The role of cryptochrome 2 in flowering in Arabidopsis

We have investigated the genetic interactions between cry2 and the various flowering pathways in relation to the regulation of flowering by photoperiod and vernalization. For this, we combined three alleles of CRY2, the wild-type CRY2-Landsberg erecta (Ler), a cry2 loss-of-function null allele, and the gain-of-function CRY2-Cape Verde Islands (Cvi), with mutants representing the various photoreceptors and flowering pathways. The analysis of CRY2 alleles combined with photoreceptor mutants showed that CRY2-Cvi could compensate the loss of phyA and cry1, also indicating that cry2 does not require functional phyA or cry1. The analysis of mutants of the photoperiod pathway showed epistasis of co and gi to the CRY2 alleles, indicating that cry2 needs the product of CO and GI genes to promote flowering. All double mutants of this pathway showed a photoperiod response very much reduced compared with Ler. In contrast, mutations in the autonomous pathway genes were additive to the CRY2 alleles, partially overcoming the effects of CRY2-Cvi and restoring day length responsiveness. The three CRY2 alleles were day length sensitive when combined with FRI-Sf2 and/or FLC-Sf2 genes, which could be reverted when the delay of flowering caused by FRI-Sf2 and FLC-Sf2 alleles was removed by vernalization. In addition, we looked at the expression of FLC and CRY2 genes and showed that CRY2 is negatively regulated by FLC. These results indicate an interaction between the photoperiod and the FLC-dependent pathways upstream to the common downstream targets of both pathways, SOC1 and FT.     

New Arabidopsis recombinant inbred lines (Landsberg erecta x Nossen) reveal natural variation in phytochrome-mediated responses

We used 52 Arabidopsis (Arabidopsis thaliana) accessions and developed a new set of 137 recombinant inbred lines between Landsberg erecta (Ler) and Nossen (No-0) to explore the genetic basis of phytochrome-mediated responses during deetiolation. Unexpectedly, most accessions showed weak or moderate hypocotyl growth and cotyledon unfolding responses to pulses of far-red light (FR). Crosses between Columbia and No-0, two accessions with poor response, segregated seedlings with unfolded cotyledons under pulsed FR, suggesting the occurrence of accession-specific loci in the repression of morphological responses to weak light signals. Confirming the latter expectation, mapping of responses to pulsed FR in the Ler x No-0 lines identified novel loci. Despite its weak response to pulsed FR, No-0 showed a response to continuous FR stronger than that observed in Ler. By mapping the differential effect of pulsed versus continuous FR, we identified two high-irradiance response loci that account for the steeper response to continuous FR in No-0. This underscores the potential of the methodology to identify loci involved in the regulation of the shape of signal input-output relationships. Loci specific for a given phytochrome-mediated response were more frequent than pleiotropic loci. Segregation of these specific loci is predicted to yield different combinations of seedling responsivity to light. Such flexibility in combination of responses is observed among accessions and could aid in the adjustment to different microenvironments.     

GIGANTEA regulates phytochrome A-mediated photomorphogenesis independently of its role in the circadian clock

GIGANTEA (GI) is a nuclear protein involved in the promotion of flowering by long days, in light input to the circadian clock, and in seedling photomorphogenesis under continuous red light but not far-red light (FR). Here, we report that in Arabidopsis (Arabidopsis thaliana) different alleles of gi have defects in the hypocotyl-growth and cotyledon-unfolding responses to hourly pulses of FR, a treatment perceived by phytochrome A (phyA). This phenotype is rescued by overexpression of GI. The very-low-fluence response of seed germination was also reduced in gi. Since the circadian clock modulates many light responses, we investigated whether these gi phenotypes were due to alterations in the circadian system or light signaling per se. In experiments where FR pulses were given to dark-incubated seeds or seedlings at different times of the day, gi showed reduced seed germination, cotyledon unfolding, and activity of a luciferase reporter fused to the promoter of a chlorophyll a/b-binding protein gene; however, rhythmic sensitivity was normal in these plants. We conclude that while GI does not affect the high-irradiance responses of phyA, it does affect phyA-mediated very-low-fluence responses via mechanisms that do not obviously involve its circadian functions.     

