INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Flavonoids are important plant secondary metabolites that have several functions, including as pigments, signaling molecules, protectants against biotic and abiotic stresses, and in fertility (Dixon and Paiva, 1995; Weisshaar and Jenkins, 1998; Winkel-Shirley, 2001). Flavonoid biosynthesis is regulated both spatially and temporally in plant development and is induced by a variety of environmental and endogenous stimuli, including light, pathogen attack, several abiotic stresses, metabolites, and plant growth regulators (Dixon and Paiva, 1995; Mol et al., 1996). Chalcone synthase (CHS), which catalyzes the first committed step in the flavonoid pathway, has become a focus of research to understand the regulation of flavonoid biosynthesis. The signaling pathways regulating CHS gene expression interact, for instance, synergistically (Fuglevand et al., 1996) or negatively (Lozoya et al., 1991). Such interactions permit the integration of responses to different stimuli (Trewavas and Malhó, 1997; Jenkins, 1999). Hence, to understand the regulation of CHS expression and flavonoid biosynthesis, it is important to identify regulators of the relevant signaling pathways. The application of a genetic approach is key to progress in this field.

Several classes of mutants altered in flavonoid biosynthesis have been identified, principally in maize (Zea mays), petunia (Petunia hybrida), Antirrhinum majus, and Arabidopsis. One class, including many of the tt (Arabidopsis transparent testa) mutants, is deficient in particular enzymes of the flavonoid biosynthesis pathway (Shirley et al., 1995; Winkel-Shirley, 2001). Other mutants are regulatory, and include several defective in transcription factors that regulate particular structural genes of the pathway. Among these are the maize R/B and C1/Pl transcription factors, Petunia AN2 and JAF13 and A. majus DELILA (Mol et al., 1996, 1998). In addition, Arabidopsis TT2 (Nesi et al., 2001), TT8 (Nesi et al., 2000), and HY5 (Ang et al., 1998) encode transcription factors that regulate steps in flavonoid biosynthesis. The genetic identification of transcription factors regulating flavonoid biosynthesis genes has proceeded in parallel with research on the promoters of the structural genes, which has identified regulatory sequence elements and transcription factors that interact with them (e.g. in parsley [Petroselinum crispum]; Feldbrügge et al., 1994, 1997).

Some regulatory flavonoid biosynthesis mutants are not altered in transcription factors. For instance, Arabidopsis ttg1 (transparent testa glabra1; Walker et al., 1999) and petunia an11 (de Vetten et al., 1997) identify WD-40 repeat proteins that appear to regulate the action of specific transcription factors. A further class of regulators of flavonoid biosynthesis genes was identified through characterization of the Arabidopsis det, cop, and fus mutants. These have a constitutive photomorphogenic phenotype in darkness, including hypocotyl growth suppression, cotyledon expansion, anthocyanin accumulation, and the expression of normally light-induced genes such as CHS and several encoding photosynthetic proteins (Hardtke and Deng, 2000). A number of the COP/DET/FUS proteins identified to date are nuclear localized and function as downstream components of several signaling pathways.

It should be emphasized that flavonoid biosynthesis is subject to tissue-specific control. Some mutants altered in flavonoid biosynthesis are also altered in development, particularly in development of epidermal tissues in the seed, leaf, and/or root. Examples include ttg1 (Walker et al., 1999), anl2 (anthocyaninless2; Kubo et al., 1999), and tt1 (Sagasser et al., 2002). TTG1 is involved in the control of both condensed tannin and anthocyanin accumulation in seeds and leaves and the normal production of leaf trichomes and root hairs. ANL2 encodes a homeodomain protein whose principle role appears to be in the specification of subepidermal cells in the leaf and root; mutation causes altered root cellular organization and altered anthocyanin accumulation in the leaf. TT1 is involved specifically in controlling differentiation of the seed endothelial cells that accumulate condensed tannins; mutant seeds fail to accumulate these pigments. Therefore, it becomes difficult to know whether a given regulator controls flavonoid biosynthesis and cellular differentiation independently or whether flavonoid biosynthesis is affected as a consequence of altered development. Clearly, the two processes are intimately associated.

