VEGETOS: An International Journal of Plant ResearchOnline ISSN: 2229-4473
Print ISSN: 0970-4078

Research Article, Vegetos Vol: 30 Issue: 1

Elucidating Variable Traits of Flower Pigments in Clivian Plants' Species

Achilonu C Conrad* and Maleka F Mathabatha
Department of Genetics, University of the Free State, P.O. BOX 339, Bloemfontein 9300, Free State, Republic of South Africa
Corresponding author : Achilonu C Conrad
Department of Genetics, University of the Free State, P.O. BOX 339, Bloemfontein 9300, Free State, Republic of South Africa
Tel: 27 51 401 9111
E-mail: [email protected]
Received: August 11, 2016 Accepted: December 16, 2016 Published: December 22, 2016
Citation: Conrad AC, Mathabatha MF (2017) Elucidating Variable Traits of Flower Pigments in Clivian Plants’ Species. Vegetos 30:1. doi: 10.5958/2229-4473.2017.00003.9

Abstract

Elucidating Variable Traits of Flower Pigments in Clivian Plants' Species

Colour patterns of flowers are vital commercial characteristics that influence floricultural plants and industries around the globe because of the multiple colour pigmentation produced by flowering plants. Flower colour patterns comprising different sets of colour can be found in many plant species and their cultivars. The varieties of an ornamental cultivated flowering plant like Clivia have also improved significantly in the floricultural industry worldwide. Clivias were recently subjected to a notable deal of retail market among conventional breeders to enhance varieties of variable flower colour traits, thus allowing the idea to understand and explore the origin and development new plant varieties. However, these ideas will be more credible if innovative studies are done on flower modification of pigments. Nonetheless, the review was focused on Clivia biology, flavonoid pigments, and factors affecting anthocyanins accumulation and colour in flowers, anthocyanin biosynthetic structural genes and CHS and ANS gene expression in flowering plants. In addition, the review concentrated on known important species of clivia, specifically on Clivia miniata, and the general studies of flavonoid biosynthetic genes from other plants in regards to flower pigmentation....

Keywords: Clivian plants; Chalcone; Anthocyanidin synthase; Flowering plants

Keywords

Clivian plants; Chalcone; Anthocyanidin synthase; Flowering plants

Introduction

Clivia belongs to the family Amaryllidaceae, a taxonomic group with only six species namely: C. gardenii- Hook [1], C. miniata (Lindl.) – Regel [2], C. caulescens - R.A. Dyer [3], C. mirabilis – Rourke [4], C. nobilis - Lindl. [4] and C. robusta - B.G. Murray et al. [5] (Figure 1). Among these species, C. miniata Regel is the most commonly cultivated species and it is commercially in demand in floriculture market around the globe. For many years, various flowering plants including Clivia have been studied by chemists, botanists and molecular biologists, all with the aim to understand variable traits that influence flower colour [6,7,8,9,10].
Figure 1: Clivia miniata as one of the six species (A) C. miniata var. miniata and (B) C. miniata var. citrina (Clivia Society of South Africa), (C) C. nobulis (Dr.LoukieViljoen), (D) C. gardenii (www.pacificbulbsociety.org), (E) C. caulescens (Clivia Society of South Africa), (F) C. mirabilis (www.davesgarden.com), (G) C. robusta.
The colouration of various flower plants is influenced due to the pigmentation affecting the commercial market of floricultural plants and it is also a key fundamental interest in biology [11,12]. The biological interest of flowers and pigmentation has grown extensively with investigations of flower colour variations using molecular techniques to enhance floriculture market and industry. The mechanism of flower colour pattern is understood to be influenced by biosynthesis of flavonoid pigments. Thus, understanding the diversity of variable colour traits, structural genes involved in anthocyanin biosynthesis in flowering plants will pave the way to manipulate traits of multiple colours which will generate quality plant species.
In this paper, we will be addressing the following; Clivia biology and flavonoid pigments. We went on to discuss factors affecting anthocyanins production and colour formation in flowering plants, anthocyanin biosynthetic structural genes and the CHS and ANS gene expression in flowering plants. Our attention will be on pigmentation as one of the major components in the process of flower development. They are different types of pigments that play key roles in colour formation in flowering plants and they are betalains [13], carotenoids [14,15] and flavonoids are the three important types of pigments responsible for colour in plants. Thus, this review entails understanding the enzymes that catalyse the reactions in anthocyanin biosynthesis, as well as the corresponding structural genes which specifically partake in the central flavonoid pathway that leads to pigment biosynthesis. In addition, we discussed further on the anthocyanin biosynthetic genes associated with gene expression in C. miniata and not the other species; since C. miniata is endemic to Southern Africa [16] and is the most commonly cultivated species in various parts of the world.

Clivia Species

The genus Clivia belongs to a monocot flowering plant family of Amaryllidaceae, which is endemic to southern Africa [16]. The family consists of approximately 59 genera and about 850 species [17]. Clivia shares common features with the other members of the subfamily of the monocot flowering plants known as Amaryllidoideae. These features are green strap-like leaves, three sepals and three petals, all very alike (but the sepals are typically thinner than the petals) and it’s called tepals [18], (Figure 2). The flower morphology is generally bellshaped and a cluster of stalks of nearly equal length from a common centre and an oval surface in the foliage, and also its colour pattern ranges from yellow through orange to red.
Figure 2: Flower anatomy in Clivia miniata.
There are six species currently recognised in the genus Clivia namely, C. nobilis [4], C. miniata [2], C. gardenii [1], C. caulescens [3], C. mirabilis [19] and C. robusta [5], (Figure 1). Amongst these species, Clivia miniata Regel is the most commonly cultivated in various parts of the globe, especially in countries including Australia, China, Belgium, Japan, New Zealand and USA [18,20,21,22]. Furthermore, C. miniata shares common features with other family members of Amaryllidaceae and they are found in South Africa, Mozambique and Swaziland [23].
Clivia miniata regel (1854)
Bush lily, Boslelie (Afrikaans) or Umayime (Zulu) are the common names for Clivia miniata species and it is endemic to South Africa and Swaziland [17]. The Latin word ‘epithet’ miniata means flowers having a red lead-like colour when it was first discovered in its natural habitat [18,20]. However in 1864, E. Regel went on to classify a specimen that was similar to the already known C. nobilis as C. miniata and this genus is the main driving force behind the success of the floricultural industry. In South Africa, populations of C. miniata were found within isolated areas along Kei River and Transkei region; through the Eastern Cape and KwaZulu-Natal Provinces, extending to Mpumalanga and into Swaziland (Figure 3) [21,24,25].
Figure 3: Clivia miniata distribution in South Africa. The orange box indicates the location in South Africa; the light-orange box represents clivia location in Swaziland.
Normally C. miniata grows as a cluster of plants with a height of about 80 cm [18], which include features like a dark green stem, strapshaped leaves that rise from the fleshy underground stem. The base of stems has a compact mass roots which seldom changes above ground level. The shoots arise from the base of the stem and sometimes form clumps if undisturbed. Koopowitz [18,26,27] and the Clivia Society revealed the leaves of a wild C. miniata are narrow (5 cm), smoothedged, strap-like and it hardly grows up to 9 cm wide.
The colours of C. miniata range from cream to sporadic occurrences of pure yellowed-flowered forms, and C. miniata var. miniata described by Aubrey [28] revealed different pastel oranges, bright and dark red colour. The orange-coloured form of C. miniata flowers also shows contrasting cream-yellow shape, revealing slight green traits that may vary in colour (Figure 1a and Figure1b). Its seeds are enveloped in berries containing one or more than 20 seeds in a berry, although 10 seeds per berry are normal in this genus plant (cliviasociety.co.za).

