flowers in earlysummer and more yellow flowers in late summer. This may be dueto the need for yellow flowers to stand out from the denser greenvegetation of late summer. There are indeed some yellow springflowers, but many do not need pollinating by animals. Dandelions(Taraxacum officinale (L.) Weber) are apomictic, daffodils (Narcis-

sus species) propagate predominantly by bulbs, and basswoodpollen (Tilia species) is spread by wind.

Those yellow spring flowers which require pollination byanimals may have yellow flavonoids which absorb ultraviolet andwould appear as dark holes to bees in the bright spring sunlight,whilst autumn flowers have carotenoids which fluoresce andappear brighter in the shorter days of autumn. The blue/purplespring flowers containing flavonoids would also absorb.

Petal colours often change over petal lifespan, for example, thefrequent light colours when flowers are first emerging oftendarken later, and sometimes this is associated with pollination.Viola cornuta L. changes from white to purple after pollination.Light-dependent anthocyanin synthesis occurs and this is in-dependent of the normal senescence of petals.

Even species age seems to be important for flower colours.Streisfield and Rausher [12] have recently shown evidence fordecreased constraint by regulatory genes, and therefore morerapid change, as a major factor operating during the evolution ofplants and their floral pigments. Rausher [13] has put forward thetheory that during evolution major transitions from blue to redand complete loss of pigments may be occurring simply due to theage of the lineage. Species may show changes in anthocyaninpathways late in their evolutionary history, rendering thatparticular lineage closer to extinction.

3.3. Pigments and pollinators

There are at least 20,000 species of bees and they are one of thelargest groups of pollinators [9]. Bee-pollinated flowers aregenerally blue or yellow but rarely red since it is believed thatred appears black to bees. Bees do not see the primary colours wedo, but they are able to see in ultraviolet light. In UV, they seethe green world as shades of grey and flowers with brilliantultraviolet reflection (due to carotenoids) and strong black or‘cyan’ ultraviolet absorption ‘holes’ (due to flavonoids) against thegrey foliage. An interesting example is the marsh marigold flower.This flower has a corolla with yellow carotenoids at the edge and ayellowish flavonoid in the centre. To us in visible light the flowerlooks yellow throughout. However, in ultraviolet light, which beescan detect, there is a shiny yellow edge and a duller, darker centre.Many flowers have ‘honey guides’ to lead the bees to the nectar,thus ensuring that they accumulate pollen for transfer to anotherflower. Some of these honey guides can only be seen in ultravioletlight (Figs. 11–13).

Beetle-pollinated flowers are usually white or dull and it isthought that beetles have poor visual senses. Fly-pollinatedflowers tend to be dull red or brown in colour and perhapssimulate the colour of meat. Examples are found in Stapelias, thecarrion flowers from South Africa. These flowers also smell ofrotting meat which provides, perhaps, more of an attraction.Moth-pollinated flowers are white or yellow which ensures thatthey stand out in dwindling light, when the moths are active.Butterflies can detect more colours than bees, and thus visit awide range of coloured flowers even red ones. Hummingbirds in

Fig. 11. (a and b) Yellow sorrel (Oxalis fontana Bunge) in visible and UV light.Fig. 12. (a and b) Epilobium angustifolium L. in visible and UV light.

R. Miller et al. / Optics & Laser Technology 43 (2011) 282–294288

the Americas and sunbirds in Africa use vision principally infinding flowers. They tend to go to bright red or yellow flowerswith little scent, large sizes, and copious quantities of nectar. Bat-pollinated flowers are dull in colour but large in size or flowers arefound in large inflorescences (Fig. 14).

It is important to realise that more than one group of animalsmay pollinate a particular plant species, and there may becompetition between groups for nectar, for example, betweenbeetles and bees. Within the orchid family all groups ofpollinators may be attracted. Orchids are probably the mosthighly evolved plant family with respect to attracting pollinators.For example, the bee orchid, Ophris apifera Huds., ‘imitates’ thecolours and patterns of its principal pollinator. There is a mutantalbino form of this flower, but there is little known about theefficacy or otherwise of its pollination (Fig. 15a and b).

