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In 1723, Anthonie van Leeuwenhoek, the "master of fleas and father of microbiology", died at the age of 90 [...].
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A plant's growth and development are shaped by its genome and the capacity to negotiate its environment for access to light, water, and nutrients. There is a vital need to understand the interactions between the plant, its physical environment, and the fertilizers used in agriculture. In this study, a commercially available volcanic ash fertilizer, Azomite®, characterized as dacitic (rhyolitic) tuff breccia, was tested for its effect on promoting early seedling vigor. Early growth and photomorphogenesis processes are well studied in Arabidopsis. Seedling assays under different light conditions were used to dissect the underlying mechanisms involved. These assays are well established and can be translated to agriculturally important crop plants. The volcanic ash fertilizer was tested at different concentrations on seedlings grown on basic media lacking sucrose either in continuous darkness (Dc), continuous Red (Rc), Far-Red (FRc), or White Light (WLc). Micronutrients in the volcanic ash significantly increased seedling growth under Rc and WLc, but not under Dc and FRc, indicating that photosynthetically active radiation was required for the observed growth increase. Furthermore, red-light photoreceptor mutant, phyB-9, lacked the growth response, and higher amount of fertilizer reduced growth in all conditions tested. These data suggest that light triggers the ability of the seedling to utilize micronutrients in volcanic ash in a dose-dependent manner. The methods described here can be used to establish mechanisms of activity of various nutrient inputs and, coupled with whole-genome expression profiling, can lead to better insights into optimizing nutrient field applications to improve crop production.
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The above-ground (phyllosphere) plant microbiome is increasingly recognized as an important component of plant health. We hypothesized that phyllosphere bacterial recruitment may be disrupted in a greenhouse setting, and that adding a bacterial amendment would therefore benefit the health and growth of host plants. Using a newly developed synthetic phyllosphere bacterial microbiome for tomato (Solanum lycopersicum), we tested this hypothesis across multiple trials by manipulating microbial inoculation of leaves and measuring subsequent plant growth and reproductive success, comparing results from plants grown in both greenhouse and field settings. We confirmed that greenhouse-grown plants have a relatively depauperate phyllosphere bacterial microbiome, which both makes them an ideal system for testing the impact of phyllosphere communities on plant health and important targets for microbial amendments as we move towards increased agricultural sustainability. We find that the addition of the synthetic microbial community early in greenhouse growth leads to an increase in fruit production in this setting, implicating the phyllosphere microbiome as a key component of plant fitness and emphasizing the role that these bacterial microbiomes likely play in the ecology and evolution of plant communities.
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In a recent Review Article on Gregor Mendel's (1822-1884) work with pea (Pisum sativum)-plants, it was proposed that this crop species should be re-vitalized as a model organism for the study of cell- and organ growth. Here, we describe the effect of exogenous gibberellic acid (GA3) on the growth of the second internode in 4-day-old light-grown pea seedlings (Pisum sativum, large var. "Senator"). lnjection of glucose into the internode caused a growth-promoting effect similar to that of the hormone GA3. Imbibition of dry pea seeds in GA3, or water as control, resulted in a drastic enhancement in organ development in this tall variety. Similar results were reported for dwarf peas. These "classical" experimental protocols are suitable to study the elusive effect of gibberellins (which act in coordination with auxin) on the regulation of plant development at the biochemical and molecular levels.
