Phytochrome B and at Least One Other Phytochrome Mediate the Accelerated Flowering Response of Arabidopsis thaliana L. to Low Red/Far-Red Ratio

We have investigated the involvement of phytochrome B in the early-flowering response of Arabidopsis thaliana L. seedlings to low red:far-red (R/FR) ratio light conditions. The phytochrome B-deficient hy3 (phyB) mutant is early flowering, and in this regard it resembles the shade-avoidance phenotype of its isogenic wild type. Seedlings carrying the hy2 mutation, resulting in a deficiency of phytochrome chromophore and hence of active phytochromes, also flower earlier than wild-type plants. Whereas hy3 or hy2 seedlings show only a slight acceleration of flowering in response to low R/FR ratio, seedlings that are doubly homozygous for both mutations flower earlier than seedlings carrying either phytochrome-related mutation alone. This additive effect clearly indicates the involvement of one or more phytochrome species in addition to phytochrome B in the flowering response as well as indicating the presence of some functional phytochrome B in hy2 seedlings. Seedlings that are homozygous for the hy3 mutation and one of the fca, fwa, or co late-flowering mutations display a pronounced early-flowering response to low R/FR ratio. A similar response to low R/FR ratio is displayed by seedlings doubly homozygous for the hy2 mutation and any one of the late-flowering mutations. Thus, placing the hy3 or hy2 mutations into a late-flowering background has the effect of uncovering a flowering response to low R/FR ratio. Seedlings that are triply homozygous for the hy3, hy2 mutations and a late-flowering mutation flower earlier than the double mutants and do not respond to low R/FR ratio. Thus, the observed flowering responses to low R/FR ratio in phytochrome B-deficient mutants can be attributed to the action of at least one other phytochrome species.     

Phytochrome-Deficient hy1 and hy2 Long Hypocotyl Mutants of Arabidopsis Are Defective in Phytochrome Chromophore Biosynthesis

The hy1 and hy2 long hypocotyl mutants of Arabidopsis contain normal levels of immunochemically detectable phytochrome A, but the molecule is photochemically nonfunctional. We have investigated the biochemical basis for this lack of function. When the hy1 and hy2 mutants were grown in white light on a medium containing biliverdin IX[alpha], a direct precursor to phytochromobilin, the phytochrome chromophore, the seedlings developed with a morphological phenotype indistinguishable from the light-grown wild-type control. Restoration of a light-grown phenotype in the hy1 mutant was also accomplished by using phycocyanobilin, a tetrapyrrole analog of phytochromobilin. Spectrophotometric and immunochemical analyses of the rescued hy1 and hy2 mutants demonstrated that they possessed wild-type levels of photochemically functional phytochrome that displayed light-induced conformational changes in the holoprotein indistinguishable from the wild type. Moreover, phytochrome A levels declined in vivo in response to white light in rescued hy1 and hy2 seedlings, indicative of biliverdin-dependent formation of photochemically functional phytochrome A that was then subject to normal selective turnover in the far-red-light-absorbing form. Combined, these data suggest that the hy1 and hy2 mutants are inhibited in chromophore biosynthesis at steps prior to the formation of biliverdin IX[alpha], thus potentially causing a global functional deficiency in all members of the phytochrome photoreceptor family.     

The Metabolism of Gibberellin A20 to Gibberellin A1 by Tall and Dwarf Mutants of Oryza sativa and Arabidopsis thaliana

The purpose of this study was to demonstrate the metabolism of gibberellin A20 (GA20) to gibberellin A1 (GA1) by tall and mutant shoots of rice (Oryza sativa L.) and Arabidopsis thaliana (L.) Heynh. The data show that the tall and dx mutant of rice and the tall and ga5 mutant of Arabidopsis metabolize GA20 to GA1. The data also show that the dy mutant of rice and the ga4 mutant of Arabidopsis block the metabolism of GA20 to GA1. [17-13C,3H]GA20 was fed to tall and the dwarf mutants, dx and dy, of rice and tall and the dwarf mutants, ga5 and ga4, of Arabidopsis. The metabolites were analyzed by high-performance liquid chromatography and full-scan gas chromatography-mass spectrometry together with Kovats retention index data. For rice, the metabolite [13C]GA, was identified from tall and dx seedlings; [13C]GA1 was not identified from the dy seedlings. [13C]GA29 was identified from tall, dx, and dy seedlings. For Arabidopsis, the metabolite [13C]GA1 was identified from tall, ga5, and ga4 plants. The amount of [13C]GA1 from ga4 plants was less than 15% of that obtained from tall and ga5 plants. [13C]GA29 was identified from tall, ga5, and ga4 plants. [13C]GA5 and [13C]GA3 were not identified from any of the six types of plant material.     