We isolated the Arabidopsis icx1 (increased chalcone synthase expression 1) mutant by using a transgene expression screen (Jackson et al., 1995). M₂ plants derived from a transgenic line containing a CHS promoter--glucuronidase (GUS) fusion were screened for altered GUS activity in the light. icx1 had an enhanced response to white light in the induction of both GUS and endogenous CHS transcript levels, but had very low GUS activity and CHS expression in darkness, like the wild type (Jackson et al., 1995). In addition, transcript levels of other flavonoid biosynthesis genes were elevated in the light and there was a 2- to 3-fold increase in anthocyanin induction (Jackson et al., 1995). Therefore, the ICX1 gene product functions as a negative regulator, constraining the light induction of flavonoid biosynthesis genes in the wild type. A further feature of the icx1 mutant is that it has a pleiotropic visible phenotype: It has altered leaf shape, fewer trichomes, altered leaf epidermal morphology, and abnormal root development (Jackson et al., 1995; J.A. Jackson, R.A. Brown, and G.I. Jenkins, unpublished data). These aspects of the phenotype of icx1 can be explained in terms of altered epidermal development. Moreover, because CHS is predominantly expressed in the epidermal layers in mature leaves (Schmelzer et al., 1988; Chory and Peto, 1990), we hypothesized that ICX1 is a regulator of several aspects of epidermal gene expression and development.

Our aim in the present study was to determine the extent to which ICX1 regulates gene expression. Through detailed characterization of the icx1 mutant, we show that the ICX1 gene product is a negative regulator of several pathways regulating CHS expression. We further show that ICX1 acts in both seedlings and mature leaf tissue and that its action in the epidermis is not confined to flavonoid biosynthesis genes.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

ICX1 Is a Negative Regulator of the Cryptochrome1 and UV-B Induction of CHS

The initial characterization of icx1 involved measurements of gene expression in white light (Jackson et al., 1995). In the present study, we wanted to determine whether ICX1 acts as a negative regulator of one or more specific phototransduction pathways regulating flavonoid biosynthesis genes. In mature leaf tissue in wild-type Arabidopsis, CHS is induced by distinct UV-B and UV-A/blue photoperception systems; there is no induction by red or far-red light (Fuglevand et al., 1996; Wade et al., 2001). Cryptochrome 1 (cry1) mediates CHS induction by UV-A light and most of the response to blue light (Fuglevand et al., 1996). The detection system for UV-B is unknown, but is not cry1 or cry2 (Wade et al., 2001).

Therefore, we examined CHS expression in icx1 in different light qualities in comparison with wild type. In these experiments, as in our previous studies (Jackson et al., 1995; Fuglevand et al., 1996; Wade et al., 2001), plants were grown for 3 weeks in a low fluence rate of white light (20 µmol m² s¹) that does not significantly induce CHS transcript accumulation. Plants were then transferred to inductive light treatments. Time courses, shown in Figure 1, A and B, reveal that icx1 accumulates higher levels of CHS transcripts in mature leaf tissue in both UV-A (cry1 activation) and UV-B light than wild type. In UV-A light, CHS transcripts increase after a few hours and reach a maximal level about 12 h after illumination before declining. In UV-B, a more rapid initial increase and earlier peak in transcript level is seen. icx1 shows similar kinetics to the wild-type in both light qualities, but a greater maximal level of induction. We conclude that ICX1 is a negative regulator of both the cry1 and UV-B inductive pathways regulating CHS in mature leaf tissue.

View larger version (69K): [in this window] [in a new window]   Figure 1.   ICX1 is a negative regulator of the cry1 and UV-B induction of CHS expression in mature Arabidopsis leaf tissue. A, icx1 shows enhanced cry1 mediated induction of CHS. Wild-type and icx1 plants were grown for 3 weeks in 20 µmol m2 s1 white light and then transferred to 80 µmol m2 s1 UV-A light. Leaf tissue was harvested at the times indicated and CHS and TUB (-TUBULIN) transcript levels were measured by sequential hybridization of DNA probes to blots of total RNA. B, icx1 shows enhanced UV-B induction of CHS. Plants were grown as above and transferred to 3.0 µmol m2 s1 UV-B light for the times indicated. CHS transcript levels were measured as in A; rRNA stained with ethidium bromide is shown as a control.

Jackson et al. (1995) reported that transcript levels of genes encoding other flavonoid biosynthesis enzymes were elevated in white light in icx1. We observed increases in PAL, CHI, and DFR transcript levels in UV-A and UV-B light qualities in icx1, along with increased levels of anthocyanin and flavonols (data not shown). Therefore, ICX1 does not regulate CHS alone.

