Phytochrome B affects responsiveness to gibberellins in Arabidopsis.

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. Arabidopsis thaliana seedlings lacking one of the phytochromes, phyB, have elongated hypocotyls and other tissues, suggesting that they may have an alteration in hormone physiology. We have studied the possibility that phyB mutations affect seedling gibberellin (GA) perception and metabolism by testing the responsiveness of wild-type and phyB seedlings to exogenous GAs. The phyB mutant elongates more than the wild type in response to the same exogenous concentrations of GA3 or GA4, showing that the mutation causes an increase in responsiveness to GAs. Among GAs that we were able to detect, we found no significant difference in endogenous levels between wild-type and phyB mutant seedlings. However, GA4 levels were below our limit of detectability, and the concentration of that active GA could have varied between wild-type and phyB mutant seedlings. These results suggest that, although GAs are required for hypocotyl cell elongation, phyB does not act primarily by changing total seedling GA levels but rather by decreasing seedling responsiveness to GAs.     

Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. In Arabidopsis thaliana, there are genes encoding at least five phytochromes, and it is of interest to learn if the different phytochromes have overlapping or distinct functions. To address this question for two of the phytochromes in Arabidopsis, we have compared light responses of the wild type with those of a phyA null mutant, a phyB null mutant, and a phyA phyB double mutant. We have found that both phyA and phyB mutants have a deficiency in germination, the phyA mutant in far-red light and the phyB mutant in the dark. Furthermore, the germination defect caused by the phyA mutation in far- red light could be suppressed by a phyB mutation, suggesting that phytochrome B (PHYB) can have an inhibitory as well as a stimulatory effect on germination. In red light, the phyA phyB double mutant, but neither single mutant, had poorly developed cotyledons, as well as reduced red-light induction of CAB gene expression and potentiation of chlorophyll induction. The phyA mutant was deficient in sensing a flowering response inductive photoperiod, suggesting that PHYA participates in sensing daylength. In contrast, the phyB mutant flowered earlier than the wild type (and the phyA mutant) under all photoperiods tested, but responded to an inductive photoperiod. Thus, PHYA and PHYB appear to have complementary functions in controlling germination, seedling development, and flowering. We discuss the implications of these results for possible mechanisms of PHYA and PHYB signal transduction.     

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.     

The gibberellic acid biosynthesis mutant ga1-3 of Arabidopsis thaliana is responsive to vernalization.

The Arabidopsis mutant ga1-3 contains a deletion in an enzyme that catalyzes an early step in the synthesis of gibberellic acid. It has been shown that ga1-3 mutant plants cannot flower under 8-h short-day (SD) conditions, even after vernalization. In this article, we present data demonstrating that the ga1-3 mutation does not block the response to vernalization in intermediate photoperiods or in long-day conditions in a late-flowering, vernalization-responsive background. Thus, GA may not have a direct role in the vernalization response in Arabidopsis, but it may be required for an alternate pathway that promotes flowering in noninductive photoperiods.     

Phytochromes A1 and B1 have distinct functions in the photoperiodic control of flowering in the obligate long-day plant Nicotiana sylvestris

The obligate long-day plant Nicotiana sylvestris with a nominal critical day length of 12 h was used to dissect the roles of two major phytochromes (phyA1 and phyB1) in the photoperiodic control of flowering using transgenic plants under-expressing PHYA1 (SUA2), over-expressing PHYB1 (SOB36), or cosuppressing the PHYB1 gene (SCB35). When tungsten filament lamps were used to extend an 8 h main photoperiod, SCB35 and SOB36 flowered earlier and later, respectively, than wild-type plants, while flowering was greatly delayed in SUA2. These results are consistent with those obtained with other long-day plants in that phyB has a negative role in the control of flowering, while phyA is required for sensing day-length extensions. However, evidence was obtained for a positive role for PHYB1 in the control of flowering. Firstly, transgenic plants under-expressing both PHYA1 and PHYB1 exhibited extreme insensitivity to day-length extensions. Secondly, flowering in SCB35 was completely repressed under 8 h extensions with far-red-deficient light from fluorescent lamps. This indicates that the dual requirement for both far-red and red for maximum floral induction is mediated by an interaction between phyA1 and phyB1. In addition, a diurnal periodicity to the sensitivity of both negative and positive light signals was observed. This is consistent with existing models in which photoperiodic time measurement is not based on the actual measurement of the duration of either the light or dark period, but rather the coincidence of endogenous rhythms of sensitivity - both positive and negative - and the presence of light cues.     