Anthocyanins: A Class of Flavonoids

Anthocyanins are water-soluble vacuolar pigments that may look red, purple, or blue depending on the pH. They are naturally occurring subgroup of flavonoids (a secondary metabolite) which is synthesised via the phenylpropanoid pathway and in addition, other major subgroups include chalcones, flavones, flavonols [29,30]. Moreover, there are so many secondary metabolites with vast functions; flavonoids play a major role in colour formation in plants, and are vital for the protection against damage from UV (ultraviolent) radiation [29,31,32] and in attraction of animal pollinators [33,34]. The secondary metabolite structure of flavonoids is complex than primary metabolites. The secondary metabolic compounds is a member of diverse classes of alkaloids, terpenes and terpenoids, polyisoprene, phenolic compounds, glycosides, tannins, rare amino acids, and amines . Some secondary metabolites are characteristic of a species; others are common to a group of plants [35]. Among these secondary metabolites, anthocyanins represent an important group of phenolic compounds.
In plants, the important group of anthocyanins are the cyanidin glycosides; because of its 50% wide range of pigment composition in fruits [36]. Anthocyanins function as pigments in plants and regulates the red bright colours, purple or blue of the flowers, skin seeds, fruits and some leaves; based on the different concentrations and compositions depending on internal (genetic) or environmental factors [37]. Besides the role of colour formation of plants, anthocyanins also constitute important food beverages for plant products like fruit juices, red wine and grains [38]. Anthocyanins have promising benefits for human health, having numerous studies which revealed that anthocyanins have anti-oxidative and anti-inflammatory [39], anti-carcinogenic [40] and anti-microbial activities [41], and can stop diseases such as cardiovascular diseases [42], diabetes and can enhance vision [43]. In addition, anthocyanins are end-products in the flavonoid pathway, and they are synthesised under the regulation of active enzymes encoded by multiple genes at the transcriptional level [44].
While flavonoids are typically located within the cellular cytosol and vacuole or on the surfaces of different plant organs [45], anthocyanins are found in the vacuoles of different plant cells in the form of glycosides. Mazza et al. [46] discovered close to 400 known anthocyanic glycosides, a form of sugar group. Anthocyanins do not belong to the sugar group (glycone), but the non-sugar group aglycone or genin part of the glycoside. While glycone can consist of a single sugar group (monosaccharide) or several sugar groups (oligosaccharide), a glycone is the compound formed after the glycosyl group on a glycoside is substituted by a hydrogen atom (www.chem.qmul.ac.uk/iupac). The aglycones are non-sugar groups also found in very small amounts in the fresh plant. The aglycon (known as anthocyanidin) is heterocyclic derivative (ring structure compound) derived from α-cromen or α-benzopyran [46]. The most common anthocyanins are pelargonidin, delphinidin, cyanidin, peonidin, petunidin and malvidin. Generally, there are 3-glycosides and 3, 5-diglycosides and the most common being glucose, and also other carbohydrates (rhamnose, xylose, galactose, arabinose, and rutinose).
Biosynthetic pathway of anthocyanins in plants
Anthocyanin biosynthetic pathway is the main branch of the general phenylpropanoid pathway which begins with phenylalanine. In general, the biosynthetic pathway from phenylalanine to anthocyanins can be divided into three stages: beginning steps of the general phenylpropanoid pathway, early steps of the flavonoid pathway and the late steps of the anthocyanin pathway.
The starting steps of the phenylpropanoid pathway comprise of three following steps from phenylalanine through cinnamic acid and coumaric acid to 4-coumaroyl CoA, then catalysed by phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumaroyl CoA: ligase (4CL), respectively (Figure 4). Furthermore to flavonoid biosynthesis, these three steps of the phenylpropanoid pathway also progress to the formation of hydroxycinnamic acid derivatives such as sinapate esters and monolignols. The genes encoding PAL, C4H and 4CL have been characterised from Arabidopsis, four genes have been identified to encode isomers of PAL [47]. The knockout mutant and gene expression experiment involved nitrogen depletion and low temperature conditions revealed PAL1 and PAL2 isomers are involved in the flavonoid pathway in Arabidopsis [48]. Gene expression profile and enzyme properties have shown that 4CL3 appears to be associated with the flavonoid pathway, whereas 4CL1 and 4CL2 are likely involved in the production of hydroxycinnamic acid [49]. Baek et al. [50] measured the flavonoid content of the isolated full-length cDNA of the C4H gene from the Korean black raspberry (Rubus sp.) and found that this gene existed as a single gene.
The early steps of the flavonoid pathway start from 4-coumaryol CoA through chalcone and naringenin to dihydroflavonol. The three steps are catalysed by chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H) respectively, thus, dihydrokaempferol which is characterised by a hydroxyl group at C4 (carbon 4) in the B-ring is formed. The following hydroxylation of dihydrokaempferol at C3 (carbon 3) catalysed by the flavonoid 3’-hydroxylase (F3’H) leads to the production of dihydroquercetin. The genes encoding this enzyme pathway have been biochemically and genetically characterised plants. The knockout mutations of genes led to the lack of production of both anthocyanins and proanthocyanidins in seeds which form transparent testa [29,51]. In petunia flowers, the white marginal picotee pattern revealed the expressed gene encoding chalcone synthase (CHS) distinctively repressed [6], and genes encoding other anthocyanin biosynthetic enzymes were expressed at similar stages in coloured and white tissues.
The late steps of the anthocyanin pathway involve steps from dihydroflavonols via leacoanthocyanidins to anthocyanidins also further modifications of anthocyanidins. The steps from dihydroflavonols to anthocyanidins are continuously catalysed by dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS), also known as leucoanthocyanidin dioxygenase (LDOX). In addition, dihydroflavonol 4-reductase (DFR) and anthocyanindin synthase (ANS) subsequently use dihydroflavonols, a substrate for the synthesis of anthocyanidins [52], and these enzymes are encoded by a single gene respectively. In Arabidopsis (Arabidopsis thaliana), the knockout transparent test (tt) mutants with an altered seed coat colour define most reactions in flavonoid biosynthesis and also involved in (PA) proanthocyanidin biosynthesis [53,54]. The tt genes required for PA biosynthesis include the transcription factors TT8, TT2, and TTG1 [55,56,57]; three genes, TT19,TT12 and AHA10 which take part in transport processes [58,59]. Nonetheless, there are several other structural genes encoding anthocyanin biosynthetic enzymes discussed further below.

