RESUMEN
Cold stress is one of the major environmental factors that limit growth and yield of plants. However, it is still not fully understood how plants account for daily temperature fluctuations, nor how these temperature changes are integrated with other regulatory systems such as the circadian clock. We demonstrate that REVEILLE2 undergoes alternative splicing after chilling that increases accumulation of a transcript isoform encoding a MYB-like transcription factor. We explore the biological function of REVEILLE2 in Arabidopsis thaliana using a combination of molecular genetics, transcriptomics, and physiology. Disruption of REVEILLE2 alternative splicing alters regulatory gene expression, impairs circadian timing, and improves photosynthetic capacity. Changes in nuclear gene expression are particularly apparent in the initial hours following chilling, with chloroplast gene expression subsequently upregulated. The response of REVEILLE2 to chilling extends our understanding of plants immediate response to cooling. We propose that the circadian component REVEILLE2 restricts plants responses to nocturnal reductions in temperature, thereby enabling appropriate responses to daily environmental changes.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Relojes Circadianos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relojes Circadianos/genética , Ritmo Circadiano/genética , Regulación de la Expresión Génica de las Plantas , TemperaturaRESUMEN
Phototropin (phot) receptor kinases play important roles in promoting plant growth by controlling light-capturing processes, such as phototropism. Phototropism is mediated through the action of NON-PHOTOTROPIC HYPOCOTYL3 (NPH3), which is dephosphorylated following phot activation. However, the functional significance of this early signaling event remains unclear. Here, we show that the onset of phototropism in dark-grown (etiolated) seedlings of Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) is enhanced by greening (deetiolation). Red and blue light were equally effective in promoting phototropism in Arabidopsis, consistent with our observations that deetiolation by phytochrome or cryptochrome was sufficient to enhance phototropism. Increased responsiveness did not result from an enhanced sensitivity to the phytohormone auxin, nor does it involve the phot-interacting protein, ROOT PHOTOTROPISM2. Instead, deetiolated seedlings showed attenuated levels of NPH3 dephosphorylation and diminished relocalization of NPH3 from the plasma membrane during phototropism. Likewise, etiolated seedlings that lack the PHYTOCHROME-INTERACTING FACTORS (PIFs) PIF1, PIF3, PIF4, and PIF5 displayed reduced NPH3 dephosphorylation and enhanced phototropism, consistent with their constitutive photomorphogenic phenotype in darkness. Phototropic enhancement could also be achieved in etiolated seedlings by lowering the light intensity to diminish NPH3 dephosphorylation. Thus, phototropism is enhanced following deetiolation through the modulation of a phosphorylation rheostat, which in turn sustains the activity of NPH3. We propose that this dynamic mode of regulation enables young seedlings to maximize their establishment under changing light conditions, depending on their photoautotrophic capacity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Etiolado/fisiología , Fototropismo/fisiología , Arabidopsis/efectos de los fármacos , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Criptocromos/metabolismo , Etiolado/efectos de los fármacos , Etiolado/efectos de la radiación , Proteínas Fluorescentes Verdes/metabolismo , Hipocótilo/efectos de los fármacos , Hipocótilo/fisiología , Hipocótilo/efectos de la radiación , Ácidos Indolacéticos/farmacología , Luz , Modelos Biológicos , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Fototropismo/efectos de los fármacos , Fototropismo/efectos de la radiación , Fitocromo/metabolismo , Agregado de Proteínas , Plantones/efectos de los fármacos , Plantones/fisiología , Plantones/efectos de la radiaciónRESUMEN
The circadian clock regulates the timing of many aspects of plant physiology, and this requires entrainment of the clock to the prevailing day:night cycle. Different plant cells and tissues can oscillate with different free-running periods, so coordination of timing across the plant is crucial. Previous work showed that a major difference between the clock in mature shoots and roots involves light inputs. The objective of this work was to define, in Arabidopsis thaliana, the operation of the root clock in more detail, and in particular how it responds to light quality. Luciferase imaging was used to study the shoot and root clocks in several null mutants of clock components and in lines with aberrant expression of phytochromes. Mutations in each of the components of the evening complex (EARLY FLOWERING 3 and 4, and LUX ARRHYTHMO) were found to have specific effects on roots, by affecting either rhythmicity or period and its response to light quality. The data suggest that the evening complex is a key part of the light input mechanism that differs between shoots and roots and show that roots sense red light via phytochrome B.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Relojes Circadianos/genética , Ritmo Circadiano/genética , Regulación de la Expresión Génica de las Plantas , LuzRESUMEN
Circadian clocks allow the temporal compartmentalization of biological processes. In Arabidopsis, circadian rhythms display organ specificity but the underlying molecular causes have not been identified. We investigated the mechanisms responsible for the similarities and differences between the clocks of mature shoots and roots in constant conditions and in light : dark cycles. We developed an imaging system to monitor clock gene expression in shoots and light- or dark-grown roots, modified a recent mathematical model of the Arabidopsis clock and used this to simulate our new data. We showed that the shoot and root circadian clocks have different rhythmic properties (period and amplitude) and respond differently to light quality. The root clock was entrained by direct exposure to low-intensity light, even in antiphase to the illumination of shoots. Differences between the clocks were more pronounced in conditions where light was present than in constant darkness, and persisted in the presence of sucrose. We simulated the data successfully by modifying those parameters of a clock model that are related to light inputs. We conclude that differences and similarities between the shoot and root clocks can largely be explained by organ-specific light inputs. This provides mechanistic insight into the developing field of organ-specific clocks.