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Giberelinas , Pisum sativum , Giberelinas/farmacología , Ácidos Indolacéticos/farmacología , Semillas , Plantones , Hormonas/farmacologíaRESUMEN
Food crops are grown with fertilizers containing nitrogen, phosphorus, and potassium (macronutrients) along with magnesium, calcium, boron, and zinc (micronutrients) at different ratios during their cultivation. Soil and plant-associated microbes have been implicated to promote plant growth, stress tolerance, and productivity. However, the high degree of variability across agricultural environments makes it difficult to assess the possible influences of nutrient fertilizers on these microbial communities. Uncovering the underlying mechanisms could lead us to achieve consistently improved food quality and productivity with minimal environmental impacts. For this purpose, we tested a commercially available fertilizer (surface-mined volcanic ash deposit Azomite) applied as a supplement to the normal fertilizer program of greenhouse-grown tomato plants. Because this treatment showed a significant increase in fruit production at measured intervals, we examined its impact on the composition of below-ground microbial communities, focusing on members identified as "core taxa" that were enriched in the rhizosphere and root endosphere compared to bulk soil and appeared above their predicted neutral distribution levels in control and treated samples. This analysis revealed that Azomite had little effect on microbial composition overall, but it had a significant, temporally selective influence on the core taxa. Changes in the composition of the core taxa were correlated with computationally inferred changes in functional pathway enrichment associated with carbohydrate metabolism, suggesting a shift in available microbial nutrients within the roots. This finding exemplifies how the nutrient environment can specifically alter the functional capacity of root-associated bacterial taxa, with the potential to improve crop productivity. IMPORTANCE Various types of soil fertilizers are used routinely to increase crop yields globally. The effects of these treatments are assessed mainly by the benefits they provide in increased crop productivity. There exists a gap in our understanding of how soil fertilizers act on the plant-associated microbial communities. The underlying mechanisms of nutrient uptake are widely complex and, thus, difficult to evaluate fully but have critical influences on both soil and plant health. Here, we presented a systematic approach to analyzing the effects of fertilizer on core microbial communities in soil and plants, leading to predictable outcomes that can be empirically tested and used to develop simple and affordable field tests. The methods described here can be used for any fertilizer and crop system. Continued effort in advancing our understanding of how fertilizers affect plant and microbe relations is needed to advance scientific understanding and help growers make better-informed decisions.
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Microbiota , Solanum lycopersicum , Productos Agrícolas , Fertilizantes/análisis , Solanum lycopersicum/microbiología , Micronutrientes/análisis , Rizosfera , Suelo , Microbiología del Suelo , Erupciones VolcánicasRESUMEN
The year 2020 marks the 150th anniversary of the elucidation of the process of plant organ growth at the cellular level by Julius Sachs (1870). In this Addendum to a Review Article in Molecular Plant, we describe this fundamental discovery and argue that the etiolated grass coleoptile still represents the system of choice for the experimental analysis of auxin (indole-3-acetic acid, IAA)-action. With reference to the phenomenon of 'tissue tension', we discuss the acid-growth hypotheses of IAA-induced wall loosening and the process of vacuolar expansion, respectively. IAA-mediated elongation appears to be independent of wall acidification, and may be regulated via the secretion of glycoproteins into the outer epidermal wall, whereby turgor (and tissue) pressure provides the 'driving force' for growth. As predicted by the "acid growth-hypothesis", the fungal phytotoxin Fusicoccin (Fc) induces organ elongation via the rapid secretion of protons. We conclude that "cell elongation" can only be understood at the level of the entire organ that displays biomechanical features not established by single cells. This systems-level approach can be traced back to the work of Sachs (1870).
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Cotiledón/metabolismo , Ácidos Indolacéticos/metabolismo , Zea mays/metabolismo , Cotiledón/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Zea mays/crecimiento & desarrolloRESUMEN
Despite growing interest in utilizing microbial-based methods for improving crop growth, much work still remains in elucidating how beneficial plant-microbe associations are established, and what role soil amendments play in shaping these interactions. Here, we describe a set of experiments that test the effect of a commercially available soil amendment, VESTA, on the soil and strawberry (Fragaria x ananassa Monterey) root bacterial microbiome. The bacterial communities of the soil, rhizosphere, and root from amendment-treated and untreated fields were profiled at four time points across the strawberry growing season using 16S rRNA gene amplicon sequencing on the Illumina MiSeq platform. In all sample types, bacterial community composition and relative abundance were significantly altered with amendment application. Importantly, time point effects on composition are more pronounced in the root and rhizosphere, suggesting an interaction between plant development and treatment effect. Surprisingly, there was slight overlap between the taxa within the amendment and those enriched in plant and soil following treatment, suggesting that VESTA may act to rewire existing networks of organisms through an, as of yet, uncharacterized mechanism. These findings demonstrate that a commercial microbial soil amendment can impact the bacterial community structure of both roots and the surrounding environment.
