1 IFEVA, Facultad de Agronomía, Universidad de Buenos Aires, Avenida San Martín 4453, 1417-Buenos Aires, Argentina
2 Instituto de Investigaciones Bioquímicas Fundación Campomar, Avenida Patricias Argentinas 435, 1405-Buenos Aires, Argentina
Present address: The Salk Institute for Biological Studies, La Jolla, California, 92037, USA
*Author for correspondence (e-mail: casal{at}ifeva.edu.ar)
Accepted March 27, 2001
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SUMMARY |
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Key words: Arabidopsis thaliana, Canalisation, Cryptochromes, Flowering, Lhc, Phytochromes
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INTRODUCTION |
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Arabidopsis thaliana has five phytochromes (phyA through phyE; Quail et al., 1995). Phytochromes are red light/far-red light photoreceptors that are present in the cytoplasm in the inactive form and migrate to the nucleus upon activation by light (Gil et al., 2000; Sakamoto and Nagatani, 1996). In the nucleus, phytochrome interacts with DNA-binding proteins (Ni et al., 1999). Active phytochrome present in the cytoplasm phosphorylates protein substrates (Fankhauser et al., 1999). Both in the cytoplasm and in the nucleus, phytochromes may interact with nucleoside diphosphate kinase 2 (Choi et al., 1999).
Two cryptochromes (cry1 and cry2) are present as blue-UV-A photoreceptors in Arabidopsis thaliana (Cashmore et al., 1999). cry2, and at least in darkness cry1, localise to the nucleus (Cashmore et al., 1999; Guo et al., 1999; Kleiner et al., 1999). Cryptochromes are not exclusive to plants. They are also involved as photoreceptors in the input to circadian clocks in Drosophila (Emery et al., 2000), and as components of both the central clock and the photoperception mechanisms in mammals (Selby et al., 2000). Cryptochromes show considerable homology to photolyases but possess a unique C-terminal extension and lack photolyase activity. The mechanisms of action are only beginning to be understood (Yang et al., 2000).
The CRY2 gene was isolated by cross-hybridisation with CRY1 cDNA as the probe (Lin et al., 1996). CRY1 had been cloned from a T-DNA mutant with impaired hypocotyl growth inhibition by blue light (Ahmad and Cashmore, 1993), and is allelic to the previously known hy4 mutant (Koornneef et al., 1980). Transgenic plants overexpressing the CRY2 gene were found to be hypersensitive to very low fluence rates of blue light and this observation was used to design a screening for plants lacking the cry2 photoreceptor (Lin et al., 1998). The cry2 mutants showed reduced hypocotyl growth and cotyledon unfolding only at low fluence rates of continuous blue light (Lin et al., 1998) and flowered later than the wild type (Guo et al., 1998). Arabidopsis plants flower earlier under long photoperiods than they do under short photoperiods (Martinez-Zapater and Somervile, 1990) but the cry2 mutant shows a severe loss of response to photoperiod (Guo et al., 1998). cry2 is allelic to fha (Guo et al., 1998) a late-flowering mutant previously isolated by Koornneef et al. (Koornneef et al., 1991).
Genetic experiments have demonstrated that the effects mediated by a given photoreceptor can be strongly affected by the activity of the others (reviewed by Casal, 2000). These interactions depended on light, temperature and developmental stage (Casal and Mazzella, 1998; Cerdán et al., 1999; Mazzella et al., 2000). cry2 has been shown to interact with phyB in the regulation of the transition between vegetative and reproductive stages (Mockler et al., 1999). Using fluorescent resonance energy transfer microscopy, phyB and cry2 have recently been shown to interact in specific nuclear speckles that are formed in a light-dependent manner (Más et al., 2000). However, cry2 interactions with other photoreceptors and at other developmental stages have not been documented. We have recently observed that the phyA phyB cry1 cry2 quadruple mutant is severely impaired during de-etiolation but retains a robust circadian rhythm of leaf movement that can be synchronised by light (Yanovsky et al., 2000). The aim of the present work was to investigate how severely growth and development could be affected by the absence of the four most important photoreceptors, and to elucidate the role of cry2 and its interaction with phyA, phyB and cry1. For this propose, growth and development were investigated in phyA, phyB, cry1 and cry2 single mutants and all double, triple and quadruple mutant combinations.