Asunto(s)
Arabidopsis/fisiología , Arabidopsis/efectos de la radiación , Relojes Circadianos/efectos de la radiación , Ritmo Circadiano/efectos de la radiación , Luz , Raíces de Plantas/fisiología , Brotes de la Planta/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Oscuridad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Modelos Biológicos , Especificidad de Órganos/genética , Especificidad de Órganos/efectos de la radiación , Fotoperiodo , Raíces de Plantas/genética , Raíces de Plantas/efectos de la radiación , Brotes de la Planta/genética , Brotes de la Planta/efectos de la radiaciónRESUMEN
Cauliflower mosaic virus (CaMV) encodes a 520 aa polypeptide, P6, which participates in several essential activities in the virus life cycle including suppressing RNA silencing and salicylic acid-responsive defence signalling. We infected Arabidopsis with CaMV mutants containing short in-frame deletions within the P6 ORF. A deletion in the distal end of domain D-I (the N-terminal 112 aa) of P6 did not affect virus replication but compromised symptom development and curtailed the ability to restore GFP fluorescence in a GFP-silenced transgenic Arabidopsis line. A deletion in the minimum transactivator domain was defective in virus replication but retained the capacity to suppress RNA silencing locally. Symptom expression in CaMV-infected plants is apparently linked to the ability to suppress RNA silencing. When transiently co-expressed with tomato bushy stunt virus P19, an elicitor of programmed cell death in Nicotiana tabacum, WT P6 suppressed the hypersensitive response, but three mutants, two with deletions within the distal end of domain D-I and one involving the N-terminal nuclear export signal (NES), were unable to do so. Deleting the N-terminal 20 aa also abolished the suppression of pathogen-associated molecular pattern-dependent PR1a expression following agroinfiltration. However, the two other deletions in domain D-I retained this activity, evidence that the mechanisms underlying these functions are not identical. The D-I domain of P6 when expressed alone failed to suppress either cell death or PR1a expression and is therefore necessary but not sufficient for all three defence suppression activities. Consequently, concerns about the biosafety of genetically modified crops carrying truncated ORFVI sequences appear unfounded.
Asunto(s)
Caulimovirus/patogenicidad , Estructura Terciaria de Proteína/genética , Interferencia de ARN/efectos de los fármacos , Ácido Salicílico/metabolismo , Transducción de Señal/efectos de los fármacos , Transactivadores/genética , Transactivadores/farmacología , Secuencia de Aminoácidos , Arabidopsis/virología , Caulimovirus/genética , Caulimovirus/metabolismo , Datos de Secuencia Molecular , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , Eliminación de Secuencia , Transactivadores/química , Transactivadores/metabolismo , Replicación ViralRESUMEN
The core of the plant circadian clock involves multiple interlocking gene expression loops and post-translational controls along with inputs from light and metabolism. The complexity of the interactions is such that few specific functions can be ascribed to single components. In previous work, we reported differences in the operation of the clocks in Arabidopsis shoots and roots, including the effects of mutations of key clock components. Here, we have used luciferase imaging to study prr7 mutants expressing CCA1::LUC and GI::LUC markers. In mature shoots expressing CCA1::LUC, loss of PRR7 radically altered behaviour in light:dark cycles and caused loss of rhythmicity in constant light but had little effect on roots. In contrast, in mature plants expressing GI::LUC, loss of PRR7 had little effect in light:dark cycles but in constant light increased the circadian period in shoots and reduced it in roots. We conclude that most or all of the circadian input to the CCA1 promoter in shoots is mediated by PRR7 and that loss of PRR7 has organ-specific effects. The results emphasise the differences in operation of the shoot and root clocks, and the importance of studying clock mutants in both light:dark cycles and constant light.