To determine whether ICX1 action is confined to mature leaves, we initially examined the UV-B and cry1 mediated induction of CHS in seedlings. As reported by other workers (Feinbaum et al., 1991; Kubasek et al., 1992; Kaiser et al., 1995), dark-grown Arabidopsis seedlings induce CHS in response to UV-B and UV-A/blue light. However, we found little difference between icx1 and wild-type seedlings in the increase in CHS transcript levels in response to both light qualities, as shown in Figure 2. Nevertheless, ICX1 does act in seedlings as shown in the experiments described below.

View larger version (74K): [in this window] [in a new window]   Figure 2.   ICX1 has no significant effect on the cry1 and UV-B induction of CHS in seedlings. Wild-type and icx1 plants were grown in darkness for 4 d and then illuminated with either 100 µmol m2 s1 UV-A/blue or 3.5 µmol m2 s1 UV-B light. Seedlings were harvested at the times indicated and CHS transcript levels were measured by hybridization of a DNA probe to blots of total RNA. rRNA stained with ethidium bromide is shown as a control.

ICX1 Is a Negative Regulator of the Phytochrome Induction of CHS in Seedlings

We wished to test whether the function of ICX1 was restricted to the UV/blue phototransduction pathways. To examine the phytochrome induction of CHS we used very young dark-grown seedlings. This phytochrome response is confined to seedlings less than 6 d old (Kaiser et al., 1995). As shown in Figure 3, in 4 d-old dark-grown seedlings the red light induction of CHS transcripts is minimal, but there is strong induction by far-red light. Both responses are mediated by phytochrome A (Barnes et al., 1996). icx1 shows a marked increase in the far-red light induction of CHS compared with wild type. The kinetics of the response were unaltered. Thus, ICX1 is also a negative regulator of the phytochrome A induction of CHS in seedlings.

View larger version (99K): [in this window] [in a new window]   Figure 3.   ICX1 is a negative regulator of the phytochrome induction of CHS in seedlings. Wild-type and icx1 plants were grown in darkness for 4 d and then illuminated with either 100 µmol m2 s1 red or 75 µmol m2 s1 far-red light. Seedlings were harvested at the times indicated and CHS transcript levels were measured by hybridization of a DNA probe to blots of total RNA; rRNA stained with ethidium bromide is shown as a control.

icx1 Does Not Show Altered Expression of CHS When UV-B Induction Is Enhanced Synergistically by Blue or UV-A Light

The induction of CHS in UV-B light is further increased by synergistic interactions with separate blue and UV-A light signaling pathways (Fuglevand et al., 1996). These synergism-specific pathways are not dependent on cry1 or cry2 (Wade et al., 2001). As shown in Figure 4A, icx1 retains the synergistic increases in CHS transcript levels under both UV-B plus blue light and UV-B plus UV-A light. Interestingly, we repeatedly observed that the CHS transcript levels in icx1 and wild type were identical under synergistic treatments, which is in contrast to inductive conditions. Moreover, the kinetics of CHS transcript accumulation under synergistic conditions were the same in the wild type and mutant, as shown in Figure 4B.

View larger version (65K): [in this window] [in a new window]   Figure 4.   icx1 does not have altered levels of CHS expression in response to the synergistic light treatments UV-A plus UV-B and blue plus UV-B. A, icx1 retains the synergistic increases in CHS expression. Wild-type and icx1 plants were grown for 3 weeks in 20 µmol m2 s1 white light (LW) and then transferred to either 87 µmol m2 s1 UV-A, 87 µmol m2 s1 blue, or 3.0 µmol m2 s1 UV-B light, or to UV-B plus blue or UV-B plus UV-A light at the same fluence rates. Leaf tissue was harvested after 6 h and CHS transcript levels were measured by hybridization of a DNA probe to a blot of total RNA; rRNA stained with ethidium bromide is shown as a control. Note: The differences between icx1 and wild-type in blue, UV-A, and UV-B cannot be seen in this autoradiograph without overexposing the lanes showing synergistic increases in CHS expression. B, CHS transcript accumulation under synergistic conditions is unaltered in icx1. Plants grown as above were transferred to 3.2 µmol m2 s1 UV-B plus either 80 µmol m2 s1 UV-A or 60 µmol m2 s1 blue light. Leaf tissue was harvested at the times indicated and CHS and TUB transcript levels measured by sequential hybridization of DNA probes to blots of total RNA.