Regulation of flowering in the long-day grass Lolium temulentum by gibberellins and the FLOWERING LOCUS T gene

Seasonal control of flowering often involves leaf sensing of daylength coupled to time measurement and generation and transport of florigenic signals to the shoot apex. We show that transmitted signals in the grass Lolium temulentum may include gibberellins (GAs) and the FLOWERING LOCUS T (FT) gene. Within 2 h of starting a florally inductive long day (LD), expression of a 20-oxidase GA biosynthetic gene increases in the leaf; its product, GA(20), then increases 5.7-fold versus short day; its substrate, GA(19), decreases equivalently; and a bioactive product, GA(5), increases 4-fold. A link between flowering, LD, GAs, and GA biosynthesis is shown in three ways: (1) applied GA(19) became florigenic on exposure to LD; (2) expression of LtGA20ox1, an important GA biosynthetic gene, increased in a florally effective LD involving incandescent lamps, but not with noninductive fluorescent lamps; and (3) paclobutrazol, an inhibitor of an early step of GA biosynthesis, blocked flowering, but only if applied before the LD. Expression studies of a 2-oxidase catabolic gene showed no changes favoring a GA increase. Thus, the early LD increase in leaf GA(5) biosynthesis, coupled with subsequent doubling in GA(5) content at the shoot apex, provides a substantial trail of evidence for GA(5) as a LD florigen. LD signaling may also involve transport of FT mRNA or protein because expression of LtFT and LtCONSTANS increased rapidly, substantially (>80-fold for FT), and independently of GA. However, because a LD from fluorescent lamps induced LtFT expression but not flowering, the nature of the light response of FT requires clarification.     

Genetic Regulation of Development in Sorghum bicolor (X. Greatly Attenuated Photoperiod Sensitivity in a Phytochrome-Deficient Sorghum Possessing a Biological Clock but Lacking a Red Light-High Irradiance Response)

The role of a light-stable, 123-kD phytochrome in the biological clock, in photoperiodic flowering and shoot growth in extended photoperiods, and in the red light-high irradiance response was studied in Sorghum bicolor using a phytochrome-deficient mutant, 58M (ma3R ma3R), and a near-isogenic wild-type cultivar, 100M (Ma3 Ma3). Since chlorophyll a/b-binding protein mRNA and ribulose bisphosphate carboxylase small subunit mRNA cycled in a circadian fashion in both 58M and 100M grown in constant light, the 123-kD phytochrome absent from 58M does not appear necessary for expression or entrainment of a functional biological clock. Although 58M previously appeared photoperiod insensitive in 12-h photoperiods, extending the photoperiod up to 24 h delayed floral initiation for up to 2 weeks but did not much affect shoot elongation. Thus, although 58M flowers early in intermediate photoperiods, a residual photoperiod sensitivity remains that presumably is not due to the missing 123-kD phytochrome. Since rapid shoot elongation persists in 58M under extended photoperiods despite delayed floral initiation, long photoperiods uncouple those processes. The observed absence of a red light-high irradiance response in 58M, in contrast to the presence of the response in 100M, strengthens the suggestion that the 123-kD phytochrome missing from 58M is a phyB.     