There is a range of pollinators of flowering plants, and eachpollinator has its preferences. There are no perfect correlationsbetween floral pigments and the spectra pollinators can detect.But perhaps we should not expect this. The different varieties ofthe same flower species and slight differences in ecosystems canaffect the pollinator: flower relationship. There are also otherfactors besides colour involved in the visual match betweenpollinator and flower—e.g., contrast between flower and leaves,corolla and pollen contrast, symmetry; and there are also scentand tactile clues. It is also important to remember that flavonoids,carotenoids and betalains may and do have multiple functions inflower petals, some unrelated pollination. On the other hand,colour does seem to be a priority for attracting most pollinators,

and maximising pollinator attraction is one of the principalfunctions of flowers.

3.4. Pigments and pollen

Coloured compounds in pollen may have more than oneattribute. For example, saffron pollen has 3 different ‘apocarote-noids’ which have been implicated in food storage, bitter taste,aroma and colour. Flavonoids can account for 2–5% of pollen dryweight, and maize pollen can have up to 10 different flavonoidslinked with sugars. The roles of pollen colour in pollination havebeen investigated. Lunau [14] looked at 67 plant species from 28families and measured the spectral reflectance of the pollengrains. The majority of the species (75%) belonged to one of twocategories: ‘human yellow’ pollen with strong reflection in greenand red but low reflection in UV and blue, and ‘human whitish’pollen with strong reflection in green and red as well as blue andUV. Bees could distinguish between these. However, other factorsneed to be considered, such as whether the pollen is hidden oreasily seen and whether it matches or contrasts with the corolla.Jorgensen et al. [15] have observed dimorphism in pollen colourand variety in pollen colour frequencies in two subspecies ofNigella degenii Vierhapper. Pollinators did show preferences butthis varied with dates and locations. In this case, pollen donorplants with darker pollen had a reproductive advantage but thiswas not due to more fertile pollen. Heuschen et al. [16] studiedthe innate colour preferences of bumblebees. They found that the

Fig. 13. (a and b) Angelica sylvestris L. in visible and UV light.

Fig. 14. Flower of Mucuna macropoda Baker f., with translucent-white stigma and

style, being pollinated by a bat.

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bumblebees prefer dummy flowers with corolla centres similar incolour to the pollen (the centres are mimicking the pollen). Whenthey investigated 162 plant species, they found that the innercolours of most flowers appear to be very similar to the pollen,and the colour was much less diverse than the peripheral colours.

3.5. Coloured nectar

Nectar also can be coloured (see Table 2). Hansen et al. [2]looked at the distribution, ecology, and evolution of this‘enigmatic floral trait’. Although coloured nectar has been knownsince 1785, it has been little studied. The authors identified67 taxa worldwide, with colours yellow, red, brown, black, greenand blue. The intensity of colour was seen to vary, even within onetaxon, and also the contrast with petal colour varies. The authorsbelieve that coloured nectar has evolved at least 15 times inangiosperms.

The authors provide evidence that coloured nectar is mainlyassociated with three principal factors:

Pollination by vertebrates—either bird or bat;Species insularity either on islands or from isolated regions oflarger land masses;Species growing at higher altitudes.

The coloured nectar can be useful as an ‘honest signal’ topollinators, leading to high pollination efficiency; or in order to

deter nectar thieves and/or inefficient pollinators, or as anti-microbials. This has still to be investigated.

3.6. Pigment location and stacking in cells

In Caesalpinia pulcherrima (L.) Sw., a transverse section of thestyle shows many deposits in the cell vacuoles, not only in theepidermal cells but even in the central tissues. These centraltissues are not exposed to light, but may change as pollen tubesgrow through them (Fig. 16).