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MATERIALS AND METHODS |
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Transgenic plants of Arabidopsis thaliana ecotype Landsberg erecta carrying the promoter of the light harvesting complex gene Lhcb1*2 from Nicotiana plumbaginifolia (from 752 to +67) fused to the gusA gene have been described previously (Cerdán et al., 1999). This line was used to introgress the transgene in the cry1, cry2, cry1 cry2, phyA phyB, phyA phyB cry1 and phyA phyB cry2 backgrounds described above by crosses followed by screenings under restricted light fields, PCR tests and kanamycin resistance.
Hypocotyl growth and cotyledon unfolding
Seeds of each genotype were sown on 0.8% (w/v) agar in clear plastic boxes (40 mm x 33 mm x 15 mm height), incubated at 6°C for 3 days and transferred to white light provided by high pressure sodium lamps (Philips SON; 300 µmol/m2/second between 400 and 700 nm) for 7 days before measurements. Photoperiod was 16 hours and temperature 20°C. A group of chilled seeds was given only a pulse of red light and transferred to darkness for 7 days. These seedlings were used as dark controls to ensure that differences among genotypes were due to differential responses to light. Hypocotyl length was measured to the nearest 0.5 mm with a ruler. The angle between the cotyledons was recorded with a protractor.
Lhcb1*2 gene expression and chlorophyll levels in de-etiolating seedlings
Approximately 100 seeds were sown in the clear plastic boxes and incubated in darkness at 6°C as described above. Chilled seeds were given a pulse of red light, incubated 24 hours in darkness and transferred to continuous white light (400 µmol/m2/second 20°C) or darkness for 24, 48 or 72 hours before harvest. In some experiments, the blue light component (100 µmol/m2/second) of the light field was eliminated by placing a combination of one yellow and one orange acetate filter (for spectra see Casal and Boccalandro, 1995).
For the measurements of ß-glucuronidase (GUS) activity, the seedlings were harvested under dim green light, homogenized in 50 µl ice-cooled extraction buffer, and microcentrifuged at 4°C. The supernatant was stored at -80°C (usually for less than a week). GUS activity was measured according to the method of Jefferson et al. (Jefferson et al., 1987) using 4-methylumbelliferyl-ß-D-glucuronide (from Sigma, St Louis) as substrate. The standard curves were prepared with 4-methylumbelliferone (4-MU from Sigma, St Louis). Protein content was measured as described previously (Lowry et al., 1951).
For comparative purposes chlorophyll levels were measured in separate sets of seedlings grown under the same conditions. The seedlings were harvested in 1 ml of N,N'-dimethylformamide and incubated in darkness at 20°C for at least 3 days. Absorbance was measured at 647 and 664 nm, and chlorophyll levels were calculated according to the method of Moran (Moran, 1982).
Leaf production and flowering
To investigate later developmental stages, the seedlings were left for 3 days on agar under white light, and were subsequently transplanted to plastic pots (7 cm height x 4 cm diameter) filled with a mixture of soil and perlite (3/1). Plants were exposed to long days (photoperiod=16 hours). In some experiments, plants were exposed either to short days (photoperiod=7 hours) or to short days, between day 0 and 21, followed by 5 long days and returned to short days. All experiments were conducted at 20°C.
The number of visible leaves (i.e. leaves larger than 2 mm) was recorded every other day (in most experiments) and the final number of leaves (rosette plus stem leaves) was used as a measurement of flowering time on a biological scale (Koornneef et al., 1991). The time when the first flower bud became externally visible to the naked eye and the time of anthesis of the first flower were recorded for each plant.
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RESULTS |
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Lhcb1*2-gusA expression during de-etiolation
In the wild type, continuous white light caused a dramatic promotion of GUS activity driven by the promoter of the tobacco Lhcb1*2 gene (Fig. 2A). Over the same time frame used for light-grown seedlings, in dark controls, relative GUS activity was at most 0.01 (data not shown). In the presence of phyA and phyB, the cry1 mutation caused only a transient reduction of GUS activity (P<0.01, for 1 and 2 days of treatment; Fig. 2A) that was no longer detectable after 3 days under continuous white light. The cry2 mutation caused a more severe reduction of GUS activity (P<0.0001) (Fig. 2A). The effects of these mutations were not additive as the double mutant behaved initially like the cry2 single mutant and, after prolonged exposure to white light (day 3), showed stronger GUS activity than the cry2 single mutant (P<0.05; Fig. 2A).
