Characterisation of the procera mutant of tomato and the interaction of gibberellins with end-of-day far-red light treatments

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1 PHYSIOLOGIA PLANTARUM 106: Copyright Physiologia Plantarum 1999 Printed in Ireland all rights reser ed ISSN Characterisation of the procera mutant of tomato and the interaction of gibberellins with end-of-day far-red light treatments A. Van Tuinen a, A. H. L. J. Peters b, R. E. Kendrick c, J. A. D. Zeevaart d and M. Koornneef b, * a Laboratoire de Biologie Cellulaire, INRA, Route de Saint-Cyr, F Versailles Cedex, France b Laboratory of Genetics, Wageningen Agricultural Uni ersity, Dreijenlaan 2, NL-6703 HA Wageningen, The Netherlands c Laboratory of Plant Physiology, Wageningen Agricultural Uni ersity, Arboretumlaan 4, NL-6703 BD Wageningen, The Netherlands d MSU-DOE Plant Research Laboratory, Michigan State Uni ersity, East Lansing, MI 48824, USA *Corresponding author, maarten.koornneef@botgen.el.wau.nl Received 4 September 1998; revised 21 January 1999 The tomato (Solanum lycopersicum L.) slender mutant procera (pro) was analysed for its relationship with gibberellin (GA) by combining it with GA deficiency due to the gib-1 mutation. The sensitivity to GA biosynthesis inhibitors and the GA content were measured in the pro gib-1 double mutant. In the gib-1 mutant background, the pro mutation strongly re- duced the GA requirement for seed germination and stem growth and almost fully restored the morphological leaf de- fects of the gib-1 mutant. An end-of-day far-red light treat- ment, when applied to the various genotypes, indicated that GAs are required for a response to this treatment, but that it act independently of the Pro gene product. Introduction Gibberellins (GAs) are a group of diterpenoid compounds controlling a wide variety of growth and developmental responses in plants. These include important roles in seed germination and cell division and elongation (Hooley 1994). The biosynthetic pathway of GAs has been elucidated and several genes, which encode enzymes for particular steps in the pathway, have been cloned (Hedden and Kamiya 1997). To achieve this, mutants defective in the biosynthetic pathway have played an important role (Ross et al. 1997). However, the mode of action of GAs is less well understood. Some components of GA-response pathways have been identified recently by the molecular identification of GA-regulated transcription activators for -amylase induction (Gubler et al. 1995). Furthermore, mutants have been identified that affect the responsiveness to GAs (Ross et al. 1997). Two major classes of GA-response mutants are known. The first class represents mutants with the phenotype of GA-deficient mutants, but they are not responsive to applied GAs. The most extensively studied mutant of this type is the dominant gain of function gai mutant of Arabidopsis (Koornneef et al. 1985). The GAI gene has recently been cloned and appears to be a transcriptional activator of the VHIID regulatory protein family (Peng et al. 1997). It appears that the loss-of-function phenotype of this gene, studied in revertants of the dominant mutant, confers a slight resistance to inhibitors of GA biosynthesis, such as paclobutrazol (Peng et al. 1997). This indicates that the wild-type (WT) GAI gene product reduces the sensitivity to GAs. In its loss-of-function phenotype, GAI resembles the second class of genes, whose mutants have a GA-hypersensitive phenotype. Mutants in this group, characterised by a slender phenotype, were isolated on the basis of their resistance to paclobutrazol (Jacobsen and Olszewski 1993), in the case of the Arabidopsis spy mutant, or as revertants of GA-deficient ga1 mutants, in the case of the rga1 mutants and additional spy alleles (Silverstone et al. 1997). Recently, the SPY (Jacobsen et al. 1996) and RGA1 (Silverstone et al. 1998) genes have been cloned. SPY encodes a serine/threonine O-linked N-acetylglucosamine transferase that can modify various other proteins by glycosylation (Kreppel et al. 1997). RGA1 shows sequence similarity with GAI. In addition to the Arabidopsis mutants described above, mutants with a slender phenotype have also been described in barley (Lanahan and Ho 1988) and pea (Reid et al. 1983). In the latter species, the slender phenotype is present when Abbre iations AC: tomato cv. Ailsa Craig; EODFR: end-of-day far-red; FR: far-red; MM: tomato cv. Moneymaker; WL: white light; WT: wild-type. Physiol. Plant. 106,