The site of pigment storage in cells is yet another factoraffecting colour in flowers. Pigments are often stacked orpackaged [1]. The packaging of chlorophylls and carotenoids inphotosynthetic tissues has been studied widely but some-what less is known about floral pigments. It is known thatcarotenoids are usually separated from flavonoids as the formerare mostly in plastids and the latter are found in cell vacuoles. Insome species the flavonoid anthocyanins are stored in specificbodies in the vacuoles (anthocyanoplasts), and these bodiesmay or may not be membrane bound. This is true in carnationswhere membraneless proteinaceous matrixes are presentwhich are thought to ‘trap’ anthocyanins. Other evidence forcolour change links to packaging includes the rose cultivarRhapsody in Blue, where changes from red purple to bluishpurple with age are linked with more packaging structures foranthocyanins observed in the vacuoles. In Lisianthus, where there

Fig. 15. (a and b) Oprhys apifera Huds. in its coloured and white forms.

Table 2Nectar colour (selected from Table 1, Hansen et al. [2]).

Species Flower size (mm) Flower colour Nectar colour Pollinator

Asphodelaceae: Aloe alooides (Bolus) van Druten 9 Yellow Clear-red Bird, insect

Fabaceae: Calliandra calothyrsus Meisn. 6–8, 40–60 Green-red Yellow Mammal, insect, bird

Melianthaceaae: Melianthus elongatus Wijnands 15–22�8 Green Black Bird

Malvaceae: Dombeya kefaensis Friis and Bidgood 13–17 White-pale pink Red Unknown

Malvaceae: Trochetia blackburniana Bojer 15–25�18–25 Pale-pink to red Clear-amber Bird, lizard, insect

Bromeliaceae: Puya alpestris Gay 50 Blue Pale-pink-blue Bird, insect

Proteaceae: Banksia grossa A.S. George 34–45 Brown Yellow-green Insect, mammal, bird?

Solanaceae: Capsicum baccatum L. 3.5–7 White-cream Clear-yellow Unknown

Fig. 16. Transverse section through a style of Caesalpinia pulcherrima (L.) Sw.

R. Miller et al. / Optics & Laser Technology 43 (2011) 282–294290

is seen to be more packaging of pigments, there is a darker purplecolour (base of petals as compared to outer zones). In maize(Zea mays L.), the amount of light can affect the amount ofpackaging [3].

3.7. Mechanisms of pigment colour change

3.7.1. Temperature

Temperature affects colour. For example, anthocyanin accu-mulation in ornamental asters varies with temperature. They havemore pigments at lower temperatures but the colours may bealtered with metals [17]. Stiles et al. [18] have shown tempera-ture-dependent anthocyanin production in flowers of the weedyperennial Plantago lanceolata L. Here, also there are darker flowersat cooler temperatures. Seventeen different anthocyanins weredetected, and most increased at cooler temperatures. There aredifferent genotypes, and those which show the greater responsesto temperatures also seem to have greater plasticity in colourproduction.

3.7.2. Vacuolar pH

Even in roses which have been cut, there is a turn from red toblue as the pH of the vacuole changes and this occurs well beforesenescence sets in [19]. In other flowers, pigments fade or darken(Fig. 17). These time-dependent changes can occur with any groupof pigments, but are most common with anthocyanins [3].

Soil pH affects the colour of hydrangea flowers due to changesin metal uptake from the soil. Pink hydrangeas can be convertedto blue by the addition of acidifiers that enable the uptakeof aluminium. Aluminium is sequestered into the flower cellvacuoles, because it is toxic, and there it turns the pinkanthocyanin blue. A more rapid way of achieving this colour

Fig. 17. Polystachya laxiflora Lindl. (Orchidaceae)-petals are green, white or brown,

depending on age.

R. Miller et al. / Optics & Laser Technology 43 (2011) 282–294 291

change is to apply aluminium in solution directly onto leavesor petals. When aluminium is mixed with a synthetic model ofan anthocyanin in solution, complexes occur. Over different pHranges (2–5), there can be seen colour amplification, colourchange or even loss of colour. As in the hydrangea flower, theanthocyanin passes from a red flavylium form to deep purplequinonoidal form when coordination with Al3+ occurs [20].

Many pigments are stored in vacuoles, and vacuolar pH haslarge effects on pigments and colours. Generally the vacuole ismore acidic than the cell cytoplasm. In Petunia, the more acidicthe vacuolar pH, the more red the flowers will be. A mutant thatcannot acidify has a shift towards the blue. In Ipomea, the bluepigments in the vacuole require a more alkaline environment.There is a protein, believed to be involved in a Na/K pump andlocalised on the vacuole membrane, which can adjust the pH ofthe vacuolar contents [3].