Light-Stimulated Cotyledon Expansion in Arabidopsis Seedlings (The Role of Phytochrome B)

Leaf and cotyledon expansion in dicotyledonous plants is a light-dependent developmental process. The unique role of phytochrome B has been tested by investigating expansion of cotyledons in wild-type and phytochrome-deficient mutants of Arabidopsis thaliana (L.) Heynh. A relatively rapid method for measuring cotyledon area was developed to quantify growth in large populations (average n [greater than or equal to] 100) of wild-type or mutant seedlings under different light and chemical treatments. Three-day-old wild-type (La-er) Arabidopsis seedlings, grown in saturating, low-fluence red light (2-4 [mu]mol m-2 s-1), showed a >250% increase in cotyledon area after 48 h of bright-red light when compared with the phytochrome mutants hy1, hy2, and hy3. An increase in epidermal cell area was observed in wild-type cotyledons but not in hy3, indicating that light-stimulated growth is due in part to cell expansion. The mutant phenotype was rescued by feeding the chromophore precursor biliverdin to the chromophore biosynthesis mutants hy1 and hy6. This treatment did not rescue the hy3 mutant. Since the hy3 lesion is specific to phytochrome B, we conclude that this pigment is involved in the enhancement of cotyledon cell expansion in bright-red light.     

The Arabidopsis ELF3 gene regulates vegetative photomorphogenesis and the photoperiodic induction of flowering

Flowering in Arabidopsis thaliana is promoted by long-day (LD) photoperiods such that plants grown in LD flower earlier, and after the production of fewer leaves, than plants grown in short-day (SD) photoperiods. The early-flowering 3 ( elf 3) mutant of Arabidopsis , which is insensitive to photoperiod with regard to floral initiation has been characterized. elf 3 mutants are also altered in several aspects of vegetative photomorphogenesis, including hypocotyl elongation. When inhibition of hypocotyl elongation was measured, elf 3 mutant seedlings were less responsive than wild-type to all wavelengths of light, and most notably defective in blue and green light-mediated inhibition. When analyzed for the flowering-time phenotype, elf 3 was epistatic to mutant alleles of the blue-light receptor encoding gene, HY 4. However, when elf 3 mutants were made deficient for functional phytochrome by the introduction of hy 2 mutant alleles, the elf 3 hy 2 double mutants displayed the novel phenotype of flowering earlier than either single mutant while still exhibiting photoperiod insensitivity, indicating that a phytochrome-mediated pathway regulating floral initiation remains functional in elf 3 single mutants. In addition, the inflorescences of one allelic combination of elf 3 hy 2 double mutants form a terminal flower similar to the structure produced by tfl 1 single mutants. These results suggest that one of the signal transduction pathways controlling photoperiodism in Arabidopsis is regulated, at least in part, by photoreceptors other than phytochrome, and that the activity of the Arabidopsis inflorescence and floral meristem identity genes may be regulated by this same pathway.     