Expression of heterologous phytochromes A, B or C in transgenic tobacco plants alters vegetative development and flowering time

In this study, oat phytochrome A (phyA), Arabidopsis phytochrome B (phyB) or Arabidopsis phytochrome C (phyC) were expressed in both day-neutral and photo-period-sensitive (short-day) tobacco (Nicotiana tabacum cv. Hicks). Introgression of the Maryland Mammoth (MM) gene into cv Hicks was used to confer short-day photo-periodic sensitivity. Expression of oat phyA led to characteristic hypersensitivity of hypocotyls to red light (R) and far-red light (FR) and an overall dwarfing of the mature plant. Expression of Arabidopsis phyB enhanced the sensitivity of hypocotyls to R and caused even more marked dwarfing of the mature plant. In contrast, the expression of Arabidopsis phyC had no detectable consequences for the photocontrol of hypocotyl elongation. However, phyC expression did lead to a R-dependent increase in cotyledon expansion in de-etiolating seedlings and to a significant increase in leaf area in mature plants. This provides the first experimental evidence that phyC is biologically active. The flowering time of cv Hicks plants grown under 8 h photoperiods was virtually unaffected by a 30 min white light (W) night break given 8 h into the dark period. In contrast, cv Hicks MM plants responded to a night break with a delay in flowering. Expression of phyA or phyB led to a night break-dependent delay in flowering in cv Hicks plants. For cv Hicks MM plants, the expression of any of phyA, phyB or phyC caused a marked enhancement of the flower-delaying effect of a night break. These observations indicate that transgenic phyA, phyB or phyC can interact with the endogenous mechanisms controlling flowering time in tobacco.     

Photoperiod Control of Gibberellin Levels and Flowering in Sorghum

Regulation of rhythmic peaks in levels of endogenous gibberellins (GAs) by photoperiod was studied in the short-day monocot sorghum (Sorghum bicolor [L.] Moench). Comparisons were made between three maturity (Ma) genotypes: 58M (Ma₁Ma₁, Ma₂Ma₂, phyB-1phyB-1, and Ma₄Ma₄ [a phytochrome B null mutant]); 90M (Ma₁Ma₁, Ma₂Ma₂, phyB-2phyB-2, and Ma₄Ma₄); and 100M (Ma₁Ma₁, Ma₂Ma₂, PHYBPHYB, and Ma₄Ma₄). Plants were grown for 14 d under 10-, 14-, 16-, 18-, and 20-h photoperiods, and GA levels were assayed by gas chromatography-mass spectrometry every 3 h for 24 h. Under inductive 10-h photoperiods, the peak of GA₂₀ and GA₁ levels in 90M and 100M was shifted from midday, observed earlier with 12-h photoperiods, to an early morning peak, and flowering was hastened. In addition, the early morning peaks in levels of GA₂₀ and GA₁ in 58M under conditions allowing early flowering (10-, 12-, and 14-h photoperiods) were shifted to midday by noninductive (18- and 20-h) photoperiods, and flowering was delayed. These results are consistent with the possibility that the diurnal rhythm of GA levels plays a role in floral initiation and may be one way by which the absence of phytochrome B causes early flowering in 58M under most photoperiods.     

Establishment of the vernalization-responsive, winter-annual habit in Arabidopsis requires a putative histone H3 methyl transferase

Winter-annual accessions of Arabidopsis thaliana are often characterized by a requirement for exposure to the cold of winter to initiate flowering in the spring. The block to flowering prior to cold exposure is due to high levels of the flowering repressor FLOWERING LOCUS C (FLC). Exposure to cold promotes flowering through a process known as vernalization that epigenetically represses FLC expression. Rapid-cycling accessions typically have low levels of FLC expression and therefore do not require vernalization. A screen for mutants in which a winter-annual Arabidopsis is converted to a rapid-cycling type has identified a putative histone H3 methyl transferase that is required for FLC expression. Lesions in this methyl transferase, EARLY FLOWERING IN SHORT DAYS (EFS), result in reduced levels of histone H3 Lys 4 trimethylation in FLC chromatin. EFS is also required for expression of other genes in the FLC clade, such as MADS AFFECTING FLOWERING2 and FLOWERING LOCUS M. The requirement for EFS to permit expression of several FLC clade genes accounts for the ability of efs lesions to suppress delayed flowering due to the presence of FRIGIDA, autonomous pathway mutations, or growth in noninductive photoperiods. efs mutants exhibit pleiotropic phenotypes, indicating that the role of EFS is not limited to the regulation of flowering time.     

