Chalcone synthase and its functions in plant resistance
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Chalcone synthase (CHS, EC 184.108.40.206) is a key enzyme of the flavonoid/isoflavonoid biosynthesis pathway. Besides being part of the plant developmental program the CHS gene expression is induced in plants under stress conditions such as UV light, bacterial or fungal infection. CHS expression causes accumulation of flavonoid and isoflavonoid phytoalexins and is involved in the salicylic acid defense pathway. This review will discuss CHS and its function in plant resistance.
KeywordsChalcone synthase Flavonoids Plant resistance
During their life cycle, plants respond actively to stress by producing phytoalexins and other stress metabolites. Such stress can result from injuries caused by the attack of insects and microbes or by mechanical wounding, and can induce many distinctive biochemical changes. These include the production of protective compounds either at the site of injury, or systemically in distant unwounded tissues (Kuhn 1988; Bowles 1990; Ryan 1990). In plants, phenylalanine is derived from the precursor chorismate and leads to the flavonoid, phenylpropanoid and stilbenoid biosynthetic pathways. All are interesting in connection with plant defense but in this review we will focus on the flavonoid biosynthesis pathway and its key enzyme chalcone synthase (CHS).
CHS is a member of the plant polyketide synthase superfamily, which also includes stilbene synthase (STS), acridone synthase, pyrone synthase, bibenzyl synthase, and p-coumaroyltriacetic acid synthase (Sanchez 2008). Chalcone synthases, the most well known representatives of this family, provide the starting materials for a diverse set of metabolites (flavonoids) which have different and important roles in flowering plants, such as providing floral pigments, antibiotics, UV protectants and insect repellents (Hahlbrock and Scheel 1989). Flavonoids also have benefits for human health, as they exhibit amongst others cancer chemopreventive (Jang et al. 1997), antimitotic (Edwards et al. 1990), estrogenic (Gehm et al. 1997) antimalarial (Li et al. 1995) antioxidant (Jang et al. 1997) and antiasthmatic (Zwaagstra et al. 1997) activities.
In this review we will evaluate the present understanding about CHS and its regulation in plant resistance.
Structure and mechanism of chalcone synthase
5.14 ± 0.30
6.1 ± 1.3
4.58 ± 0.24
4.7 ± 1.1
1.04 ± 0.17
5.2 ± 0.9
2.52 ± 0.22
4.1 ± 1.2
2.17 ± 0.35
5.1 ± 0.7
1.73 ± 0.21
2.2 ± 0.2
14.9 ± 0.2
8.0 ± 0.2
Control of CHS activity
In plants, CHS is activated by a wide range of environmental and developmental stimuli. Theoretically, there are many ways to regulate CHS activity in vivo, from metabolic control to the control of initiation of transcription of the CHS gene (Martin 1993).
There are many studies showing that CHS is inhibited noncompetitively by flavonoid pathway products like naringenin, chalcone naringenin and the other end products of CoA esters. For example, the parsley CHS is 50% inhibited by 100 μM naringenin and 10 μM CoA esters (Hinderer and Seitz 1985; Kreuzaler and Hahlbrock 1975), the flavonoids luteolin and apigenin are inhibitory to rye CHS in vitro (Peters et al. 1988), whereas in carrot, among the range of flavonoids tested, only naringenin and chalcone narigenin can inhibit CHS at 100 μM (Hinderer and Seitz 1985). It seems that flavonoids accumulate in the cytosol to a level that blocks CHS activity to avoid toxic levels for the plant (Whitehead and Dixon 1983), though there is no direct evidence that this inhibition happens in vivo.
Control of CHS turnover
In plants, CHS may always be present in the cells but is only activated under certain specific conditions. The statement “CHS may always be present in the cells but is only activated under certain specific conditions” means that CHS is activated at the protein level. However, it has been shown that UV light and biotic elicitors induce the flavonoid biosynthetic pathway at the transcriptional level and that CHS is not detectable before the onset of the various stress situations. Studies on parsley cell cultures showed that the induction of CHS activity by UV light was the result of de novo synthesis and active enzyme subsequently decayed with a half-life of 6 h, whereas inactive enzyme decayed more slowly with a half-life of 18 h (Schröder and Schäafer 1980). Inactive CHS could be detected by CHS antibodies and the size of the protein was not changed. In another study about accumulation of CHS during UV induction, Chappell and Hahlbrock (1984) concluded that the accumulation of flavonoid end products is presumably determined by activity of the rate-limiting step(s) in flavonoid biosynthesis and may not precisely reflect the dynamics of CHS activity in vivo.