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Bacterias/genética , Fragaria/crecimiento & desarrollo , Fragaria/microbiología , Microbiota/genética , Raíces de Plantas/microbiología , Microbiología del Suelo , Producción de Cultivos/métodos , ADN Bacteriano/genética , Secuenciación de Nucleótidos de Alto Rendimiento , ARN Ribosómico 16S/genética , Rizosfera , Análisis de Secuencia de ADN , Suelo/químicaRESUMEN
In 1963, a monograph by Thomas D. Luckey entitled Germfree Life and Gnotobiology was published, with a focus on animals treated with microbes and reference to the work of Louis Pasteur (1822-1895). Here, we review the history and current status of plant gnotobiology, which can be traced back to the experiments of Jean-Baptiste Boussingault (1801-1887) published in 1838. Since the outer surfaces of typical land plants are much larger than their internal areas, embryophytes "wear their guts on the outside." We describe the principles of gnotobiological analyses, with reference to epiphytic metylobacteria, and sunflower (Helianthus annuus) as well as Arabidopsis as model dicots. Finally, a Californian field experiment aiming to improve crop yield in strawberries (Fragaria ananassa) is described to document the practical value of this novel research agenda.
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Agricultura , Fenómenos Fisiológicos Bacterianos , Fragaria/microbiología , Helianthus/microbiología , SimbiosisRESUMEN
Reorganization of the cortical microtubule cytoskeleton is critical for guard cell function. Here, we investigate how environmental and hormonal signals cause these rearrangements and find that COP1, a RING-finger-type ubiquitin E3 ligase, is required for degradation of tubulin, likely by the 26S proteasome. This degradation is required for stomatal closing. In addition to regulating the cytoskeleton, we show that cop1 mutation impaired the activity of S-type anion channels, which are critical for stomatal closure. Thus, COP1 is revealed as a potential coordinator of cytoskeletal and electrophysiological activities required for guard cell function.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/fisiología , Fenómenos Electrofisiológicos , Microtúbulos/metabolismo , Estomas de Plantas/anatomía & histología , Ubiquitina-Proteína Ligasas/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Calcio/metabolismo , Fenómenos Electrofisiológicos/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Imagen Molecular , Mutación , Estomas de Plantas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Crop yield is a highly complex quantitative trait. Historically, successful breeding for improved grain yield has led to crop plants with improved source capacity, altered plant architecture, and increased resistance to abiotic and biotic stresses. To date, transgenic approaches towards improving crop grain yield have primarily focused on protecting plants from herbicide, insects, or disease. In contrast, we have focused on identifying genes that, when expressed in soybean, improve the intrinsic ability of the plant to yield more. Through the large scale screening of candidate genes in transgenic soybean, we identified an Arabidopsis thaliana B-box domain gene (AtBBX32) that significantly increases soybean grain yield year after year in multiple transgenic events in multi-location field trials. In order to understand the underlying physiological changes that are associated with increased yield in transgenic soybean, we examined phenotypic differences in two AtBBX32-expressing lines and found increases in plant height and node, flower, pod, and seed number. We propose that these phenotypic changes are likely the result of changes in the timing of reproductive development in transgenic soybean that lead to the increased duration of the pod and seed development period. Consistent with the role of BBX32 in A. thaliana in regulating light signaling, we show that the constitutive expression of AtBBX32 in soybean alters the abundance of a subset of gene transcripts in the early morning hours. In particular, AtBBX32 alters transcript levels of the soybean clock genes GmTOC1 and LHY-CCA1-like2 (GmLCL2). We propose that through the expression of AtBBX32 and modulation of the abundance of circadian clock genes during the transition from dark to light, the timing of critical phases of reproductive development are altered. These findings demonstrate a specific role for AtBBX32 in modulating soybean development, and demonstrate the validity of expressing single genes in crops to deliver increased agricultural productivity.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Glycine max/genética , Semillas/crecimiento & desarrollo , Semillas/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Relojes Biológicos/genética , Proteínas Portadoras/genética , Regulación del Desarrollo de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducción/genética , Supresión GenéticaRESUMEN