Phytochrome chromophore deficiency leads to overproduction of jasmonic acid and elevated expression of jasmonate-responsive genes in Arabidopsis

An Arabidopsis mutant line named hy1-101 was isolated because it shows stunted root growth on medium containing low concentrations of jasmonic acid (JA). Subsequent investigation indicated that even in the absence of JA, hy1-101 plants exhibit shorter roots and express higher levels of a group of JA-inducible defense genes. Here, we show that the hy1-101 mutant has increased production of JA and its jasmonate-related phenotype is suppressed by the coi1-1 mutation that interrupts JA signaling. Gene cloning and genetic complementation analyses revealed that the hy1-101 mutant contains a mutation in the HY1 gene, which encodes a heme oxygenase essential for phytochrome chromophore biosynthesis. These results support a hypothesis that phytochrome chromophore deficiency leads to overproduction of JA and activates COI1-dependent JA responses. Indeed, we show that, like hy1-101, independent alleles of the phytochrome chromophore-deficient mutants, including hy1-100 and hy2 (CS68), also show elevated JA levels and constant expression of JA-inducible defense genes. We further provide evidence showing that, on the other hand, JA inhibits the expression of a group of light-inducible and photosynthesis-related genes. Together, these data imply that the JA-signaled defense pathway and phytochrome chromophore-mediated light signaling might have antagonistic effects on each other.     

Multiple heme oxygenase family members contribute to the biosynthesis of the phytochrome chromophore in Arabidopsis

The oxidative cleavage of heme by heme oxygenases (HOs) to form biliverdin IXalpha (BV) is the committed step in the biosynthesis of the phytochrome (phy) chromophore and thus essential for proper photomorphogenesis in plants. Arabidopsis (Arabidopsis thaliana) contains four possible HO genes (HY1, HO2-4). Genetic analysis of the HY1 locus showed previously that it is the major source of BV with hy1 mutant plants displaying long hypocotyls and decreased chlorophyll accumulation consistent with a substantial deficiency in photochemically active phys. More recent analysis of HO2 suggested that it also plays a role in phy assembly and photomorphogenesis but the ho2 mutant phenotype is more subtle than that of hy1 mutants. Here, we define the functions of HO3 and HO4 in Arabidopsis. Like HY1, the HO3 and HO4 proteins have the capacity to synthesize BV from heme. Through a phenotypic analysis of T-DNA insertion mutants affecting HO3 and HO4 in combination with mutants affecting HY1 or HO2, we demonstrate that both of the encoded proteins also have roles in photomorphogenesis, especially in the absence of HY1. Disruption of HO3 and HO4 in the hy1 background further desensitizes seedlings to red and far-red light and accelerates flowering time, with the triple mutant strongly resembling seedlings deficient in the synthesis of multiple phy apoproteins. The hy1/ho3/ho4 mutant can be rescued phenotypically and for the accumulation of holo-phy by feeding seedlings BV. Taken together, we conclude that multiple members of the Arabidopsis HO family are important for synthesizing the bilin chromophore used to assemble photochemically active phys.     

Immunochemically detectable phytochrome is present at normal levels but is photochemically nonfunctional in the hy 1 and hy 2 long hypocotyl mutants of Arabidopsis

The hy 1 and hy 2 long hypocotyl mutants of Arabidopsis thaliana contain less than 20% (the detection limit) of the phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. In contrast, spectral measurements for the hy 3, hy 4, and hy 5 long hypocotyl mutants indicate that they each contain levels of phytochrome equivalent to the wild-type parent. Immunoblot analysis using a monoclonal antibody directed against the chromophore-bearing region of etiolated-oat phytochrome demonstrates that extracts of all mutant and wild-type Arabidopsis tissues, prepared by extraction of proteins into hot SDS-containing buffer, have identical levels of one major immunodetectable protein (116 kDa). An assay involving controlled in vitro proteolysis, known to produce distinctive fragmentation patterns for Pr and Pfr (Vierstra RD, Quail PH, Planta 156: 158–165, 1982), indicates that the 116 kDa polypeptide from the wild-type parent represents Arabidopsis phytochrome. The 116 kDa protein from either hy 3, hy 4, or hy 5 displays the same fragmentation pattern found for the wild type. Together with the spectral data, these results indicate that the mutant phenotype of these variants does not involve lesions in the polypeptide sequence that lead to gross conformational aberrations, and suggest that the genetic lesions may affect steps in the transduction chain downstream of the photoreceptor. In contrast, this same analysis for hy 1 and hy 2 has revealed that the 116 kDa protein from either of these mutants is not degraded differently in response to the different wavelengths of irradiation given in vitro. Moreover, whereas immunoblot analysis of tissue extracts from light-grown wild-type seedlings show that the 116 kDa phytochrome protein level is greatly reduced relative to dark-grown tissue as expected, similar extracts of light-grown hy 1 and hy 2 seedlings contain the 116 kDa polypeptide in amounts equivalent to those of dark-grown tissue. Combined, these data indicate that the hy 1 and hy 2 mutants both produce normal levels of immunochemically detectable phytochrome that is photochemically nonfunctional.     