Functional/Biological Significance of Anthocyanins in Plants

Pigmentation
Light beam on a wide range of light spectrum can be absorbed by flavonoid compounds [60]. The absorbance the flavonoid compounds is measured via spectrophotometer shifts towards longer wavelengths as the conjugation of the three planar ring structures increases and saturation decreases. In addition, the modified form of anthocyanins is composed of maximum absorbance across the visible spectrum ranging from 500-550 nm [61]. An additional modification by pH effect and interactions with metal ions and co-pigments produce a visual signal that promotes the primary functions of flavonoids in flowers, seeds and fruits being the initiator of pollinators and seed dispersers [62].
Nonetheless, the efficiency of photo-protection by anthocyanins depends on their localization within plant tissue [63,64,65]; and it can be assessed in regards to external filtering when photoprotective pigment(s) function as a screen and/or internal filtering when such pigments compete with light absorption by chlorophyll within a leaf. A notable feature of higher plant leaves shows chlorophyll pigments contribute to light absorption in the red region of the visible spectrum; absorption at shorter wavelengths, specifically in the green to blue region, thus, can be resulted to other principal leaf pigments: carotenoids and flavonoids - including anthocyanins [66,67,68]. Moreover, because of the attribution of plant pigmentation, flower colouration results to great artistic value to humans, so comprehensible biotechnological concept would be innovative to create novel colours in flowers.
Stress protection against ultra-violet light
The ability of flavonoids to absorb ultra-violet (UV) light further shows its role in UV protection. The characteristics of UV-absorbance in the epidermal layers of susceptible tissues have shown significant function as ‘sunscreens’ against harmful UV radiation [69,70]. Studies on Petunia and Arabidopsis have shown that the synthesis of flavonols with higher hydroxylation levels in induced firmly by exposure to UV-B radiation, suggesting a UV stress response [71]. Furthermore, anthocyanins in plants play vital roles which include the red pigments of plants, prevent photo-inhibition and photo-damage via the absorption of extreme solar radiation that would else be absorbed by chloroplast pigment [68,72,73]. Anthocyanin in leaves functions as indirect protection against excess light through their oxy-radical scavenging properties. Subsequently to a mechanical damage to the red and green portions of Pseudowintera colourata leaves, Gould et al. [63] observed that a necrotic lesion and intense anthocyanin band had formed at these injured areas. The anthocyanin level was high due to the accumulation of H2O2 rate that is associated with real-time imaging of the injured palisade mesophyll cells with fluorochromes [74,75].
Structural genes encoding anthocyanin biosynthetic enzymes
Many structural genes encoding anthocyanin biosynthetic enzymes have been cloned and characterised (Table 1), and also include the summarised genetic loci and genes isolated from different plants. In addition, [76] revealed the enzymes in the flavonoid pathway show different characteristics in substrate specificity or preference in various plant species.
Table 1: Loci of flavonoid biosynthetic genes in plants.
Chalcone synthase (CHS): Chalcone synthase (CHS) is the first committed enzyme in all flavonoid biosynthesis. The enzyme is a member of type III polyketide synthase, which is one of the general polyketide synthases and it forms a catalytically –active single polypeptide [77]. The CHS catalyses claisen-ester (carbon-carbon bond between two ester molecules) condensation connected with CO2 which originated from malonyl-CoA and acyl-thioester (i.e., -coumaroyl-CoA), see (Figure 4). In addition, CHS catalyses the formation of a triketide intermediate from -coumaroyl-CoA and three molecules of malonyl-CoA, then progress to cyclize triketide intermediate which further result to naringenin chalcone.
Figure 4: Pathway representing flavonoid biosynthesis in plants. The enzymes for each step are identified in capital letters and intermediates are indicated in the rectangular boxes which are also found in Clivia. The synthesis starts from 4-coumaroyl-CoA to the production of anthocyanin. CHS, chalcone synthase, CHI chalcone isomerase; F3H, flavonone 3-hydroxylase; F3’H flavonoid-3’-hydroxylase; F3’5’H flavonoid 3’ 5’–hydroxylase; FLS flavonol synthase; DFR dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; GT glycosyltransferase; AT acyltransferase; MT methyltransferase.
There are CHS genes involved in anthocyanin biosynthesis in plants. The first flavonoid biosynthetic gene isolated was the CHS gene from parsley [78]. The isolated CHS cDNA from parsley was further used as a molecular probe to isolate clones of two different CHS genes from petunia [79]. Furthermore, CHS multi-gene family provides a vital model to understand the functional importance and the differential expression patterns of the gene family members. This gene structure is not complicated because it is generally conserved across taxa which consist of two exons separated by one intron; except from other research study done on Antirrhinum and Arabidopsis, which has three exons and two introns [80]. Studies done on Viola cornuta flowers, a different genus showed dramatic developmental colour change over eight days from early stage to maturity (ontogeny) and CHS influences two environmental stimuli; light and pollination [81]. In addition, chalcone synthase genes offer a high standard of sequence homology at the amino acid level and have been the main focus of various studies in dicotyledonous plants, and where [81,82].
Chalcone synthase is also referred to as type III polyketide synthase enzyme (PKS), and it is structurally the simplest that functions as a homodimeric iterative enzyme (a repetitive process which is composed of two polypeptide chains that are identical in the order, number, and kind of their amino acid residues); a monomer size of 40-45 kDa with two independent active sites which catalyses series of decarboxylation, condensation and cyclization reactions [83,84]. Ferrer et al. [85] and Jez et al. [86] revealed each alfalfa
