Genetic dissection of blue-light sensing in tomato using mutants deficient in cryptochrome 1 and phytochromes A, B1 and B2

Several novel allelic groups of tomato (Solanum lycopersicum L.) mutants with impaired photomorphogenesis have been identified after gamma-ray mutagenesis of phyA phyB1 double-mutant seed. Recessive mutants in one allelic group are characterized by retarded hook opening, increased hypocotyl elongation and reduced hypocotyl chlorophyll content under white light (WL). These mutants showed a specific impairment in response to blue light (BL) resulting from lesions in the gene encoding the BL receptor cryptochrome 1 (cry1). Phytochrome A and cry1 are identified as the major photoreceptors mediating BL-induced de-etiolation in tomato, and act under low and high irradiances, respectively. Phytochromes B1 and B2 also contribute to BL sensing, and the relative contribution of each of these four photoreceptors differs according to the light conditions and the specific process examined. Development of the phyA phyB1 phyB2 cry1 quadruple mutant under WL is severely impaired, and seedlings die before flowering. The quadruple mutant is essentially blind to BL, but experiments employing simultaneous irradiation with BL and red light suggest that an additional non-phytochrome photoreceptor may be active under short daily BL exposures. In addition to effects on de-etiolation, cry1 is active in older, WL-grown plants, and influences stem elongation, apical dominance, and the chlorophyll content of leaves and fruit. These results provide the first mutant-based characterization of cry1 in a plant species other than Arabidopsis.     

Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato

The role of phytochrome B2 (phyB2) in the control of photomorphogenesis in tomato (Solanum lycopersicum L.) has been investigated using recently isolated mutants carrying lesions in the PHYB2 gene. The physiological interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phyB2 have also been explored, using an isogenic series of all possible mutant combinations and several different phenotypic characteristics. The loss of phyB2 had a negligible effect on the development of white-light-grown wild-type or phyA-deficient plants, but substantially enhanced the elongated pale phenotype of the phyB1 mutant. This redundancy was also seen in the control of de-etiolation under continuous red light (R), where the loss of phyB2 had no detectable effect in the presence of phyB1. Under continuous R, phyA action was largely independent of phyB1 and phyB2 in terms of the control of hypocotyl elongation, but antagonized the effects of phyB1 in the control of anthocyanin synthesis, indicating that photoreceptors may interact differently to control different traits. Irradiance response curves for anthocyanin synthesis revealed that phyB1 and phyB2 together mediate all the detectable response to high-irradiance R, and, surprisingly, that the phyA-dependent low-irradiance component is also strongly reduced in the phyB1 phyB2 double mutant. This is not associated with a reduction in phyA protein content or responsiveness to continuous far-red light (FR), suggesting that phyB1 and phyB2 specifically influence phyA activity under low-irradiance R. Finally, the phyA phyB1 phyB2 triple mutant showed strong residual responsiveness to supplementary daytime FR, indicating that at least one of the two remaining phytochromes plays a significant role in tomato photomorphogenesis.     

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     

HYPERSENSITIVE TO RED AND BLUE 1 and its C-terminal regulatory function control FLOWERING LOCUS T expression

The red and far-red light-absorbing phytochromes and UV-A/blue light-absorbing cryptochromes regulate seedling de-etiolation and flowering responses. The signaling steps that mediate the photoreceptor regulation on key flowering genes remain largely unknown. We report that a previously identified photomorphogenic mutant, hypersensitive to red and blue 1 (hrb1), flowered late and showed attenuated expression of FLOWERING LOCUS T (FT) over both long days and short days. Transgenic plants that overexpress the full-length HRB1, or its C-terminal half, flowered early and accumulated more FT messages under short-day conditions. The transgenic plants also displayed hyposensitive de-etiolation phenotypes, and the expression of these phenotypes requires the action of PIF4. The double mutant of hrb1/cry2 showed a flowering phenotype and an FT expression pattern similar to hrb1 under long-day conditions, suggesting that HRB1 may function downstream of cry2 under long-day conditions. In contrast, hrb1/phyB-9 showed a flowering phenotype and an FT expression pattern similar to phyB-9 over both long days and short days, indicating a modulatory role of HRB1 in the flowering pathway mediated by phyB. Overexpression of HRB1 did not affect the expression of the central clock oscillators, TOC1 and CCA1. HRB1 therefore represents a signaling step that regulates FT expression downstream of red and blue light perception.     

