Present address: Department of Plant Biology, University of Georgia, Athens, GA 30602, USA.
Phytochromes differentially regulate seed germination responses to light quality and temperature cues during seed maturation
Article first published online: 15 MAY 2009
© 2009 Blackwell Publishing Ltd
Plant, Cell & Environment
Volume 32, Issue 10, pages 1297–1309, October 2009
How to Cite
DECHAINE, J. M., GARDNER, G. and WEINIG, C. (2009), Phytochromes differentially regulate seed germination responses to light quality and temperature cues during seed maturation. Plant, Cell & Environment, 32: 1297–1309. doi: 10.1111/j.1365-3040.2009.01998.x
- Issue published online: 7 SEP 2009
- Article first published online: 15 MAY 2009
- Received 2 February 2009; received in revised form 20 April 2009; accepted for publication 21 April 2009
- Arabidopsis thaliana;
- foliar shade;
- maternal effects;
- maternal environment;
- natural variation;
- red to far-red ratio (R : FR);
The ratio of red to far-red light (R : FR) experienced by seeds during maturation affects germination, but the genetic regulation of this effect is poorly understood. In Arabidopsis thaliana, responses to R : FR are governed by five phytochrome photoreceptors, PHYA–PHYE. PHYA, PHYB and PHYE mediate germination, but their roles in germination response to the seed maturation environment are largely unknown. Seeds of A. thaliana phytochrome mutants and natural accessions were matured in a factorial combination of cold (16 °C) and warm (24 °C) temperatures and high (R : FR = 1) and low (R : FR = 0.6) R : FR environments, resembling sunlight and foliar shade, respectively. Germination was observed in resulting seeds. All five phytochromes mediated germination responses to seed maturation temperature and/or R : FR environment. PHYA suppressed germination in seeds matured under cold temperature, and PHYB promoted germination under the same conditions. PHYD and PHYE promoted germination of seeds matured under warm temperature, but this effect diminished when seeds matured under reduced R : FR. The A. thaliana natural accessions exhibited interesting variation in germination responses to the experimental conditions. Our results suggest that the role of individual PHY loci in regulating plant responses to R : FR varies depending on temperature and provide novel insights into the genetic basis of maternal effects.
Plants rely on environmental cues to direct life-history processes. In species that disperse seeds close to the maternal plant, the seed maturation environment may be a reliable indicator of the environmental conditions in subsequent generations (Donohue & Schmitt 1998). The timing of seed germination is highly sensitive to several aspects of the seed maturation (also termed maternal) environment, including water availability, soil nutrients, photoperiod, temperature and light quality (reviewed in Gutterman 2000; Finch-Savage & Leubner-Metzger 2006; Contreras et al. 2008; Donohue et al. 2008). Light quality, that is, the ratio of red (R) to far-red (FR) light (R : FR), experienced by the maternal plant during seed development is predictive of canopy conditions; a low R : FR indicates below-canopy or competitive conditions, because chlorophyll selectively absorbs light in the red region of the spectrum while transmitting FR light (Smith 1982). A high R : FR indicates open sunlit gaps, which are favourable for germination and plant growth. Canopy conditions during seed maturation influence germination timing in a number of species (Schmitt, Niles & Wulff 1992; Donohue & Schmitt 1998; Gutterman 2000; Orozco-Segovia et al. 2000), and these effects have recently been shown to be adaptive in the wild (Galloway & Etterson 2007).
In Arabidopsis thaliana, responses to R : FR are governed by a family of five phytochrome photoreceptors, PHYA–PHYE (Sharrock & Quail 1989). Their gene products fall into two classes: Type 1 includes phyA, which increases to high levels in dark-grown seeds and seedlings and then rapidly declines in light. All remaining phytochrome proteins are Type 2 and are relatively stable in R light (Sharrock & Clack 2002). The phytochrome protein is synthesized in the inactive, R light absorbing form (Pr), which then photoconverts to the active, FR light absorbing form (Pfr) in response to R light. Pfr reverts to Pr in FR light or over a dark period. Unique functions for each phytochrome species have been identified at several stages of plant development, such as seed germination, growth and flowering (reviewed in Quail 2002).
Photoreversible phytochrome effects on seed germination were identified several decades ago (Borthwick et al. 1952). More recently, roles for specific phytochromes in regulating germination responses have been demonstrated. In A. thaliana, PHYA induces germination under very low fluence (VLFR; <1 µmol m−2) light ranging in wavelength from 300 to 780 nm (Shinomura et al. 1996) and under heavy canopy shade (R : FR < 0.05) in the field (Botto et al. 1996). Under continuous FR light, this VLFR is also dependent on the presence of PHYE (Hennig et al. 2002). PHYA and PHYB interact to mediate germination under low fluence R light (LFR, >10 µmol m−2) in A. thaliana (Botto, Sanchez & Casal 1995; Botto et al. 1996; Shinomura et al. 1996). In tomato, PHYB appears to be solely responsible for LFR, whereas PHYA inhibits germination under continuous R or FR light (Appenroth et al. 2006).