CHS2 structural monomer consists of two structural domains. The upper domains consist of four amino acids (Cys164, Phe215, His303 and Asn336) and are currently situated at the active site defined as a catalytic tool of CHS. The lower domain of CHS consists of a large active site; make space for the tetraketide required for chalcone synthesis (i.e., naringenin and resveratrol) from one ρ-coumaroyl-CoA and three malonyl-CoA molecules which are then catalysed by CHS.
Chalcone isomerase (CHI): In vascular plants, chalcone isomerase (CHI) catalyses the conversion of chalcone to chiral (S)- flavanone which is the initiation step in the production of plant flavonoids (Figure 4); the CHI compounds contributes to the attraction of pollinators, defence and development of higher plants [87,88]. Chalcone isomerase converts the yellow chalcone into the corresponding flavanone’s, a pigment known as the colourless naringenin [89]. Thus, an intramolecular reaction is blocked during the C-ring, increasing a stereo-chemically intramolecular cyclisation reaction results to a biologically active (S)-isomer [90].
Molecular studies have revealed that disruption of CHI and dihydroflavonol 4-reductase (DFR) caused by transposon insertions give rise to yellow flowers carnation [91]. Thus, the absence of CHI activity looks important for the production of yellow-flowered carnations. van Tunen et al. [92] showed Petunia (po) mutant lines without chiA promoter activity in anthers are also identified to have yellow or greenish pollen (Table 1). The production of gold bulb colour in an onion mutant (Allium cepa) by the accumulation of chalcone derivatives including a yellow pigment is caused by the inactivation of CHI [93]. Hence, it’s requirement to prevent CHI activity to produce yellow-flowered plants by pigmentation of chalcone or chalcone derivatives. Nonetheless, the isolation of CHI have been a mystery; reason being that there is the apparent absence of a related protein from primary metabolism [94].
However, Grotewold and Peterson [89] reported the first cloning of chalcone flavonone isomerase gene (ZmCHI1) from maize (Table 1). In addition, the analyses indicated that the maize CHI gene (ZmCHI1) is regulated in the pericarp by the P gene - Myb homologue. The CHI1 gene for maize has four exons and an intron-exon structure identical to the CHI-B gene of Petunia hybrida. Two CHI isoenzymes have been identified; (1) commonly the CHI1-type that can make use of 6’-hydroxychalcone substrates, and (2) the CHI2-type that can catalyse the isomerisation of both 6’-hydroxy- and 6’- deoxychalcones [95]. The clusters of the tandem gene of both types occurring in Lotus japonicas by Ralston et al. [96] suggested that type 2 CHIs evolved from an ancestral type 1 CHI by gene duplication.
Flavanone 3-hydroxylase (F3H): Flavanone 3-hydroxylase (F3H) is a family of 2-oxoglutarate-dependent dioxygenases (2-ODDs) [97], catalysing the oxygenation at carbon-3 position of flavonone (2S)- naringenin to produce dihydroflavonol (dihydrokaempferol) with the production of CO2 and succinate from oxygen and oxoglutarate as co-substrates (Figure 4). All the F3Hs were featured with two conserved motifs: (His233, Asp235, and His289) for binding FeII and (Arg299 and Ser301) for binding 2-OG (2–oxoglutarate) [98,99]. The F3H enzyme also catalyses the stereo-specific hydroxylation of (2S)- naringenin and (2S)-eriodictyol to form (2R,3R)-dihydrokaempferol and (2R,3R)-dihydroquercetin, respectively [100]. Britsch et al. [101] reported that F3H has amino acids that play a role in Fe2+ and 2-oxogluturate binding.
A reported high F3H activity in tea leaves by Punyasiri et al. [102] suggested that F3H is important in the biosynthesis of catechins in tea (Camellia sinensis). Thus, studies have shown that F3H do not regulate only the types and quantities of flavonoids, colours, flavours of flowers and fruits, but also plays a vital role in the resistance to abiotic stress [103,104,105]. Furthermore, Watkinson et al. [106] showed F3H expression level was increased when potato (Solanum tuberosum ssp. Andigena) was exposed to drought stress during growth. The F3H was significantly increased Reaumuria soongorica, a desert plant when exposed to UV-B radiation and drought stress [107]. There is also an increase of F3H expression produced in Arabidopsis thaliana grown in nutrient-deficient soil [108,109,110]. Nevertheless, the F3H genes have been cloned and characterized from many plants, including Saussurea medusa - SmF3H [111], Ginkgo biloba - GbF3H [112], Scutellaria viscidula - SvF3H [113], Arabidopsis thaliana – AtF3H [114] and Camellia sinensis - CsF3H [115] respectively.
Flavonoid-3’-hydroxylase (F3‘H) and Flavonoid 3’,5’-hydroxylase (F3’ 5’ H): Flavonoid 3’-hydroxylase (F3’H) and flavonoid 3’,5’-hydroxylase (F3’5’H) are members of cytochrome CYP75 protein superfamily of P450 enzymes [116,117]. For the P450 enzymes to be active, it must be coupled to an electron donor [116], it can either be a cytochrome P450 reductase or cytochrome b5. In addition, the reductase is anchored to the surface of the endoplasmic reticulum via its N- or C-terminus. The F3’H and F3’, 5’H catalyse the hydroxylation of flavonoid B-ring at the 3’ or the 3’,5’ positions. Seitz et al. [118] showed how to hydroxylate a broad range of flavonoid substrates in vitro, including naringenin, dihydrokaempferol, kaempferol and apigenin, allowing the production of 3’,4’- and 3’,4’,5’-hydroxylated intermediates and flavonoid end products through multiple pathways (Figure 4). For instance, F3’H and F3’, 5’H compete for the production of delphinidin and cyanidin, the precursors of blue and red anthocyanins [119].
Studies showed that F3’H and F3’5’H are examined of its putative role in flavonoid metabolism in plants such as Petunia hybrida [120], Catharanthus roseus [121], Vitius vinifera [122], Campanula medium [123] and Solanum lycopersicum [76], respectively. The F3’5’H gene was first isolated from Catharanthus roseus [121] using heterologous screening cDNA with the CYP75 Hf1 genes from Petunia hybrid (Table 1). The genes encoding F3’5’H in grape shows expression in different parts of the plant, which accumulate flavonoids specifically in the skin of the ripened berries where the highest levels of anthocyanins are synthesised [124].