Antagonistic but complementary actions of phytochromes A and B allow seedling de-etiolation.

Using dichromatic radiation, we show that the actions of phytochromes A and B (phyA and phyB) in Arabidopsis thaliana are antagonistic in mediating red and far-red radiation effects on seedling de-etiolation and yet act in a complementary manner to regulate de-etiolation, irrespective of spectral composition. At low phytochrome photoequilibria inhibition of hypocotyl extension was strong, because of the action of a far-red high-irradiance response mediated by phyA. At high phytochrome photoequilibria inhibition of hypocotyl extension was also strong, because of the action of phyB. At intermediate photoequilibria hypocotyl inhibition was less strong. In their natural environment, this dual action will strongly retard hypocotyl growth and promote cotyledon opening and expansion both in open daylight and under dense vegetation. Overlapping action by phyA and phyB will substantially promote de-etiolation in sparse vegetation. The antagonistic and complementary actions of phyA and phyB, therefore, allow the optimum regulation of seedling growth after emergence from the soil.     

The gigas mutant in pea is deficient in the floral stimulus

Identification of a gene acting in the floral stimulus pathway should provide a basis for determining the identity of this elusive substance. Our tests indicate the Gi (gigas) gene in pea (Pisum sativum L.) acts in this manner. The gigas mutant was selected by Dl M. Vassiteva following gamma radiation of the late flowering, quantitative long day cultivar Virtus. The gigas trait showed single gene recessive inheritance and the mutant allele was symbolised gi consistent with our preliminary report. Gigas plants were later flowering than the initial line in all conditions tested and they showed an enhanced response to photoperiod and vernalisation. Unvernalised gigas plants did not flower under a 24-h photoperiod comprising 8 h of daylight and 16 h of weak (3μmol m^?2 s^?1) incandescent light and they took on a phenotype similar to the vegl (vegetative) mutant in pea. However, genetic tests showed the two mutants were not allelic. Three or four weeks vernalisation at 4^?C resulted in 100% flowering of gigas plants under the 24-h photoperiod. Applied gibberellin A₃ inhibited flowering in gigas plants given partial cold induction. Grafting studies showed the promotive effect of vernalisation occurred in the shoot. Grafting studies were also used to examine the physiological basis of delayed flowering in the gigas mutant. These studies indicated that gigas plants produced normal levels of flower inhibitor and they responded in a normal manner to the floral stimulus, Reciprocal grafts were made between the gigas mutant and the wild-type initial line. Under the 24-h photoperiod, either a wild-type root-stock with cotyledons or a wild-type shoot induced flowering in a gigas graft partner. However, under a 9-h photoperiod, flowering was only induced if the wild-type partner possessed both roots and a shoot. We conclude that gigas plants are deficient in the floral stimulus or a precursor which can be supplied across a graft union by a wild-type donor. Of the 12 major flowering genes known in pea, Gi is the first found to act on the synthesis pathway for the floral stimulus.     

Phytochrome control of flowering is temperature sensitive and correlates with expression of the floral integrator FT

In Arabidopsis flowering is accelerated by reduced red:far-red (R:FR) ratio which signals the presence of neighbouring vegetation. Hastened flowering is one component of the shade-avoidance syndrome of responses, which alter many aspects of development in response to the threat of potential competition. Of the red/far-red-absorbing photoreceptors it is phyB that plays the most prominent role in shade-avoidance, although other related phytochromes act redundantly with phyB. It is well established that the phyB mutant has a constitutively early flowering phenotype. However, we have shown that the early flowering phenotype of phyB is temperature-dependent. We have established that this temperature-sensitive flowering response defines a pathway that appears to be independent of the autonomous-FLC pathway. Furthermore, we have demonstrated that the phytochromes control the expression of the floral promoter FT. We have also shown that other phyB-controlled responses, including petiole elongation, are not sensitive to the same temperature change. This suggests that discrete pathways control flowering and petiole elongation, components of the shade-avoidance response. This work provides an insight into the phytochrome and temperature interactions that maintain flowering control.     