One way of interpreting the synergistic interactions is that blue and UV-A light cause the removal of specific negative regulators associated with the UV-B signaling pathway inducing CHS. Because ICX1 is a negative regulator of CHS expression, it is possible that it encodes a component removed by synergistic light treatments in the wild type. This would explain why the wild-type and icx1 mutant have an identical level of expression under synergistic conditions. However, ICX1 cannot be the only target negative regulator removed in a particular synergistic interaction (UV-B and blue light or UV-B and UV-A light), otherwise the mutant would not be expected to show the observed synergistic increases in expression under those particular conditions. Therefore, we suggest that ICX1 is one of several negative regulators removed under synergistic light treatments to produce the elevated level of expression seen in the wild type.

ICX1 Regulates Expression of an Epidermally Expressed LTP Gene, But Not rbcS and CAB, in Photosynthetic Tissues

CHS expression is largely confined to the epidermis in mature leaf tissue (Schmelzer et al., 1988; Chory and Peto, 1990). Therefore, the question arises as to whether ICX1 functions specifically in the epidermis to regulate flavonoid biosynthesis genes and possibly other genes, or whether it is likely to function in a range of tissues to control a variety of genes. To address this question we examined the expression of three gene families unrelated to flavonoid biosynthesis, one epidermally expressed and the others expressed in photosynthetic tissue.

Genes encoding nonspecific lipid transfer proteins (LTPs) are principally expressed in the epidermis in mature leaf tissue in Arabidopsis (Thoma et al., 1994) and other species (Kader, 1996). We recently found that LTP transcript accumulation in Arabidopsis and Brassica napus is stimulated by red and blue light, but not by UV-B (Sohal et al., 1999). When we examined LTP1 transcript levels in icx1, we observed that the mutant had elevated induction under blue light compared with wild type, as shown in Figure 5A. Similar results were seen in red light (not shown) but no induction was seen in UV-B. This indicates that ICX1 functions in the epidermis to regulate at least one class of genes unrelated to flavonoid biosynthesis. Because icx1 shows several alterations in epidermal development throughout the plant (Jackson et al., 1995; R.A. Brown, J.A. Jackson, and G.I. Jenkins, unpublished data), we hypothesize that the mutant may be altered in the expression of a range of epidermally expressed genes.

View larger version (78K): [in this window] [in a new window]   Figure 5.   icx1 has elevated expression of epidermally expressed LTP transcripts but unaltered expression of transcripts expressed in photosynthetic tissues. A, icx1 has elevated light-induction of epidermally expressed LTP1 transcripts but is not altered in the regulation of CAB expression by blue and UV-B light. Wild-type and icx1 plants were grown for 3 weeks in 20 µmol m2 s1 white light (LW) and then transferred to 100 µmol m2 s1 blue or 3.5 µmol m2 s1 UV-B light. Leaf tissue was harvested after 6 h and LTP1, CAB, and TUB transcript levels measured by sequential hybridization of DNA probes to blots of total RNA. B, icx1 is not altered in the regulation of rbcS expression by UV-A light. Plants grown as above were transferred to 80 µmol m2 s1 UV-A or 45 µmol m2 s1 red light for 6 h. Leaf tissue was harvested and rbcS and TUB transcript levels measured as above.

Jackson et al. (1995) reported that icx1 was unaltered in the expression of CAB transcripts in white light. CAB genes, or lhcb1 genes, encode the major light-harvesting chlorophyll protein of thylakoid membranes. We extended our previous observations by examining the levels of CAB transcripts in response to regulatory light treatments. In addition, we examined the expression of rbcS genes, which encode the small subunit of Rubisco. Both rbcS and CAB genes are expressed predominantly in leaf photosynthetic tissues rather than epidermal tissue. Figure 5A shows that blue light reduces the level of CAB transcripts relative to that in low-fluence rate white light in the wild type. This is most likely a response to maximize light capture under limiting conditions. UV-B also reduces CAB expression, as reported by others (Jordan et al., 1994). As shown in Figure 5B, rbcS transcript levels are stimulated by UV-A light, consistent with previous reports (Sawbridge et al., 1994). This response permits increased accumulation of Rubisco under conditions favorable for carbon fixation. In each of the CAB and rbcS responses we examined, icx1 behaves identically to the wild type.