Control of CHS through trans-genes
The activity of CHS can be controlled by antisense or sense genes. The studies on expression of antisense genes in Petunia (e.g. Van der Krol et al. 1988; Van der Meer et al. 1993), tobacco (Wang et al. 2006), Gerbera hybrida (Elomaa et al. 1996) and Arabidopsis (Le Gall et al. 2005) have shown that the presence of antisense CHS could inhibit the expression of the endogenous CHS in plants. In flowers of antisense CHS transgenic Petunia, the antisense construct was able to inhibit expression of the endogenous CHS genes to varying degrees, which is observed phenotypically as an inhibition of anthocyanin production to give completely acyanic or patterned flowers. In the cyanic sectors and flowers, transcripts of the endogenous CHS genes were under the detection limit, but the antisense transcripts were also barely detectable (Van der Krol et al. 1990b). The antisense effect most likely involves homologous pairing between the transcripts of endogenous CHS genes and transcripts of the introduced antisense CHS gene to form double stranded RNA that is very rapidly degraded, thus inhibiting CHS transcript accumulation and hence CHS activity.
Introducing a heterologous CHS gene in sense orientation can inhibit CHS activity in transgenic plants. This phenomenon is called co-suppression since it involves the reduction of transcriptional level of both endogenous and introduced genes in tissues where the endogenous gene is normally expressed (Napoli et al. 1990; Jorgensen 1995). This is known as gene silencing in which the transgene triggered not only its own silencing but also the endogenous chalcone synthase gene (Hammond et al. 2001). But on the other hand the introduced CHS gene may be expressed to high levels in tissue where the endogenous CHS genes are not expressed, such as in leaves of Petunia (Van der Krol et al. 1990a). Some studies have shown that co-suppression correlates with DNA methylation of the silenced sequences, presumably leading to a blockade at the transcriptional level or/and failure of transcript to accumulate in the cytoplasm resulting in a lack of enzyme activity (Ingelbrecht et al. 1994; Furner et al. 1998; Amedeo et al. 2000). Nowadays, the molecular mechanism of co-suppression of gene expression is thought to be related to the RNAi mechanism (Hannon 2002).
CHS localization and dynamics
The CHS protein in buckwheat (Fagopyrum esculentum) hypocotyls is located in the cytosol and associates with the cytoplasmic face of the rough endoplasmic reticulum (rER), but not with nuclei, plastids, mitochondria, Golgi, or tonoplasts (Hrazdina and Jensen 1992). Saslowsky and Winkel (2001) examined the subcellular location of CHS and CHI in Arabidopsis roots. High levels of both enzymes were found in the epidermal and cortex cells of the elongation zone and the root tip, consistent with the accumulation of flavonoid endproducts at these sites. Co-localization of CHS and CHI was observed at the endoplasmic reticulum and tonoplast in these cells.
However, there is evidence that flavonoids located in the nucleus may be synthesized in situ (Saslowsky and Winkel 2001). Several recent reports describe the accumulation of flavonoids in the nucleus in such diverse species as Arabidopsis thaliana, Brassica napus, Flaveria chloraefolia, Picea abies, Tsuga canadensis, and Taxus baccata (Buer and Muday 2004; Feucht et al. 2004; Grandmaison and Ibrahim 1996; Hutzler et al. 1998; Kuras et al. 1999; Peer et al. 2001). For the enzymes of the flavonoid pathway, several mechanisms may be involved. In the cytoplasm, flavonoid enzyme complexes are believed to assemble at the ER and in electron dense particles through the association of operationally-soluble enzymes such as CHS and CHI with the membrane-bound P450 hydroxylase, flavonoid 3′-hydroxylase (Saslowsky and Winkel 2001; Hrazdina and Wagner 1985). CHS possesses sequences resembling a classic nuclear localization signal (NLS). This signal is located on the surface, on the opposite side of the protein from the dimerization interface and could function to direct CHS, and perhaps associated enzymes into the nucleus. The localization of end products such as flavonol sulfate esters and flavan-3-ols to the nucleus suggests that additional flavonoid enzymes are also present in the nucleus (Grandmaison and Ibrahim 1996; Feucht et al.2004).
There is an immuno gold-labeling study in grape berry showing that CHS was localized in rough endoplasmic reticulum (ER) and cytoplasm of the skin cells, while few gold particles were found on the cell wall. Besides, two novel sites of CHS were observed within cells of developing grape berry, one is in the plastids which remain unchanged throughout all stages of berry development. At the ripening stage of grape berry, CHS is present in the vacuole and in the vacuole membrane (tonoplast) (Tian et al. 2008). It is suggested that in grape berries, the synthesis of flavonoids in the ripening stage may occur in the vacuole.