Low temperature spectrophotometry of the phototransformation of Pfr to Pr in pelletable pea phytochrome

Manabe, K. 1987. Low temperature spectrophotometry of the phototransformation of P_fr to P_r, in pelletable pea phytochrome.
Low temperature spectrophotometry was used to study the phototransformation of P_fr to P_r in 1000–7000 g pelletable fractions extracted from dark grown pea ( Pisum sativum L. cv. Alaska) epicotyls which had been irradiated with red and then far-red light. At -170°C, far-red irradiation of the pelletable phytochrome which had been pre-irradiated with saturating fluence of red light before freezing caused formation of an intermediate (named I₆₆₀), the difference spectrum of which showed a marked ab-sorbance decrease at 740 nm and a concomitant small increase at about 660 nm. The inermediate I₆₆₀ was converted to another intermediate (I₆₆₀) when it was warmed above -80°C. The difference spectrum of this intermediate showed a positive peak at 670 nm. This intermediate was photoconverted to P_fr by red irradiation and also underwent dark reversion to P_fr at -60°C. I₆₆₀ formed P_r if the temperature was above -10°C. The basic features of the phytochrome intermediates resemble those obtained in vivo and in degraded purified phytochrome.     

Mapping-by-Sequencing Identifies HvPHYTOCHROME C as a Candidate Gene for the early maturity 5 Locus Modulating the Circadian Clock and Photoperiodic Flowering in Barley

Phytochromes play an important role in light signaling and photoperiodic control of flowering time in plants. Here we propose that the red/far-red light photoreceptor HvPHYTOCHROME C (HvPHYC), carrying a mutation in a conserved region of the GAF domain, is a candidate underlying the early maturity 5 locus in barley (Hordeum vulgare L.). We fine mapped the gene using a mapping-by-sequencing approach applied on the whole-exome capture data from bulked early flowering segregants derived from a backcross of the Bowman(eam5) introgression line. We demonstrate that eam5 disrupts circadian expression of clock genes. Moreover, it interacts with the major photoperiod response gene Ppd-H1 to accelerate flowering under noninductive short days. Our results suggest that HvPHYC participates in transmission of light signals to the circadian clock and thus modulates light-dependent processes such as photoperiodic regulation of flowering.     

Arabidopsis phytochromes A and B synergistically repress SPA1 under blue light

In Arabidopsis, although studies have demonstrated that phytochrome A(phyA) and phyB are involved in blue light signaling, how blue light-activated phytochromes modulate the activity of the CONSTITUTIVELY PHOTOMORPHOGENIC1(COP1)-SUPPRESSOR OF PHYA-105(SPA1) E3 complex remains largely unknown. Here, we show that phyA responds to early and weak blue light, whereas phyB responds to sustainable and strong blue light. Activation of both phyA and phyB by blue light inhibits SPA1 activity.Specifically,...     

Genetic analysis of the roles of phytochromes A and B1 in the reversed gravitropic response of the lz-2 tomato mutant

The lz-2 mutation in tomato ( Lycopersicon esculentum ) causes conditional reversal of shoot gravitropism by light. This response is mediated by phytochrome. To further elicit the mechanism by which phytochrome regulates the lz-2 phenotype, phytochrome-deficient lz-2 plants were generated. Introduction of au alleles, which severely block chromophore biosynthesis, eliminated the reversal of hypocotyl gravitropism in continuous red and far-red light. The fri ¹ and tri ¹ alleles were introduced to specifically deplete phytochromes A and B1, respectively. In dark-grown seedlings, phytochrome A was necessary for response to high-irradiance far-red light, a complete response to low fluence red light, and also mediated the effects of blue light in a far-red reversible manner. Loss of phytochrome B1 alone did not significantly affect the behaviour of lz-2 plants under any light treatment tested. However, dark-grown lz-2 plants lacking both phytochrome A and B1 exhibited reduced responses to continuous red and were less responsive to low fluence red light and high fluence blue light than plants that were deficient for phytochrome A alone. In high light, full spectrum greenhouse conditions, lz-2 plants grew downward regardless of the phytochrome deficiency. These results indicate that phytochromes A and B1 play significant roles in mediating the lz-2 phenotype and that at least one additional phytochrome is involved in reversing shoot gravitropism in this mutant.     

Phytochrome in Pharbitis nil during and after de-etiolation

Phytochrome (P) was measured by in vivo spectrophotometry in the cotyledons of Pharbitis nil Choisy cv. Violet. In etiolated plants exposure to red light (R) results in a rapid fall in P content by destruction of the far-red absorbing form of phytochrome (Pfr). In continuous R or white light the P content falls as a result of destruction to a stable 3% of that originally present. In Norflurazon-treated white light de-etiolated plants, Pfr undergoes reversion in darkness to the red absorbing form of phytochrome (Pr) with a half life of 4 h at 19°C, while total P remains constant. Synthesis of Pr after de-etiolation occurs at a slow rate and is enhanced by terminating the de-etiolation light treatment with far-red light. The results are discussed in relation to the P control of flowering.     

Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice

We have isolated phytochrome B (phyB) and phyC mutants from rice (Oryza sativa) and have produced all combinations of double mutants. Seedlings of phyB and phyB phyC mutants exhibited a partial loss of sensitivity to continuous red light (Rc) but still showed significant deetiolation responses. The responses to Rc were completely canceled in phyA phyB double mutants. These results indicate that phyA and phyB act in a highly redundant manner to control deetiolation under Rc. Under continuous far-red light (FRc), phyA mutants showed partially impaired deetiolation, and phyA phyC double mutants showed no significant residual phytochrome responses, indicating that not only phyA but also phyC is involved in the photoperception of FRc in rice. Interestingly, the phyB phyC double mutant displayed clear R/FR reversibility in the pulse irradiation experiments, indicating that both phyA and phyB can mediate the low-fluence response for gene expression. Rice is a short-day plant, and we found that mutation in either phyB or phyC caused moderate early flowering under the long-day photoperiod, while monogenic phyA mutation had little effect on the flowering time. The phyA mutation, however, in combination with phyB or phyC mutation caused dramatic early flowering.     

The composition and function of thylakoid membranes from pea plants grown under white or green light with or without far-red light

Pea plants ( Pisum sativum L. ev. Greenfeast) were grown for 2 to 3 weeks in while (˜ 50 μmol photons m⁻² s⁻¹; 400–700 nm) or green (˜ 30 μmol photons m⁻² s ⁻¹ 400–700 nm) light (16 h day/8 h night), with or without far-red light. Supplementary far-red light decreased leaf area and increased internodal length in both white and green light, demonstrating that phytochrome influenced leaf size and plant growth. However, there was no effect of far-red light on chlorophyll a /chlorophyll b ratios, chlorophyll-protein composition, the stoichiometry of electron transport complexes or photosynthetic function of isolated thylakoids. These results suggest that phytochrome is ineffective in modulating the composition and function of thylakoids in pea plants grown at low irradiance. One possible explanation of the ineffectiveness of phytochrome on thylakoids is discussed in terms of the drastic attenuation of red relative to far-red light in green tissue.     

The effect of daylength on the transition to flowering in phytochrome-deficient, late-flowering and double mutants of Arabidopsis thaliana

The effect of daylength on flowering was investigated in the following mutants of Arabidopsis thaliana : phytochrome B deficient ( hy3=phyB ); phytochrome chromophore deficient ( hy2 ); late-flowering ( co, gi. fca and fwa ); the hy2 and hy3 , late-flowering double mutants and the hy2, hy3 , late-flowering triple mutants. The hy mutants flower with fewer rosette leaves than the Landsberg erecta wild type under both long day and short day conditions and express this effect to a different degree in all late-flowering mutant backgrounds and under both daylengths, with the exception of fca under short days. The number of cauline leaves and days to flowering is less affected by the hy genotype. The hy2, hy3 double mutants flower with even fewer rosette leaves than the hy2 and hy3 monogenic mutants, suggesting an inhibitory role for phytochrome B and other stable phytochromes on flowering. The complex interaction between phytochrome, daylength and the effect of the late-flowering genes on the various parameters that describe the transition to flowering in Arabidopsis is discussed.     

Connecting the sun to flowering in sunflower adaptation

Species living in seasonal environments often adaptively time their reproduction in response to photoperiod cues. We characterized the expression of genes in the flowering-time regulatory network across wild populations of the common sunflower, Helianthus annuus, that we found to be adaptively differentiated for photoperiod response. The observed clinal variation was associated with changes at multiple hierarchical levels in multiple pathways. Paralogue-specific changes in FT homologue expression and tissue-specific changes in SOC1 homologue expression were associated with loss and reversal of plasticity, respectively, suggesting that redundancy and modularity are gene network characteristics easily exploited by natural selection to produce evolutionary innovation. Distinct genetic mechanisms contribute to convergent evolution of photoperiod responses within sunflower, suggesting regulatory network architecture does not impose strong constraints on the evolution of phenotypic plasticity.