lnduction of Anthocyanin Accumulation by Cytokinins A ... - NCBI

Plant Physiol. (1995) 108: 47-57

lnduction of Anthocyanin Accumulation by Cytokinins in A rabidopsis thaliana' Jill

D e i k m a n * and

Philip E.

Hammer2

Department of Biology and Biotechnology Institute, Pennsylvania State University, University Park, Pennsylvania 16802

Cytokinin-regulated genes are often controlled by factors in addition to cytokinins. For example, mRNAs that accumulate within 4 h of addition of cytokinin to a cytokininstarved soybean suspension culture are also induced by auxin in auxin-starved cells (Crowell et al., 1990). The nitrate reductase gene is controlled by light and nitrate as well as by cytokinins (Chen, 1989). Cytokinins and light interact to influence chloroplast development and associated gene expression (Chen, 1989). The complexity of cytokinin control of gene expression is also illustrated by the fact that cytokinins act at different steps. For example, cytokinin control of cab and rbcS genes in Lemna gibba was shown to be primarily at a posttranscriptional level (Flores and Tobin, 1986), whereas cytokinins induce nitrate reductase gene transcription (Lu et al., 1990). Defining the molecular details of control of gene expression by cytokinins has potential for helping to elucidate the elements involved in cytokinin signal transduction. One response many plants have to cytokinin treatment is to accumulate anthocyanins. Cytokinins have been shown to cause an increase in anthocyanin accumulation in tissue culture and in parts of intact plants. For example, anthocyanin accumulation in response to cytokinins was shown in carrot (Daucus carota) suspension culture cells (Ozeki and Komamine, 1981), callus of D. carota and Heliantkus tuberosus (Ibrahim et al., 19711, and cultures of Haplopappus grucilis (Constabel et al., 1971), Rosa multiflora, and Malus pumila (Ibrahim et al., 1971). In intact plants, cytokinins have been shown to promote anthocyanin synthesis in petals of Impatiens balsamina (Klein and Hagen, 1961) and in Rosa kybrida (Nakamura et al., 1980) and Brassica oleracea seedlings (Pecket and Bassim, 1974). Anthocyanins are pigmented flavonoids that are responsible for most of the red, pink, purple, and blue colors found in plants (Taiz and Zeiger, 1991). They function to attract animals for pollination and seed dispersal, and they are believed to protect plant cells from UV radiation. Related flavonoids serve as antibiotics against microbial pathogens and as insect repellents and are involved in signaling in plant-microbe and pollen-pistil interactions (Hahlbrock and Scheel, 1989; Mo et al., 1992). Accumulation of flavonoids, including anthocyanins, is stimulated by various environmental stresses including UV light, high-

Arabidopsis thaliana plants treated with exogenous cytokinins accumulate anthocyanin pigments. We have characterized this response because it is potentially useful as a genetic marker for cytokinin responsiveness. Levels of mRNAs for four genes of the anthocyanin biosynthesis pathway, phenylalanine ammonia lyase 1 (PAL1), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) were shown to increase coordinately in response to benzyladenine (BA). However, nuclear run-on transcription experimentssuggested that although CHS and DFR are controlled by BA at the transcriptional level, PALl and CHI are controlled by BA posttranscriptionally.CHS mRNA levels increased within 2 h of BA spray application, and peaked by 3 h. Levels of PALl mRNA did not increase within 6 h of BA spray. We also showed that PAL1, CHS, CHI, and DFR mRNA levels fluctuate during a 24-h period and appear to be controlled by a circadian clock. The relation between cytokinin regulation and light regulation of CHS gene transcription is discussed.

Cytokinins are important regulators of many aspects of plant development, including cell division, nutrient mobilization, senescence, chloroplast development, and apical dominance (Binns, 1994). Despite the widely acknowledged importance of cytokinin in plant development, very little is known about its mechanism of action at the molecular level. Cytokinins have been shown to affect the expression of specific genes by both increasing and decreasing the abunA partia1 list of dance of particular proteins or "As. genes that are up-regulated by cytokinin includes nitrate reductase, rbcS, cab, hydroxypyruvate reductase in excised pumpkin cotyledons, a "multiple stimulus response" gene in Nicotiana plumbaginifolia cells, the early nodulin gene SrEnod2 from Sesbania rostrata, and a number of unidentified cDNAs from cultured soybean cells (Flores and Tobin, 1986; Chen, 1989; Crowell et al., 1990; Stabel et al., 1990; Dehio and de Bruijn, 1992; Dominov et al., 1992). Expression of phytochrome and a cucumber gene encoding a cDNA called CR9 are repressed by cytokinin (Cotton et al., 1990; Teramoto et al., 1994). ~

~

' This work was funded by National Science Foundation grant No. IBN-9210221 and by the Pennsylvania State University. Present address: Ciba Geigy Corporation, P.O. Box 12257, Research Triangle Park, NC 27709-2257. * Corresponding author; e-mail jxdl40psuvm.psu.edu; fax: 1-814-865-9131.