Phytochromes are not only sensitive to the germination environment, but they may also mediate the effects of light quality during seed maturation on subsequent germination. A. thaliana seeds matured under a high R : FR germinate in darkness, but seeds that matured under environments rich in FR light require light to germinate, and this effect is R : FR reversible (McCullough & Shropshire 1970; Hayes & Klein 1974). A possible mechanism for this effect is that the photostationary state of the phytochrome protein (Pr or Pfr) in the dry seed is set by the R : FR environment during seed maturation (Cresswell & Grime 1981). Seeds show little response to photoreversible light cues when dry but become increasingly responsive over imbibition, which suggests that the phytochrome state in the dry seed is relatively stable and may determine the amount of light that is required for germination (Kendrick & Frankland 1969; Kendrick & Spruit 1977; Cresswell & Grime 1981). Accordingly, interspecific differences in a light requirement for seed germination are partially attributable to the timing of chlorophyll breakdown in the maternal tissues relative to seed maturation (Cresswell & Grime 1981). Species that require light for germination retain chlorophyll in the maternal tissues surrounding the seeds, and therefore, their seeds experience a low R : FR throughout maturation. Effects of light quality during seed maturation on germination are likely phytochrome-mediated, but the roles of individual phytochrome loci in these effects are unknown.
Phytochrome effects are also sensitive to temperature. For example, a temperature reduction of 6 °C revealed novel roles for PHYA, PHYD and PHYE in flowering (Halliday & Whitelam 2003). Phytochrome function also varies depending on the temperature seeds experience during germination and maturation. Heschel et al. (2007) showed that although PHYB influences germination across a range of germination temperatures, the effects of PHYA and PHYE become more significant at warmer and cooler temperatures, respectively. In addition, phytochromes differentially regulate seed germination depending on the temperature during seed maturation (Donohue et al. 2008). For example, PHYB and PHYD are important for breaking cold-induced dormancy, whereas PHYA contributes to maintaining dormancy.
In addition to using mutants to characterize the roles of the individual phytochrome loci, recent work has shown that natural allelic variation in phytochrome genes affects hypocotyl elongation and flowering time (Aukerman et al. 1997; Maloof et al. 2001; Balasubramanian et al. 2006; Filiault et al. 2008). These studies suggest that natural selection may target phytochrome genes, and that allelic variation in phytochrome loci likely contributes to phenotypic differences among natural populations of A. thaliana. Natural allelic variation in phytochrome genes may also affect seed germination, but this has not yet been examined.
In this study, we compare the germination responses of multiple phytochrome mutant plants to their wild-type background in order to investigate which, if any, phytochromes regulate the effects of light quality and temperature cues during seed maturation on subsequent germination. In addition, we examine germination responses to the same environmental cues in several A. thaliana natural accessions in order to test for genetic variation and more generally investigate the effects of the seed maturation treatments on germination in A. thaliana.
MATERIALS AND METHODS
In order to investigate the role of each phytochrome in mediating the effects of seed maturation environment on germination, we compared seed germination in A. thaliana wild-type genotypes Landsberg erecta[Ler, Arabidopsis Biological Resource Center (ABRC) stock number: CS20], Columbia (Col-0, CS6673) and Wassilewskija (Ws, CS915) to mutants that exhibit loss of function in one or more phytochrome genes. Single mutants of phyA, phyB and phyE were generated in the Ler and/or Col-0 backgrounds through forward and backward genetic screens (Koornneef, Rolff & Spruit 1980; Nagatani, Reed & Chory 1993; Reed et al. 1993, 1994; Devlin, Patel & Whitelam. 1998). We included multiple phyA and phyB deficient mutant alleles in the Ler background; among these, phyA-203 (CS6221), phyA-205 (CS6222), phyB-4 (CS6212) and phyB-7 (CS6215) have been shown to exhibit weak phytochrome-mediated phenotypic responses compared with phyA-201 (CS6219), phyA-202 (CS6220), phyB-1 (CS6211), phyB-5 (CS6213) and phyB-9 (CS6217) (Reed et al. 1993, 1994). The phyD-1 mutation is a natural null allele that was identified in Ws and crossed into the Ler background (Aukerman et al. 1997). Mutants deficient in phyC were identified from T-DNA insertion lines in the Col-0 (phyC-2) and Ws (phyC-3) backgrounds (Monte et al. 2003). Ws is naturally deficient in phyD, and therefore, phyC-3 lacks phyC and phyD. Double and triple mutants were obtained by crossing (Devlin et al. 1998, 1999). All single phyA and phyB mutants were obtained from ABRC. PhyC-2, phyC-3, phyD-1 phyA-201/phyB-5, phyA-201/phyD-1, phyB-1/phyD-1 and phyA-201/phyB-1/phyD-1 were obtained from Robert A. Sharrock. PhyE-1, phyA-201/phyE-1, phyB-1/phyE-1, phyB-1/phyD-1/phyE-1, phyA-201/phyB-1/phyE-1 and phyA-201/phyB-1/phyD-1/phyE-1 were obtained from Gary C. Whitelam.