2 the plant is homozygous for both the la and cry s mutations (Potts et al. 1985). In tomato, the elongated procera (pro) mutant resembles the latter mutants (Jones 1987, Jupe et al. 1988). Genetically, the pro mutant is monogenic and recessive and maps to the top of chromosome 11 (Van Tuinen et al. 1998). The cellular phenotype of pro and various aspects of its GA-related physiology have been described previously (Jones 1987, Jupe et al. 1988), as well as the effect of this mutation on peroxidase activity (Jupe and Scott 1992) and its responsiveness to fusicoccin (Woodhead et al. 1997). Although the pro mutant was shown to be a phenocopy of GA-treated WT plants, the GA dose-response curves did not allow a definitive conclusion on the responsiveness of the mutant (Jones 1987). The experiments described by Jones (1987) showed a clear response to applied GA, which was not excessive, compared to the response of WT. This might be an indication that the GA saturation level was approached. In the present study, we have analysed the GA responsiveness of pro in more detail by analysing several additional traits. For this, we also constructed a double mutant of pro and the GA-deficient gib-1 mutant (Koornneef et al. 1990). In mutants that are completely deficient in GA biosynthesis, a treatment that changes growth of this mutant after the application of GAs can be assumed to act solely through its effect on GA sensitivity. Therefore, the use of the pro gib-1 double mutant allows a better analysis of the changes in GA sensitivity due to the pro mutation, which, in addition to the possible saturation of the GA response, is also complicated by the effect of the pro mutant on GA levels, as reported by Jones (1987). Changes in GA sensitivity can also be due to physiological stimuli, which affect the perception of GAs. These environmental effects can then be mimicked by the application of GAs. One such response, mediated by phytochrome in light-grown plants, is the shade-avoidance syndrome, which results in a promotion of stem elongation in order to avoid the shade light of other plants, which is relatively rich in far-red (FR) light. Depending on the plant species, the shade-avoidance process is also associated with early flowering, as is the case in Arabidopsis (Smith and Whitelam 1997), or reduced anthocyanin synthesis, as is the case in tomato (López-Juez et al. 1990). A simple method used in the laboratory to trigger this phytochrome-mediated shadeavoidance process is to expose plants briefly to FR at the end of the daily photoperiod (end-of-day far-red [EODFR]) (Downs et al. 1957). A study of phytochrome mutants has indicated that B-type phytochromes play a major role in these responses, although there is some redundancy with other members of the phytochrome gene family (Smith and Whitelam 1997). The involvement of GAs in the EODFR light response was proposed by Downs et al. (1957) and it has been suggested that it acts either through changes in the level or the responsiveness to plant hormones, such as GA (Garcia-Martinez et al. 1987, López-Juez et al. 1995). The various genotypes available made it possible to analyse the interaction of the EODFR light treatment with GAs and give further insight into the way this treatment in tomato exclusively affects GA sensitivity, as suggested by most recent research in other plants (reviewed by Swain and Olszewski [1996] and Chory and Li [1997]). Materials and methods Plant material The pro mutant was isolated after X-ray radiation of the Solanum lycopersicum L. cv. Condine Red (Stubbe 1957). The pro mutant, introgressed into the cv. Ailsa Craig (AC) by repeated backcrossing, was described by Maxon Smith and Ritchie (1983) and this genotype (GCR 380) was used in the present experiments. The gib-1 mutant (isolation number W335) in the genetic background of the cv. Moneymaker (MM) has been described by Koornneef et al. (1990). The pro gib-1 double mutant was obtained as shown by the scheme in Fig. 1. Tetcyclacis experiment Seeds of the different genotypes were germinated in Petri dishes and germinating seeds were planted in 500-ml plastic containers in a mixture of peat and sand (volume ratio 1:1). Seeds of the gib-1 and pro gib-1 genotypes were germinated on 15 M GA 4+7 on filter paper until the radicles appeared. The plants were initially grown in a growth chamber with 16 h of light from fluorescent and incandescent lamps at 300 mol m 2 s 1 at 23 C and 8 h of darkness at 20 C. Treatment with the GA biosynthesis inhibitor tetcyclacis and stem length measurements were started 2 weeks after Fig. 1. Scheme describing the isolation of the pro gib-1 double mutant. 122 Physiol. Plant. 106, 1999