A B-box zinc finger protein, B-BOX32 (BBX32), was identified as playing a role in determining hypocotyl length during a large-scale functional genomics study in Arabidopsis (Arabidopsis thaliana). Further analysis revealed that seedlings overexpressing BBX32 display elongated hypocotyls in red, far-red, and blue light, along with reduced cotyledon expansion in red light. Through comparative analysis of mutant and overexpression line phenotypes, including global expression profiling and growth curve studies, we demonstrate that BBX32 acts antagonistically to ELONGATED HYPOCOTYL5 (HY5). We further show that BBX32 interacts with SALT TOLERANCE HOMOLOG2/BBX21, another B-box protein previously shown to interact with HY5. Based on these data, we propose that BBX32 functions downstream of multiple photoreceptors as a modulator of light responses. As such, BBX32 potentially has a native role in mediating gene repression to maintain dark adaptation.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Fototransducción/efectos de la radiación , Luz , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Oscuridad , Perfilación de la Expresión Génica , Genes de Plantas/genética , Hipocótilo/crecimiento & desarrollo , Hipocótilo/efectos de la radiación , Fototransducción/genética , Modelos Biológicos , Unión Proteica/efectos de la radiaciónRESUMEN
The E3 ubiquitin ligase COP1 (CONSTITUTIVE PHOTOMORPHOGENIC1) plays a key role in the repression of the plant photomorphogenic development in darkness. In the presence of light, COP1 is inactivated by a mechanism which is not completely understood. This leads to accumulation of COP1's target transcription factors, which initiates photomorphogenesis, resulting in dramatic changes of the seedling's physiology. Here we use a mathematical model to explore the possible mechanism of COP1 modulation upon dark/light transition in Arabidopsis thaliana based upon data for two COP1 target proteins: HY5 and HFR1, which play critical roles in photomorphogenesis. The main reactions in our model are the inactivation of COP1 by a proposed photoreceptor-related inhibitor I and interactions between COP1 and a CUL4 (CULLIN4)-based ligase. For building and verification of the model, we used the available published and our new data on the kinetics of HY5 and HFR1 together with the data on COP1 abundance. HY5 has been shown to accumulate at a slower rate than HFR1. To describe the observed differences in the timecourses of the "slow" target HY5 and the "fast" target HFR1, we hypothesize a switch between the activities of COP1 and CUL4 ligases upon dark/light transition, with COP1 being active mostly in darkness and CUL4 in light. The model predicts a bi-phasic kinetics of COP1 activity upon the exposure of plants to light, with its restoration after the initial decline and the following slow depletion of the total COP1 content. CUL4 activity is predicted to increase in the presence of light. We propose that the ubiquitin ligase switch is important for the complex regulation of multiple transcription factors during plants development. In addition, this provides a new mechanism for sensing the duration of light period, which is important for seasonal changes in plant development.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/efectos de la radiación , Modelos Biológicos , Morfogénesis/efectos de la radiación , Fotoperiodo , Ubiquitina-Proteína Ligasas/metabolismo , Algoritmos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Simulación por Computador , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/fisiología , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Cinética , Morfogénesis/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Regulación hacia Arriba/efectos de la radiaciónAsunto(s)
Proteínas de Arabidopsis/clasificación , Terminología como Asunto , Factores de Transcripción/clasificación , Dedos de Zinc/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Teorema de Bayes , Proteínas de Unión al ADN/clasificación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Filogenia , Estructura Terciaria de Proteína/genética , Proteómica/métodos , Análisis de Secuencia de Proteína/métodos , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