We also included six additional A. thaliana natural accessions (for a total of nine accessions, six plus Col-0, Ler and Ws) in order to preliminarily examine natural variation in A. thaliana germination responses to the seed maturation environments tested in this study. All accessions were obtained from the ABRC and included: Col-4 (CS933); Ga-2 (CS6715); HOG (CS6178); JI-3 (CS6745); Lm-2 (CS6784); and Me-0 (CS1364). These accessions were chosen in two steps. Firstly, 25 accessions were chosen from a set of ∼180 that were part of a larger association mapping experiment (C. Weinig, unpublished data) and exhibited significant variation in adult shade-avoidance phenotypes. From these, 10 accessions were chosen because they clustered either with phyA or phyB in hypocotyl responses to red or far-red light (Maloof et al. 2001). The additional 15 were randomly chosen to represent the latitudinal range of A. thaliana. In a pilot experiment, these accessions were matured under simulated foliar-shade and neutral-shade treatments (see following methods) in a greenhouse and germinated under the same treatments. For further investigation, we chose the 10 natural accessions that expressed the greatest variation in germination percentage when seeds had been matured under simulated foliar shade. One accession, Bla-11, did not set seed in all four seed maturation treatments and was excluded from analyses. All mutants and natural accessions are highly inbred and were allowed to self-pollinate. Maternal, paternal and direct genetic effects cannot be differentiated in this experiment.
Seed maturation and germination conditions
For seed maturation, four sets of plants were established by randomly planting two replicate individuals of each genotype into every other pot cavity (5 cm diameter × 5 cm deep) of two Araflats (Betatech, Belgium) in Sunshine Mix, LP5 soil (Sun Gro Horticulture, Vancouver, Canada). Plants were stagger-planted based on the estimated days to seed maturation for each combination of genotype and seed maturation treatment. For example, in a prior experiment, Ler plants had taken on average of 1 week longer to mature seeds in cold/neutral shade than in warm/neutral shade treatments. Therefore, Ler seeds were planted 1 week earlier in cold/neutral shade than in the warm/neutral shade. Stagger-planting ensured that seed collection could be done simultaneously and that after-ripening would not vary among genotypes or across seed maturation treatments. Seeds were cold-stratified at 4 °C for 4 d and then placed into two Conviron E7/2 growth chambers (Controlled Environments Ltd, Winnipeg, Canada) under a 12 h photoperiod and constant 20 °C. Growth chambers were fitted with eight white fluorescent lights (F038/871, 32W, Sylvania Octron, Danvers, MA, USA), two infrared lights (Geneva Scientific, Williams Bay, WI, USA) and four incandescent lights (Sylvania Double Life, Soft White, 120V/75W). In the treatments, total PAR = 210 µmol m−2 s−1 and R : FR = 1.1, as measured using a LI-250A light sensor (Li-Cor, Lincoln, NE, USA) and SKR 110, 660/730 nm sensor (Skye Instruments Ltd, Powys, UK), respectively. At bolting, growth chambers were reset to cold (17 °C day/14 °C night) and warm (26 °C day /22 °C night) temperatures, and white (#214, Full Touch Spun) and green (#730, Liberty Green) filters (Lee filters, Burbank, Canada) were placed over two flats in each growth chamber to impose simulated foliar shade (R : FR = 0.6, PAR = 170 µmol m−2 s−1) and neutral shade (R : FR = 1.1, PAR = 175 µmol m−2 s−1) light environments. This experimental set-up generated four seed maturation treatments: cold/simulated foliar shade (C/FS); cold/neutral shade (C/NS); warm/simulated foliar shade (W/FS); and warm/neutral shade (W/NS). The temperature treatments in this study simulate the average cold and warm temperatures that wild A. thaliana plants would likely experience in early or late spring, respectively. The simulated foliar shade seed maturation environment simulates R : FR conditions typical of a competitive environment or under a plant canopy. The treatments were applied at bolting for two reasons. Firstly, A. thaliana plants in the wild germinate early in the spring when there is often little plant competition, and begin to experience shading at bolting when neighboring A. thaliana individuals are also bolting and flowering and have large rosettes. Secondly, several studies provide strong evidence that effects of maternal light quality on seed germination are perceived during seed maturation, possibly by the seed itself (McCullough & Shropshire 1970; Hayes & Klein 1974; Cresswell & Grime 1981; Contreras et al. 2008), and that changes in light quality perceived by the maternal plant before seed maturation have little or no direct effect on seed germination.