When Quintana et al. [21] investigated the causes of colourvariation in violet, lilac and red hybrids of Anagallis monelli L.(Primulaceae), they found that there were indeed differences inthe pigments found in the different colour flowers. Upper andlower petal epidermal cells differed in colour and pigments, butalso the pHs of the upper and lower epidermis varied in each case.The pH was seen to be a major determinant of colour.

3.7.3. Metals

Metals affect pigment structure and thus petal colour. Peoplehave been using floral pigments for a long time, and it wasestablished early on that ‘mordanting’ was needed in order tostabilise and/or alter colours. Traditionally, according to Cannonand Cannon [22] urine, wood ash, plant galls, tannins, and crabapple juice have been used. The best mordants are iron (nails),alum, potassium dichromate, copper sulphate, and ferroussulphate. For example, French marigold (Tagetes patula L.) flowersare light yellow naturally, with alum they become golden yellow,chrome gives dark orange, and copper gives brownish tones. Thisis due to alterations in flavonoids related to quercetin. Africamarigold (Tagetes erecta L.), which can provide a brilliant orangeon wool or silk requires tin as a mordant. This has a quercetin-likeflavonoid and also the carotenoid lutein.

More recently, Shoji et al. [23] have investigated the bluecolour development in the bottom of the perianth of the tulip cv.

Murasakizuisho. The whole tulip is purple but the bottom isblue. Protoplasts were prepared from both the blue and purpleregions of the flower. Flavonoid composition, vacuolar pH andmetal element contents were determined. It was found that theanthocyanin and flavonol compositions of the different protoplasttypes were the same and the pH differed little. However, the Fe3+content was 25 times higher in blue protoplasts than in purpleones. If Fe3+ was added to a solution of the purple pigments thenthey turned blue.

Cornflowers and Himalayan poppies are both blue due to thepresence of normally red anthocyanins that are complexed withmagnesium, and thus turn blue. Nissan-Levi et al. [17] studied therole of magnesium in increasing colour in the flowers of severalornamental plants. Adding magnesium led to higher anthocyanincontent, even at high temperatures, in Anigozanthos with redflowers, Limonium with blue bracts, Gypsophila with pink flowersand Aconitum with blue flowers. Thus different plants, withdifferent anthocyanins, could have increased colour (15–70%)with increased magnesium. These effects were even stronger athigher temperatures and were effective even in detached flowerbuds. The authors hypothesise that magnesium can stabilise all ofthe different pigments involved.

3.7.4. Sugars

Many pigments are linked in cell vacuoles to sugars. Bowles etal. [24] have reviewed the evidence for enzymes involved intransferring sugar residues to small molecules such as pigmentsin plant cells. Glycosylation can give plasticity to these molecules.Mostly glucose is added, although other monosaccharides can alsobe used. The sugars link to –OH, –COOH, –NH2, –SH and –C–Cgroups. Addition of sugars generally makes the pigment mole-cules more water-soluble and more stable and the final step in thesynthesis of pigments is usually glycosylation. Sugars are thoughtto be added in the cytoplasm of cells but then there are carriersthat transport the products across the tonoplast membrane, usinga glutathione pump, into the vacuole, where the glycosylatedcompounds can accumulate. The enzymes that carry out theglycosylations are ubiquitous and generally not too specific.

With respect to flavonoids (flavonols and anthocyanins) andbetalains, sugar addition stabilises the molecules and removalof the sugar usually leads to degradation and the formationof breakdown products. When maize mutants have deficientglycosylated flavonoid synthesis the pollen is sterile because afunctional pollen tube is not produced. These glycosylatedflavonoids are probably associated with pollen membranes.