Abbreviations: CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol reductase; PAL, phenylalanine ammonia lyase; rDNA, DNA encoding rRNA. 47

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Plant Physiol. Vol. 08, 1995

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intensity light, wounding, pathogen attack, drought, and nutrient deficiency (McClure, 1975). The flavonoid biosynthetic pathway is illustrated in Figure 1. Arabidopsis :genes encoding PAL, CHS, CHI, and DFR have been cloned (Feinbaum and Ausubel, 1988; Dong et al., 1991; Shirley et al., 1992). In Arabidopsis, PAL is encoded by a small gene family (Ohl et al., 19901, whereas CHS, CHI, and DFR are single-copy genes (Feinbaum and Ausubel, 1988; Shirley et al., 1992). Cytokinin induction of anthocyanin production is reminiscent of the classical cytokinin bioassay of betacyanin induction in Amarantkus seedlings (Biddington and Thomas, 1973). Betacyanins are chemically unrelated to anthocyanins and are produced by only a few plant families that do not produce anthocyanins (Piatelli, 1981). Betacyanins have a physiological role similar to that of anthocyanins, and they are induced by a similar set of signals (light, wounding, and development) (Mabry, 1980; Piatelli, 1981). Cytokinin induction of anthocyanin biosynthesis could be a valuable probe for studying the mechanism of cytokinin action because it is a readily observable response and a great deal is known about the molecular biology and genetics of anthocyanin biosynthesis (Dooner et al., 1991; Martin and Gerats, 1993; van der Meer et al., 1993). Furthermore, although many cytokinin responses are considered essential to plant development, it is unlikely that elimination of cytokinin-induced anthocyanin biosynthesis would be lethal to a plant. Therefore, it should be possible to identify mutants in this response. For these reasons, we have characterized cytokinin induction of anthocyanins in

Arabidopsis, an excellent model organism for cai rying out a molecular genetic analysis of cytokinin action. We have found that cytokinin stimulates a large accumulation of anthocyanin in Arabidopsis and thíit this increase is due to the coordinate increased accumulation of mRNAs encoded by four genes in the anthocyan n biosynthesis pathway. Interestingly, two of these genes appear to be regulated by cytokinin at the transcriptional level and two at the posttranscriptional level. The implications of this finding to the relation between cytokinin and light regulation of gene expression are discussed. MATERIALS A N D METHODS Plant Material

Arabidopsis tkaliana plants (Wassilewskija ecot ype) were grown under sterile conditions on nutrient agar (one-halfstrength Murashige and Skoog salts [Gibco], 1 %SUC,B, vitamins [ l O O mg/L myo-inositol, 10 mg/L thiamine HC1,l mg/L nicotinic acid, and 1 mg/L pyridoxine SCl], and 0.8% tissue culture-grade agar, pH 5.7). Plant hormones (purchased from Sigma) were added to the meclium after autoclaving at the concentrations indicated. ?'o surface sterilize seeds before plating, the seeds were soaked in 95% ethanol for 5 min and 10% bleach for 5 min and were then rinsed five times with sterile water. The plated seeds were held at 4°C for 2 d and were then moved to a lighted, temperature-controlled growth room. Plants were grown at 22°C with an 18-h day/6-h night photoperiod. L,ght intensity was 60 to 100 pmol m-' spl. The Arabidopsis mutant etr was obtained from the Arabidopsis Biological Resource Center at Ohio State University.

L-phenylalanine

PAL

4

++

Anthocyanin Measurement

3 malonyl-COA

p-coumaroyl-COA

y Chalcone

4

CHI

Flavanone

4 4

Anthocyanins were extracted from preweigh ed shoots with propanol:HCl:H,O (18:1:81 [percent voluniel) as described by Schmidt and Mohr (1981).For the dose-response experiment, shoots of five 10-d-old plants were pooled in 1 mL of extraction solution. The samples were boiled for 3 min and then incubated overnight at 25°C. A,,, and A,,, of the extraction solution were measured, and the difference between these two readings was used as a nieasure of anthocyanins. Four replicates were measured for each treatment.

Di hydroflavonol DFR

++ +

Leucoanthocyanidin

Anthocyanin

Figure 1. A simplified representation of the flavonoid biosynthesis pathway (Dooner et al., 1991). The genes that have been cloned from Arabidopsis are indicated: PAL1 (Dong et al., 1991), CHS (Feinbaum and Ausubel, 1988), CHI (Shirley et al., 1992), and DFR (Shirley et al., 1992).

Twenty-Four-Hour BA and Ethylene Treatments

For the 24-h BA treatment, plants were grown on Whatman No. 1 filter paper that was on top of nutrient agar. When the plants were 9 d old, the filters holding the plants were transferred to a fresh Petri plate containir g 3 mL of buffered nutrient solution with either no hormone or 11 p~ BA. After 24 h, anthocyanins were extracted from the shoots. Ethylene treatment was carried out 011 10-d-old plants grown on nutrient agar. Tape was removed from the plates and they were placed in a bell jar. Ethylene was injected into the jar at a final concentration of 1 pL/L. The ethylene concentration was verified by measuring a sample

lnduction of Anthocyanin Accumulation by Cytokinins

49

with a gas chromatograph at the beginning and at the end of the treatment. Anthocyanins were extracted from shoot samples harvested after 24 h of ethylene treatment.

sequencing project led by T. Newman at the Michigan State University-Department of Energy Plant Research Laboratory (East Lansing, MI).