Flavonol synthase (FLS): The biosynthetic pathway to flavonoids is well established and flavonol synthase (FLS) is one of the first committed enzymes, which catalyses dihydroflavonols into flavonols which results to quercetin 6 [38]; in addition, the enzyme branches from the main pathway route leading to the formation of anthocyanin (Figure 4), [125,126]. Since FLS is a 2-oxoglutarate dependent dioxygenase (2-ODD), it requires 2-oxoglutarate (2-OG) and ferrous iron for catalytic function. The crystallographic studies of 2-ODDs [99] have established a characteristic arrangement of the double-stranded beta helix (DSBH) in this protein family. The DSBH comprises of two sets of four beta sheets that are arranged in an anti-parallel fashion, forming a sandwich-like structure. In addition, the rigid structure of the beta sheet offers structural support for accommodation two conserved motifs [99]. The motif is known to be vital for attaching the ferrous iron required as a source for dioxygen activation and subsequent catalysis [98].
The FLAVONOL SYNTHASE1 (AtFLS1, At5g08640), was the first identified from Arabidopsis thaliana as the FLS gene encoding the catalytic active protein [127,128]. In the Arabidopsis genome, there are five additional genes with high sequence similarity to FLS. While the AtFLS1 gene mainly influence flavonoid levels in Arabidopsis [129,130], thus, the mutant deficient AtFLS1 still build-up significant amounts of flavonols [131]. In addition, the over-expression of CnFLS1 in Nicotiana tabacum resulted in the colour alteration from the floral organs into white or light yellow; and the metabolic analysis revealed a significant increase of flavonols and reduction of anthocyanins in transgenic plants [132]. Moreover, other FLS-like genes may have different non-catalytic functions, however, it remains yet unidentified.
Dihydroflavonol-4-reductase (DFR): The first reaction leading to anthocyanin and proanthocyanidin formation starts by DFR enzyme catalysing the production of flavan-3,4-diols (leucoanthocyanidins) through the reduction of three colourless, corresponding dihydroflavonols, dihydrokaempferol (DHK), dihydroquercetin (DHQ), and dihydromyricetin (DHM) [133], which are also intermediates of flavonol biosynthesis via the flavonol synthase reaction (Figure 4). The DFR is also known to affect the biosynthesis of other flavonoids, example, flavonols and proanthocyanidin [134,135,136]. The dihydroflavonol substrate of DFR can be catalysed by FLS which results in flavonols and leucoanthocyanidins respectively, and it can successively be converted to proanthocyanidin by leucoanthocyanidin reductase (LAR) [137,138,139]. Moreover, these two enzymes are key branches in flavonoid pathways. Hence, understanding more of DFR function in the regulation of flavonoid biosynthesis in plants is of importance.
Dihydroflavonol-4-reductase uses NADPH (Nicotinamide adenine dinucleotide phosphate) as a cofactor to catalyse the reduction of dihydroflavonols to their unstable colourless leucoanthocyanidins, a known precursors for anthocyanin and proanthocyanidin biosynthesis [140,141,142]. The DFR is also responsible for NADPHdependent reduction of the dihydroflavonols, dihydroquercetin (DHQ), dihydrokaempferol (DHK) and dihydromyricetin (DHM) becoming three sub-pathways leading to orange pelargonidins, pink anthocyanins and purple delphidins respectively [133]. Due to the crucial role of DFR in the flavonoid pathway, DFR genes have been isolated from various species. These includes grape (Vitis vinifera) [143,144], apple (Malus domestica) [145], strawberry (Fragaria ananassa) [146] , purple sweet potato (Ipomoea batatas Lam) [147], maize (Zea mays) [148] and petunia (Petunia hybrida) [149].
Anthocyanidin synthase (ANS) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT or 3GT): Anthocyanidin synthase (ANS) or leucocyanidin oxygenase (LDOX) being the penultimate step in the biosynthetic pathway that leads to the production of anthocyanins (a form of flavonoids); from the colourless leucoanthocyanidin to the coloured form of anthocyanidins (Figure 4), [150,151]. The UDPglucose: flavonoid 3-O-glucosyltransferase (UGFT) is the final enzyme in the anthocyanin pathway. In addition, the transfer of the glucosyl moiety from UDP-glucose to the 3-hydroxyl group of anthocyanidins by UFGT was revealed to be important for anthocyanidin stability, water solubility and improving stability by external hydrogen bonding of sugar residues with the surrounding water molecules in the vacuole [152,153].
The ANS proteins are members of 2-oxoglutarate-dependent dioxygenases (2-ODDs) family of proteins which also comprises of flavonoid enzymes known as flavanone 3-β hydroxylase (F3H), flavonol synthase (FLS) and flavone synthase I (FS I) proteins. These proteins use molecular oxygen as the co-substrate and are characterised with its co-factor requirements of ferrous iron (Fe2+), 2-oxoglutarate and/ or ascorbate [153]. However, these dioxygenases and it’s widely spread unlimited substrate specificities are involved in the pathways of flavonoids, gibberellins and alkaloids to penicillin’s and cephalosporin’s [154]. In addition, the ANS contains multicomponent active site containing metal, co-substrate, and dihydroquercetin (i.e. two molecules of a substrate analog) - based on the crystal structure of the enzyme in Arabidopsis [155]. In grapevines, the downstream in the pathway of UFGT level is the main point for anthocyanin quantitative variation [156,157]. The cDNA clone (Ih3GT) encoding 3GT was isolated from a cDNA library of flower buds from Iris hollandica by screening with a 3GT cDNA clone of Antirrhinum majus [152].
Fewer studies on characterization of ANS gene have been done in plants. But in Ginkgo biloba, all full-length cDNA and genomic DNA of ANS was successfully isolated and cloned. The genomic DNA analysis revealed that GbANS gene had three exons and two introns [158]. In blackcurrant (Ribes nigrum L.), the RnANS1 gene isolated and the full-length cDNA of 1427bp showed similar conserved domain of the protein as compared to other plants such as P. suffruticosa and P. lactifora [153,159,160]. Another report on the isolation of cDNA clone coding for ANS gene from Saussurea medusa (SmANS1). Cheng et al. isolated and characterised cDNA clone library obtained from a red callus line of S. medusa, where the SmANS1 controls the overall metabolic flux to the target products leading to both red and yellow S. medusa cell cultures at different levels. The RnANS1 gene of a blackcurrant (Ribes nigrum L.) shrub, a family of Grossulariaceae were studied [160,161]. The clone and expression analysed from ANS homolog RnANS1 in various fruit developmental stages revealed a clarified understanding of anthocyanin biosynthetic pathway and the manipulation of anthocyanin production in blackcurrant plant.