ICX1 Is a Negative Regulator of Non-Light Pathways Regulating CHS and Anthocyanin Accumulation

Because CHS expression and anthocyanin biosynthesis are regulated by a range of environmental and endogenous (metabolic and hormonal) factors, we investigated whether the function of ICX1 was restricted to light responses. Exposure to low temperature stimulates CHS expression provided that the plants are exposed to light (Leyva et al., 1995). To determine whether the mutant was altered in this response, we transferred icx1 and wild-type plants from 21°C to 7°C. They were kept in 20 µmol m² s¹ white light, which on its own is insufficient to induce CHS. Under these conditions, CHS transcripts increase in the wild type in response to cold within a few hours, as shown in Figure 6, and are readily detectable after 8 h. In icx1, the level of CHS induction over the same time course is much greater. Therefore, we conclude that ICX1 is a negative regulator of the low-temperature induction of CHS.

View larger version (86K): [in this window] [in a new window]   Figure 6.   ICX1 is a negative regulator of the low-temperature induction of CHS expression. Wild-type and icx1 plants were grown for 3 weeks in 20 µmol m2 s1 white light at 21°C and then transferred to 7°C in the same light conditions. Leaf tissue was harvested at the times indicated and CHS and TUB transcript levels measured by sequential hybridization of DNA probes to a blot of total RNA.

In addition, we examined whether icx1 is altered in responses to endogenous signals by measuring anthocyanin accumulation. Suc is reported to stimulate anthocyanin accumulation and CHS expression in Arabidopsis (Tsukaya et al., 1991; Mita et al., 1997) and other species (Urwin and Jenkins, 1997; Chalker-Scott, 1999). To test the response of icx1 to Suc, we grew plants in 100 µmol m² s¹ white light on agar plates containing either 0% or 2% (w/v) Suc. As shown in Figure 7A, anthocyanin accumulation is much greater in plants grown in the presence of Suc than in its absence. In fact, the action of Suc and light is synergistic (see Fig. 7B for the response to Suc in darkness). icx1 shows greater anthocyanin accumulation than the wild type, indicating that ICX1 does negatively regulate the response to Suc. The elevated response to Suc in icx1 was also observed at the level of CHS transcripts (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 7.   ICX1 is a negative regulator of the Suc and cytokinin induction of anthocyanin accumulation. A, icx1 has enhanced anthocyanin accumulation in response to Suc. Wild-type and icx1 seedlings were germinated on agar containing 0% (S) or 2% (+S; w/v) Suc and grown in 100 µmol m2 s1 white light for 4 d. Anthocyanin was measured per unit fresh weight of tissue ± SE. B, icx1 has enhanced anthocyanin accumulation in darkness in response to cytokinin. Wild-type and icx1 seedlings were grown in darkness on agar containing 2% (w/v) Suc and varying concentrations of kinetin for 7 d. Anthocyanin was measured per unit fresh weight of tissue ± SE.

In the above experiments the responses to cold and Suc were tested in the presence of light. Hence, we wanted to be sure that the enhanced responsiveness of icx1 to non-light stimuli was genuine and not the product of enhanced responsiveness to light. Therefore, we decided to examine the effects of an inductive stimulus in darkness and chose to measure anthocyanin accumulation after the addition of cytokinin. Cytokinin stimulates CHS expression and anthocyanin accumulation in Arabidopsis (Deikman and Hammer, 1995) and is reported to partly phenocopy the det1 mutant phenotype in darkness (Chory et al., 1991). We grew seedlings in darkness on media containing 2% (w/v) Suc and cytokinin at various concentrations. As shown in Figure 7B, cytokinin had no significant effect on anthocyanin accumulation in wild-type seedlings, whereas icx1 showed a strong increase in response to cytokinin. This demonstrates, firstly, that ICX1 acts as a negative regulator of the cytokinin induction of anthocyanin accumulation and, secondly, that light is not essential to observe a hyper-responsive phenotype in the mutant.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The data presented show that ICX1, either directly or indirectly, negatively regulates multiple pathways inducing flavonoid biosynthesis gene expression in Arabidopsis: light induction mediated by UV-B, cry1, and phyA, and non-light responses to environmental (cold), metabolic (Suc), and hormonal (cytokinin) stimuli. Moreover, ICX1 acts in both seedlings and mature leaf tissue. To our knowledge, no other regulator of flavonoid biosynthesis has been shown to act as ubiquitously. Though most of our data relate to CHS expression, it is evident that icx1 has elevated transcript levels of other phenylpropanoid/flavonoid biosynthesis genes as well as increased accumulation of anthocyanin and other flavonoids in response to various light and non-light stimuli (Jackson et al., 1995; Wade, 1999). However, the action of ICX1 is not confined to flavonoid biosynthesis genes because we have shown that LTP1 transcripts are elevated in icx1 in response to specific light treatments and the icx1 mutant has a visible phenotype (Jackson et al., 1995). Hence, ICX1 is involved in several aspects of epidermal gene expression and development.