BA Spray Treatment

Nuclear Run-On Transcription Assay

In preliminary experiments we determined that a BA concentration of 2.2 p~ was optimal for causing an increase in anthocyanin concentration within 16 h in young rosettes. BA was dissolved initially in a small volume of 1 N KOH and was then diluted to 2.2 (LM BA with water. Control plants were sprayed with the same final concentration of KOH, which was 0.01 N. For the short-term time course, plants were grown on nutrient agar for 10 d in our standard growth-room conditions. They were sprayed at 2:OO PM with the BA or control solutions using a Preval (Precision Valve Corp., Yonkers, NY) spray unit. Each plate was sprayed for about 3 s at a distance of about 25 cm. The leaves were thoroughly wetted by this treatment. The plants were then returned to the growth room until harvest. Tissue harvesting was accomplished by flash-freezing in liquid nitrogen by pouring liquid nitrogen onto the agar plates and then scraping the frozen shoots into a cooled test tube. Shoots were harvested O, 1, 2, 3, 4, and 6 h after the spray treatment.

Plants were grown on nutrient agar with O or 0.44 PM BA at 22°C for 10 d. Shoots were harvested between 1:OO and 2:OO PM by flash-freezing in liquid nitrogen. The tissue was divided; most (about 6-8 g) was used for isolation of nuclei and the rest was used for mRNA isolation. Nuclei isolation was carried out as described by DellaPenna et al. (1989). This procedure involves a Perco11 gradient with a 2.2 M Suc pad, and the majority of the nuclei banded above the Suc layer. [32P]UTPincorporation was inhibited by 60% in the presence of 2 mg/pL a-amanitin. The amount of DNA in a 10-pL aliquot of each nuclei preparation was determined by incubation with 40 pg/mL RNase for 1 h at 37"C, followed by a 1-h incubation at 37°C in 20 pg/mL proteinase K (predigested). The samples were then extracted with phenol/chloroform and with chloroform alone, and the DNA was precipitated with ethanol. The DNA was resuspended in water and quantified with a spectrophotometer. The amount of DNA recovered was used to estimate the relative concentration of nuclei in each preparation. Nuclear run-on transcription was carried out and [3ZP]mRNAwas purified as described by DellaPenna et al. (1989). The [32P]nRNA was hybridized to plasmid DNAs that had been digested to release their inserts, separated by agarose gel electrophoresis, and transferred to a GeneScreen membrane following procedures, recommended by New England Nuclear. An equal number of cpm incorporated into [32P]nRNA was used for each hybridization. Hybridization and washing conditions were as described (DellaPenna et al., 1989). Radioactivity hybridizing to each insert was quantified with a PhosphorImager.

RNA Cel Blots

RNA was isolated from frozen shoots by the procedure of Rochester et al. (1986). Total RNA was denatured with formaldehyde, separated by electrophoresis on agaroseformaldehyde gels, blotted onto GeneScreen membranes (New England Nuclear), and hybridized with 32P-labeled DNA as described by Sambrook et al. (1989).Hybridization was carried out at 42°C in a buffer containing 5X SSPE (Sambrook et al., 19891, 50% formamide, 5X Denhardt's solution (Sambrook et al., 1989), 1%SDS, and 100 pg/mL denatured salmon sperm DNA. After hybridization, the blots were washed at 55OC in 0.1X SSPE, 0.05% sarcosine, and 0.01% sodium pyrophosphate. Hybridization signals were quantified with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) or a Betascope model603 Blot Analyzer (Betagen Corp., Waltham, MA). Hybridizing probe DNA was removed from the blot by treatment for 10 min at 100°C with 1%SDS in 10 p~ Tris, pH 8, 0.1 p~ EDTA, and blots were reprobed. Hybridization of each blot with 32P-labeled rDNA was carried out, quantified as above, and used to determine the relative amount of RNA present in each lane. D N A Probes

DNAs used as probes were isolated inserts from genomic clones of PALl (Dong et al., 1991) and CHS (Feinbaum and Ausubel, 1988), kindly provided by Dr. Frederick Ausubel, and CHI (Shirley et al., 1992) and DFR (Shirley et al., 1992), which were generously provided by Dr. Brenda Shirley. The tubulin (identification No. B64XP) and ubiquitin (identification No. 4E10T7P) probes were cDNAs obtained from the Arabidopsis Biological Resource Center at Ohio State University. These clones were generated by the cDNA

RESULTS Effects of Exogenous Cytokinin on Arabidopsis Growth and Anthocyanin Accumulation

To determine the effect of exogenous cytokinin on Arabidopsis growth and development, plants were grown on nutrient agar in the presence of different concentrations of the synthetic cytokinin BA for 10 d. The effect of different doses of BA on general growth and morphology is shown in Figure 2. Even the lowest concentration of BA (0.044 p ~ ) caused some stunting of shoot growth, although leaf expansion occurred at this concentration. Leaf expansion was completely suppressed at 22 p~ BA. A readily observed effect of BA treatment in Arabidopsis seedlings is the accumulation of red pigmentation (Fig. 2). This coloration appears most markedly on the hypocotyl, stem, and petioles. We extracted anthocyanins from shoots of plants grown for 10 d on different concentrations of BA. The amount of anthocyanins in the shoots increased slightly with 0.044 p~ BA, and anthocyanin accumulation was maximal at 0.44 PM BA (Fig. 3A). At that concentration there was a 50-fold increase in anthocyanins when anthocyanins were calculated on a per gram fresh weight basis.