Factors Affecting Anthocyanins Concentration and Colour in Flowers

pH level and metal ions
The level of pH in plants is known to influence the colour of flowers. Generally, plant species, the colouration of plant tissues are formed from the accumulation of anthocyanin pigments in the vacuoles of (sub) epidermal cells. The change in colour of anthocyanins is influenced by the pH, co-existing colourless compounds such as (co-pigments, typically flavones and flavonols), and metal ions. Tanaka et al. [162] demonstrated an in vitro assay where the pH 3 resulted in red anthocyanidin pigments which stabilise the flavilium cation, and it is colourless under mildly acidic conditions (pH 3-6), thus, its blue and unstable as the quinonoidal base forms beyond pH 6. Furthermore, Fukada-Tanaka et al. [163] revealed that this colour change in plants and the increase of pH vacuole needs a putative Na+/ H+ exchanger encoded by the PURPLE gene. The PURPLE protein transports sodium ions and protons into and out the vacuole, causing a less acidic vacuole and a blue colour of Ipomoea nil petals.
In morning glory (Ipomoea tricolour) petals, the vacuolar pH is low when the flower bud opens, which results in a red pigmentation, but during the process of maturation, the vacuolar pH increases and the petals incurs a darker blue colour [164]. The pH level in Petunia hybrida flowers is usually lower than in Ipomoea flowers, while the wildtype flower colour of Petunia hybrida remains reddish; which implies a low pH value. Spelt et al. [165] showed that an increase in pH levels from the petal extracts of Petunia hybrida indicates the disappearance of anthocyanins and the fading of flower colour. In addition, metal ions such as Al3+ and Fe3+, play a vital role in the production of blue flowers in hydrangea Hydrangea macrophylla [166] and Tulipa gesneriana [167], respectively. On the other hand, the decrease of anthocyanin pigments due to inactivation of enzymes of the anthocyanin pathway such as CHS (in co-suppressed lines) affecting anthocyanin synthesis, mutation also occurs in anthocyanin1 genes (An1, An2, and An11) in the petals of petunia; because of an increase of Ph [165]. In addition, the switch of pH can be associated with the increased vacuolar pH from the bluish flower colour, thus the mutated An1 loci stopped the activity to activate vacuolar acidification and could yet stimulate transcriptional activation of anthocyanin biosynthesis.
Co-pigmentation
Anthocyanin co-pigmentation is a medium to improve the stability of anthocyanins in plant tissues. Pigments and other non-coloured organic components form chemical complexes with other flavonoids, a phenomenon known as ‘intermolecular co-pigmentation’ [168,169]. Co-pigmentation is known to have much colour variability of bluish flowers and for stable wine colours in Iris ensata Thunb [170], and Agapanthus africanus [171]. Co-pigmentation are responsible for colour in young red wines between 30% and 50% he [172]. While Dobrei et al. [173] and Blanco-Vega et al. [174] further revealed that during storage or aging a decline in co-pigmented anthocyanins and an increase in polymerized anthocyanins are observed for all types of red wine with changes in colour characteristics of the product.
Intramolecular co-pigmentation is another type mechanism for co-pigmentation which represents the covalent acylation of the anthocyanin molecule, and can stabilize the pigments [168]. The covalent bond of anthocyanin molecules occurs with an organic acid, an aromatic acyl group, or a flavonoid [175]. However, the stability of these anthocyanins in weakly acidic or neutral solution is due to its structure, where the aromatic residue of acyl groups are linked with the pyrylium ring of the flavylium cation; so, this binding decreases the hydration at the C-2 and C-4 position [176]. The threshold of the hydration reaction reduces and the stability of the chromophores strongly increases, since the proton transfer reaction seemly remains unaffected. Generally, intramolecular co-pigmentation is known to be stronger and more effective unlike the intermolecular co-pigmentation, because of the covalent bonding being stronger than the non-covalent binding [176]. Intramolecular co-pigmentation has been noted to stabilize the colour in many plants, like, radishes, red potatoes, red cabbage, black carrots, and purple potatoes and other plant materials, which contain high amounts of acylated anthocyanins [177].
Chalcone synthase (CHS) and anthocyanidin synthase (ANS)
Chalcone synthase (CHS) is an enzyme that catalyses the first committed step of flavonoid biosynthesis. The CHS condenses a phenylpropanoid CoA ester (e.g., p-coumaroyl-CoA) with three acetate units from malonyl-CoA molecules, and cyclizes the resulting intermediate to produce a chalcone (e.g., naringenin chalcone) (Figure 4), [127,178]. Chalcone synthase is a member of the CHS superfamily; also known as type III polyketide synthases (III PKS) located in all plant species [77,179,180]. In addition, enzymes of the CHS superfamily reveals homology in sequence, structure and general catalytic forms, indicating homodimers of 40-45 kDa subunits and all contain a Cys-His-Asn catalytic triad in the active site [77,181].
There’re other gene members of the superfamily found in plants such as stilbene synthase (STS), acridone synthase, pyrone synthase, bibenzyl synthase and p-coumaroyltriacetic acid synthase clone [182]. However, chalcone synthase often displayed a complex system of regulation and quite a number of potential regulatory elements. The activity of CHS was first reported in extracts of parsley (Petroselium crispum) in Freiburg by [183]. The enzyme was initially named flavanone synthase, in view of the fact that chalcone was quick to convert flavanone in a non-enzymatic reaction that was not identifiable as the earliest product. It was later rectified few year later with immense techniques, though the wrong name was included in the publications as at that time [183].
The chalcone synthase (CHS) is encoded by a multigene family in plants, thus the expression of CHS genes serves as one of the factors influencing anthocyanin biosynthesis. For example, at least eight CHS genes have been described for Petunia hybrida [184] and pea (Pisum spp.) [185], nine gene family members are present in morning glories (Ipomoea spp.) [186,187]; two genes (CHSD and CHSE) encode enzymes functional for flavonoid biosynthesis, and CHSD is primarily responsible for pigmentation in the floral limb. In addition, the mutation of Ipomoea CHSD gene results in an albino phenotype that is not compensated by other CHS family members [187]. Moreover, there are three CHS-like genes (Chs1,Chs3 and Chs3) which exist in Arabidopsis thaliana [188]. In peas, eight members of the CHS genes were expressed during development and in response to so many environmental factors such as elicitor induction, and UV responsiveness [185]. Between the three CHS genes in grapevine (Vitis vinifera), CHS1 is accumulated in berry skin and young leaves [189], and CHS2 and CHS3 were mostly expressed in the skins of the berry during pigmentation [190].
Researchers often channel their interest in the promoter region of the CHS gene due to the fact that the gene has a wellcharacterized promoter in plants and its expression is induced by multiple signals. For instance, data from several studies of the Physcomitrella patens transcriptome and genome have been published [191,192,193,194,195]. More so, 480 Mb haploid genome of the plant has been sequenced [196], and the annotated version contains about 35,938 gene models distributed among 2,106 scaffolds, which composed of 19,136 contigs. Jiang et al. [197] revealed the genome of P. patens was shown to contain as many as 19 putative genes that could encode enzymes of the CHS superfamily. The occurrence of multiple CHS superfamily genes in plants is common and seems to spread widely across taxa. Moreover, four CHS superfamily genes (PnJCHS, PnICHS, PnLCHS and PnPCHS) have been identified and cloned from in Psilotum nudum using a reverse transcriptionpolymerase chain reaction strategy [198].