Control of CHS gene expression
In Arabidopsis, parsley, and snapdragon only a single copy of the CHS gene has been found. In most angiosperms CHS has been shown to be encoded by a multigene family, such as in petunia (violet 30) (Koes et al. 1987), morning glories (Ipomoea) (Durbin et al. 2000), Gerbera (Helariutta et al. 1996), leguminous plants (Ryder et al. 1987; Wingender et al. 1989; Ito et al. 1997), and Cannabis sativa (Sanchez 2008).
Regulation of CHS gene expression
Many studies have shown that the CHS gene is constitutively expressed in flowers, but also its expression can be induced by light/UV light and in response to phytopathogens, elicitors or wounding in different parts of the plant, resulting in enhanced production of flavonoids (Koes et al. 1987; Ryder et al. 1984, 1987; Bell et al. 1986; Burbulis et al. 1996). CHS expression is also regulated by the circadian clock (Thain et al. 2002).
The level of CHS gene expression is reflected by the level of the CHS transcripts in plant cells. In order for transcription to take place, the RNA polymerase II must attach to specific DNA sequences in the CHS promoter in the vicinity of the TATA box and must be activated by specific DNA-binding proteins (transcription factors) binding to response elements further upstream in the promoter. The CHS promoter was studied extensively in Phaseolus vulgaris, Antirrhinum, Arabidopsis, and parsley (Dixon et al. 1994; Faktor 1997; Feinbaum et al. 1991; Lipphardt et al. 1988).
The CHS promoter contains the nucleotide sequence CACGTG regulatory motif known as G-box, which has been found to be important in the response to light/UV light (Kaulen et al. 1986; Staiger et al. 1989; Dixon et al. 1994; Schulze et al. 1989). Besides the G-box there are other domains in the CHS promoter involved in the light activation of CHS transcription. Those domains have been identified in the parsley CHS promoter as Box I, Box II, Box III, Box IV or three copies of H-box (CCTACC) in the Phaseolus vulgarisCHS15 promoter. These boxes play a role as core promoter together with the G-box and all are required for light inducibility (Block et al. 1990; Lawton et al. 1990; Weisshaar et al. 1991).
The PetuniaCHSA promoter was studied by van der Meer et al. (1990, 1993) to understand the role of the promoter in tissue-specific CHS expression. The studies showed that the promoter sequence between +1 and −67 confers flower specific CHS gene expression. Another study on the Antirrhinum CHS promoter has shown that the sequences between +1 and −39 allow CHS expression in root and stems, whereas sequences between −39 and −197 are required for expression in petals and seeds (Fritze et al. 1991).
The regulators of CHS in plants are controlled by some specific loci. In maize, there are four loci, cl, r, vp, and clf, involved in the regulation of CHS expression (Dooner 1983). Multiple regulatory loci for CHS expression have also been described for the petunia regulatory mutant Red Star. The phenotype of this mutant of red and white sectors in the flower petals is thought to depend on at least four regulatory genes, all of which regulate CHS expression in trans (Mol et al. 1983). In the CHS gene family of Phaseolus vulgaris, the regulation is via the a and a2 loci though they regulate different CHS members in different ways. The CHS genes might have different combinations of cis elements that determine their response to the products of these regulatory loci. The expression of CHSl in flower tissue has an absolute requirement for the products of both the a and a2 loci, whereas, in root tissue, the products of these loci are not required. It is possible that the CHSl gene interacts with one or more factors present in roots, which are absent in flowers, that can substitute for the products of the a and a2 loci. CHS3 expression in flower tissue is more complicated: it requires the product of the a2 locus, but has a lower level of expression in a mutants compared with wild type. This suggests that CHS3 interacts with both the a2 and a locus products, but, unlike the CHSl gene, it may also interact with other products, allowing transcription at a low level in a mutants. CHS2 is expressed in roots but not in petal tissue, suggesting that it may not be able to interact with the products of a and a2 loci in petal tissue (Harker et al. 1990).