PNZIP Is a Novel Mesophyll-Specific cDNA That Is Regulated by Phytochrome and a Circadian Rhythm and Encodes a Protein with a Leucine Zipper Motif

Single, double, and triple null combinations of Arabidopsis mutants lacking the photoreceptors phytochrome (phy) A (phyA-201), phyB (phyB-5), and cryptochrome (cry) 1 (hy4-2.23n) were examined for de-etiolation responses in high-fluence red, far-red, blue, and broad-spectrum white light. Cotyledon unhooking, unfolding, and expansion, hypocotyl growth, and the accumulation of chlorophylls and anthocyanin in 5-d-old seedlings were measured under each light condition and in the dark. phyA was the major photoreceptor/effector for most far-red-light responses, although phyB and cry1 modulated anthocyanin accumulation in a phyA-dependent manner. phyB was the major photoreceptor in red light, although cry1 acted as a phyA/phyB-dependent modulator of chlorophyll accumulation under these conditions. All three photoreceptors contributed to most blue light deetiolation responses, either redundantly or additively; however, phyB acted as a modulator of cotyledon expansion dependent on the presence of cry1. As reported previously, flowering time in long days was promoted by phyA and inhibited by phyB, with each suppressing the other's effect. In addition to the effector/modulator relationships described above, measurements of hypocotyls from blue-light-grown seedlings demonstrated phytochrome activity in blue light and cry1 activity in a phyAphyB mutant background.     

The Effect of Epibrassinolide on Arabidopsis Seedling Morphogenesis and Hormonal Balance under Green Light

We studied the effect of 24-epibrassinolide (EB) on the levels of endogenous hormones and photomorphogenesis of Arabidopsis thaliana (L.) Heynh wild-type (Ler) and mutant (hy4) seedlings. This mutant is deficient in the cryptochrome 1 (CRY1) synthesis. CRY1, which is a product of the HY4 gene, is a blue light photoreceptor in wild-type plants, but is sensitive to green light as well. In dark-grown seven-day-old mutant seedlings, the ABA/zeatin ratio differed from this ratio in wild-type seedlings. Thehy4 mutant exhibited a lower zeatin and higher free-ABA contents, which could retard its hypocotyl growth in darkness. EB retarded the growth of hypocotyls in etiolated hy4 seedlings and enlarged their cotyledons more efficiently than in wild-type seedlings. Green light (GL) did not affect the growth of hypocotyls but enlarged cotyledons of hy4 seedlings, which might be associated with some increase in the level of free IAA and a considerable decrease in free ABA and also with a decrease in the cytokinin level in seedlings. The hy4 cotyledon response to GL depended evidently on photoreceptors other than CRY1. GL enhanced the effects of EB on the morphogenesis of both Ler and hy4 seedlings, which was coupled with changes in the balance of endogenous IAA, ABA, and cytokinins. We may suppose that EB is involved in the control of photomorphogenesis by interaction with endogenous hormones, which are involved in the transduction of a light signal absorbed by the GL photoreceptors.     

Action spectrum for cryptochrome-dependent hypocotyl growth inhibition in Arabidopsis