3 sowing. The main light period was shortened to 10 h, followed by 1 h of light from incandescent bulbs at 10 mol m 2 s 1. Tetcyclacis (a gift from Dr W. Rademacher, BASF, Limburgerhof, Germany) applications (10 ml of a M solution) were made to the soil on alternate days. Stem height was measured from the cotyledonary node to the shoot tip. Gibberellin measurements Plant material for GA extraction was grown in a growth chamber with a 16-h photoperiod as described for the tetcyclacis experiment. One-month-old plants were harvested, at which time the tenth leaf had started to expand. Tops of the plants down to the youngest fully expanded leaf were harvested, frozen in liquid nitrogen and lyophilised. GAs were extracted and purified as described (Talon et al. 1991). [ 2 H]GAs (a gift from Dr L. Mander, Australian National University, Canberra, Australia) were added as internal standards. Following purification, the samples were methylated and trimethylsilylated and GAs in the derivatised material were analysed by GC-selected ion monitoring with magnetic field switching using a JEOL AX505 double-focusing mass spectrometer equipped with a 5890 Hewlett-Packard gas chromatograph. The amounts of GAs were calculated from the ratio of M + and M + +2 for each GA with appropriate correction for the percentage [ 2 H]-enrichment. The experiment was repeated once with similar results. Germination experiment Seeds of MM, the gib-1 mutant and the pro gib-1 double mutant were sown in glass Petri dishes (25 seeds per Petri dish) on two layers of filter paper soaked with a solution containing 0 (H 2 O) or M GA 4+7 (ICI, Bracknell, UK) dissolved in 1 M KOH and adjusted to the right concentration in a 10 diluted phosphate citrate buffer (0.05 M K 2 HPO 4, 0.05 M citric acid, ph 4.8). Petri dishes were wrapped individually in plastic bags, which were closed with a rubber band. The Petri dishes were then incubated in darkness in a light-tight box at 25 C for 1 week, after which germination was scored. Two dishes were used for each treatment and the experiment was repeated three times. The results are presented as the means of all experiments. Growth of plant material for the dose-response and end-of-day far-red light experiments For the dose-response (Figs. 5 and 6) and EODFR light (Figs. 7 and 8) experiments, seeds of all genotypes were sown in Petri dishes on two layers of filter paper soaked with2mlof1 M GA 4+7 and placed in a darkroom at 25 C for 4 days. The germinated seeds were then transferred to trays filled with a mixture of potting compost and sand (volume ratio 4:1) and grown for 9 (EODFR experiment 2) or 13 days (dose-response and EODFR experiment 1) in a phytotron with a daily irradiation schedule of 16 h of white light (WL; PAR 190 mol m 2 s 1 )/8 h of darkness at 25 C and relative humidity (RH) of 65 70%. At day 13 (EODFR experiment 2) or day 17 (dose-response and EODFR experiment 1), the seedlings were transplanted into 11-cm square plastic pots (filled with the above-mentioned soil mixture) and further grown in the phytotron (dose-response experiment) or, for the EODFR light experiments, transferred to growth cabinets (16 h of WL; PAR 145 [125 in experiment 2] mol m 2 s 1 /8 h of darkness cycle), where they grew for another week. The plants were then selected for uniform height and the respective GA or EODFR light treatment was started (day 0). Dose-response experiment At day 0 (see growth of plant material), stem length, from the cotyledons to the shoot tips, was measured with a ruler. After measuring, the plants were placed at random on trolleys, with all growing tips at the same distance from the light source. A 30- l drop containing 0 (H 2 O), 0.3,3or30 g GA 4+7 (dissolved as described above) with 5% (v/v) ethanol and 0.05% (v/v) Tween 80 was then placed with a micropipette on the youngest visible developing leaf. At day 12, stem height was measured again and the increase in height was calculated. The length of the leaf below the treated leaf was measured. Four punches were taken from this leaf with a cork borer and weight and leaf thickness were determined. During the experiment, trolleys were turned daily and height of trolleys and plants adjusted to keep all growing tips at the same distance from the light source, which was provided by TL40/33 fluorescent tubes. Six replicates per genotype were measured per treatment. End-of-day far-red light experiments The EODFR light experiments were carried out in cabinets fitted with white fluorescent tubes (Philips TL40/33) and FR was provided by Sylvania F48T12/232/VHO tubes wrapped with one layer of dark-green and one layer of primary red filter (Lee, Flashlight Sales BV, Utrecht, The Netherlands). Results from two different experiments are presented. Experiment 1 is shown in Fig. 7 and experiment 2 is shown in Fig. 8. After measuring the stem length, these plants were placed at random in growth cabinets, with all growing tips at the same distance from the light source and GA 4+7 (3 g in experiment 1, 5 g in experiment 2) was applied as described above. After the 16-h daily WL period, the plants were either submitted to an immediate 8-h dark period or given a 20-min FR pulse (4.6 mol m 2 s 1 ) before the dark period. In experiment 2, a second dose of GA 4+7 was applied on day 6. Stem length was measured again at day 10 (experiment 1) or 12 (experiment 2) and any increase was calculated. For anthocyanin determination (Fig. 7) at day 10, the growing tips were harvested, weighed and extracted with 1.8 ml acidified (1% [w/v] HCl) methanol for 48 h in darkness with shaking. A Folch partitioning (Folch et al. 1957) was Physiol. Plant. 106,