The precursor molecules for anthocyanins are called antho-cyanidins. These are converted to anthocyanins (blue, red, andpurple pigments) mainly due to the addition of sugars. The 3-O-glucosides of pelargonidin, cyanidin and delphinidin create thebrick-red, red, and blue colours of pelargoniums, petunias anddelphiniums, respectively. After this step, other modifications canalso occur—further glycosylations, methylations, acylations, addi-tion of metal ions, vacuolar pH changes, and the presence of co-pigments such as flavonols and flavones.

Flavonols and betalains can also be glycosylated, and at leastsome specific betanidin glycotransferase enzymes have beenpartially purified. One has been cloned—it can also work onflavonoids. There must be a communality of structures betweenbetalains and flavonoids which allow enzymes to work.

Saito et al. [25] have determined that glycosylations, varying intime and space, determine the presence of colour or non-colour ina white-centred petunia cultivar. The white sites have quercetinwith 7-O and 30-O glucosides, while the coloured sites do notpossess these. In boundary areas cells have variable colourdensities.

R. Miller et al. / Optics & Laser Technology 43 (2011) 282–294292

There are also co-pigments involved in petal colour. These arecolourless, but affect the brightness and brilliance of colours.Many are flavones and flavonols, but organic acids also can playthis role. Toyama-Kato et al. [26] have used designed acylquinicacid derivatives in order to achieve the blue colour of hydrangeasin an aqueous solution. Their supramolecular metal-complexpigment contained the flavonoid delphinidin 3-glucoside, plus5-O- caffeoylquinic acid and 5-O-p-couramoylquinic acids as co-pigments, as well as Al3+. They provide evidence that the 1-COON,1-OH and 5-O-ester of quinic acid are required for blue colour.

3.7.5. Conical cells/cell shape

Much petal colour is localised in the epidermal cells, and, asQuintana et al. [21] have noted, there may be significantdifferences in pigments between the upper and lower epidermalcells [1]. Epidermal cells also differ in shape, e.g., conical, flat orpointed (Fig. 18). In 1994, Noda et al. reported on how the shapesof epidermal cells can affect flower colour. Conical cells increasethe proportion of incident light that enters epidermal cells,enhancing light absorption by the pigments and thus the intensityof colour.

A mutant Antirrhinum (snapdragon) called ‘mixta’ leads to lossof the conical form of the inner epidermal cells and a flat cell formis the result which, in turn, leads to a loss of intensity of reflected

Fig. 18. Scanning electron micrograph of the cone cells of the petals of Stongylodon

macrobotrys A. Gray.

Table 3Some genes recently linked with flower colour.

Plant Locus/Gene

Petunia integrifolia (Hook.) Schinz and Thell. and

Petunia axillaris (Lam.) B.S.P

Transcription factor anthoc

Soybean W1

Snapdragon Myb transcription factor m

Snapdragon AmMYBML2, R2R3 myb tra

Petunia Phmyb 1, R2R3 myb trancri

Arabidopsis AtMYB16, R2R3 myb transc

Lotus japonicus (Regel) K. Larsen CrtW gene from marine bac

Rose F 30 ,50H (flavonoid hydroxy

overexpression of iris DFR (

reductase) gene, downregul

DFR gene

colour (Table 3). As a result of the mixta mutation, flowers arepink and not the usual magenta. Bees are known to discriminateagainst white mutant flowers as these appear to be similar tothe green leaves. They also, initially, show a bias against mixtaflowers. However, the bees do learn, as colour is mainly anadvertisement not an innate attractant for experienced pollinators[27]. Conical cells enhance colour saturation but they mayalso provide tactile clues, affect petal reflexing, increase petaltemperature and affect gaseous emissions. Lee [1] has recentlyreviewed some of the evidence about the effects of epidermal cellshape on flower (and leaf) function.

Conical cells are widely distributed in petal epidermis, andMartin et al. [28] have suggested that their presence or absenceis linked with nectar and/or pollen gatherers. There also havebeen multiple times that conical-shaped cells have been lost bymutation. For example, in the genus Solanum, most species haveconical cells but Solanum dulcamara L. (woody nightshade) has flatcells. This can lead to petal reflexing, and thus allow for differentpollinators.