Seeds of mutant plants and natural accessions were matured and collected under the four seed maturation treatments. To control for variation in seed maturation, six apical siliques at roughly the same maturity level were collected from the primary inflorescence of each replicate plant and pooled by genotype and seed maturation treatment. Seeds were stored in the dark at room temperature for 1 month and then six replicates of 10–20 seeds for each combination of genotype by seed maturation treatment were planted, under a green safe light, onto 0.5% agar in 4.5-cm-diameter plastic Petri dishes. Seeds imbibed water for 24 h in darkness and were placed into the same growth chambers in a 20 °C, 12 h photoperiod, and the same neutral shade treatment as the seed maturation generation. Seeds were checked for germination every day for 10 d. Germination was scored when a seed's cotyledons were green and had begun to unfold. We scored germination in this manner for ecological relevance. In several instances, the radical would emerge 1–2 mm from a seed but no more by the end of the 10 d (and the seedling appeared to be no longer viable); these seeds would not have established in a natural environment. One caveat of this method is that we cannot explicitly separate the effects of radicle emergence and greening, although we do not expect our results to differ greatly for these two germination measures. In fact, mean germination percentages differed by at most 1% if seeds that germinated but failed to open cotyledons were included in the analyses versus if they were not (results not shown).
Seed mass was also determined by weighing two groups of 10 seeds for each genotype by seed maturation treatment combination. To evaluate the repeatability of our results, the entire experiment (seed maturation and germination) was completed twice, each time in two different Conviron E7/2 growth chambers. The six additional A. thaliana natural accessions, as well as phyC-3, phyA-211 and phyA-211/phyB-9 were only included in the second experimental trial. Therefore, the results of the second trial are reported with discussion of the consistencies and differences between trials.
Germination percentage was calculated for each replicate by dividing the number of seeds germinated by the number of seeds planted. This value is presented for all genotype by seed maturation treatment combinations that exhibited >90% germination. If the germination percentage of a genotype by seed maturation treatment combination was <90%, we estimated maximum possible germination by germinating two replicates of 10–20 seeds on 0.5% agar supplemented with 100 µm GA4+7. All other experimental conditions were as described earlier. GA4+7 promotes germination in A. thaliana; seeds that do not germinate with GA4+7 are unlikely to be viable. GA4+7 has the additional advantage of testing for possible suppression of germination by our seed maturation treatments, because GA regulates germination downstream of the phytochromes (Yamaguchi et al. 1998). Germination with GA4+7 was >90% for all genotype by seed maturation treatment combinations tested except phyA/phyE (C/FS), phyA/phyB/phyE (C/FS, C/NS), phyA/phyB/phyD (C/NS) and phyA-211/phyB-9 (Col-0) (W/FS). Their germination percentages ranged from 65 to 85%. These results suggest that most seeds were viable and capable of germination and that the seed maturation treatments did not suppress germination in a manner independent of the mutant alleles. Germination percentage data were adjusted using the results of the GA4+7 experiment as the maximum possible germination (instead of 100%). The GA4+7 adjustment had very little effect on the results, because generally, seeds that germinated to <90% with GA4+7 exhibited very low germination (<10%) in all experiments.
Germination day was calculated by assigning a germination day to each seed that germinated and calculating mean days to germination for each replicate. Germination percentage displayed constant variance. Days to germination violated the analysis of variance (anova) assumption of homoscedasticity. As suggested by a Box–Cox procedure (PROC TRANSREG, SAS 2001), germination day was transformed by taking the inverse. After transformation, germination day displayed constant variance, and the transformed values were used in subsequent analyses.
To test for differences in the effects of seed maturation treatment on germination between wild-type genotypes and phytochrome mutants, we used an anova model (PROC GLM) in which we split the analysis by seed maturation treatment (light quality and temperature) and then tested for the fixed effects of growth chamber (during seed germination) and genotype on germination percentage and germination day. All mutants in a wild-type background were included in a model and a priori contrasts between each mutant genotype and its respective wild-type genotype and between specific mutant pairs were applied. A significant effect of genotype indicates that the two genotypes differed in their response to the specified seed maturation treatment combination (Donohue et al. 2008). Bonferroni corrections for multiple tests, based on the total number of tests in one mutant background and seed maturation treatment, were applied to the results.