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Figure 2. Effects of increasing doses of BA on Arabidopsis growth and development. Plants were grown for 10 d on nutrient agar containing (from left to right) 0, 0.044, 0.44, 4.4, or 22 JIM BA.

We also computed anthocyanin accumulation on a per plant basis and found that the results were qualitatively similar; the increase at 0.44 /J.M BA was 14-fold when values were calculated per plant. Similar effects were observed with two other cytokinins, zeatin and N6-(A2 isopentenyl)adenine (data not shown). Effect of Auxin and Ethylene on Anthocyanin Accumulation

To investigate whether cytokinin-induced anthocyanin production was likely to be a general stress response to hormone overexposure, the accumulation of anthocyanins in response to exposure to two other hormones, auxin and ethylene, was examined. Plants grown for 10 d with different concentrations of IAA up to 10 fj.M did not accumulate anthocyanins (Fig. 3B). A small amount of anthocyanin was produced by plants grown on 114 /U.M IAA, but growth of these plants was almost completely inhibited, with no leaf expansion or root elongation following germination (not shown). Plants treated with the lower doses of IAA exhibited reduced root elongation and an increase in root branching (not shown), demonstrating that the IAA was active and effective in altering plant growth patterns. Thus, although BA caused maximal anthocyanin production at doses that were only moderately inhibitory to growth (Figs. 2 and 3A), anthocyanins accumulated in response to IAA only at extremely inhibitory doses (Fig. 3B and data not shown). Many effects of plant stress are mediated by ethylene (Hyodo, 1991), and cytokinins have been shown to cause ethylene production (Suttle, 1986; Woodson and Brandt, 1991). To investigate whether cytokinin-regulated anthocyanin production could be an ethylene-mediated stress response, we examined the effect of ethylene on anthocyanin accumulation. Ten-day-old plants were treated with 11 JAM BA or with 1 ju,L/L ethylene for 24 h, and anthocyanin levels were determined (Table I). Anthocyanin levels increased 6-fold in response to the 24-h cytokinin treatment but did not increase in response to the ethylene treatment.

We also examined the ability of cytokinins to induce anthocyanin production in the ethylene-insensitive mutant etr. The etr mutant is defective in every ethylene response measured (Bleecker et al, 1988; Chang et al., 1993), but did produce anthocyanins in response to cytokinins (Table II). In this case the values are expressed on a per plant basis instead of per gram fresh weight because the etr plants became vitrified on the cytokinin medium and so comparison to wild type in terms of fresh weight was not possible. These results indicate that cytokinin-regulated anthocyanin production is not mediated by stress ethylene.

B • 12 10-

IAA (nM)

Figure 3. Anthocyanin accumulation in response to hormone application. Plants were grown for 10 d in the presence of the indicated hormones, and anthocyanins were extracted from whole shoots.

51

Induction of Anthocyanin Accumulation by Cytokinins

Table I. Anthocyanin accumulation in plants treated with ethylene or with BA for 24 h so values are shown in parentheses. Treatment

Anthocyanins A535/g fresh wt

Ethylene OjuL/L 1 /xL/L Fold induction BA

0.59 (0.15) 0.66(0.16) 1.1 0.30(0.12) 1.82(1.28) 6.1

0 AIM 1 1 /AM

Fold induction

Regulation of Genes of the Anthocyanin Biosynthesis Pathway by a Circadian Rhythm

In studying regulation of expression of genes in the anthocyanin biosynthesis pathway in response to cytokinin, we found that mRNA levels for these genes are not present at constant levels throughout the day. To examine fluctuations in mRNA levels, 10-d-old plants grown on nutrient agar in the normal 18-h light/6-h dark photoperiod were harvested at approximately 3-h intervals during the day from 8:00 AM until 2:00 AM and then the following morning at 8:00 AM. RNA was harvested from the shoots, and mRNA levels for Arabidopsis PALI (Dong et al., 1991), CHS (Feinbaum and Ausubel, 1988), CHI (Shirley et al., 1992), and DFR (Shirley et al., 1992) were determined by gel blot analysis. mRNA levels for PALI, CHS, CHI, and DFR were lowest in the late afternoon, rose in the evening, and were highest at night and in the early morning (Fig. 4). For example, the mRNA level for PALI was 7.5 times higher at 8:00 AM than at 5:00 PM. The mRNA level for CHS was 9.7 times higher at 8:00 AM than at 5:00 PM. To test whether the diurnal variation in mRNA concentrations was due to a circadian rhythm, we examined the persistence of the diurnal pattern in the absence of light/ dark cueing. For this experiment, 8-d-old plants were removed from the 18-h light/6-h dark condition and were grown for an additional 48 h in either continuous light or in darkness. Shoots were then harvested every 6 h for a 24-h period. These treatments were carried out at the same time as the experiment described above to examine mRNA levels in the 18-h light/6-h dark photoperiod, so the different treatments are directly comparable. RNA was isolated from these shoots and levels of PALI, CHS, CHI, and DFR mRNAs were determined as described above. Plants grown in continuous light had a similar pattern of variation Table II. Anthocyanin accumulation in ethylene-insensitive mutant (etr) or wild-type (wt) plants grown on nutrient agar with or without hormones for 10 d at 25° C so values are shown in parentheses. Anthocyanins Genotype No hormone Columbia wt etr