Cryptochrome blue-light photoreceptors are found in both plants and animals and have been implicated in numerous developmental and circadian signaling pathways. Nevertheless, no action spectrum for a physiological response shown to be entirely under the control of cryptochrome has been reported. In this work, an action spectrum was determined in vivo for a cryptochrome-mediated high-irradiance response, the blue-light-dependent inhibition of hypocotyl elongation in Arabidopsis. Comparison of growth of wild-type, cry1cry2 cryptochrome-deficient double mutants, and cryptochrome-overexpressing seedlings demonstrated that responsivity to monochromatic light sources within the range of 390 to 530 nm results from the activity of cryptochrome with no other photoreceptor having a significant primary role at the fluence range tested. In both green- and norflurazon-treated (chlorophyll-deficient) seedlings, cryptochrome activity is fairly uniform throughout its range of maximal response (390-480 nm), with no sharply defined peak at 450 nm; however, activity at longer wavelengths was disproportionately enhanced in CRY1-overexpressing seedlings as compared with wild type. The action spectrum does not correlate well with the absorption spectra either of purified recombinant cryptochrome photoreceptor or to that of a second class of blue-light photoreceptor, phototropin (PHOT1 and PHOT2). Photoreceptor concentration as determined by western-blot analysis showed a greater stability of CRY2 protein under the monochromatic light conditions used in this study as compared with broad band blue light, suggesting a complex mechanism of photoreceptor activation. The possible role of additional photoreceptors (in particular phytochrome A) in cryptochrome responses is discussed.     

Blue light-dependent in vivo and in vitro phosphorylation of Arabidopsis cryptochrome 1

Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light responses in animals and plants. Arabidopsis cryptochrome 1 (cry1) is the major photoreceptor mediating blue light inhibition of hypocotyl elongation. The initial photochemistry underlying cryptochrome function and regulation remain poorly understood. We report here a study of the blue light-dependent phosphorylation of Arabidopsis cry1. Cry1 is detected primarily as unphosphorylated protein in etiolated seedlings, but it is phosphorylated in plants exposed to blue light. Cry1 phosphorylation increases in response to increased fluence of blue light, whereas the phosphorylated cry1 disappears rapidly when plants are transferred from light to dark. Light-dependent cry1 phosphorylation appears specific to blue light, because little cry1 phosphorylation is detected in seedlings treated with red light or far-red light, and it is largely independent from phytochrome actions, because no phytochrome mutants tested significantly affect cry1 phosphorylation. The Arabidopsis cry1 protein expressed and purified from insect cells is phosphorylated in vitro in a blue light-dependent manner, consistent with cry1 undergoing autophosphorylation. To determine whether cry1 phosphorylation is associated with its function or regulation, we isolated and characterized missense cry1 mutants that express full-length CRY1 apoprotein. Mutant residues are found throughout the CRY1 coding sequence, but none of these inactive cry1 mutant proteins shows blue light-induced phosphorylation. These results demonstrate that blue light-dependent cry1 phosphorylation is closely associated with the function or regulation of the photoreceptor and that the overall structure of cry1 is critical to its phosphorylation.     

Pleiotropic effects of the Arabidopsis cryptochrome 2 allelic variation underlie fruit trait-related QTL

The previous molecular identification of a flowering time QTL segregating in the Arabidopsis L er x Cvi cross, demonstrated that natural allelic variation at the blue light photoreceptor CRY2 gene affects flowering time (El-Assal et al., 2001). In addition, previous works on the same cross have mapped several QTL affecting other unrelated life history traits in the CRY2 genomic region. In the present report, we have used a set of Arabidopsis L er transgenic plants carrying four different functional CRY2 transgenes for phenotypic analyses, with the aim of exploring the extent of pleiotropy of CRY2 allelic variation. It is concluded that previously identified QTL affecting fruit length, ovule number per fruit, and percentage of unfertilized ovules are caused by this same Ler/Cvi CRY2 allelic variation. In addition, dose effects of the CRY2-L er allele are detected for fruit length. A seed weight QTL at the map position of CRY2 could not be confirmed and also no effect on seed dormancy was observed. Thus, it is shown that transgenic plants carrying different alleles can be a useful tool to attribute QTL for different complex traits to a specific locus, even when the relationship among the traits has not been previously suggested.     