We used a second anova model (PROC GLM) to test for effects of seed maturation treatment on seed mass, germination percentage and germination day among the nine A. thaliana natural accessions. This model included genotype and temperature and light quality during seed maturation, and all possible interactions as fixed effects. The three-way interaction between genotype, seed maturation temperature and light quality was not significant for germination percentage or day, and was removed from the model for those traits.
PhyA mutant seeds derived from all seed maturation treatments except warm/neutral shade germinated to higher percentages than wild-type, although the significance of these effects differed by mutant allele (Fig. 1; Table 1). PhyA-201 and phyA-203 exhibited significantly higher germination than Ler when seeds matured under the cold seed maturation treatment. PhyA-202, phyA-205 (only in neutral shade) and phyA-211 exhibited the same trend, but they were not significant after Bonferroni correction (Figs 1 & 2). The one exception was that phyA-205 seeds matured under cold and simulated foliar shade germinated to a lower percentage than Ler. PhyA-205 is a documented weak mutant (Reed et al. 1994), and this result was not repeatable, that is, in the first experimental trial all phyA mutants (including phyA-205) germinated to a higher percentage than Ler when seeds were matured in cold temperature. Germination in phyA deficient seeds ranged from 12 to 25% higher than wild-type genotypes in seeds matured in cold and neutral shade and 22–55% higher in seeds matured in cold and simulated foliar shade (excluding phyA-205). Germination day was accelerated by 2–5 d over wild-type genotypes in phyA mutant seeds matured under warm temperature and simulated foliar shade (Fig. 3; Table 2). Two phyA mutants, phyA-201 and phyA-203, also exhibited accelerated germination in seeds matured under cold temperature and either light quality treatment (Table 2). Similar patterns of germination were observed in the first experimental trial, with the exception that phyA-211 was not tested.
|Seed maturation treatment|
|Cold temperature||Warm temperature|
|Simulated foliar shade||Neutral shade||Simulated foliar shade||Neutral shade|
|F ratio||F ratio||F ratio||F ratio|
|Mutants versus wild-type genotypes|
|Multiple mutant comparisons|
|phyA-201/phyD-1 versus phyA-201||0.04||0.61||6.43*||0.72|
|phyB-1/phyD-1 versus phyB-1||0.35||0.14||3.30||8.99**|
|phyA-201/phyB-1/phyD-1 versus phyA-201/phyB-5||0.10||0.03||5.60*||1.97|
|phyA-201/phyE-1 versus phyA-201||24.03***||124.01***||28.35***||58.01***|
|phyB-1/phyE-1 versus phyB-1||0.54||0.15||19.31***||16.54***|
|phyA-201/phyB-1/phyE-1 versus phyA-201/phyB-5||0.11||0.00||4.04*||4.18*|
|phyB-1/phyD-1/phyE-1 versus phyB-5/phyD-1||0.06||0.26||1.30||0.20|
|Seed maturation treatment|
|Cold temperature||Warm temperature|
|Simulated foliar shade||Neutral shade||Simulated foliar shade||Neutral shade|
|F ratio||F ratio||F ratio||F ratio|
|Mutants versus Ler|
In general, Ler mutants deficient in phyB exhibited reduced germination when compared with Ler, and this was most significant when seeds matured under neutral shade (Fig. 1; Table 1). These trends were weaker in documented weak mutants, phyB-4 and phyB-7 (Reed et al. 1993) than phyB-1 and phyB-5. Seed germination was also reduced in phyB-9 (Col-0 background) seeds matured in cold temperature and neutral shade, as well as warm temperature and simulated foliar shade (Fig. 2). This difference between phyB-9 and phyB mutants in the Ler background under warm treatments is largely attributable to differing germination patterns between Col-0 and Ler; the Col-0 genotype exhibited 100% germination in seeds matured under warm/simulated foliar shade (Fig. 2). In addition, germination was accelerated by 1.5–3 d in phyB-1, phyB-4 and phyB-5 seeds matured under simulated foliar shade and either temperature treatment (Fig. 3; Table 2). Differences in germination between phyB alleles and wild-type genotypes followed the same trends in the first experimental trial (data not shown).
Germination was reduced in phyA/phyB (Ler and Col-0 backgrounds) mutant seeds matured under all four seed maturation treatments (Figs 1 & 2; Table 1). In addition, phyA-201/phyB-5 seeds matured under warm temperature (and either light quality treatment) germinated 2 d earlier that Ler (Fig. 3; Table 2). These results were repeated in both experimental trials.