4.4 /XM BA

A535/p/ant 0.0001 (0.0003) 0.027 (0.008) 0.0003 (0.0009) 0.042 (0.02)

6:00

9:30 13:00 16:30 20:00 23:30 3:00 6:30 10:00

Time of Day Figure 4. Fluctuation in mRNA levels of anthocyanin biosynthesis genes in a 24-h period. A, RNA gel blot probed with PAL, CHS, CHI, DFR, and rDNA. mRNAs were isolated at the indicated times of day. B, mRNA levels were quantified as described in "Materials and Methods." The bar along the x axis indicates whether lights were on (white bars) or off (black bar).

of mRNA levels, but the pattern was shifted by about 3 h (Fig. 5). Plants grown in the dark contained very little mRNA for any of the genes (Fig. 5A). These results indicate that the daily fluctuations in mRNA levels for genes in the anthocyanin biosynthesis pathway are controlled by a circadian clock, although gene expression also requires light. The tight coordinate regulation of these genes is apparent in this phenomenon. Expression of Anthocyanin Biosynthesis Genes in Response to Cytokinins

To examine whether anthocyanin accumulation in response to cytokinin was due to an increase in mRNA concentrations for genes encoding anthocyanin biosynthesis enzymes, RNA was isolated from plants treated with the same concentrations of BA as the plants shown in Figure 2, and a gel blot hybridization experiment was carried out. All plant material was harvested simultaneously by freezing in liquid nitrogen, when mRNA levels for the anthocyanin biosynthesis genes were lowest, at about 4 PM. Figure 6 shows that PALI, CHS, CHI, and DFR mRNA levels all increased in response to 0.44 /AM BA. Induction of CHS gene expression was greatest and the

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Deikman and Hammer

highest at night (Fig. 7). However, the difference between mRNA levels at the high and low points of the day was smaller in the presence of BA than in its absence. For example, in the absence of exogenous cytokinin there was 21.9 times more CHS mRNA at 9:00 AM than at 6:00 PM, but in the presence of BA there was only 1.9 times more CHS mRNA at 9:00 AM than at 6:00 PM. The results with PALI were similar, with a 13.8-fold difference in the absence of exogenous BA but a 2.6-fold difference in the presence of BA. Since diurnal fluctuations continued in the presence of saturating amounts of BA, these data suggest that endogenous cytokinins are not involved in regulating gene expression in response to the circadian clock. The dampening of the diurnal fluctuations by BA could be due to effects of BA on either transcription or translation of these genes. The amount of increase in steady-state mRNA levels caused by BA was different at different times of the day. Specifically, for CHS, BA caused a 35.6-fold increase in mRNA levels at 6:00 PM but caused only a 3.1-fold increase in mRNA levels at 9:00 AM. The results for PALI mRNA were similar, with a 7.3-fold increase at 6:00 PM and a slight, 1.4-fold increase at 9:00 AM.

6:40

10:00 13:20 16:40 20:00 23:20 26:40 30:00 33:20

Time of Day Figure 5. mRNA levels in a 24-h period in continuous light or continuous darkness. A, RNA gel blot. mRNAs were isolated at the indicated times from plants that were placed in continuous light for 48 h or continuous darkness for 48 h. B, Quantification of signals from Figures 4A (18-h light, 6-h dark photoperiod) and 5A (24 h light only) for PAL and CHS.

amount of CHS mRNA produced was maximal at 0.44 /XM BA. CHS mRNA increased 8-fold at 0.44 /MM BA compared to water controls. PALI mRNA was increased by about 3.7-fold with 0.44 /MM BA, but its level continued to increase with higher concentrations of BA. The pattern of CHS mRNA accumulation resembles that of anthocyanin accumulation in peaking with 0.44 /MM BA (compare Figs. 3A and 6). Relation of Cytokinin Induction of Gene Expression to the Circadian Regulation

It seemed possible that endogenous cytokinin could be involved in controlling the diurnal fluctuations in mRNA levels for the anthocyanin biosynthesis genes. If so, treatment with high levels of cytokinins should mask the diurnal variation in mRNA levels, since the induction of mRNA accumulation was maximal with 4.4 ;UM BA. Therefore, we examined the levels of CHS and PALI mRNA in shoots treated with or without 4.4 /MM BA at different times during the day. We found that levels of CHS and PALI mRNA were elevated at every time during the day in the presence of exogenous BA but that the amounts of these mRNAs were still lowest in the late afternoon to early evening and

0.44

0.044

BA Figure 6. Accumulation of PAL1, CHS, CHI, and DFR mRNAs in response to BA dose. A, RNA gel blot showing hybridization of RNAs from plants grown on 0, 0.044, 0.44, 4.4, and 44 /MM BA for 10 d. B, Quantification of the data from A.