The serine-rich N-terminal domain of oat phytochrome a helps regulate light responses and subnuclear localization of the photoreceptor

Phytochrome (phy) A mediates two distinct photobiological responses in plants: the very-low-fluence response (VLFR), which can be saturated by short pulses of very-low-fluence light, and the high-irradiance response (HIR), which requires prolonged irradiation with higher fluences of far-red light (FR). To investigate whether the VLFR and HIR involve different domains within the phyA molecule, transgenic tobacco (Nicotiana tabacum cv Xanthi) and Arabidopsis seedlings expressing full-length (FL) and various deletion mutants of oat (Avena sativa) phyA were examined for their light sensitivity. Although most mutants were either partially active or inactive, a strong differential effect was observed for the Delta6-12 phyA mutant missing the serine-rich domain between amino acids 6 and 12. Delta6-12 phyA was as active as FL phyA for the VLFR of hypocotyl growth and cotyledon unfolding in Arabidopsis, and was hyperactive in the VLFR of hypocotyl growth and cotyledon unfolding in tobacco, and the VLFR blocking subsequent greening under white light in Arabidopsis. In contrast, Delta6-12 phyA showed a dominant-negative suppression of HIR in both species. In hypocotyl cells of Arabidopsis irradiated with FR phyA:green fluorescent protein (GFP) and Delta6-12 phyA:GFP fusions localized to the nucleus and coalesced into foci. The proportion of nuclei with abundant foci was enhanced by continuous compared with hourly FR provided at equal total fluence in FL phyA:GFP, and by Delta6-12 phyA mutation under hourly FR. We propose that the N-terminal serine-rich domain of phyA is involved in channeling downstream signaling via the VLFR or HIR pathways in different cellular contexts.     

Chimeric proteins between cry1 and cry2 Arabidopsis blue light photoreceptors indicate overlapping functions and varying protein stability.

A blue light (cryptochrome) photoreceptor from Arabidopsis, cry1, has been identified recently and shown to mediate a number of blue light-dependent phenotypes. Similar to phytochrome, the cryptochrome photoreceptors are encoded by a gene family of homologous members with considerable amino acid sequence similarity within the N-terminal chromophore binding domain. The two members of the Arabidopsis cryptochrome gene family (CRY1 and CRY2) overlap in function, but their proteins differ in stability: cry2 is rapidly degraded under light fluences (green, blue, and UV) that activate the photoreceptor, but cry1 is not. Here, we demonstrate by overexpression in transgenic plants of cry1 and cry2 fusion constructs that their domains are functionally interchangeable. Hybrid receptor proteins mediate functions similar to cry1 and include inhibition of hypocotyl elongation and blue light-dependent anthocyanin accumulation; differences in activity appear to be correlated with differing protein stability. Because cry2 accumulates to high levels under low-light intensities, it may have greater significance in wild-type plants under conditions when light is limited.     

Phase-shifting the fruit fly clock without cryptochrome

The blue light photopigment cryptochrome (CRY) is thought to be the main circadian photoreceptor of Drosophila melanogaster. Nevertheless, entrainment to light-dark cycles is possible without functional CRY. Here, we monitored phase response curves of cry(01) mutants and control flies to 1-hour 1000-lux light pulses. We found that cry(01) mutants phase-shift their activity rhythm in the subjective early morning and late evening, although with reduced magnitude. This phase-shifting capability is sufficient for the slowed entrainment of the mutants, indicating that the eyes contribute to the clock's light sensitivity around dawn and dusk. With longer light pulses (3 hours and 6 hours), wild-type flies show greatly enhanced magnitude of phase shift, but CRY-less flies seem impaired in the ability to integrate duration of the light pulse in a wild-type manner: Only 6-hour light pulses at circadian time 21 significantly increased the magnitude of phase advances in cry(01) mutants. At circadian time 15, the mutants exhibited phase advances instead of the expected delays. These complex results are discussed.     