In the Col-0 background, PHYC had no effect on germination in seeds matured in the warm temperature, that is, differences between phyC-2 and Col-0 were marginal in the second experimental trial and were not detected in the first experimental trial (Fig. 2; Table 1). In contrast, phyC-3 seeds germinated to a higher percentage than Ws when matured under warm temperature or simulated foliar shade (Fig. 2; Table 1). PHYC had some effect on germination in Col-0 seeds matured in cold temperature, but the direction of the effect differed between experimental trials, making it difficult to draw any conclusions. The phyA/phyB/phyD/phyE mutant showed some germination (2–5%) in seeds matured under all treatments except in cold/neutral shade (Fig. 4), suggesting that phyC plays some role in germination, or alternatively, A. thaliana seeds can germinate without phytochrome.
PHYD had weak effects on germination percentage in seeds matured under cold temperature and phyD seeds matured under cold temperature or simulated foliar shade germinated 1.5–2.5 d earlier than Ler. More specifically, PhyD seeds matured in cold temperature did not differ significantly in germination percentage from Ler, and germination differences were not significant between phyA/phyD and phyA, phyB/phyD and phyB, and phyA/phyB/phyD and phyA/phyB (Fig. 5, Table 1). In contrast, phyD germination was significantly lower than Ler in seeds matured under warm temperature, and this effect was most pronounced in seeds also matured under neutral shade. Seeds matured under warm temperature and either light quality treatment germinated to a lower percentage for phyB/phyD than phyB or phyD, and for phyA/phyB/phyD than phyA/phyB (Fig. 5; Table 1). PhyD results were consistent across both experimental trials (data not shown).
Seeds deficient in phyE germinated significantly less than Ler when matured under neutral shade and either temperature (Fig. 4; Table 1) and significantly later than Ler when matured under neutral shade and warm temperature (Fig. 3; Table 2). PHYE also appeared to counteract the effects of PHYA across all seed maturation treatments, except warm/simulated foliar shade, that is, phyA/phyE germination was >75% less than phyA germination. Mutant seeds matured in cold temperature that were deficient in phyB and phyE (phyB/phyE, phyB/phyD/phyE, phyA/phyB/phyE, and phyA/phyB/phyD/phyE) exhibited very low germination when compared with Ler (Fig. 4). In addition, phyB/phyE seeds matured under warm temperature and neutral shade germinated >30% less than phyB or phyE under the same conditions. Patterns of germination in phyE seeds were consistent across both experimental trials (data not shown).
Cold maturation temperatures reduced germination percentages by almost half when compared with seeds matured under warm temperatures (Table 3). Light quality during seed maturation had no overall effect on germination percentage, but natural accessions differed in their responsiveness to seed maturation temperature and light quality for both germination percentage and day (Figs 6 & 7; Table 3a). Note that, for instance, Ler germinates to a higher percentage under neutral than simulated foliar shade, and Col-0 shows the opposite pattern. Temperature and light environment, as well as their interaction, had a strong effect on seed mass, and accessions differed in the response of seed mass to these treatments (Table 3a). In general, seeds produced in cold temperature and/or simulated foliar shade were heavier than seed produced in warm temperature and/or neutral shade (Table 3b).
|(a)||Seed mass (mg)||Germination percent||Germination day|
|Source||d.f.||F ratio||F ratio||F ratio|
|Light quality (LQ)||1||104.04***||0.02||9.64***|
|Seed maturation treatment||Least-squared means ± 1 SE|
|Cold/simulated foliar shade||0.274 ± 0.004||31.472 ± 2.563||5.850 ± 0.174|
|Cold/neutral shade||0.204 ± 0.004||32.053 ± 2.537||6.431 ± 0.178|
|Warm/simulated foliar shade||0.187 ± 0.004||60.507 ± 2.563||5.985 ± 0.159|
|Warm/neutral shade||0.182 ± 0.004||59.248 ± 2.512||6.431 ± 0.141|
In this study, we examined how temperature and light quality during seed maturation influenced germination in A. thaliana phytochrome mutants and natural accessions. Our results suggest that all five A. thaliana phytochromes partially mediate germination in response to seed maturation temperature and/or light quality. In general, functional PHYA inhibited germination; whereas PHYB, PHYD and PHYE generally promoted germination, but the phenotypes of individual phytochrome mutants differed by seed maturation treatment. In addition, we demonstrated significant natural variation in germination responses to the tested seed maturation environments.
