CCA1 and ELF3 Interact in the control of hypocotyl length and flowering time in Arabidopsis

The circadian clock is an endogenous oscillator with a period of approximately 24 h that allows organisms to anticipate, and respond to, changes in the environment. In Arabidopsis (Arabidopsis thaliana), the circadian clock regulates a wide variety of physiological processes, including hypocotyl elongation and flowering time. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is a central clock component, and CCA1 overexpression causes circadian dysfunction, elongated hypocotyls, and late flowering. EARLY FLOWERING3 (ELF3) modulates light input to the clock and is also postulated to be part of the clock mechanism. elf3 mutations cause light-dependent arrhythmicity, elongated hypocotyls, and early flowering. Although both genes affect similar processes, their relationship is not clear. Here, we show that CCA1 represses ELF3 by associating with its promoter, completing a CCA1-ELF3 negative feedback loop that places ELF3 within the oscillator. We also show that ELF3 acts downstream of CCA1, mediating the repression of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5 in the control of hypocotyl elongation. In the regulation of flowering, our findings show that ELF3 and CCA1 either cooperate or act in parallel through the CONSTANS/FLOWERING LOCUS T pathway. In addition, we show that CCA1 represses GIGANTEA and SUPPRESSOR OF CONSTANS1 by direct interaction with their promoters, revealing additional connections between the circadian clock and the flowering pathways.     

Suppressors of an Arabidopsis thaliana phyB mutation identify genes that control light signaling and hypocotyl elongation.

Ambient light controls the development and physiology of plants. The Arabidopsis thaliana photoreceptor phytochrome B (PHYB) regulates developmental light responses at both seedling and adult stages. To identify genes that mediate control of development by light, we screened for suppressors of the long hypocotyl phenotype caused by a phyB mutation. Genetic analyses show that the shy (short hypocotyl) mutations we have isolated fall in several loci. Phenotypes of the mutants suggest that some of the genes identified have functions in control of light responses. Other loci specifically affect cell elongation or expansion.     

Absence of phyB inhibits hypocotyl elongation in dark-grown Ih cucumber seedlings: an active role for PrB

Cucumber (Cucumis sativus L.) seedlings carrying the long hypocotyl (Ih) mutation, which confers a lack of B-type phytochrome (phyB), were significantly shorter than their near-isogenic wild-type counterparts when grown in complete darkness. Relative growth rates determined for 5 mm hypocotyl regions were lower in Ih seedlings in all growing regions, and the zone of elongation was less extensive in Ih hypocotyls. Digital imaging microscopy revealed that the pattern of epidermal cell lengths along the stem axis differed between the Ih mutant and the iso-genic wild-type. These findings (and the fact that experiments were conducted under conditions where phytochrome photoconversion to the far-red-absorbing form does not occur) suggest that the red-absorbing form of phyB (PrB) is an active positive regulator of development in etiolated plants.     

Interaction between the Late Elongated hypocotyl (LHY) and Early flowering 3 (ELF3) genes in the Arabidopsis circadian clock

The circadian clock governs rhythms with 24 hours that allow organisms to anticipate daily changing environmental time cues. In Arabidopsis, the circadian clock is conceptually composed of three parts; input pathways for light and temperature signals, oscillators and output pathways for physiological processes including leaf movement, gene expression rhythms and flowering time. Oscillators consist of three interlocking loops, named morning, central and evening loops. Components of the central oscillator contain LHY, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and TIMING OF CAB EXPRESSION (TOC1). The oscillator can be reset by light signals through input pathways. Genetic studies have revealed the components involved in light input pathways. The elf3 (early flowering 3) mutant was isolated by insensitivity to photoperiod showing long hypocotyls, elongated petioles and pale leaves characteristic of plants defective in light perception. Therefore the ELF3 has been proposed to act on light input pathways. The aim of this study is to test whether LHY and ELF3 encode interacting components of a circadian light input pathway. To address this possibility, lhy-1 elf3-1 (LHY overexpressing-mutant X ELF3 loss of function-mutant) and lhy-11 elf3-1 (LHY loss of function-mutant X ELF3 loss of function-mutant) double mutants were constructed. Their visual phenotypes and CAB (Chlorophyll a/b binding protein) expression patterns demonstrate that LHY may function downstream of ELF3 and that this interaction is disrupted when LHY expression is placed under the control of the 35S promoter. In addition, ELF3 is required for vigorous rhythms of LHY gene expression and LHY protein levels.     