Noda et al. [29] reported cloning of the MIXTA gene and it wasdescribed as a myb-type gene involved with transcription controlof cell shape. In 2007, the same group Baumann et al. [30]compared MIXTA with other myb transcription factors affectingcell (and petal) shape. MIXTA is a myb transcription factor genefound to control cell shape in Antirrhinum majus L., but there is arelated gene, AmMYBML2 (a R2R3 myb factor) also expressed inthis species. AmMYBML2 is expressed at low levels in the corollamaximally just when flowers open, which is later than MIXTA.

PhMYB1 is a mixta-related gene from Petunia hybrida Vilm. It isexpressed in both inner and outer epidermes of the corolla.AtMYB16 is a mixta-related gene from Arabidopsis thaliana (L.)Heynh. AtMYB16 is expressed most highly in petals.

The phmyb1 mutant in Petunia has a flower colour and formwhich is different from the wild-type plant. The flowers are lighterand the petals have a different form since they are reflexed, curvedbackwards and downwards. The Petunia mutant is unstable, andthus revertant sectors appear on the petals. The revertant sectorsare darker in colour and have no reflexing. The shape of cells isalso affected. In wild-type Petunia, the petal inner and outerepidermal cells are conical/papillate. In the mutant, the innerepidermal cells have small cones and the outer epidermal cells areflat. Mutant cells occupy a larger surface area than wild-type cells,and thus the petals reflex.

Mixta mutants also show some reflexing, and an unstablemixta mutant showed a mixture of mutant and wild-typephenotypes. In A. majus and several other species of Antirrhinum

Pigment/other effect

yanin 2 Mauve to light pink

Need to form delphinidin-3-glycoside, colour shift

from purple to white

ixta Change in conical cell shape, flowers change from

magenta to pink

sncription factor Expressed after mixta, changes shapes of cells

ption factor Change in conical cell shape, flower colour and

shape of flower—revertant sectors are seen

ription factor Helps to establish flower shape

terium inserted Beta-carotene ketolase synthesized, pink

carotenoids change to red, flowers from light

yellow to orange

lase) from viola,

dihydroflavonol 4-

ation of endogenous

Blue colour (95% delphinidin produced)

R. Miller et al. / Optics & Laser Technology 43 (2011) 282–294 293

where MIXTA is more expressed, the petal epidermal cells aremore conical and there is less reflexing of petals.

When all the genes were expressed in tobacco, it wasdetermined that the Petunia PhMYB1, the Arabidopsis AtMYB16,and the Antirrhinum AmMYBML2 genes share homologousfunctions and they are separate from MIXTA. Baumann et al.[30] conclude that MIXTA is thought to initiate the changes in cellgrowth direction and in its absence cells are flat. The other 3 genesfunction later to help establish a complete flower shape. They allcan affect petal colour and thus pollination effectiveness.

3.8. Genes and pigments

There is at present a great deal of research into pigments, thegenes required for their synthesis, and how these may be modified(Table 3). Within the last year:

�

Zabala and Vodkin [31] have identified a DNA rearrangementresulting in small tandem repeats in a gene from white ascompared with purple soybeans. This W1 gene locus isrequired for flower pigmentation. It is needed to form theanthocyanin delphinidin-3-glucoside. One gene at this site ispresent in a single copy and is expressed at low levels in alltissues including the flower but its expression is sufficient toaccount for all the delphinidin-based anthocyanins andproanthocyanins in the purple soybean flower. Thus, a genepresent in small numbers and expressed at low levels can bestudied. � Cunningham and Gantt [32] have produced a portfolio of

plasmids in E.coli, using Adonis aestivalis L. cDNA, whichaccount for 9 different enzymes of carotenoid and isoprenoidsynthesis. The enzymatic activity of the gene products hasbeen verified. Using this portfolio, more can be learnt aboutcarotenoid gene expression.

� Suzuki et al. [33] have overexpressed one carotenoid gene from

the marine bacteria Agrobacterium aurantiacum (encodingbeta-carotene ketolase) in Lotus japonicus (Regel) K. Larsen,thus modifying the carotenoid biosynthetic pathway in thisplant. As a result, pink carotenoids were changed to red ones inlarger quantities and flower petals changed from light yellowto deep yellow or orange. Novel carotenoids were synthesizedwhich can be studied further.