Transcription factors involved in of CHS gene expression
Trans-acting factors of bean CHS15 that bind to two short sequences centered on the G-box and H-box also make major contributions to the in vivo transcription of the promoter (Arias et al. 1993; Yu et al. 1993). Trans activation required both a MYB-binding site and a G-box like element (Sablowski et al. 1994). MYB305, one of the MYB-like proteins that have been implicated in the transcriptional control of tissue-specific CHS gene expression, is also recognized by a cis element of the light-regulatory unit 1 (LRUI) of CHS in parsley (Feldbrügge et al. 1997). G-box/H-box binding factor 1(G/HBF-1), a basic leucine zipper (bZIP) protein, that binds to both the G-box and the adjacent H-box in the proximal region of the CHS15 bean promoter, is rapidly phosphorylated in elicited soybean cells, this happen also to the CHS15, CHS7, and CHS1 promoter (Dröge et al. 1997; Yoshida et al. 2008). Protein and mRNA levels of G/HBF-l do not change during the induction of CHS genes following pathogen attack (Yoshida et al. 2008) but CHS gene expression is strongly stimulated following phosphorylation responding to fungal elicitor treatment in vitro (Dröge et al. 1997).
CHS activity in plant resistance
Chalcone synthase expression in plant under stress conditions
Phaseolus vulgaris cells
Ryder et al. (1984)
UV-B and UV-A/blue light
Christie and Jenkins (1996)
Leyva et al. (1995)
UV-B, UV-A, and blue Light
Feinbaum and Ausubel (1988)
SA, ethylene, methyl jasmonate Alternaria brassicicola
Schenk et al. (2000)
Koes et al. (1989)
Shvarts et al. (1997)
Petroselinum hortense cells
Kreuzaler et al. (1983)
Phenolic compounds, flavonoids, catechin
Schnitzler et al. (1996)
Nagy et al. (2004)
Ophiostoma polonicum and wounding
Brignolas et al. (1995)
Haussuehl et al. (1996)
Christensen et al. (1998)
Harrison and Dixon (1993)
Junghans et al. (1993)
Dalkin et al. (1990)
Lipphardt et al. (1988)
Staiger et al. (1989)
Pseudomonas syringae pv glycinea
Dhawale et al. (1989)
Phytophthora megasperma f. sp. Glycinea
Wounding, JA, MeJ
Richard et al. (2000)
UV, Pythium aphanidermatum
Gläßgen et al. (1998)
Zhou et al. (2007)
Sorghum mesocotyl, juvenile sorghum tissues
3-Deoxyanthocyanidins, apigeninidin luteolinidin
Phytoalexins are antimicrobial metabolites produced by plants in response to microbial attack (or biotic and abiotic elicitors) (Dixon 1986). Phytoalexins come from many different metabolite classes such as flavonoids, stilbenoids, sesquiterpenoids, steroids and alkaloids. CHS can help the plant to produce more flavonoids, isoflavonoid-type phytoalexins and other related metabolites to protect it against stress. Accumulation of flavonoids and isoflavonoids in response to pathogen attack is seen in many plant species, and their importance as antimicrobial phytoalexins is well established (Matthews and Matthews 1989; Van Etten and Pueppke 1976). Flavonoid phytoalexins have been described in legumes, cereals, sorghum, rice, Cephalocereus senilis,Beta vulgaris (Hipskind et al. 1990; Johnson et al. 1976; Kodama et al. 1992; Pare et al. 1992). Some isoflavonoids were increased in Lupin luteus after infection with Fusarium oxysporum such as genistein, wighteone and luteon (Morkunas et al. 2005). The isoflavones, daidzein, genistein and glycitein, in soybean were strongly increased after infection by Sclerotinia sclerotiorum (Wegulo et al. 2005). Stilbenes are known as the phytoalexins in peanut (Ingham 1976) and grapes (Langcake and Pryce 1977a, b). There is also evidence that stilbene synthase (STS) has developed from CHS several times in the evolution (Tropf et al. 1994).
Van Etten et al. (1995) defined phytoanticipins as low molecular weight, antimicrobial compounds that are constitutively expressed in plants without the need for infection with fungal pathogens or are produced after infection solely from preexisting constituents. The distinction between phytoalexins and phytoanticipins is not always clear as some compounds may be phytoalexins in one species and phytoanticipins in another species. Phytoanticipins also are classed into several chemical groups such as flavonoids, terpenoids, steroids, glucosinolates, and alkaloids.
The flavonoid epicatechin plays an important role as phytoanticipin in avocado fruits (Guetsky et al. 2005) and antimicrobial isoflavones desmodianones A, B and C have been isolated from Desmodium canum (Monache et al. 1996). Anthocyanins as products of the flavonoid metabolism are, for example responsible for the red to purple and blue colors of many fruits, vegetables, flowers, and cereal grains. In plants they serve as attractants for pollination and seed dispersal, give constitutive protection against the harmful effects of UV irradiation, and as phytoanticipins provide antiviral and antimicrobial activities in plants (Wrolstad 2000). Genotypes of Ipomoea purpurea with nonfunctional copies of chalcone synthase (CHS) received greater herbivore damage and twice the intensity of infection by the fungal pathogen Rhizoctonia solani than the wild type (Zufall and Rausher 2001).