Induction of Anthocyanin Accumulation by Cytokinins A

Radiolabeled transcripts were hybridized to blotted coding sequences for PALI, CHS, CHI, and DFR (Fig. 9). Signals on the gel blot were quantified with a Phosphorlmager, and the induction by BA for each transcript is shown (Table III). RNA was also isolated from these plants, and the mRNA was analyzed by gel blot hybridization. The ratio of mRNA in BA-treated compared to untreated plants is shown in Table III. It is interesting that although mRNA levels for PALI, CHS, CHI, and DFR all increase in response to BA (Fig. 6; Table III), only CHS and DFR were transcriptionally activated by BA. PALI and CHI appear to be regulated by BA posttranscriptionally. The ratio of CHS mRNA in BA-treated plants compared to untreated plants was greater than the ratio of run-on transcription under these two conditions. This discrepancy indicates that BA may influence CHS mRNA levels by effects on both transcriptional and posttranscriptional processes.

PAL

•••••t

CHS

rDNAj

§ § § § § § § § § § § O ) C \ J L D O ) r - - < t C n < M i n c O T -

f-

»-

»-

CM OJ

i-

»-

Control

»-

CM

BA

B c

O

.Q

s: CD ~ ^0 CD

53

50000,

DISCUSSION

QC

Cytokinins Cause Increased Anthocyanin Production 9:00

12:00

15:00

18:00

21:00

24:00

Time of Day Figure 7. Effect of BA on the diurnal fluctuation in mRNA levels. A, RNA gel blot. Plants were grown on nutrient medium with no hormones (control) or with 4.4 J^M BA. RNA was isolated at the indicated times. B, Quantification of the signals for CHS mRNA from A.

Our results demonstrate that cytokinins cause a substantial increase in accumulation of anthocyanins in Arabidopsis plants. The maximal, 50-fold accumulation occurs at BA doses that are only moderately inhibitory to growth (Figs. 2 and 3A). In contrast, a smaller, 4-fold, anthocyanin accu-

Time Course of Cytokinin-lnduced CHS Gene Expression

The rapidity of BA induction of expression of PALI and CHS gene expression was investigated by spraying plants with BA and then examining mRNA accumulation. Preliminary experiments in which plants were sprayed with different concentrations of BA and then anthocyanins were measured after 16 h or more indicated that 2.2 JUM BA produced a consistent response of increased anthocyanin accumulation (data not shown). We believe that this high concentration is necessary because very little hormone penetrates the target tissues (the hypocotyls and petioles) with a spray application. There is no discernible effect on plant growth by this treatment. For RNA analysis, 10-d-old plants grown on nutrient agar were sprayed with 2.2 /U.M BA in 0.01 N KOH or with 0.01 N KOH, and shoots were harvested after 0, 1,2, 3, 4, and 6 h. Plants were sprayed at 2:00 PM, when mRNA levels for these genes were at a low level. RNA was extracted and analyzed by blot hybridization as before. An increase in CHS mRNA levels was detectable after 2 h and was maximal at 3 h, with a 2.4-fold increase in CHS mRNA. CHS mRNA levels then declined, and they were comparable to control levels by 6 h after treatment (Fig. 8). Levels for PALI mRNA, on the other hand, did not increase in response to BA, but appear to have decreased slightly (Fig. 8). Nuclear Run-On Transcription

]

Nuclei were isolated from Arabidopsis plants grown with or without 0.44 JUM BA for 10 d, and nuclear run-on transcription was carried out in the presence of [32P]UTP.

0

1

2

3

4

6

time after sorav fhl

1

2

3

4

5

Time after spray (h) Figure 8. Effect of short-term BA treatment on PAL and CHS mRNA accumulation. Plants were sprayed with 2.2 JIM BA in 0.01 N KOH or with 0.01 N KOH, and RNA was isolated at the indicated times. A, RNA gel blot. B, PAL and CHS mRNA levels were quantified as described in "Materials and Methods." The ratios of mRNA levels in plants sprayed with BA compared with plants sprayed with the control solution are plotted.

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Deikman and Hammer

Run-on transcription

RNA gel blot

BA: PAL CHS CHI DFR rDNA

Tubulin Ubiquitin Figure 9. Nuclear run-on transcription in plants grown in the presence or absence of BA. Plants were grown on nutrient agar for 10 d in the presence of 0.44 JAM BA or without hormone. The plants were used for isolation of both nuclei and mRNA (see "Materials and Methods"). Run-on transcription, [ 3 2 P]RNA synthesized in nuclei isolated from these plants was hybridized to blotted cDNA clones for PALI, CHS, CHI, DFR, tubulin, ubiquitin, and rDNA. The latter three cDNAs serve as controls for genes whose transcription rates are not altered by cytokinins. RNA gel blot, Replicate blots were hybridized with the indicated cloned DNAs. See Table III for a quantitative analysis of these data.

mulation is stimulated only by culture at levels of the auxin IAA that almost completely inhibit growth and development after germination (Fig. 3B and data not shown). Experiments with other auxins need to be carried out to generalize about the effect of auxins on anthocyanin production. However, these results suggest that anthocyanin production in response to cytokinins is not simply a general response to severe hormonal stress. Our data indicate that the cytokinin effect on anthocyanin accumulation is not mediated by stress ethylene. First, exogenous application of 1 /xL/L ethylene for 24 h did not cause increased anthocyanin production, whereas a 24-h treatment with 11 /J.M BA caused a 6-fold increase in anthocyanins (Table I). Although the ethylene dose used was shown to inhibit growth of Arabidopsis roots and hypocotyls (Bleecker et al., 1988), it is possible that higher amounts of ethylene could cause anthocyanin accumulation. However, the ethylene-insensitive mutant etr was shown to be defective in every ethylene response tested (Bleecker et al., 1988), yet it accumulated a normal amount of anthocyanins in response to BA (Table II). Thus, the ability to sense ethylene is not necessary for cytokinin induction of anthocyanin accumulation. Coordinate Induction of Anthocyanin Biosynthesis Genes by Cytokinins