PHYTOCHROME-INTERACTING FACTOR5 (PIF5), a basic helix-loop-helix transcription factor, interacts specifically with the photoactivated form of phytochrome B (phyB). Here, we report that dark-grown Arabidopsis thaliana seedlings overexpressing PIF5 (PIF5-OX) exhibit exaggerated apical hooks and short hypocotyls, reminiscent of the triple response induced by elevated ethylene levels, whereas pif5 mutants fail to maintain tight hooks like those of wild-type seedlings. Silver ions, an ethylene receptor blocker, rescued the triple-response phenotype, and we show that PIF5-OX seedlings express enhanced levels of key ethylene biosynthesis enzymes and produce elevated ethylene levels. Exposure of PIF5-OX seedlings to prolonged continuous red light (Rc) promotes hypocotyl elongation relative to dark controls, the reciprocal of the Rc-imposed hypocotyl inhibition displayed by wild-type seedlings. In contrast with this PIF5-OX hyposensitivity to Rc, pif5 mutant seedlings are hypersensitive relative to wild-type seedlings. We show that this contrast is due to reciprocal changes in phyB protein levels in prolonged Rc. Compared with wild-type seedlings, PIF5-OX seedlings have reduced, whereas pif5 mutants have increased, phyB (and phyC) levels in Rc. The phyB degradation in the overexpressors depends on a functional phyB binding motif in PIF5 and involves the 26S proteasome pathway. Our data thus indicate that overexpressed PIF5 causes altered ethylene levels, which promote the triple response in darkness, whereas in the light, the interaction of photoactivated phyB with PIF5 causes degradation of the photoreceptor protein. The evidence suggests that endogenous PIF5 negatively regulates phyB-imposed hypocotyl inhibition in prolonged Rc by reducing photoreceptor abundance, and thereby photosensory capacity, rather than functioning as a signaling intermediate.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Etilenos/biosíntesis , Fitocromo/fisiología , Transducción de Señal/fisiología , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Oscuridad , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Hipocótilo/genética , Hipocótilo/metabolismo , Hipocótilo/efectos de la radiación , Immunoblotting , Luz , Mutación , Fenotipo , Fitocromo/metabolismo , Fitocromo B/metabolismo , Fitocromo B/fisiología , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Plantones/genética , Plantones/metabolismo , Plantones/efectos de la radiación , Transducción de Señal/genéticaRESUMEN
The phytochrome (phy) family of sensory photoreceptors (phyA-phyE in Arabidopsis thaliana) induces changes in target-gene expression upon light-induced translocation to the nucleus, where certain members interact with selected members of the constitutively nuclear basic helix-loop-helix transcription factor family, such as PHYTOCHROME-INTERACTING FACTOR3 (PIF3). Previous evidence indicates that the binding of the photoactivated photoreceptor molecule to PIF3 induces rapid phosphorylation of the transcription factor in the cell prior to its degradation via the ubiqitin-proteosome system. To investigate whether this apparent primary signaling mechanism can be generalized to other phy-interacting partners, we have examined the molecular behavior of a second related phy-interacting member of the basic helix-loop-helix family, PIF5, during early deetiolation, immediately following initial exposure of dark-grown seedlings to light. The data show that red light induces very rapid phosphorylation and subsequent degradation (t(1/2) < 5 min) of PIF5 via the proteosome system upon irradiation. Photobiological and genetic evidence indicates that the photoactivated phy molecule acts within 60 s to induce this phosphorylation of PIF5, and that phyA and phyB redundantly dominate this process, with phyD playing an apparently minor role. Collectively, the data support the proposal that the rapid phy-induced phosphorylation of PIF3 and PIF5 may represent the biochemical mechanism of primary signal transfer from photoactivated photoreceptor to binding partner, and that phyA and phyB (and possibly phyD) may signal to multiple, shared partners utilizing this common mechanism.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Luz , Fitocromo A/metabolismo , Fitocromo B/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Fosforilación/efectos de la radiación , Fitocromo A/genética , Fitocromo B/genética , Unión Proteica , Desnaturalización ProteicaRESUMEN
In previous time-resolved microarray-based expression profiling, we identified 32 genes encoding putative transcription factors, signaling components, and unknown proteins that are rapidly and robustly induced by phytochrome (phy)-mediated light signals. Postulating that they are the most likely to be direct targets of phy signaling and to function in the primary phy regulatory circuitry, we examined the impact of targeted mutations in these genes on the phy-induced seedling deetiolation process in Arabidopsis thaliana. Using light-imposed concomitant inhibition of hypocotyl and stimulation of cotyledon growth as diagnostic criteria for normal deetiolation, we identified three major mutant response categories. Seven (22%) lines displayed statistically