Functional PHYA inhibited and slowed germination in seeds matured under cold temperature or simulated foliar shade. The only mutants not to show this response were phyA-205 (under cold/simulated foliar shade) and phyA-211 (under warm/simulated foliar shade). PhyA-205 is a documented weak mutant (Reed et al. 1994); and Col exhibited 100% germination among seeds matured under warm temperature and simulated foliar shade, making it hard to discern effects of phyA under that treatment. Also, the results of phyA-211 should be interpreted with caution, because it was included in only one experimental trial. The inhibitory effect of PHYA is surprising, because previous research has almost exclusively shown that PHYA promotes germination in A. thaliana (Botto et al. 1996; Shinomura et al. 1996; Heschel et al. 2007). The contrasting results are likely caused by our study's focus on the effects of the seed maturation environment on germination, whereas most previous work examined only the environment during seed germination. Consistent with this hypothesis, the only other study to investigate the effects of seed maturation environment on germination in A. thaliana phytochrome mutants likewise demonstrated that PHYA inhibited germination in seeds matured in cold temperature, although only in a phyD background (Donohue et al. 2008). We found that functional PHYC inhibited germination in a phyD deficient background (Ws) in seeds matured in warm temperature or simulated foliar shade. This suggests a new role for PHYC in regulating seed germination that is more similar to PHYA than to PHYB, PHYD, or PHYE. Although caveats include the fact that the phyC-3 results were not replicated and that mutants in Ws are not directly comparable with Ler. This relationship between PHYC and PHYA is not completely unexpected, because PHYA and PHYC arose from a duplication event and share more sequence similarity with each other than with PHYB, PHYD or PHYE (Donoghue & Mathews 1998).
Functional PHYB and PHYE acted antagonistically to PHYA to promote germination. All phyB mutants in the Ler background germinated to a significantly lower percentage than wild-type in seeds matured under cold and neutral shade. Although not significant, this pattern was also observed in phyB-9 (Col). Germination was also significantly inhibited in seeds of phyB-1 and phyB-5 matured under cold and warm temperatures and neutral shade. PhyB-4 and phyB-7 seeds exhibited more variable germination responses, but these mutants are known to exhibit only weak phenotypic effects (Reed et al. 1993). Germination in the phyE single mutant was also significantly inhibited when seeds were matured under neutral shade. Also, mutants deficient in phyB and phyE counteracted the effects of phyA; that is, double and triple mutants deficient in phyA and phyB or phyE germinated to lower percentages than wild-type genotypes and the phyA single mutants. PHYB has previously been shown to have an antagonistic effect on PHYA in germination, but in that example PHYB inhibited PHYA-mediated germination in FR (Hennig et al. 2001). Our phyB results are consistent with Donohue et al. (2008), who also found that mutants deficient in phyB generally exhibited lower germination than wild-type genotypes. However, that study found no significant effect of seed maturation treatment on germination in phyE, possibly because light quality effects were not examined. Functional PHYD also increased germination percentage in seeds matured under warm temperatures but did not counteract the effects of PHYA. Donohue et al. (2008) also found that PHYD promoted germination in seeds that matured under warm temperatures and were subjected to a warm stratification treatment; our seeds received no stratification, but were matured under warm temperatures.
Interestingly, the differences in germination percentages between wild-type genotypes and seeds deficient in phyB, phyD and/or phyE were generally most pronounced when the seeds were matured under neutral shade. It is unlikely that these results are solely attributable to differences in the state of the phytochrome proteins in the seeds, because under that hypothesis one would expect that seeds matured under a low R : FR, as in the simulated foliar shade treatment, would have less Pfr than seeds matured under the neutral shade treatment. Therefore, seeds matured under simulated foliar shade should germinate more slowly and to a lower percentage than seeds matured under neutral shade. This pattern is observed in the Ler wild-type background, but the opposite is observed in mutants deficient in phyB, phyD and/or phyE.
A possible explanation for the effects of phyB, phyD and phyE seeds matured under a low R : FR is that the light quality during seed maturation influences the total amount of phytochrome in the dry seed, therefore, altering the seed's sensitivity to light. A. thaliana seeds matured under a high R : FR germinate to a much higher percentage in dark than seeds matured under a very low R : FR, but they also require over a 300× larger dosage of R light than seeds matured under low R : FR to reach 50% germination (McCullough & Shropshire 1970). Seeds that matured under high R : FR retain viability and regain germinability to the level of seeds matured under low R : FR after 9 months of storage. These results suggest that seeds matured under a high R : FR express a reduced sensitivity to R light, which gradually increases over dark storage. Although differences in R : FR between seed maturation treatments are less severe in our study than in the aforementioned work, they should be enough to alter total phytochrome levels in seeds and may explain why seeds deficient in phyB, phyD and phyE exhibited lower germination when matured under a high R : FR than when matured under a low R : FR. It remains to be tested if and how total phytochrome levels of the various phytochrome genes were affected by seed maturation treatment.