Different Roles for Phytochrome in Etiolated and Green Plants Deduced from Characterization of Arabidopsis thaliana Mutants

We have isolated a new complementation group of Arabidopsis thaliana long hypocotyl mutant (hy6) and have characterized a variety of light-regulated phenomena in hy6 and other previously isolated A. thaliana hy mutants. Among six complementation groups that define the HY phenotype in A. thaliana, three (hy1, hy2, and hy6) had significantly lowered levels of photoreversibly detectable phytochrome, although near wild-type levels of the phytochrome apoprotein were present in all three mutants. When photoregulation of chlorophyll a/b binding protein (cab) gene expression was examined, results obtained depended dramatically on the light regime employed. Using the red/far-red photoreversibility assay on etiolated plants, the accumulation of cab mRNAs was considerably less in the phytochrome-deficient mutants than in wild-type A. thaliana seedlings. When grown in high-fluence rate white light, however, the mutants accumulated wild-type levels of cab mRNAs and other mRNAs thought to be regulated by phytochrome. An examination of the light-grown phenotypes of the phytochrome-deficient mutants, using biochemical, molecular, and morphological techniques, revealed that the mutants displayed incomplete chloroplast and leaf development under conditions where wild-type chloroplasts developed normally. Thus, although phytochrome may play a role in gene expression in etiolated plants, a primary role for phytochrome in green plants is likely to be in modulating the amount of chloroplast development, rather than triggering the initiation of events (e.g., gene expression) associated with chloroplast development.     

Photoresponses of Light-Grown phyA Mutants of Arabidopsis (Phytochrome A Is Required for the Perception of Daylength Extensions)

Several aspects of the photophysiology of wild-type Arabidopsis thaliana seedlings were compared with those of a phytochrome A null mutant, phyA-1, and a mutant, fhy1, that is putatively involved in the transduction of light signals from phytochrome A. Although phyA seedlings display a near wild-type phenotype when grown in white light (W), they nevertheless display several photomorphogenic abnormalities. Thus, whereas the germination of wild-type and fhy1 seeds is almost fully promoted by a pulse of red light (R) or by continuous far-red light (FR), phyA seed germination is responsive only to R. Following growth under day/night cycles, but not under continuous W, the hypocotyls of light-grown phyA and fhy1 seedlings are more elongated than those of wild-type seedlings. For seedlings grown under low red/far-red (R/FR) ratio light conditions, phyA and fhy1 seedlings display a more marked promotion of hypocotyl elongation than wild-type seedlings. Similarly, seedlings that are doubly null for phytochrome A and phytochrome B(phyA phyB) also have more elongated hypocotyls under low R/FR ratio conditions than phyB seedlings. This indicates that phytochrome A action in light-grown seedlings is antagonistic to the action of phytochrome B. Although wild-type, fhy1, and phyA seedlings flower at essentially the same time under both short-day and long-day conditions, an obvious consequence of phytochrome A deficiency is a pronounced late flowering under conditions where a short day of 8 h of fluorescent W is extended by 8 h of low-fluence-rate incandescent light. The evidence thus indicates that phytochrome A plays a role in seed germination, in the control of elongation growth of light-grown seedlings, and in the perception of daylength.     