EAF1 regulates vegetative-phase change and flowering time in Arabidopsis.

We have identified a new locus that regulates vegetative phase change and flowering time in Arabidopsis. An early-flowering mutant, eaf1 (early flowering 1) was isolated and characterized. eaf1 plants flowered earlier than the wild type under either short-day or long-day conditions, and showed a reduction in the juvenile and adult vegetative phases. When grown under short-day conditions, eaf1 plants were slightly pale green and had elongated petioles, phenotypes that are observed in mutants altered in either phytochrome or the gibberellin (GA) response. eaf1 seed showed increased resistance to the GA biosynthesis inhibitor paclobutrazol, suggesting that GA metabolism and/or response had been altered. Comparison of eaf1 to other early-flowering mutants revealed that eaf1 shifts to the adult phase early and flowers early, similarly to the phyB (phytochrome B) and spy (spindly) mutants. eaf1 maps to chromosome 2, but defines a locus distinct from phyB, clf (curly leaf), and elf3 (early-flowering 3). These results demonstrate that eaf1 defines a new locus involved in an autonomous pathway and may affect GA regulation of flowering.     

Flowering of the grass Lolium perenne: effects of vernalization and long days on gibberellin biosynthesis and signaling

Almost 50 years ago, it was shown that gibberellin (GA) applications caused flowering in species normally responding to cold (vernalization) and long day (LD). The implication that GAs are involved with vernalization and LD responses is examined here with the grass Lolium perenne. This species has an obligatory requirement for exposure to both vernalization and LD for its flowering (inflorescence initiation). Specific effects of vernalization or LD on GA synthesis, content, and action have been documented using four treatment pairs: nonvernalized or vernalized plants exposed to short days (SDs) or LDs. Irrespective of vernalization status, exposure to two LDs increased expression of L. perenne GA 20-oxidase-1 (LpGA20ox1), a critical GA biosynthetic gene, with endogenous GAs increasing by up to 5-fold in leaf and shoot. In parallel, LD led to degradation of a DELLA protein, SLENDER (within 48 h of LD or within 2 h of GA application). There was no effect on GA catabolism or abscisic acid content. Loss of SLENDER, which is a repressor of GA signaling, confirms the physiological relevance of increased GA content in LD. For flowering, applied GA replaced the need for LD but not that for vernalization. Thus, GAs may be an LD, leaf-sourced hormonal signal for flowering of L. perenne. By contrast, vernalization had little impact on GA or SLENDER levels or on SLENDER degradation following GA application. Thus, although vernalization and GA are both required for flowering of L. perenne, GA signaling is independent of vernalization that apparently impacts on unrelated processes.     

Independent control of gibberellin biosynthesis and flowering time by the circadian clock in Arabidopsis

Flowering of the facultative long-day plant Arabidopsis is controlled by several endogenous and environmental factors, among them gibberellins (GAs) and day length. The promotion of flowering by long days involves an endogenous clock that interacts with light cues provided by the environment. Light, and specifically photoperiod, is also known to regulate the biosynthesis of GAs, but the effects of GAs and photoperiod on flowering are at least partially separable. Here, we have used a short-period mutant, toc1, to investigate the role of the circadian clock in the control of flowering time by GAs and photoperiod. We show that toc1 affects expression of several floral regulators and a GA biosynthetic gene, but that these effects are independent.     