Analysis of steady-state mRNA levels for four genes in the anthocyanin biosynthesis pathway indicated that ex-


pression of these genes is coordinately controlled by exogenous cytokinin. mRNA levels for PALI, CHS, CHI, and DFR were all increased by BA treatment (Fig. 6). The BA dose response for mRNA accumulation was similar to that for anthocyanin accumulation (Fig. 3A), indicating that mRNA accumulation is responsible for the BA-stimulated increase in anthocyanin accumulation. The sensitivity of this response is in the same range as other cytokinin responses such as bud formation in moss protonemata. Both responses are saturated at about 1 JUM BA (Kende, 1971). Although BA caused a coordinate increase in mRNA accumulation of PALI, CHS, CHI, and DFR, it caused a substantial increase in the transcription rate of CHS and DFR but not of PALI and CHI. PALI and CHI appear to be regulated by BA only at the posttranscriptional level. It is likely that cytokinin affects stability or processing of the CHS mRNA as well as the CHS transcription rate, since the increase in CHS mRNA was larger than the increase in CHS run-on transcription (Fig. 9; Table III). The question of how the levels of mRNAs for these different genes are adjusted by different mechanisms to result in synchronous gene expression will be interesting to pursue. Genes of the anthocyanin biosynthesis pathway are coordinately regulated in maize kernel aleurone, and these genes are all controlled by the transcription factors R and Cl (Martin and Gerats, 1993). The R gene has been shown to be active in Arabidopsis and can restore anthocyanin production in the ttg mutant, suggesting that R is a functional homolog of ttg (Lloyd et al., 1992). We show here that PALI, CHS, CHI, and DFR are also coordinately regulated in response to the circadian clock (Figs. 4 and 5). However, there is precedence for separate regulatory mechanisms of this set of genes. Genes of the anthocyanin biosynthesis pathway are under the control of at least two discrete regulatory mechanisms in flowers of dicots (Martin and Gerats, 1993). Also, during early seedling growth of Arabidopsis there are temporal differences in expression of these genes, and the mRNAs for these genes increase in the order that the enzymes act in the flavonoid biosynthesis pathway (Kubasek et al., 1992). The control of anthocyanin biosynthesis gene expression by cytokinins is the first case we are aware of in which it has been demonstrated that

Table III. Comparison of induction of transcription versus mRNA accumulation Transcription refers to transcript synthesized in nuclear run-on transcription reactions. mRNA indicates transcript accumulated in plants from which the nuclei were isolated, n.d., Not determined. -, Not applicable. Transcription

Gene PAL CHS

CHI DFR Tubulin Ubiquitin rDNA

mRNA

BA/no

BA/no

hormone

hormone

1.15

3.7

10.30 1.42 34.17 1.18 0.85 0.64

56.1 9.4 5.1 n.d.

n.d. -

lnduction of Anthocyanin Accumulation by Cytokinins coordinate regulation of these genes occurs via different regulatory mechanisms. Time Course of Cytokinin lnduction of Gene Expression

We detected an increase in CHS mRNA within 2 h of BA spray application but did not see any increase in PALl mRNA levels even after 6 h (Fig. 8). Perhaps there was no increase in PALl mRNA after the BA spray because the diurna1 variation in gene expression caused PALl mRNA levels to be so low at the time of day we sprayed that stabilization of PALl mRNA had no significant effect. The results might have been different if we had sprayed plants at a time of day when the background PALl mRNA leve1 was high. The increase in CHS mRNA levels was only transient. It is likely that the cytokinin applied in this spray treatment was not taken up efficiently by the plants, was not transported to the target tissues (the hypocotyl and petiole bases), and may have been rapidly degraded. The rapid effect of BA on anthocyanin accumulation was also supported by the experiment in which anthocyanins were measured after 24 h (Table I). In that case there was a 6-fold increase in anthocyanin levels, even though anthocyanin accumulation requires many steps, including gene transcription, protein synthesis, and the coordinated activity of severa1 enzymes. lnteraction of Light and Cytokinins in Control of Gene Expression