significant, reciprocal, aberrant photoresponsiveness in the two organs, suggesting disruption of normal deetiolation; 13 (41%) lines displayed significant defects either unidirectionally in both organs or in hypocotyls only, suggesting global effects not directly related to photomorphogenic signaling; and 12 (37%) lines displayed no significant difference in photoresponsiveness from the wild type. Potential reasons for the high proportion of rapidly light-responsive genes apparently unnecessary for the deetiolation phenotype are discussed. One of the seven disrupted genes displaying a significant mutant phenotype, the basic helix-loop-helix factor-encoding PHYTOCHROME-INTERACTING FACTOR3-LIKE1 gene, was found to be necessary for rapid light-induced expression of the photomorphogenesis- and circadian-related PSEUDO-RESPONSE REGULATOR9 gene, indicating a regulatory function in the early phy-induced transcriptional network.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Fitocromo/fisiología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Cotiledón/genética , Cotiledón/crecimiento & desarrollo , Cotiledón/metabolismo , ADN Bacteriano , Perfilación de la Expresión Génica , Hipocótilo/genética , Hipocótilo/crecimiento & desarrollo , Hipocótilo/metabolismo , Luz , Mutagénesis Insercional , Mutación , Fitocromo/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
Evidence has been presented that a negative transcriptional feedback loop formed by the genes CIRCADIAN CLOCK ASSOCIATED (CCA1), LATE ELONGATED HYPOCOTYL (LHY) and TIMING OF CAB (TOC1) constitutes the core of the central oscillator of the circadian clock in Arabidopsis. Here we show that these genes are expressed at constant, basal levels in dark-grown seedlings. Transfer to constant red light (Rc) rapidly induces a biphasic pattern of CCA1 and LHY expression, and a reciprocal TOC1 expression pattern over the first 24 h, consistent with initial induction of this synchronous oscillation by the light signal. We have used this assay with wild-type and mutant seedlings to examine the role of these oscillator components, and to determine the function of ELF3 and ELF4 in their light-regulated expression. The data show that whereas TOC1 is necessary for light-induced CCA1/LHY expression, the combined absence of CCA1 and LHY has little effect on the pattern of light-induced TOC1 expression, indicating that the negative regulatory arm of the proposed oscillator is not fully functional during initial seedling de-etiolation. By contrast, ELF4 is necessary for light-induced expression of both CCA1 and LHY, and conversely, CCA1 and LHY act negatively on light-induced ELF4 expression. Together with the observation that the temporal light-induced expression profile of ELF4 is counter-phased to that of CCA1 and LHY and parallels that of TOC1, these data are consistent with a previously unrecognized negative-feedback loop formed by CCA1/LHY and ELF4 in a manner analogous to the proposed CCA1/LHY/TOC1 oscillator. ELF3 is also necessary for light-induced CCA1/LHY expression, but it is neither light-induced nor clock-regulated during de-etiolation. Taken together, the data suggest (a) that ELF3, ELF4, and TOC1 all function in the primary, phytochrome-mediated light-input pathway to the circadian oscillator in Arabidopsis; and (b) that this oscillator consists of two or more interlocking transcriptional feedback loops that may be differentially operative during initial light induction and under steady-state circadian conditions in entrained green plants.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Fitocromo/farmacología , Factores de Transcripción/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Ritmo Circadiano/efectos de la radiación , Proteínas de Unión al ADN/genética , Retroalimentación Fisiológica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luz , Factores de Transcripción/genéticaRESUMEN
The phytochrome (phy) family of sensory photoreceptors (phyA to phyE) in Arabidopsis thaliana control plant developmental transitions in response to informational light signals throughout the life cycle. The photoactivated conformer of the photoreceptor Pfr has been shown to translocate into the nucleus where it induces changes in gene expression by an unknown mechanism. Here, we have identified two basic helix-loop-helix (bHLH) transcription factors, designated PHYTOCHROME-INTERACTING FACTOR5 (PIF5) and PIF6, which interact specifically with the Pfr form of phyB. These two factors cluster tightly with PIF3 and two other phy-interacting bHLH proteins in a phylogenetic subfamily within the large Arabidopsis bHLH (AtbHLH) family. We have identified a novel sequence motif (designated the active phytochrome binding [APB] motif) that is conserved in these phy-interacting AtbHLHs but not