3.8.1. Genetic modification (GM)

Certainly the most widely publicised recent research effortsinto flower pigments are those associated with GM flowers.Florigene (now owned by the Japanese company Suntory)announced in 2004 that, after 20 years research, it had produceda blue rose. Blue roses have long been produced by putting whiteroses into dyes but roses naturally lack the enzymes which wouldallow for blue colour production. In 1991, Calgene isolated thegene responsible for blue colour in Petunia. In 1993, Calgenemerged with Florigene, and since then many genetically modifiedornamental flowers have been produced. These include long-lifeand disease-resistant carnations, new morphologies of gerberas,and new colours for carnations, roses and chrysanthemums.Florigene was bought by Suntory in 2003.

Chandler and Tanaka (2007) [34] have reviewed the methodsused to genetically modify plants and flowers especially to alterflower colour. Manipulation of the anthocyanin biosynthesispathway in carnations has led to the production of novel-colouredflowers (the Moon series), and this has led to GM flowers beingsold in Australia, Japan and the US. The authors deal with the needto adhere to intellectual property rights, regulations concerningthe growth of GM plants and they claim environmental concerns

are unwarranted. Katsumoto et al. [35] have published theevidence of how a rose was engineered to be blue. The processis not simple since genes for flavonoid enzymes from Viola and Iris

needed to be introduced into the rose and a rose endogenous genehad to be downregulated. Thus, the delphinidin flavonoid pigmentaccumulated and this trait was inherited. The blue rose was due tobe introduced in 2008.

Public reaction to the blue rose has been discussed in AmyStewart’s recent [36] book, Flower Confidential, and on the website of GM Watch. Amounts differ, but something like $20 billionwere spent in 2006 in the US on ornamental plants. So far,the only really successful GM ornamental plants are Florigene(Suntory)’s carnations, of which 75 million have been sold in thelast 10 years in Australia, Japan and the US as ‘Moondust’,‘Moonshadow’, ‘Moonlite’, and ‘Moonshade’. Blue roses are beingproduced using the same technologies of gene insertion, down-regulation and overexpression, and 90% of all the pigment inthe blue rose is the introduced blue pigment delphinidin. Thetechnology that has been used has become known due to the needfor patenting and the granting of Australian legal permission togrow the rose in greenhouses.

Grotewold [3] has reviewed most of the recent literatureconcerning the genetics and biochemistry of floral pigments.Some of his conclusions are:

�

Historically there has been a great deal of study of theanthocyanins. We are now beginning to study the otherpigments in the same detail. � Biotechnology methods are well advanced in studies of

anthocyanins.

� The plants that have been most studied are Petunia, Anti-

rrhinum, Zea mays L. and Arabidopsis. Other species are beinginvestigated with respect to the ecological regulation of colour.

4. Conclusions

We have already accumulated a great deal of knowledge aboutsome of the pigments found in the flowers of higher plants.The diversity of compounds, the plant species and plant organswhere are found, their synthesis and genetic regulation, and theirecological significance are still being studied. We are beginning tounderstand more about the nature of the pigments and howchemical and physical factors such as cell shape and pigmentpackaging can influence colour. There is still much work to bedone but the knowledge of floral pigments and how colour iscommunicated will be useful in understanding interactionsbetween organisms, how organisms evolve, and how we mayutilise natural products.

Copyrights of illustrations

Fig. 1. Simon J. Owens; Figs. 2–9 and 15–18, The Trustees of theRoyal Botanic Gardens, Kew; Figs. 11–13 Bjørn Rørslett; Fig. 14Helen Fortune-Hopkins.

Acknowledgments

Helen Fortune-Hopkins (Mucuna), Tim Fulcher (scanning slides),Madeline Harley (Achillea millefolium L.), Roger Joiner (scanningslides), Riikka Lundahl (scanning slides), Chrissie Prychid (Strongy-

lodon macrobotrys A. Gray).

R. Miller et al. / Optics & Laser Technology 43 (2011) 282–294294

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