Regulation of genes in the anthocyanin biosynthesis pathway by light has been well documented (van der Meer et al., 1993). In cultured parsley cells, there is evidence for regulation of CHS gene expression by a UV-B light receptor, a blue light receptor, and phytochrome (Bruns et al., 1986; Ohl et al., 1989). We have found that BA does not cause an increase in anthocyanin accumulation in the absence of light (data not shown). There could be many steps involved in anthocyanin biosynthesis that are light-requiring, so it would be interesting to determine whether BA can cause an increase in CHS transcription in the absence of light. Alternatively, BA may simply enhance CHS transcription that has already been activated by light. Cytokinins and light interact to influence a large number of processes. For the most part these light-regulated events are mediated by phytochrome. The similarity of cytokinin and red light effects has been reported for lettuce seed germination (Miller, 1956), leaf expansion (Miller, 1956), watermelon cotyledon maturation (Lampugnani et al., 1980), amaranthin synthesis in Amarantkus tricolor (Piatelli et al., 1971), nitrate reductase activity in etiolated maize leaves (Rao et al., 1984), and chloroplast development (Parthier, 1979). Cytokinins have been shown to influence expression of light-regulated genes important in photosynthesis (Lerbs et al., 1984; Tessendier de Ia Serve et al., 1985; Flores and Tobin, 1988). Since the processes mentioned above were shown to be mediated by phytochrome, it is especially interesting that cytokinins and red light both down-regulate phytochrome mRNA (Cotton et al., 1990).

55

It has been hypothesized that phytochrome acts by increasing the concentration or effectiveness of cytokinins (Cotton et al., 1990). However, whether cytokinins are part of phytochrome signal transduction or whether cytokinins and light act in parallel has not yet been determined. The evidence that red light activates cab transcription but cytokinins primarily affect cab mRNA stability in Lemna gibba (Flores and Tobin, 1988) suggests that phytochrome and cytokinins act independently. Earlier studies on the quantitative effects of light and kinetin on Chl accumulation in excised mustard cotyledons resulted in the conclusion that kinetin and light effects are additive (Kasemir and Mohr, 1982). These results suggest that cytokinins do not mediate light action. Arabidopsis PALl and CHS have both been shown to be transcriptionally activated in response to light (Feinbaum and Ausubel, 1988; Ohl et al., 1990). Our results suggest that cytokinin regulation of anthocyanin biosynthesis gene expression occurs via a different signal transduction pathway from light, since only CHS, and not PALl, appears to be transcriptionally activated by cytokinin. A more direct way of determining whether cytokinin and light act by means of the same or different signal transduction pathways would be to characterize DNA sequences required for the transcriptional response. Analysis of the CHS promoter in transgenic Arabidopsis plants showed that sequences responsible for blue light response could be separated from sequences necessary for response to high-intensity light and suggested that the blue and high-intensity light regulatory elements are distinct (Feinbaum et al., 1991). On the other hand, the parsley CHS gene contains sequences that appear to respond to both blue and UV light in parsley protoplasts (Merkle et al., 1994). It would be of value to determine whether the same elements are involved in cytokinin- and light-regulated transcription of the CHS gene. Such information would indicate whether cytokinins and light promote the action of a common signal transduction pathway or whether they act via independent pathways.

Physiological Significance of Cytokinin Control of Anthocyanin Production

Regulation of cell division and morphogenesis are widely regarded as the most significant cytokinin functions. However, most of the cytokinin-regulated genes that have been studied are genes involved in response to light, stress, or other environmental signals (Flores and Tobin, 1986; Memlink et al., 1987; Chen, 1989; Cotton et al., 1990; Lu et al., 1990; Dehio and de Bruijn, 1992; Dominov et al., 1992). It has been argued that in some of these cases these genes are identified because exposure to exogenous cytokinin constitutes a stress. The possibility that cytokinins have a central endogenous role in controlling expression of these genes should not be overlooked. Perhaps increased production of stress-related genes is coordinated with cell division and growth because growing regions might be more susceptible to harm from UV light, pathogens, or other environmental stresses. Alternatively, it may be that

Deikman and tiammer

56

cytokinins have functions in nondividing tissue that are currently not understood and are underestimated. In this regard, it is interesting that an Arabidopsis mutant that is defective in its response to cytokinins in a root elongation assay does not produce anthocyanins in response to cytokinins (Deikman and Ulrich, 1995). The cyrl mutant has 10-fold less sensitivity to cytokinins in terms of root elongation and does not have decreased sensitivity to other plant hormones. The mutation has severe effects on shoot development. After formation of a few leaves, a single infertile flower is generally produced. Although cyrl has been observed to produce anthocyanins in some instances, such as in tissue culture, it does not produce anthocyanins in response to cytokinins. Therefore, the lesion is not in a structural gene for anthocyanin biosynthesis but in a gene that encodes a regulatory protein. The phenotype of this mutant further supports a specific role for cytokinins in controlling anthocyanin production. Studying cytokinin control of anthocyanin biosynthesis is a promising model system for investigating the mechanism of cytokinin action. The CHS gene is activated by cytokinins at the transcriptional level, and mRNA for CHS increases within 2 to 3 h of cytokinin application. Both light and cytokinins activate CHS gene transcription, :;o this gene could be used to examine the interaction between light and cytokinins at the molecular level. As more is understood about the light signal transduction pathways it will be possible to determine how cytokinins affec't their components. ACKNOWLEDCMENTS

We acknowledge the capable technical assistance o f Marie Pacella and Margaret Ulrich. We thank Drs. Frederick Ausubel and Brenda Shirley for providing cloned PALI, CHS, CHI, and DFR DNAs, and Drs. Mark Guiltinan and Benjamin Mo11 for thoughtful comments on the manuscript. Received November 1, 1994; accepted January 16, 1995. Copyright Clearance Center: 0032-0889/95/108/0047/ 11. LITERATURE ClTED

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