Contribution of gibberellin deactivation by AtGA2ox2 to the suppression of germination of dark-imbibed Arabidopsis thaliana seeds

Gibberellin levels in imbibed Arabidopsis thaliana seeds are regulated by light via phytochrome, presumably through regulation of gibberellin biosynthesis genes, AtGA3ox1 and AtGA3ox2, and a deactivation gene, AtGA2ox2. Here, we show that a loss-of-function ga2ox2 mutation causes an increase in GA(4) levels and partly suppresses the germination inability during dark imbibition after inactivation of phytochrome. Experiments using 2,2-dimethylGA(4), a GA(4) analog resistant to gibberellin 2-oxidase, in combination with ga2ox2 mutant seeds suggest that the efficiency of deactivation of exogenous GA(4) by AtGA2ox2 is dependent on light conditions, which partly explains phytochrome-mediated changes in gibberellin effectiveness (sensitivity) found in previous studies.     

Differences and similarities in the photoregulation of gibberellin metabolism between rice and dicots

In rice seedlings, elongation of leaf sheaths is suppressed by light stimuli. The response is mediated by two classes of photoreceptors, phytochromes and cryptochromes. However, it remains unclear how these photoreceptors interact in the process. Our recent study using phytochrome mutants and novel cryptochrome RNAi lines revealed that cryptochromes and phytochromes function cooperatively, but independently to reduce active GA contents in seedlings in visible light. Blue light captured by cryptochrome 1 (cry1a and cry1b) induces robust expression of GA 2-oxidase genes (OsGA2ox4-7). In parallel, phytochrome B with auxiliary action of phytochrome A mediates repression of GA 20-oxidase genes (OsGA20ox2 and OsGA20ox4). The independent effects cumulatively reduce active GA contents, leading to a suppression of leaf sheath elongation. These regulatory mechanisms are distinct from phytochrome B function in dicots. We discuss reasons why the distinct system appeared in rice, and advantages of the rice system in early photomorphogenesis.     

Gibberellin requirement for Arabidopsis seed germination is determined both by testa characteristics and embryonic abscisic acid

The mechanisms imposing a gibberellin (GA) requirement to promote the germination of dormant and non-dormant Arabidopsis seeds were analyzed using the GA-deficient mutant ga1, several seed coat pigmentation and structure mutants, and the abscisic acid (ABA)-deficient mutant aba1. Testa mutants, which exhibit reduced seed dormancy, were not resistant to GA biosynthesis inhibitors such as tetcyclacis and paclobutrazol, contrarily to what was found before for other non-dormant mutants in Arabidopsis. However, testa mutants were more sensitive to exogenous GAs than the wild-types in the presence of the inhibitors or when transferred to a GA-deficient background. The germination capacity of the ga1-1 mutant could be integrally restored, without the help of exogenous GAs, by removing the envelopes or by transferring the mutation to a tt background (tt4 and ttg1). The double mutants still required light and chilling for dormancy breaking, which may indicate that both agents can have an effect independently of GA biosynthesis. The ABA biosynthesis inhibitor norflurazon was partially efficient in releasing the dormancy of wild-type and mutant seeds. These results suggest that GAs are required to overcome the germination constraints imposed both by the seed coat and ABA-related embryo dormancy.     

Mutations causing defects in the biosynthesis and response to gibberellins, abscisic acid and phytochrome B do not inhibit vernalization in Arabidopsis fca-1

 The roles of gibberellins, abscisic acid and phytochrome B in the vernalization response were investigated by combining mutations causing defects in their biosynthesis and response with the Arabidopsis thaliana (L.) Heynh. fca-1 mutation. The fca-1 mutation confers a very late-flowering phenotype which can be reversed to wild-type flowering if the seedlings are vernalized. Vernalization was unaffected in ga1-3, gai, abi1-1, abi2-1, abi3-1 and phyB-1 backgrounds, suggesting that gibberellin action mediated via GA1 and GAI, abscisic acid action mediated through ABI1 and ABI2, and phytochrome B, function independently of vernalization. However, the mutations did interact with fca-1 to change flowering time in the absence of vernalization. The abi1 fca-1 and abi2 fca-1 double mutants flowered earlier than fca-1 implying a role for abscisic acid in floral repression. Combination of ga1-3 or gai with fca-1 unexpectedly resulted in opposite interactions, with gai partially suppressing the late flowering of fca-1. Received: 17 July 1999 / Accepted: 11 October 1999     