4 Fig. 2. Wild-type (WT, cv. Moneymaker), gib-1 and pro mutant and pro gib-1 double mutant plants grown for 5 weeks in a 16 h of white light (PAR 190 mol m 2 s 1 )/8 h of darkness cycle at 25 C. Before planting, the seeds were imbibed for 4 days on filter paper to which 1 M GA 4+7 was added. performed by adding 1.35 ml H 2 O and 3.6 ml chloroform to the extracts and centrifugating for 30 min at 3600 rpm. The absorbance (A) of the top phase was determined (DU-64; Beckman Instruments Inc., Fullerton, USA) at 535 nm (A 535 ). During the experiments, plants were turned every second day and height adjusted to keep all growing tips at the same distance from the light source. Four (experiment 1) or eight (experiment 2) replicates per genotype were measured per treatment. Results The relationship between the pro mutation and gibberellins The pro mutant is taller with less serrated leaves than WT as described by Jones (1987). The pro gib-1 double mutant resembled WT plants in its height, but still lacked leaf serration (Fig. 2). Treatment of pro plants with the GA biosynthesis inhibitor tetcyclacis only led to a 22% reduction in plant height (Fig. 3). The pro gib-1 double mutant was also relatively insensitive to tetcyclacis, especially when compared to the WT, which had a similar plant height under these conditions. The epistasis of the pro mutation over GA deficiency due to the gib-1 mutation, as well as the relative insensitivity towards the inhibiting effect of tetcyclacis, indicates that the pro mutation abolishes to a great extent the GA requirement for elongation growth. The incompleteness of the epistasis of pro, as well as the small effect of tetcyclacis in pro, indicates that lack of a GA requirement is not total. The GA levels in the various genotypes, which are presented in Table 1, show that the phenotype of pro, which resembles a GA-treated WT and the data mentioned above, cannot be explained by GA overproduction. The data indicate that GA levels are reduced compared to WT for all genotypes that were analysed, confirming the lower GA levels reported by Jones (1987) for the pro mutant and by Koornneef et al. (1990) for the gib-1 mutant. By combining gib-1 with pro, the amount of the active gibberellin, GA 1 was even lower than in the single gib-1 mutant. However, despite this fact, the pro gib-1 double mutant plants are as tall as WT. 124 Fig. 3. The effect of tetcyclacis on plant height of pro and gib-1 mutants. Tetcyclacis treatment (10 ml of M solution) on alternate days was started when the plants were 2 weeks old. Days are numbered from the beginning of treatment. GA responsiveness In tomato, GA is required for seed germination, as shown by the lack of germination in extreme GA-deficient mutants (Groot and Karssen 1987, Koornneef et al. 1990). When combined with the gib-1 mutation, the pro mutation partially releases the GA requirement for germination and leads to increased sensitivity to applied GAs of the remaining fraction of non-germinating seeds (Fig. 4). Tomato WTs, as well as the pro mutant, respond to applied GA by elongation growth. This GA responsiveness was also tested for a number of elongation parameters in the pro gib-1 double mutant and compared to the response of the monogenic mutants and WT. In Fig. 5, it is shown that all genotypes respond to applied GA. However, the amount of GA applied is not sufficient to satisfy the GA requirement of the gib-1 mutant and to completely restore WT. The strongest GA responsiveness, measured as total increase in Table 1. Gibberellin content of 1-month-old tomato wild-type and mutant plants grown in a 16 h of white light (300 mol m 2 s 1 )/8 h of darkness cycle, as measured by GC-MS-SIM with internal standards. ND, no isotopic dilution of internal standards was detected. Genotype Gibberellin content (ng g 1 DW) GA 53 GA 44 GA 19 GA 20 GA 1 GA 8 WT pro gib ND ND ND ND 1.3 pro gib ND ND 0.2 ND 1.1 Physiol. Plant. 106, 1999