Although we found overall patterns in the effects of PHYA, PHYB, and PHYE on seed germination, there was also considerable variation in phytochrome function in seeds matured under different treatments (e.g. warm versus cold). Phytochromes, particularly phyB, phyD and phyE, exhibit conditional redundancy for several phenotypes (Aukerman et al. 1997; Devlin et al. 1998, 1999; Hennig et al. 2001). Phytochromes bind to the same interacting partners (Ni, Tepperman & Quail 1998; Oh et al. 2004), and environmental variation in phytochrome function may reflect differences in optimal binding conditions among phytochrome species (Halliday & Whitelam 2003). Environmental variation in phytochrome function could also be caused by phytochrome effects on the gibberellic acid (GA) and abscisic acid (ABA) biosynthesis pathways. In response to R light, phytochromes interact with PIF3-like 5 (PIL5) to increase the amount of bioactive GAs in germinating seeds by increasing the transcription of GA-oxidase genes that convert inactive GA precursors to active GAs (Yamaguchi et al. 1998). Phytochromes also downregulate AtGA2ox2, a gene that reduces bioactive GA (Oh et al. 2006). Cold stratification increases the transcription of GA-oxidase genes by the transcription factor SPATULA (SPT) (Penfield et al. 2005). Red light irradiation also reduces ABA levels in the seed by downstream members of the phyB pathway and interactions between GA and ABA (Finch-Savage & Leubner-Metzger 2006; Seo et al. 2006; Oh et al. 2007). In addition, light conditions (in this case, photoperiod) during seed maturation have been shown to influence the amount of ABA in seeds and the seeds' sensitivity to ABA in germination (Contreras et al. 2008). Thus, the variability in seed germination response to temperature and light cues during seed maturation is likely caused by the complex interactions among the hormone signalling pathways and their environmental inputs (Holdsworth, Bentsink & Soppe 2008).
In addition to demonstrating phytochrome action in effects of seed maturation environment on germination, we examined the same germination responses in nine A. thaliana natural accessions, in order to test for ecologically relevant natural variation under our experimental conditions. One caveat to the following results is that only three of the accessions – Ler; Col-0; and Lm-2 – were included in both experimental trials. We found highly significant genetic variation for germination response to the seed maturation treatments. For example, seed germination was suppressed in Ler when seeds were matured under simulated foliar shade and suppressed in Col-0 when seeds were matured under neutral shade. Several other accessions exhibited no effect of seed maturation treatment on germination percentage (HOG, Jl-3) or only temperature effects (Lm-2, Me-0, Ws). Likewise, germination was accelerated in seeds matured under neutral shade and warm temperature for Ler, but accelerated in seeds matured under simulated foliar shade for HOG and Me-O. The large effect of the simulated foliar shade maturation treatment on HOG seeds accounts for the significant overall treatment effect of light quality on germination day (Table 3).
In addition to significant interactions between genotype and seed maturation treatment, we found overall effects of the seed maturation treatments on seed mass and germination percentage. Seeds were heavier and germination percentage was severely reduced in seeds matured under cold temperatures. Cold-induced dormancy has been found in a number of species and likely prevents seeds from germinating too early or late in the season (Lacey 1996; Gutterman 2000; Donohue et al. 2008). For the same reason, plants may produce heavier seeds under unfavourable conditions, such as cold temperatures and simulated foliar shade, if these seeds stay viable longer in the soil (although seed mass and germination percentage were not correlated in our study). Accordingly, lettuce plants produce seeds that are heavier, more dormant and stay viable longer in storage under long-day (which, similar to cold temperatures, predicts unfavourable conditions for germination) than short-day photoperiods (Contreras et al. 2008).
In conclusion, we demonstrated that A. thaliana phytochromes are important regulators of germination, and that the relative role of each phytochrome is influenced by temperature and light quality during seed maturation. We found an unexpected role for PHYA (and possibly PHYC) in inhibiting germination and showed that functional PHYB and PHYE are necessary for high germination, especially in seeds matured under neutral shade. In addition, using natural accessions of A. thaliana, we found ecologically relevant patterns of phytochrome-mediated germination responses to the tested experimental conditons.
The authors thank two anonomyous reviewers for insightful comments that improved upon an earlier version of this manuscript. We are grateful to R.A. Sharrock and G.C. Whitelam for providing the phytochrome mutant seeds. We also thank D. Brinkman, K. Dorn, A. Schutte and E. Berkas for advice on experimental techniques and help in planting and maintaining experimental plants. We also thank the National Science Foundation for grant DBI-0227103 (to C.W.).
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