Photomorphogenic mutants of Arabidopsis thaliana reveal activities of multiple photosensory systems during light-stimulated apical-hook opening

Fluence rate-response curves were generated for red-, far-red-, and blue-light-stimulated apical-hook opening in seedlings of several photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. Compared to wild-type plants, hook opening was reduced in the phytochrome-deficient hy1, hy2, and hy6 mutants in red and far-red light at all fluence rates tested, and in low-fluence blue light, but was normal under high-irradiance blue light. In contrast, the blue-light-response mutants (blu1, blu2, and blu3) lacked the high-irradiance-dependent hook-opening response in blue light while hook opening was normal in low-fluence blue light and in red and farred light at all fluence rates tested. Hook opening in the phytochrome-B-deficient hy3 mutant was similar to wild type in all light conditions tested. The effects of the different mutations on light-induced hook opening indicate that a phytochrome(s) other than phytochrome B mediates hook opening stimulated by red, far-red and lowfluence blue light, while a blue-light-absorbing photoreceptor mediates the blue-light-sensitive high-irradiance response. Although the phytochrome and blue-light photosensory systems appear to work independently for the most part, some of their signal-transduction components may interact since the hy4, and hy5 mutants showed reduced hook-opening responses under conditions dependent on the phytochrome and blue-light-photosensory systems.We thank Jeff Young and Brian Parks for their many helpful suggestions during the progress of this research. This work was supported by National Science Foundation Grant No. DCB-9106697.     

Analysis of phytochrome-deficient yellow-green-2 and aurea mutants of tomato

Two alleles of the yellow-green-2 ( yg-2) and eight different alleles of the aurea ( au ) locus of tomato ( Lycopersicon esculentum Mill.) were compared. All are characterized by a paler green colour compared with wild-type (WT), an elongated hypocotyl in red light, and low or below detection limits of spectrophotometrically active phytochrome. Hypocotyl length was variable in white light, ranging from that of WT to more elongated. Immunochemical analysis revealed that etiolated seedlings of the yg-2 mutant have approximately 25% of the WT level of phytochrome A protein (PHYA), whereas that of phytochrome B protein (PHYB) is normal. In this it resembles the au mutant. The au,yg-2 double mutant has a more extreme chlorophyll deficiency than either parent. Since the yg-2 and au mutants have a less severe phenotype at the adult stage, that is, are leaky, the additive effect can be explained by assuming that the mutants control two steps in the chromophore biosynthesis pathway. Combination, by crossing, of the yg-2 and au mutants with a transgenic tomato line that overexpresses oat phytochrome A3 (PhyA-3) essentially failed to restore the WT phenotype under white fluorescent light conditions, although under greenhouse conditions some evidence for increased sensitivity to light was observed. Immunochemically, oat PHYA-3 protein is detectable in both the yg-2,PhyA-3 and au,PhyA-3 'double' mutants. Spectrophotometrical analysis, however, revealed that holophytochrome was undetectable in the yg-2,PhyA-3 and au,PhyA-3 'double' mutants. These results are compatible with both mutants being disturbed in phytochrome chromophore biosynthesis.     

The pef mutants of Arabidopsis thaliana define lesions early in the phytochrome signaling pathway

In a screen for early-flowering mutants, a number of mutants that were early flowering under both short and long days were isolated. One such mutant, pef1, was selectively insensitive to both red and far-red light in the inhibition of hypocotyl elongation response; a classic phytochrome phenotype mediated by both PHYA and PHYB. The pef1 mutant seedlings could not be phenotypically rescued by biliverdin, a precursor of the phytochrome chromophore, nor did they fail to complement any previously identified elongated hypocotyl (hy) mutants. Difference spectra and Western blot analysis showed normal concentrations of PHYA photoreceptor apoprotein, which appeared photochemically active. It was concluded that the pef1 mutant is defective in both PHYA- and PHYB- mediated signaling pathways, and may represent a lesion in an early step of the phytochrome signal transduction pathway. Additional pef mutants deficient specifically in PHYB-mediated responses were also identified by this screen.