Catecholamine stimulation of the gibberellin action that induces lettuce hypocotyl elongation

Effects of catecholamines and their derivatives on gibberellicacid (GA)-induced lettuce hypocotyl elongation was studied,because catecholamines have a chemical structure similar tothe dihydroconiferyl alcohol that has been isolated from lettucecotyledons as a GA synergist. Epinephrine, norepinephrine, dopamineand 3,4-dihydroxymandelic acid synergistically enhanced thepromoting effect of GA on hypocotyl elongation. In contrast,metanephrine, normetanephrine, DOPA and 3-methoxy-4- hydroxymandelicacid did not enhance the GA effect. The action of catecholamineswas inhibited by trans-cinnamic acid which competitively inhibitedthe action of dihydroconiferyl alcohol; this suggests that thereceptor site for catecholamines is the same as that for dihydroconiferylalcohol. The basic ethyl acetate fraction from lettuce seedlingssynergistically enhanced the GA effect. TLC analyses of thisbasic ethyl acetate fraction revealed that the chromatographicarea corresponding to authentic catecholamines could enhancethe GA effect. From these results, a possible role for catecholamines in theregulation of lettuce hypocotyl elongation caused by GA wasposited, and is discussed here. (Received May 15, 1979; )     

Gibberellins control Arabidopsis hypocotyl growth via regulation of cellular elongation

The gibberellins (GAs) are endogenous regulators of plant growth. Experiments are described here that test the hypothesis that GA regulates hypocotyl growth by altering the extent of hypocotyl cell elongation. These experiments use GA-deficient and altered GA-response mutants of Arabidopsis thaliana (L.) Heyhn. It is shown that GA regulates elongation, in both light- and dark-grown hypocotyls, by influencing the rate and final extent of cellular elongation. However, light- and dark-grown hypocotyls exhibit markedly different GA dose-response relationships. The length of dark-grown hypocotyls is relatively unaffected by exogenous GA, whilst light-grown hypocotyl length is significantly increased by exogenous GA. Further analysis suggests that GA control of hypocotyl length is close to saturation in dark-grown hypocotyls, but not in light grown hypocotyls. The results show that a large range of possible hypocotyl lengths is achieved via dose-dependent GA-regulated alterations in the degree of elongation of individual hypocotyl cells.Key words: Arabidopsis, cell elongation, gibberellin (GA), GA mutants, hypocotyl.      

Phytochrome E influences internode elongation and flowering time in Arabidopsis.

From a screen of M2 seedlings derived from gamma-mutagenesis of seeds doubly null for phytochromes phyA and phyB, we isolated a mutant lacking phyE. The PHYE gene of the selected mutant, phyE-1, was found to contain a 1-bp deletion at a position equivalent to codon 726, which is predicted to result in a premature stop at codon 739. Immunoblot analysis showed that the phyE protein was undetectable in the phyE-1 mutant. In the phyA- and phyB-deficient background, phyE deficiency led to early flowering, elongation of internodes between adjacent rosette leaves, and reduced petiole elongation. This is a phenocopy of the response of phyA phyB seedlings to end-of-day far-red light treatments. Furthermore, a phyE deficiency attenuated the responses of phyA phyB seedlings to end-of-day far-red light treatments. Monogenic phyE mutants were indistinguishable from wild-type seedlings. However, phyB phyE double mutants flowered earlier and had longer petioles than did phyB mutants. The elongation and flowering responses conferred by phyE deficiency are typical of shade avoidance responses to the low red/far-red ratio. We conclude that in conjunction with phyB and to a lesser extent with phyD, phyE functions in the regulation of the shade avoidance syndrome.     

Mechanical inhibition of hypocotyl elongation induces radial enlargement: implications for cytokinin action

Excised elongating segments from 3-day-old soybean (Glycine max var. Wayne) seedlings radially enlarged when auxin-promoted elongation was mechanically inhibited. Growth was similar to segments treated with auxin plus cytokinin. This observation suggests that cytokinin does not necessarily directly “reorient” cell enlargement. Cytokinin-induced radial cell growth may be a secondary effect of cytokinin's inhibition of auxin-promoted elongation.     

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.     

Over-expression of an AT-hook gene, AHL22, delays flowering and inhibits the elongation of the hypocotyl in Arabidopsis thaliana

The Arabidopsis genome encodes 29 AHL (AT-hook motif nuclear localized) proteins, but the function for most of them remains unknown. We report here a study of the AHL22 gene, which was originally identified as a gain-of-function allele that enhanced the phenotype of the cry1 cry2 mutant. AHL22 is a nuclear protein with the binding activity for an AT-rich DNA sequence. AHL22 overexpression delayed flowering and caused a constitutive photomorphogenic phenotype. The loss-of-function AHL22 mutant showed no clear phenotype on flowering, but slightly longer hypocotyls. However, silencing four AHL genes (AHL22, AHL18, AHL27, and AHL29) resulted in early flowering and enhanced ahl22-1 mutant phenotype on the growth of hypocotyls, suggesting genetic redundancy of AHL22 with other AHL genes on these plant developmental events. Further analysis showed that AHL22 controlled flowering and hypocotyl elongation might result from primarily the regulation of FT and PIF4 expression, respectively.     