It is interesting that the icx1 mutant does not show constitutive expression of CHS or anthocyanin accumulation. The amplitude of the response to particular stimuli increases, not the basal level of response. This amplification occurs with little or no alteration of the kinetics of the response (see e.g. Fig. 1). Also, there is no evidence of ectopic acquisition of responses in the mutant; for instance, mature leaves still lack the red and far-red light induction of CHS found in young seedlings (Wade, 1999; data not shown). It is difficult in some responses, such as the cold induction of CHS, which requires light (Leyva et al., 1995) to examine separately the action of ICX1 on non-light and light pathways. However, the measurements of anthocyanin accumulation in response to cytokinin in darkness demonstrate that ICX1 is unequivocally a negative regulator of non-light as well as light responses.

Our findings indicate that ICX1 is a novel regulator of flavonoid biosynthesis. First, most such regulators reported to date are positive rather than negative regulators, whether transcription factors or components that modulate the activity of transcription factors. Second, most of the reported regulators control specific subsets of genes. For example, Arabidopsis TTG1, TT2, and TT8 control later steps in flavonoid biosynthesis, from DFR (Nesi et al., 2000); in maize, C1 and Pl control CHS and later genes (Mol et al., 1998). ICX1 is unusual in affecting multiple steps in the pathway, including PAL, CHS, and DFR. The icx1 phenotype has some similarity with the consequences of overexpression of Arabidopsis ATMYB75 (PAP1), which greatly elevates PAL, CHS, DFR, and other transcripts and causes gross overaccumulation of flavonoids (Borevitz et al., 2000).

The best characterized negative regulators of flavonoid biosynthesis genes are the COP/DET/FUS genes. However, icx1 is different from cop/det/fus mutants in several important respects and, therefore, is not a weak cop/det/fus allele. First, cop/det/fus mutants have increased expression of flavonoid biosynthesis genes and anthocyanin accumulation in darkness, whereas icx1 does not. Second, the cop/det/fus mutants show constitutive photomorphogenesis, whereas icx1 in darkness has an etiolated hypocotyl, unexpanded cotyledons, and an apical hook; the hypocotyl is shorter than wild type in darkness, but this is probably because of slow growth resulting from impaired root development. Third, icx1 has other visible phenotypic characteristics (e.g. altered leaf shape) that are absent in the cop/det/fus mutants. Fourth, in contrast to some cop/det/fus mutants, icx1 is not epistatic to photoreceptor mutants such as cry1 (Wade, 1999). Fifth, some cop/det/fus mutants show ectopic spatial expression of CHS, CAB, and other genes; for example, det1 has CHS expression in leaf mesophyll tissue in addition to the epidermis (Chory and Peto, 1990). In contrast, preliminary experiments with plants expressing a CHS promoter-GUS fusion indicate that the epidermal location of expression is similar in icx1 and the wild type (Wade, 1999; data not shown). Finally, the map position of ICX1 (around position 100 on chromosome 1; R.A. Brown and G.I. Jenkins, unpublished data) does not correspond to any known COP/DET/FUS locus.