Anthocyanidin synthase (ANS) or leucocyanidin oxygenase (LDOX) being the penultimate step in the biosynthetic pathway that leads to the production of anthocyanins (a form of flavonoids); from the colourless leucoanthocyanidin to the coloured form of anthocyanidins [105]. The ANS proteins are members of 2-oxoglutarate-dependent dioxygenases (2-ODDs) family of proteins which also comprises of flavonoid enzymes known as flavanone 3-β hydroxylase (F3H), flavonol synthase (FLS) and flavone synthase I (FS I) proteins. These proteins use molecular oxygen as the cosubstrate and are characterised with its co-factor requirements of ferrous iron (Fe2+), 2-oxoglutarate and/ or ascorbate [153]. The ANS contains multicomponent active site containing metal, co-substrate, and dihydroquercetin (i.e. two molecules of a substrate analogue) - based on the crystal structure of the enzyme in Arabidopsis [155]. The homologs of ANS have been cloned and characterised in so many plants such as (Spinacia oleracea) spinach and (Phytolacca americana) pokeweed [199], Saussurea medusa [161], Ginko biloba [158], Ribes nigrum [160] and Reaumuria trigyna [200]. Furthermore, the dioxygenases of ANS and its widely spread of unlimited substrate specificities are involved in the pathways of flavonoids, gibberellins and alkaloids to penicillin’s and cephalosporin’s [154]. Anthocyanins are predominantly accumulated in the skin of grape berries while the condensed tannins are present in the seeds and skin. Hence, these compounds are synthesised through pathways of which the accumulation of ANS is a significant regulatory enzyme [201,202]. Saito and Yamazaki [203] revealed that the red form of Perilla frutescens has confirmed that ANS is localised in the epidermal cells specifically correlating to the epidermis-specific accumulation trend of anthocyanins. The transcript of ANS gene in grapevine has been detected in almost all the organs such as leaves, tendril, green cane, root, seeds, berry skin and berry flesh [157,204]. Nonetheless, ANS in grapevine, like other enzymes are involved in the flavonoid biosynthetic pathway; which occurs at a low level and it’s difficult to assay [205]. Understanding the tissue-specific accumulation of specific types of flavonoid end-product led by differential gene expression and gene products such as CHS and ANS are important.
The CHS and ANS gene expression in flowering plants
The level of CHS transcripts in plant cells is reflected by the CHS gene expression levels. For transcription to occur, the RNA polymerase II must bind to specific DNA sequences in the CHS promoter region of the TATA box and should be activated by certain DNA-binding proteins known as transcription factors [206], thus, the response elements bind further upstream in the promoter. Dixon et al. [207] indicated that CHS promoter contains the nucleotide sequence CACGTG regulatory motif known as G-box, which has been found to be vital in the response to light and UV lights; meaning it plays a role in a plant containing CHS genes. Multiple regulatory loci which control CHS expression have also been explained in Petunia hybrida regulatory mutant Red Star [208]. Furthermore, the deduced phenotypic feature of this mutant of red and white parts in the flower petals is thought to depend on at least four regulatory genes, all regulates the CHS expression. Nonetheless, structural gene like CHS can be grouped into classes relating to its position in the pathway and regulation of expression: CHS, CHI and F3H are the early biosynthetic genes, while DFR and ANS genes are late biosynthetic genes [209].
In addition, Yuan et al. [210] studied six isolated putative structural genes from Tulipa fosteriana which showed tissue-specific expression profiles measure by real-time quantitative PCR. The early biosynthetic genes TfCHS1, TfCHI1 and TfF3H1 accumulated more transcripts in stems and leaves than those in floral organs (stamens, pistils, petals). The CHS, CHI and F3H are prerequisite for the synthesis of precursors of anthocyanidin and colourless flavone and flavonol derivatives [211], this is result in the stems and leaves containing more colourless flavone and flavonol derivatives than petals. Trojan et al. [212] revealed the expression of CHS gene in individual layers of the caryopsis in wheat and the gene was expressed at higher levels in genotypes with a blue aleurone layer and in the seed coats. This illustrates the fact that CHS is the key enzyme in anthocyanin biosynthetic pathway as demonstrated by Moore et al. [213]. To understand the regulation of the PmCHS gene in Polygonum minus, PmCHS gene expression in different tissues of P. minus was measured using relative qRT-PCR. In addition, the expression of PmCHS was significantly higher in the roots than in other tissues. The result was consistent with the findings of Li et al. [214], who revealed flavonoid biosynthesis genes, including CHS, was highly expressed in the lower parts of the plant, especially the roots. Viljoen et al. [215] investigated the expression of CHS and DFR in flowering tissues (tepal, carpel and stamen) of C. miniata at different stages of yellow and orange flower development and linked it to anthocyanin content. Furthermore, the anthocyanin content in orange flowers tissues was higher than that of yellow flowers. Nonetheless, having identified CHS gene families in many plant species, the mechanisms underlying their expression and regulation have not yet been entirely elucidated. To elucidate the specific expression of CHS family genes, the potential transcription factors and regulatory elements that are involved in anthocyanin biosynthesis and light responses should be studied thoroughly on a biochemical and genetic level.
Anthocyanidin synthase gene expression: Anthocyanidin synthase (ANS) is a key enzyme in the anthocyanin biosynthetic pathway complex which catalyses the terminal oxidation of leucocyanidin to coloured anthocyanins such as dihydroquercetins, utilising 2-oxoglutarate (2OG) as the substrate [133,216]. Moreover, Cheng et al. [161] isolation of cDNA clone coding for an ANS gene (SmANS1) from Saussurea medusa. The SmANS1 was obtained from cDNA libraries prepared from a red cell line of S. medusa, SmANS1 has sequence homology to similar proteins from other plants, and it is expressed in both red and yellow S. medusa cell cultures at different levels. Nevertheless, the ANS in the grapevine like other enzymes involved in the flavonoid biosynthetic pathway are present at a low level and it’s also difficult to assay [205]. Moreover, information about ANS has come from the studies on gene expression, little is yet known about the expression and regulation of ANS at the protein level in grape.
The anthocyanidin synthase (ANS) expression has been revealed to be controlled in specific tissues and cell types during plant development in response to a variety of stimuli, including stress, environmental light stimuli and [217,218,219]. Li et al. [160] reported the first cloning and expression analysis of the RnANS1 gene in in various fruit developmental stages of blackcurrant (Ribes nigrum L.). The expression of RnANS1 was upregulated during fruit maturation, and it’s associated with the accumulation of anthocyanins and soluble carbohydrates in the fruit. This RnANS1 expression profile is in contrast to that in grape berries, in which ANS transcript levels peak at an early stage of fruit development and decline gradually during the 30 to 60 days after full developmental of the grapevine [220], signifying the expression profile of ANS is species-specific. In addition to the correlation between RnANS1 transcript levels and anthocyanin content, we found that RnANS1 expression and soluble carbohydrate levels were also related, although the reason for this is unclear. Wang et al. [221] revealed the expression of ANS in early samples mostly responds to proanthocyanidins (PAs) increase, while the introduction of ANS at the onset of grape berries (Vitis vinifera L. Cabernet Sauvignon) ripening leading to anthocyanin synthesis. In addition, the ANS is controlled at both the transcriptional and protein levels and regulated in a temporal manner. Saito & Yamazaki [203] showed how the ANS mRNA was expressed in leaves and stems of the red form of Perilla frutescen but not in the green form, though the ANS gene is present also in green Perilla. Furthermore, these tissuespecific accumulations of mRNA and protein of ANS correspond well to the tissue-specific accumulation pattern of anthocyanin, signifying the importance of the contribution of ANS in anthocyanin biosynthesis.