5 Fig. 4. Gibberellin (GA 4+7 ) dose-response curve for germination of wild-type (WT, cv. Moneymaker), gib-1 mutant and pro gib-1 double mutant seeds after 7 days of darkness at 25 C. height due to GA application, is observed in the pro gib-1 double mutant, suggesting that the effect of the pro mutation is an increased sensitivity to applied and endogenous GAs. The pro and gib-1 mutations also influence other vegetative traits, such as leaf thickness and leaf length. Similarly, as observed for leaf shape, the pro mutation is almost completely epistatic to gib-1 with respect to leaf thickness (Fig. 6A) and this trait is also hardly affected by GA application. This is in contrast to leaf length, for which a significant GA response was observed in the double mutant (Fig. 6B). End-of-day far-red light responses in the pro and gib-1 mutants in relationship to gibberellin Fig. 6. Leaf thickness (A) and leaf length (B) of 36-day-old wildtype (WT1, cv. Moneymaker; WT2, cv. Ailsa Craig), gib-1 and pro mutants and the pro gib-1 double mutant grown in a 16 h of white light (PAR 190 mol m 2 s 1 )/8 h of darkness cycle at 25 C. On day 24 after sowing GA 4+7 was applied to the youngest visible leaf. The leaf below the treated leaf was used for measuring the leaf characteristics. In the present experiments, the effect of EODFR light treatment on plant height was investigated. This treatment reduces the amount of active phytochrome in the subsequent dark period and thereby leads to enhanced elongation growth. In Fig. 7A, it is shown that GA application and EODFR light treatment both promote elongation growth in WT and in the monogenic pro mutant. When these treatments are combined, they act in an additive way. However, the relative effect of GA application is stronger in the pro mutant than in WT. This GA effect is even more pronounced in the gib-1 mutant, which hardly responds to EODFR light. In contrast, the pro gib-1 double mutant shows a small, but significant, EODFR light response. However, when GA is supplied both at the start and in the middle of the EODFR light treatment (day 6), the response of the gib-1 mutant treated with GA is comparable to that of WT (Fig. 8). This indicates that GAs have to be present for the expression of the EODFR light response on elongation growth. Not all components of the phytochrome EODFR light response appear to require a detectable level of GA. This is shown in Fig. 7B, where the reduction of anthocyanin levels due to the EODFR light treatment is observed in all genotypes and particularly dramatically in the gib-1 mutant. Discussion Fig. 5. Increase in plant height 12 days after GA application on the youngest visible leaf of 24-day-old plants of wild-type (WT1, Moneymaker; WT2, Ailsa Craig), gib-1 and pro mutants and the pro gib-1 double mutant grown in a 16 h of white light (PAR 190 mol m 2 s 1 )/8 h of darkness cycle at 25 C. According to Hooley (1994) GA-constitutive-response mutants are those mutants that have a phenotype similar to WT plants treated with GA, but whose phenotype is not due to GA overproduction. The pro mutant fulfils both criteria (Jones 1987, Jupe et al. 1988). The present analysis of the pro gib-1 double mutants and the partial insensitivity of the Physiol. Plant. 106,

6 Fig. 7. Increase in plant height (A) and anthocyanin content (A 535 g 1 fresh weight) (B) of wild-type (WT1, cv. Moneymaker; WT2, cv. Ailsa Craig), gib-1 and pro mutant and pro gib-1 double mutant plants (A). All plants received a drop of H 2 O or 3 g GA 4+7, applied to the youngest visible leaf, and after the 16-h white light (PAR 145 mol m 2 s 1 ) period were either submitted to an immediate 8-h dark period or given a 20-min far-red light pulse before the dark period. Plant height was measured before and after ten daily cycles with an end-of-day far-red (EODFR) light treatment. Anthocyanin content was measured after ten daily cycles of EODFR light treatment. Bars represent the mean SE. pro mutant to inhibitors of GA biosynthesis, further confirms that the pro mutant hardly requires endogenous GAs for elongation growth. In this respect, the pro mutant resembles the barley sln mutant (Lanahan and Ho 1988), the Arabidopsis spy mutant (Jacobsen and Olszewski 1993) and Fig. 8. Increase in plant height of wild-type (WT, cv. Moneymaker) and gib-1 mutant plants. After the 16-h white light (PAR 125 mol m 2 s 1 ) period, plants were either submitted to an immediate 8-h dark period or given a 20-min far-red light pulse before the dark period. Plant height was measured before and after 12 daily cycles with an end-of-day far-red (EODFR) light treatment. At days 0 and 6 of the experiment, a drop of H 2 Oor5 g GA 4+7 was applied to the youngest visible leaf. Bars represent the mean SE. 126 the la cry s double mutant in pea (Potts et al. 1985). In addition, this is also true for other GA-dependent processes, such as seed germination. The pro mutant differs from the la cry s double mutant and from the barley sln mutant in that the GA responsiveness is not saturated. However, it should be emphasised that this saturation effect depends on the trait observed. Different plant characters apparently differ in their GA requirement. Elongation growth and seed germination for instance have a higher requirement than leaf