Phenolic compounds like flavonoids strongly absorb UV light and thus are able to protect plants from DNA damage caused by UV. Anthocyanins belong to a class of flavonoids that accumulate in leaves and stems as plant sunscreen in response to light intensity (Leyva et al. 1995). Expression of CHS genes is known to be regulated by light through a photoreceptor-mediated mechanism (Koes et al. 1989). In several cases, it was found that the photoregulated production of flavonoids is at least in part due to the transcriptional induction of CHS (Chappell and Hahlbrock 1984; Feinbaum and Ausubel 1988; van Tunen et al.1988; Taylor and Briggs 1990). Examination of CHS expression in parsley cell culture suggested that a UV-B light receptor, a blue light receptor and phytochrome may all play a role in light-induced CHS expression (Bruns et al. 1986; Ohl et al. 1989).
High intensity light and UV-A were found to regulate expression of chimeric chalcone synthase genes in transgenic Arabidopsis thaliana plants (Feinbaum et al. 1991). High-intensity light treatment of A. thaliana plants for 24 h caused a 50-fold increase in CHS enzyme activity and an accumulation of visibly detectable levels of anthocyanin pigments in the vegetative structures of these plants (Feinbaum and Ausubel 1988). The expression of CHS genes was increased with time during a 24 h exposure to UV-A on swollen hypocotyls of the red turnip ‘Tsuda’ and induced anthocyanin accumulation (Zhou et al. 2007). The flavonoids accumulate in epidermal cells of the leaves and it is specifically in these cells that CHS gene expression is induced by light stimuli (Schmelzer et al. 1988). However, in mustard the expression of two CHS genes is induced coordinately in seedlings grown in a dark environment for 36–42 h, though this induction is enhanced by supplying red or far red light (Ehmann et al. 1991).
Auxin and jasmonic acid signaling
In plant increase of CHS activity causes a high accumulation flavonoid level that inhibit polar auxin transport (Brown et al. 2001; Faulkner and Rubery 1992; Jacobs and Rubery 1988). Inhibitors of auxin transport could increase the resistance of tomato plants to Fusarium oxysporum (Davis 1954). Also other research showed that CHS is expressed in the nodule primordium and later primarily in uninfected cells of the nodule apex in Rhizobium infected legumes. This may explain the induction of nodule on infected legume roots, higher accumulation of flavonoids blocks auxin transport, causing a local accumulation of auxin, a growth hormone, which caused the induction of nodule growth and development (Estabrook and Sengupta 1991; Yang et al.1992).
Jasmonic acid and its esters, such as methyl jasmonate (MeJA) are a group of plant hormones having a signaling role in insect and disease resistance (Xu et al. 1994). They could activate CHS in soybean and parsley cell cultures (Creelman et al. 1992) and Picea glauca (Richard et al. 2000). It is thought that volatile jasmonates are released from wounded tissue; thus elicitating plants to activate CHS which cause a production of phytoalexins in advance to resist an infection.
CHS is known as the key entry enzyme commited to the production of the polyketide phenylpropanoids in plants. It seems that all plants contain at least one CHS gene and often CHS gene families in plant with different expression patterns. In certain cases evolution into genes that encode enzymes with different substrate specificity, particularly for the starter molecule (e.g. aliphatic CoA ester instead of cinamic acid derivative) give different ring closure such as in stilbenes. The flavonoid pathway genes are highly diverted and have been found to be present from the earliest plants on land (the bryophytes, liverworts and hornworts) to the highly evolved flowering plants. Chalcones, flavonols and flavones were found in the earliest plants. Those flavonoids function as sunscreen protecting against UV radiation as plants began colonizing land and also play a regulation auxin transport (Markham, 1988; Shirley, 1996; Li et al. 1993; Brown et al. 2001). Later stage of plants such as the ferns and allies are known as oldest group of plants producing proanthocyanidins, procyanidin, prodelphinidin and flavanols. Anthocyanidin, a flavonoid, plays an important role in plant pigment action and is found in gymnosperms and angiosperms. These flavonoids serve diverse functions in different plant species, e.g. as pigments, phytoalexins, UV protectants, signal molecules in plant-microbe interactions, antioxidants, and pollinator attractants or feeding deterrents. In other words these unique plant compounds play a major role in the interaction of plants with their environment (De Bruyne et al. 1999; Kong et al. 2003: Marles et al. 2003; Yilmaz and Toledo 2004).
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