Drosophila CRY is a deep brain circadian photoreceptor

cry (cryptochrome) is an important clock gene, and recent data indicate that it encodes a critical circadian photoreceptor in Drosophila. A mutant allele, cry(b), inhibits circadian photoresponses. Restricting CRY expression to specific fly tissues shows that CRY expression is needed in a cell-autonomous fashion for oscillators present in different locations. CRY overexpression in brain pacemaker cells increases behavioral photosensitivity, and this restricted CRY expression also rescues all circadian defects of cry(b) behavior. As wild-type pacemaker neurons express CRY, the results indicate that they make a striking contribution to all aspects of behavioral circadian rhythms and are directly light responsive. These brain neurons therefore contain an identified deep brain photoreceptor, as well as the other circadian elements: a central pace-maker and a behavioral output system.     

Seasonal and plant-density dependency for quantitative trait loci affecting flowering time in multiple populations of Arabidopsis thaliana

Multiple environmental cues regulate the transition to flowering. In natural environments, plants perceive seasonal progression by changes in day length and growth temperature, and plant density is monitored by changes in the light quality reflected from neighbouring vegetation. To understand the seasonal and plant-density dependence associated with natural allelic variation in flowering time, we conducted a quantitative trait loci (QTL) mapping study in Ler x Cvi, Bay x Sha and Ler x No-0 recombinant inbred line (RIL) populations of Arabidopsis thaliana. Days and total leaf number to bolting were examined under low and high plant density (200 or 1600 plants m(-2)) in autumn-winter and spring seasons. We found between 4 and 10 QTLs associated with seasonal and density variations in each RIL population. For Ler x Cvi and Bay x Sha RIL populations, a major proportion of QTLs showed seasonal and density interaction (up to 63%) and four QTLs were common to all environments (21%). Only three QTLs showed seasonal or density dependency. By aligning the linkage maps onto a common physical map, we detected at least one QTL at chromosome 2 and two QTLs at chromosome 5 that overlap between the three RIL populations, suggesting that these QTLs play a crucial role in the adaptive control of flowering time.     

FRIGIDA LIKE 2 is a functional allele in Landsberg erecta and compensates for a nonsense allele of FRIGIDA LIKE 1

The Landsberg erecta (Ler) accession of Arabidopsis (Arabidopsis thaliana) has a weak allele of the floral inhibitor FLOWERING LOCUS C (FLC). FLC-Ler is weakly up-regulated by the active San Feliu-2 (Sf2) allele of FRIGIDA (FRI-Sf2), resulting in a moderately late-flowering phenotype. By contrast, the Columbia (Col) allele of FLC is strongly up-regulated by FRI-Sf2, resulting in a very late-flowering phenotype. In Col, the FRI-related gene FRI LIKE 1 (FRL1) is required for FRI-mediated up-regulation of FLC. It is shown here that in Ler, the FRL1-related gene FRI LIKE 2 (FRL2), but not FRL1, is required for FRI-mediated up-regulation of FLC. FRL1-Ler is shown to be a nonsense allele of FRL1 due to a naturally occurring premature stop codon in the middle of the conceptual protein sequence, suggesting that FRL1-Ler is nonfunctional. Compared to FRL2-Col, FRL2-Ler has two amino acid changes in the conceptual protein sequence. Plants homozygous for FRI-Sf2, FLC-Ler, FRL1-Ler, and FRL2-Col have no detectable FLC expression, resulting in an extremely early flowering phenotype. Transformation of a genomic fragment of FRL2-Ler, but not of FRL2-Col, into a recombinant inbred line derived from these plants restores both FRI-mediated up-regulation of FLC expression and a late-flowering phenotype, indicating that FRL2-Ler is the functional allele of FRL2. Taken together, these results suggest that in the two different Arabidopsis accessions Col and Ler, either FRL1 or FRL2, but not both, is functional and required for FRI-mediated up-regulation of FLC.     