in other noninteractors. Using the isolated domain and site-directed mutagenesis, we have shown that this motif is both necessary and sufficient for binding to phyB. Transgenic expression of the native APB-containing AtbHLH protein, PIF4, in a pif4 null mutant, rescued the photoresponse defect in this mutant, whereas mutated PIF4 constructs with site-directed substitutions in conserved APB residues did not. These data indicate that the APB motif is necessary for PIF4 function in light-regulated seedling development and suggest that conformer-specific binding of phyB to PIF4 via the APB motif is necessary for this function in vivo. Binding assays with the isolated APB domain detected interaction with phyB, but none of the other four Arabidopsis phys. Collectively, the data suggest that the APB domain provides a phyB-specific recognition module within the AtbHLH family, thereby conferring photoreceptor target specificity on a subset of these transcription factors and, thus, the potential for selective signal channeling to segments of the transcriptional network.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Secuencias Hélice-Asa-Hélice/fisiología , Células Fotorreceptoras/metabolismo , Fitocromo/metabolismo , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Sitios de Unión/genética , ADN Complementario/genética , ADN Complementario/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Luz , Datos de Secuencia Molecular , Mutación/genética , Estimulación Luminosa , Fitocromo B , Unión Proteica/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificaciónRESUMEN
Different Arabidopsis phytochrome (phy) family members (phyA through phyE) display differential photosensory and/or physiological functions in regulating growth and developmental responses to light signals. To identify the genes regulated by phyB in response to continuous monochromatic red light (Rc) during the induction of seedling de-etiolation, we have performed time-course, microarray-based expression profiling of wild type (WT) and phyB null mutants. Comparison of the observed expression patterns with those induced by continuous monochromatic far-red light (FRc; perceived exclusively by phyA) in WT and phyA null-mutant seedlings suggests early convergence of the FRc and Rc photosensory pathways to control a largely common transcriptional network. phyB mutant seedlings retain a surprisingly high level of responsiveness to Rc for the majority of Rc-regulated genes on the microarray, indicating that one or more other phys have a major role in regulating their expression. Combined with the robust visible morphogenic phenotype of the phyB mutant in Rc, these data suggest that different members of the phy family act in organ-specific fashion in regulating seedling de-etiolation. Specifically, phyB appears to be the dominant, if not exclusive, photoreceptor in regulating a minority population of genes involved in suppression of hypocotyl cell elongation in response to Rc signals. By contrast, this sensory function is apparently shared by one or more other phys in regulating the majority Rc-responsive gene set involved in other important facets of the de-etiolation process in the apical region, such as cotyledon cell expansion.
Asunto(s)
Arabidopsis/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/genética , Células Fotorreceptoras , Fitocromo/genética , Factores de Transcripción , Proteínas de Arabidopsis/genética , Secuencia de Bases , Oscuridad , Regulación del Desarrollo de la Expresión Génica/genética , Luz , Iluminación , Fitocromo B , Transducción de Señal/genética , Transcripción Genética/genéticaRESUMEN
To define the functions of genes previously identified by expression profiling as being rapidly light induced under phytochrome (phy) control, we are investigating the seedling de-etiolation phenotypes of mutants carrying T-DNA insertional disruptions at these loci. Mutants at one such locus displayed reduced responsiveness to continuous red, but not continuous far-red light, suggesting a role in phyB signaling but not phyA signaling. Consistent with such a role, expression of this gene is induced by continuous red light in wild-type seedlings, but the level of induction is strongly reduced in phyB-null mutants. The locus encodes a novel protein that we show localizes to the nucleus, thus suggesting a function in light-regulated gene expression. Recently, this locus was identified as EARLY FLOWERING 4, a gene implicated in floral induction and regulating the expression of the gene CIRCADIAN CLOCK-ASSOCIATED 1. Together with these previous data, our findings suggest that EARLY FLOWERING 4 functions as a signaling intermediate in phy-regulated gene expression involved in promotion of seedling de-etiolation, circadian clock function, and photoperiod perception.