GAMYB-like Genes, Flowering, and Gibberellin Signaling in Arabidopsis

We have identified three Arabidopsis genes with GAMYB-like activity, AtMYB33, AtMYB65, and AtMYB101, which can substitute for barley (Hordeum vulgare) GAMYB in transactivating the barley alpha-amylase promoter. We have investigated the relationships between gibberellins (GAs), these GAMYB-like genes, and petiole elongation and flowering of Arabidopsis. Within 1 to 2 d of transferring plants from short- to long-day photoperiods, growth rate and erectness of petioles increased, and there were morphological changes at the shoot apex associated with the transition to flowering. These responses were accompanied by accumulation of GAs in the petioles (GA(1) by 11-fold and GA(4) by 3-fold), and an increase in expression of AtMYB33 at the shoot apex. Inhibition of GA biosynthesis using paclobutrazol blocked the petiole elongation induced by long days. Causality was suggested by the finding that, with GA treatment, plants flowered in short days, AtMYB33 expression increased at the shoot apex, and the petioles elongated and grew erect. That AtMYB33 may mediate a GA signaling role in flowering was supported by its ability to bind to a specific 8-bp sequence in the promoter of the floral meristem-identity gene, LEAFY, this same sequence being important in the GA response of the LEAFY promoter. One or more of these AtMYB genes may also play a role in the root tip during germination and, later, in stem tissue. These findings extend our earlier studies of GA signaling in the Gramineae to include a dicot species, Arabidopsis, and indicate that GAMYB-like genes may mediate GA signaling in growth and flowering responses.     

Interaction of phytochromes A and B in the control of de-etiolation and flowering in pea

The interactions of phytochrome A (phyA) and phytochrome B (phyB) in the photocontrol of vegetative and reproductive development in pea have been investigated using null mutants for each phytochrome. White-light-grown phyA phyB double mutant plants show severely impaired de-etiolation both at the seedling stage and later in development, with a reduced rate of leaf production and swollen, twisted internodes, and enlarged cells in all stem tissues. PhyA and phyB act in a highly redundant manner to control de-etiolation under continuous, high-irradiance red light. The phyA phyB double mutant shows no significant residual phytochrome responses for either de-etiolation or shade-avoidance, but undergoes partial de-etiolation in blue light. PhyB is shown to inhibit flowering under both long and short photoperiods and this inhibition is required for expression of the promotive effect of phyA. PhyA is solely responsible for the promotion of flowering by night-breaks with white light, whereas phyB appears to play a major role in detection of light quality in end-of-day light treatments, night breaks and day extensions. Finally, the inhibitory effect of phyB is not graft-transmissible, suggesting that phyB acts in a different manner and after phyA in the control of flower induction.     

Cytokinin affects circadian-clock oscillation in a phytochrome B- and Arabidopsis response regulator 4-dependent manner

In higher plants, many developmental processes, such as photomorphogenesis and flowering, are coregulated by light and the phytohormone cytokinin. Interactions between light and cytokinin pathways are presumably mediated by common signaling intermediates. However, the molecular mechanism of these interactions remains unclear. Here, we report that cytokinin specifically induces the expression of the Arabidopsis circadian oscillator genes LATE ELONGATED HYPOCOTYL ( LHY ) and CIRCADIAN CLOCK-ASSOCIATED 1 ( CCA1 ) but represses the expression of TIMING OF CAB EXPRESSION 1 in a light-dependent manner. Consistent with these observations, cytokinin causes a shifted phase of the circadian clock. Mutant studies showed that the altered clock oscillation modulated by cytokinin is dependent on phytochrome B ( PHYB ) and Arabidopsis RESPONSE REGULATOR 4 ( ARR4 ). Whereas overexpression of LHY or CCA1 renders plants slightly more sensitive to cytokinin, phyB and a lhy/cca1 double mutant are less sensitive to the hormone. These results suggest that cytokinin affects the circadian clock oscillation in a PHYB - and ARR4 -dependent manner and that cytokinin signaling is also regulated by light-signaling components, including PHYB , LHY and CCA1 . Therefore, phyB, ARR4 and the circadian oscillator may function as signaling intermediates to integrate light and cytokinin pathways.