Other mutants with superficial similarity to icx1 are the tomato (Lycopersicon esculentum) hp-1 and hp-2 (high pigment), ip (intense pigmentation), and atv (atroviolacea) mutants (Kendrick et al., 1997). All have elevated anthocyanin accumulation in response to light compared with wild type and, therefore, are altered in negative regulators. The HP and ATV genes are proposed to encode negative regulators of phytochrome signaling, whereas IP is suggested to be specific to blue light signaling. Hence, they appear more specific than ICX1 in their action. HP-2 was recently shown to encode a DET1 homolog, although significant differences in the phenotypes of tomato hp-2 and Arabidopsis det1 suggest that the proteins function somewhat differently in the two species (Mustilli et al., 1999). The Arabidopsis anl2 mutant is altered in leaf anthocyanin accumulation and root development (Kubo et al., 1999), traits also affected in icx1 (Jackson et al., 1995). However, icx1 is altered in additional aspects of epidermal development, has elevated rather than reduced levels of anthocyanin, and, again, ICX1 does not map to the same region as ANL2 or any other homeodomain protein.

On the basis of our data, we predict that ICX1 acts principally, if not entirely, in epidermal tissues. This is consistent with the observed effects on flavonoid biosynthesis gene and LTP1 gene expression in the mutant and the lack of effect on rbcS and CAB expression. Moreover, the visible phenotype of the mutant (including altered epidermal cell division/expansion, trichome number, leaf shape, root elongation, and root hair initiation; Jackson et al., 1995; J.A. Jackson, R.A. Brown, and G.I. Jenkins, unpublished data) can be explained in terms of altered epidermal development, probably as a consequence of altered gene expression. Of course, it is possible that ICX1 is a selective regulator of nonepidermally expressed genes, and that CAB and rbcS are two genes that it does not control. In addition, it is possible that ICX1 affects particular UV-B, UV-A, and blue light signaling pathways that do not regulate CAB and rbcS expression. Nevertheless, our data are consistent with the hypothesis that ICX1 acts in the epidermis to regulate the expression of different classes of genes and it is possible that it is an epidermis-specific regulator.

Because ICX1 is a negative regulator of diverse pathways regulating flavonoid biosynthesis genes, it may encode a common component of the pathways or indirectly affect the expression or activity of one or more common components. Present evidence supports the latter interpretation. A common component would most likely function downstream in the different pathways to directly affect transcription of the relevant genes. Most of the known regulators of flavonoid biosynthesis genes are either transcription factors or components that modulate the activity of transcription factors (see above). Our present map data indicate that ICX1 is unlikely to encode a transcription factor (R.A. Brown and G.I. Jenkins, unpublished data). In addition, we have genetic evidence that ICX1 functions upstream of the basic Leu zipper transcription factor HY5 that is involved in regulating the CHS promoter (Ang et al., 1998); a hy5 icx1 double mutant lacks CHS induction (H.K. Wade and G.I. Jenkins, unpublished data). Hence, we hypothesize that ICX1 acts some distance upstream of transcription factors that directly regulate flavonoid biosynthesis genes. TTG1 in Arabidopsis (Walker et al., 1999) and AN11 in petunia (de Vetten et al., 1997) are reported to modulate the activity of relevant transcription factors, but we do not know yet if ICX1 acts directly to regulate transcription factor expression or activity or functions further upstream. There are some similarities between icx1 and ttg1 in the pleiotropic nature of the phenotype, but icx1 has increased flavonoid biosynthesis gene expression, whereas ttg1 lacks expression of the late biosynthetic genes.

We do not know if ICX1 regulates genes concerned with epidermal development and flavonoid biosynthesis independently. It is possible that the alterations in flavonoid biosynthesis gene expression and LTP1 expression seen in icx1 are a consequence of altered epidermal cell differentiation. Similar conclusions have been drawn, for instance, for the action of ANL2 in leaf subepidermal cells (Kubo et al., 1999) and TT1 in the seed endothelium (Sagasser et al., 2002). In these examples, the prime function of the gene may be to specify the cell type and, hence, the alteration in flavonoid biosynthesis may be secondary. Certainly the phenotype of icx1 emphasizes the link between epidermal development and flavonoid biosynthesis. However, in icx1, the mutation amplifies rather than reduces flavonoid biosynthesis gene expression and it is difficult to envisage how a positive regulator of cell identity would achieve this effect. Moreover, the altered gene expression in icx1 does not appear to reflect an increase in the proportion of leaf cells expressing flavonoid biosynthesis genes, based on preliminary studies of the spatial distribution of GUS expression driven by the CHS promoter (Wade, 1999). In any case, such an increase would have to be substantial to account for the severalfold elevation of CHS expression seen in icx1 in response to some stimuli. Furthermore, an alteration in the number of cells expressing CHS would not explain why the mutant has increased CHS expression in response to some stimuli but not others; for example, in seedlings we found little change in UV-B and UV-A/blue light induction but large changes in far-red and Suc induction, and in leaves, we found no change in synergistic light induction but increases in UV-B, UV-A/blue, and low-temperature induction.