parameters, such as leaf shape and leaf thickness. This incomplete epistasis of pro over gib-1 for certain characters is in agreement with the leaky nature of the pro mutant phenotype and is compatible with the observation that the GA responsiveness of pro is not saturated for these traits. Incomplete epistasis to GA deficiency for elongation growth was also observed for the spy mutant (Jacobsen and Olszewski 1993). To explain this characteristic, genetic redundancy might be a more likely explanation than leakiness of the pro mutation itself, since this mutant was induced by irradiation. The situation might be similar to that in Arabidopsis, where rga1 mutants have been described, which in combination with spy lead to a saturated GA responsiveness (Silverstone et al. 1997). The latter observation indicates that redundant genes for this property are not restricted to pea. When compared to spy and rga1, pro is likely to be an orthologue of spy, since mutations at both the SPY and Pro genes lead to a recognisable single mutant phenotype, which is not the case for mutations in RGA1. However, one should not rule out that similar genes may contribute in a quantitatively different way in different species, as has been shown by the comparison of phytochrome B-type mutants in Arabidopsis and tomato (Van Tuinen et al. 1995). The availability of cloned Arabidopsis genes and knowledge about the genetic map positions of the pro mutation (Van Tuinen et al. 1998) may be used to further investigate the homology between genes that give a similar phenotype in different species when they are mutated. The comparison of Pro with the pea genes is of interest because of the allelic variation at the Cry locus, where the cry s allele alone is not sufficient to give a slender phenotype, but does modify the response to extended day length by FR light (Murfet 1990). The present experiments indicate that the Pro gene is not directly involved in light perception, because the mutant responds to changes in light quality. Furthermore, both its overall phenotype and its response to broad band spectral light sources (Peters et al. 1992; Peters and Kendrick, unpublished data) are different from the now well-characterised phytochrome mutants of tomato (Kendrick et al. 1997). However, since both photomorphogenesis and GA influence processes, such as seed germination and elongation growth, it has been suggested that photomorphogenic treatments affect GA signal transduction. The observation that the effect of applied GAs is amplified by EODFR light treatment (Fig. 7) favours the hypothesis that phytochrome acts through changes in GA responsiveness. GA signal transduction is assumed to be the site of action of the GA-constitutive-response mutants (Hooley 1994). It is possible that GA and phytochrome signal transduction interact only relatively far downstream at the level of common target genes, such as those affecting Physiol. Plant. 106, 1999

7 cell elongation processes. The fact that sufficient amounts of GAs have to be present continuously for an EODFR light treatment to be effective was also observed for the na mutant in pea (Reid et al. 1990). This dependency on GA places the EODFR light response downstream of GA action. If EODFR light acts at the level of Pro or its direct targets, one would expect an effect in the pro gib-1 double mutant, taking into account the leaky nature of the pro mutant phenotype due to genetic redundancy. Anthocyanin content of developing tomato leaves has previously been shown to be under the control of phytochrome, being reduced by EODFR light treatments (Peters et al. 1992). Application of GA reduces anthocyanin levels and the anthocyanin content is modified in the GArelated mutants described in this report (Fig. 7B). The observation that the GA and EODFR light effects are largely additive indicates that for anthocyanin accumulation these treatments act independently from each other, in contrast to elongation growth. Further analysis of the relationship between photomorphogenesis and GA metabolism can be done by investigating the genetic interactions between the various mutants modified with respect to phytochromes and GA metabolism and/or signal transduction. Genotypes that abolish both GA and phytochrome responsiveness may identify the common target. Further analysis of the Pro gene at the molecular level could provide further insight into these important signalling pathways. Acknowledgements This research was supported by a grant to A. van Tuinen from the Foundation for Life Sciences (SLW), formerly the Foundation for Biological Research (BION), which is subsidised by The Netherlands Organisation for Scientific Research (NWO). J. A. D. Zeevaart was supported by US Department of Energy grant no. 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