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Since dark controls show very low levels of GUS driven by the Lhcb1*2 promoter, the seedlings were grown under white light minus blue light to investigate whether the effects of cry1 and cry2 were either constitutive or dependent on blue light. The cry1, cry2 and cry1 cry2 mutants showed normal levels of GUS activity (P>0.1) and all the mutants in the phyA phyB background showed negligible GUS activity under orange light (i.e. white light minus blue light; Fig. 2B). It is noteworthy that in the presence of phyA and phyB, blue light did not increase GUS activity in the wild type, rather, it decreased GUS activity in the cry2 background (P<0.0001). In the absence of phyA and phyB, blue light perceived primarily by cry1 increased GUS activity (P<0.0001).
The rate of leaf appearance
The number of leaves observed at a given time depends on the rate at which the leaves are produced in the apex during the vegetative phase, the transition of the apex between the vegetative and reproductive stages (that results in the cessation of leaf production), and the early growth of the leaf primordium. The only single mutation that reduced the rate of leaf appearance in vegetative plants was phyB (wild type=0.57±0.03 leaves/day, phyB=0.31±0.09 leaves/day; Fig. 3). In the quadruple mutant the first true leaves (i.e. the leaves after the cotyledons) were detected 10 days later than in the wild type. Following this initial delay, which is consistent with the impaired de-etiolation in the quadruple mutant (see Fig. 1), the rate of leaf appearance remained lower in the phyA phyB cry1 cry2 quadruple mutant than in the wild type or even than in the phyB mutant (Fig. 3). These observations indicate that the consequences of deficient light perception on vegetative development are not restricted to de-etiolation but continue beyond this stage (see also Fig. 4A).
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The final number of leaves
Compared to the wild type, the cry2 mutation increased the final number of leaves per plant (P<0.0001) (Fig. 5A), indicating delayed transition between the vegetative and the reproductive phase under long days (Guo et al., 1998). Interestingly, under our conditions the cry1 mutation also increased leaf number (P<0.05) and the cry1 cry2 double mutant showed the additive effects of both mutations. Delayed flowering for cry1 had been observed in the Columbia but not in the Landsberg erecta background (Bagnall et al., 1996; Mockler et al., 1999). The phyB mutation reduced the final number of leaves, indicating an earlier transition between the vegetative and reproductive phase expressed in a biological scale (P<0.0001) (Fig. 5A) (see also Goto et al., 1991). The phyB mutation was epistatic to cry2 (Mockler et al., 1999) and to cry1. All the mutant combinations carrying the phyB allele, including the quadruple mutant, produced 7-8 leaves (P>0.1; Fig. 5A). This indicates that large differences in leaf appearance rate did not result in differences in the final number of leaves. All the mutant combinations carrying the phyB allele produced 11±0.3 leaves under long as well as short days in glasshouse conditions. The phyA mutation had been shown to delay flowering in Arabidopsis plants grown under short days extended with low intensities of incandescent light, which is comparatively rich in far-red light (Johnson et al., 1994). In the conditions of present experiments, however, the phyA mutant produced the same number of leaves than the wild type and the phyA mutation caused a small but statistically significant (P<0.0001) reduction in final leaf number in the background of cry1 (comparison between the phyA cry1 and cry1 mutants), cry2 (comparison between the phyA cry2 and cry2 mutants), and cry1 cry2 (comparison between the phyA cry1 cry2 and cry1 cry2 mutants) (Fig. 5A,C). This effect of the phyA mutation was observed in independently segregating lines of phyA cry2 and phyA cry1 cry2 (data not shown). Thus, the delay in flowering observed in the cry1 and cry2 mutants was reduced not only by the phyB but also by the phyA mutation.
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Time between first visible bud and anthesis
The time between the appearance of first visible bud and anthesis was analysed as a measure of the pace of development after the transition to the reproductive stage. This period was extended in the triple mutants bearing the phyB allele (i.e. phyA phyB cry1, phyA phyB cry2, phyB cry1 cry2) and in the phyA phyB cry1 cry2 quadruple mutant (P<0.0001) (Fig. 7). Compared to the wild type, the cry2 mutant showed delayed flowering (Fig. 5A,B), but was not affected in the time between first visible bud and anthesis (P>0.1). However, the effects of the cry2 mutation became evident in the comparison between phyA phyB and phyA phyB cry2 (P<0.0001) (Fig. 7) as well as between phyB cry1 and phyB cry1 cry2 (P<0.0001) (Fig. 7), indicating redundancy of cry2 with phyA, phyB and cry1.