In conclusion, this study shows that ICX1 is a novel negative regulator of diverse pathways inducing flavonoid biosynthesis gene expression in Arabidopsis. Further information about the nature and function of the gene product will come from map-based cloning of ICX1, which has been initiated.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Material

Seeds of wild-type Arabidopsis ecotype Landsberg erecta originated from Dr. Caroline Dean (John Innes Centre, Norwich, UK). Seeds of icx1 (Jackson et al., 1995) were obtained by selfing a mutant line backcrossed twice to wild type. Seeds were sown on compost and placed in darkness for 3 d at 7°C before transfer to 21°C. Plants were grown routinely in a low fluence rate (20 µmol m² s¹) of white light for 21 d before experimental treatment.

Illuminations were carried out in controlled environment rooms at 21°C. Plants were exposed to either single light treatments or, in the case of synergism experiments, to two light qualities simultaneously. The white, UV-B, UV-A, blue, red, and far-red light sources were described by Wade et al. (2001). Fluence rates and spectral qualities were measured with a spectroradiometer (model SR9910, Macam Photometrics, Livingston, UK).

Plants used for cold treatment (Fig. 6) were grown for 21 d as above before transfer to 20 µmol m² s¹ white light at 7°C for the times indicated.

Seedlings were grown on 0.8% (w/v) agar plates containing 1× Murashige and Skoog salts and 1× B5 vitamins. Suc (2% [w/v]) was added to the plates (except for -Suc). Cytokinin was added to a final concentration of either 0, 0.08, 0.5, or 2 mg L¹. Seeds were surface sterilized by a 2-min wash in 70% (v/v) ethanol, followed by a 10-min immersion in a bleach solution (10% [v/v] sodium hypochlorite and 0.01% [v/v] Triton X-100). Seeds were then washed five times with sterile distilled water. The seeds were then vernalized for 3 d (7°C). Seedlings used in the experiments in Figures 2 and 3 were grown in darkness at 21°C for 4 d after the cold treatment.

RNA Isolation and Hybridization Analysis

Samples of leaf tissue or whole seedlings were harvested into liquid nitrogen, ground with a mortar and pestle, and RNA extracted as described by Wade et al. (2001). RNA (5 or 10 µg per lane) was fractionated in 1.3% (w/v) agarose/formaldehyde gels and blotted onto nylon membrane using standard techniques (Sambrook et al., 1989). CHS transcripts were measured by hybridization of blots to a radioactively labeled homologous probe as described by Wade et al. (2001). CAB (Leutwiler et al., 1986) and rbcS (generously provided by Michael Timko, University of Virginia, Charlottesville) probes were used to measure transcript levels. Arabidopsis LTP1 cDNA (Thoma et al., 1994) was obtained from the Arabidopsis Biological Resources Centre (Columbus, OH) and hybridized to filters as described by Sohal et al. (1999). After washing and autoradiography, filters were stripped of the probe and rehybridized to the cDNA insert from pcf4-2 encoding a Chlamydomonas reinhardtii -tubulin (Silflow et al., 1985); in some cases, two hybridizing bands were seen, although both appeared constitutive. In some experiments, rRNA bands visualized using ethidium bromide were used as a loading control; the 25S band is shown as a positive image.

Anthocyanin Measurement

Whole seedlings were harvested into microcentrifuge tubes in approximately 100-mg batches and fresh weight measurements were taken. Each sample was then homogenized in 1% (v/v) HCl in methanol. The tissue was shaken at 4°C overnight before a chloroform extraction. The supernatant absorbance was quantified spectrophotometrically (A₅₃₀-A₆₅₇) and anthocyanin calculated per gram fresh weight.

Reproducibility of Experiments

All experiments were repeated at least three times. The results obtained in repeated experiments followed the same trend and representative results from individual experiments are presented.