A study of gibberellin homeostasis and cryptochrome-mediated blue light inhibition of hypocotyl elongation

Cryptochromes mediate blue light-dependent photomorphogenic responses, such as inhibition of hypocotyl elongation. To investigate the underlying mechanism, we analyzed a genetic suppressor, scc7-D (suppressors of cry1cry2), which suppressed the long-hypocotyl phenotype of the cry1cry2 (cryptochrome1/cryptochrome2) mutant in a light-dependent but wavelength-independent manner. scc7-D is a gain-of-expression allele of the GA2ox8 gene encoding a gibberellin (GA)-inactivating enzyme, GA 2-oxidase. Although scc7-D is hypersensitive to light, transgenic seedlings expressing GA2ox at a level higher than scc7-D showed a constitutive photomorphogenic phenotype, confirming a general role of GA2ox and GA in the suppression of hypocotyl elongation. Prompted by this result, we investigated blue light regulation of mRNA expression of the GA metabolic and catabolic genes. We demonstrated that cryptochromes are required for the blue light regulation of GA2ox1, GA20ox1, and GA3ox1 expression in transient induction, continuous illumination, and photoperiodic conditions. The kinetics of cryptochrome induction of GA2ox1 expression and cryptochrome suppression of GA20ox1 or GA3ox1 expression correlate with the cryptochrome-dependent transient reduction of GA(4) in etiolated wild-type seedlings exposed to blue light. Therefore we propose that in deetiolating seedlings, cryptochromes mediate blue light regulation of GA catabolic/metabolic genes, which affect GA levels and hypocotyl elongation. Surprisingly, no significant change in the GA(4) content was detected in the whole shoot samples of the wild-type or cry1cry2 seedlings grown in the dark or continuous blue light, suggesting that cryptochromes may also regulate GA responsiveness and/or trigger cell- or tissue-specific changes of the level of bioactive GAs.     

Interactive signalling by phytochromes and cryptochromes generates de-etiolation homeostasis in Arabidopsis thaliana

Single, double, triple and quadruple mutants of phyA, phyB, cry1 and cry2 were exposed to different sunlight irradiances and photoperiods to investigate the roll played by phytochrome A, phytochrome B, cryptochrome 1 and cryptochrome 2 during de-etiolation of Arabidopsis thaliana seedlings under natural radiation. Even the quadruple mutant retained some hypocotyl-growth inhibition by sunlight. Hypocotyl length was strongly affected by interactions among photoreceptors. Double phyA phyB, phyA cry1, and cry1 cry2 mutants were taller than expected from the additive action of single mutations. Some of these redundant interactions required the presence of phytochromes A and/or B. Interactions among photoreceptors resulted in a 44% reduction of the response to irradiance and a 70% reduction of the response to photoperiod. The complex network of interactions among photoreceptors is proposed to buffer de-etiolation against changes in irradiance and photoperiod, i.e light fluctuations not related to the positions of the shoot above or below soil level     

Analysis of natural allelic variation at seed dormancy loci of Arabidopsis thaliana

Arabidopsis accessions differ largely in their seed dormancy behavior. To understand the genetic basis of this intraspecific variation we analyzed two accessions: the laboratory strain Landsberg erecta (Ler) with low dormancy and the strong-dormancy accession Cape Verde Islands (Cvi). We used a quantitative trait loci (QTL) mapping approach to identify loci affecting the after-ripening requirement measured as the number of days of seed dry storage required to reach 50% germination. Thus, seven QTL were identified and named delay of germination (DOG) 1-7. To confirm and characterize these loci, we developed 12 near-isogenic lines carrying single and double Cvi introgression fragments in a Ler genetic background. The analysis of these lines for germination in water confirmed four QTL (DOG1, DOG2, DOG3, and DOG6) as showing large additive effects in Ler background. In addition, it was found that DOG1 and DOG3 genetically interact, the strong dormancy determined by DOG1-Cvi alleles depending on DOG3-Ler alleles. These genotypes were further characterized for seed dormancy/germination behavior in five other test conditions, including seed coat removal, gibberellins, and an abscisic acid biosynthesis inhibitor. The role of the Ler/Cvi allelic variation in affecting dormancy is discussed in the context of current knowledge of Arabidopsis germination.