Concluding Remarks

Some flowering plants are important to the floricultural industry and market globally. The high demands of these flowering plants such as clivia have overall been increasing in South America, Asia and Africa. However, since flower colour and their varied patterns contribute to the high commerciality in the industry, thorough molecular research should assist to enlarge the floricultural industry. The variable traits that are sought after in the floriculture industry/ market are the pigmentation of flowers. The colour pigments can be studied widely for a number of years with the intention to clarify various aspects including the biochemical properties and genetic biosynthesis of different colour traits. Nonetheless, Clivia has also improved significantly in the floricultural industry around the globe; perhaps genetic engineering could be the approach to enhance different Clivia species. Though, a detail comprehension of the specific anthocyanin biosynthetic genes resulting to colour pigmentation would be imperative.
Anthocyanin biosynthesis is an important pathway that is responsible for tissue pigmentation in flowering plants in general. However, so many genes encoding key biosynthetic enzymes have been identified and characterised, but specifically our gene of interest (CHS and ANS) [222,223]. These genes play a significant role in colour pigmentation and flower patterns. The type of flower colour pattern shows the type of information mechanism [224]. A gene whose expression level is different between tissues colour is likely a target for pigment biosynthesis regulation. In addition, when no target genes are detected, other regulatory mechanisms should be examined; like the differences in translocation, modification of enzymes may also influence anthocyanin biosynthesis. Larsen et al. [225] and Sasaki et al. [226] revealed the possibility of the differences in transport of anthocyanin into vacuoles by glutathione-S-transferase.
Furthermore, it is still vital to understand how the crosstalk between gene products and other cellular factors in a flowering plant resulting in specific colour formation. Thus, using a genetic engineering approach to modifying genes associated with colour traits in a flowering plant is essential. Interestingly, the unique appearance of anthocyanin pigmentation makes it a very good visual biological marker in research. On this note, anthocyanin can be used as a visible marker for plant transformation [227] and viral infection [228]; which in turn improves the floricultural industry and market. In addition, the co-suppression of candidate genes and anthocyanin biosynthetic/ regulatory genes in anthocyanin-enriched plant tissue produces good marker for viral-induced gene silencing [229,230]. Alternatively, progress has been made in engineering anthocyanin production in microbes [231], utilising plants or plant cultured cells to produce anthocyanins.

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