Cytokinin action is coupled to ethylene in its effects on the inhibition of root and hypocotyl elongation in Arabidopsis thaliana seedlings.

Cytokinins have profound effects on seedling development in Arabidopsis thaliana. Benzyladenine (BA) inhibits root elongation in light- or dark-grown seedlings, and in dark-grown seedlings BA inhibits hypocotyl elongation and exaggerates the curvature of apical hooks. The latter are characteristic ethylene responses and, therefore, the possible involvement of ethylene in BA responses was examined in seedlings. It was found that the inhibitory effects of BA on root and hypocotyl elongation were partially blocked by the action of ethylene inhibitors or ethylene-resistant mutations (ein1-1 and ein2-1). Ethylene production was stimulated by submicromolar concentrations of BA and could account, in part, for the inhibition of root and hypocotyl elongation. It was demonstrated further that BA did not affect the sensitivity of seedlings to ethylene. Thus, the effect of cytokinin on root and hypocotyl elongation in Arabidopsis appears to be mediated largely by the production of ethylene. The coupling between cytokinin and ethylene responses is further supported by the discovery that the cytokinin-resistant mutant ckr1 is resistant to ethylene and is allelic to the ethylene-resistant mutant ein2.     

Phytochrome D acts in the shade-avoidance syndrome in Arabidopsis by controlling elongation growth and flowering time

Shade avoidance in higher plants is regulated by the action of multiple phytochrome (phy) species that detect changes in the red/far-red ratio (R/FR) of incident light and initiate a redirection of growth and an acceleration of flowering. The phyB mutant of Arabidopsis is constitutively elongated and early flowering and displays attenuated responses to both reduced R/FR and end-of-day far-red light, conditions that induce strong shade-avoidance reactions in wild-type plants. This indicates that phyB plays an important role in the control of shade avoidance. In Arabidopsis phyB and phyD are the products of a recently duplicated gene and share approximately 80% identity. We investigated the role played by phyD in shade avoidance by analyzing the responses of phyD-deficient mutants. Compared with the monogenic phyB mutant, the phyB-phyD double mutant flowers early and has a smaller leaf area, phenotypes that are characteristic of shade avoidance. Furthermore, compared with the monogenic phyB mutant, the phyB-phyD double mutant shows a more attenuated response to a reduced R/FR for these responses. Compared with the phyA-phyB double mutant, the phyA-phyB-phyD triple mutant has elongated petioles and displays an enhanced elongation of internodes in response to end-of-day far-red light. These characteristics indicate that phyD acts in the shade-avoidance syndrome by controlling flowering time and leaf area and that phyC and/or phyE also play a role.     

Transgene-mediated auxin overproduction in Arabidopsis: hypocotyl elongation phenotype and interactions with the hy6-1 hypocotyl elongation and axr1 auxin-resistant mutants

Transgenic Arabidopsis thaliana plants constitutively expressing Agrobacterium tumefaciens tryptophan monooxygenase (iaaM) were obtained and characterized. Arabidopsis plants expressing iaaM have up to 4-fold higher levels of free indole-3-acetic acid (IAA) and display increased hypocotyl elongation in the light. This result clearly demonstrates that excess endogenous auxin can promote cell elongation in a whole plant. Interactions of the auxin-overproducing transgenic plants with the phytochrome-deficient hy6-1 and auxin-resistant axrl-3 mutations were also studied. The effects of auxin overproduction on hypocotyl elongation were not additive to the effects of phytochrome deficiency in the hy6-1 mutant, indicating that excess auxin does not counteract factors that limit hypocotyl elongation in hy6-1 seedlings. Auxin-overproducing seedlings are also qualitatively indistinguishable from wild-type controls in their response to red, far-red, and blue light treatments, demonstrating that the effect of excess auxin on hypocotyl elongation is independent of red and blue light-mediated effects. All phenotypic effects of iaaM-mediated auxin overproduction (i.e. increased hypocotyl elongation in the light, severe rosette leaf epinasty, and increased apical dominance) are suppressed by the auxin-resistant axr1-3 mutation. The axr1-3 mutation apparently blocks auxin signal transduction since it does not reduce auxin levels when combined with the auxin-overproducing transgene.