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DISCUSSION |
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Hierarchy of cry1 and cry2
Both cry1 and cry2 play recurring roles during Arabidopsis development. Of particular significance are the effects on the activity of the Lhcb1*2 promoter as the expression of Lhc genes was thought to be under the control of phytochromes but not cryptochromes (Anderson et al., 1999; Gao and Kaufman, 1994). Our results, however, are in keeping with recent observations that the C terminus of CRY1 is able to confer Lhc expression in darkness (Yang et al., 2000). Although the tobacco Lhcb1*2 promoter used here and the Arabidopsis Lhcb1*2 promoter conserve regions critical for light regulation (including CAAT and GATA boxes, the binding site for CCA1, etc) at similar positions, differential regulation of the tobacco and endogenous Lhcb1*2 promoters cannot be ruled out with available information. The hierarchy of the relative impact of cry1 and cry2 mutations was strongly dependent on developmental context. Whereas cry1 dominated over cry2 in hypocotyl growth and cotyledon unfolding, the complementary pattern was observed for the transition to the reproductive stage (Figs 5, 6; see also Mockler et al., 1999). For the expression of Lhcb1*2-gusA the effect of the cry2 mutation was stronger than that of the cry1 mutation in the PHYA PHYB background but the opposite was true in the phyA phyB background (Fig. 2A). Actually, the comparison between cry2 and cry1 cry2 mutants (Fig. 2) suggests that cry1 could be involved in the inhibition of expression of photosynthetic genes by strong light. Context specificity of homologous receptors is not uncommon and has been observed in other systems. For instance, frizzled receptors in Drosophila are redundant in the control of the pattering of the embryonic nervous system, with a higher effect of frizzled 2 than frizzled 1. However, only frizzled 1 is required for normal epithelial planar polarity (Boutros et al., 2000). cry1 and cry2 show differences in intracellular localisation, protein stability and apparent signalling capacity of their C terminus (Cashmore et al., 1999; Guo et al., 1998; Guo et al., 1999; Kleiner et al., 1999; Yang et al., 2000). The interplay between these differences and the developmental contexts could bring about the observed variation in cry1/cry2 hierarchy.
phyA can delay flowering
Under long days cry2 has been proposed to counteract the delay in flowering caused by phyB because the cry2 mutation delays flowering in the presence of active phyB (Guo et al., 1998; Mockler et al., 1999). The analysis of novel mutant combinations presented here indicates that the effects of cry2 on the final number of leaves (a measure of the transition between vegetative and reproductive development on a biological scale) were reduced not only by the phyB mutation but also by the phyA mutation (Fig. 5A). In other words, although the phyA mutation has previously been shown to delay flowering in plants grown under short days extended with low fluences of light rich in far-red (Johnson et al., 1994) (and we have observed the same phenomenon in unreported experiments), the phyA mutation caused acceleration of flowering in the cry2, cry1 and cry1 cry2 backgrounds (Figs 5A, 6). In addition, although the cry1 cry2 double mutant virtually failed to show a flowering response when exposed to 5 long days (a treatment that accelerates flowering in the wild type), the phyA mutation restored the effects of long days (Fig. 6). Using the same phyA and cry2 alleles involved in the experiments shown here but with a different light protocol, M. Blázquez and D. Weigel (personal communication) have observed delayed flowering in the double compared to the single mutants. Clearly, phyA can accelerate or delay flowering in Arabidopsis depending on the genetic background and light conditions.
Flowering is under the control of environmental and endogenous signals and we are only beginning to understand at a molecular level how these signals are integrated (Blázquez and Weigel, 2000). Light itself has a dual effect on flowering of long-day plants. (1) A positive, photoperiod-dependent effect, that involves the action of light in combination with a circadian rhythm of sensitivity whose phase is in turn light regulated. (2) A negative effect that saturates with short photoperiods (De Lint, 1960) and dominates under red light (Guo et al., 1998). The current view is that cry2 (Guo et al., 1998), phyA (Johnson et al., 1994), and cry1 (Bagnall et al., 1996), mediate the positive effect whereas phyB (Reed et al., 1993), phyD (Devlin et al., 1999), and phyE (Devlin et al., 1998) mediate the negative effect. The observations that phyA can both advance and delay flowering, and that flowering can be accelerated by the phyB mutation (Reed et al., 1993) as well as by the overexpression of phyB (Bagnall et al., 1995), suggest that the various photoreceptors could operate in a more integrated way and become involved both in negative and positive effects albeit with a different hierarchy (Fig. 8).
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ACKNOWLEDGMENTS |
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