RESUMEN
We analyzed the susceptibility of Arabidopsis mutants with defects in salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signaling to infection by Cauliflower mosaic virus (CaMV). Mutants cpr1-1 and cpr5-2, in which SA-dependent defense signaling is activated constitutively, were substantially more resistant than the wild type to systemic infection, implicating SA signaling in defense against CaMV. However, SA-deficient NahG, sid2-2, eds5-1, and pad4-1 did not show enhanced susceptibility. A cpr5 eds5 double mutant also was resistant, suggesting that resistance in cpr5 may function partially independently of SA. Treatment of cpr5 and cpr5 eds5, but not cpr1, with salicyl-hydroxamic acid, an inhibitor of alternative oxidase, partially restored susceptibility to wild-type levels. Mutants etr1-1, etr1-3, and ein2-1, and two mutants with lesions in ET/JA-mediated defense, eds4 and eds8, also showed reduced virus susceptibility, demonstrating that ET-dependent responses also play a role in susceptibility. We used a green fluorescent protein (GFP)-expressing CaMV recombinant to monitor virus movement. In mutants with reduced susceptibility, cpr1-1, cpr5-2, and etr1-1, CaMV-GFP formed local lesions similar to the wild type, but systemic spread was almost completely absent in cpr1 and cpr5 and was substantially reduced in etr1-1. Thus, mutations with enhanced systemic acquired resistance or compromised ET signaling show diminished long-distance virus movement.
Asunto(s)
Arabidopsis/inmunología , Arabidopsis/virología , Caulimovirus/fisiología , Etilenos/metabolismo , Transducción de Señal , Antimicina A/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Transporte Biológico/efectos de los fármacos , Caulimovirus/efectos de los fármacos , Proteínas Mitocondriales , Mutación/genética , Oxidorreductasas/metabolismo , Enfermedades de las Plantas/virología , Proteínas de Plantas , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión/metabolismo , Salicilamidas/farmacología , Ácido Salicílico/farmacología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Replicación Viral/efectos de los fármacosRESUMEN
Cauliflower mosaic virus (CaMV) encodes a multifunctional protein P6 that is required for translation of the 35S RNA and also acts as a suppressor of RNA silencing. Here we demonstrate that P6 additionally acts as a pathogenicity effector of an unique and novel type, modifying NPR1 (a key regulator of salicylic acid (SA)- and jasmonic acid (JA)-dependent signaling) and inhibiting SA-dependent defence responses We find that that transgene-mediated expression of P6 in Arabidopsis and transient expression in Nicotiana benthamiana has profound effects on defence signaling, suppressing expression of representative SA-responsive genes and increasing expression of representative JA-responsive genes. Relative to wild-type Arabidopsis P6-expressing transgenics had greatly reduced expression of PR-1 following SA-treatment, infection by CaMV or inoculation with an avirulent bacterial pathogen Pseudomonas syringae pv tomato (Pst). Similarly transient expression in Nicotiana benthamiana of P6 (including a mutant form defective in translational transactivation activity) suppressed PR-1a transcript accumulation in response to Agrobacterium infiltration and following SA-treatment. As well as suppressing the expression of representative SA-regulated genes, P6-transgenic Arabidopsis showed greatly enhanced susceptibility to both virulent and avirulent Pst (titres elevated 10 to 30-fold compared to non-transgenic controls) but reduced susceptibility to the necrotrophic fungus Botrytis cinerea. Necrosis following SA-treatment or inoculation with avirulent Pst was reduced and delayed in P6-transgenics. NPR1 an important regulator of SA/JA crosstalk, was more highly expressed in the presence of P6 and introduction of the P6 transgene into a transgenic line expressing an NPR1:GFP fusion resulted in greatly increased fluorescence in nuclei even in the absence of SA. Thus in the presence of P6 an inactive form of NPR1 is mislocalized in the nucleus even in uninduced plants. These results demonstrate that P6 is a new type of pathogenicity effector protein that enhances susceptibility to biotrophic pathogens by suppressing SA- but enhancing JA-signaling responses.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Inmunidad Innata/fisiología , Ácido Salicílico/metabolismo , Transducción de Señal/efectos de los fármacos , Transactivadores/farmacología , Factores de Virulencia/farmacología , Análisis de Varianza , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Botrytis , Ciclopentanos/metabolismo , Resistencia a la Enfermedad/genética , Microscopía Fluorescente , Oxilipinas/metabolismo , Enfermedades de las Plantas/microbiología , Plantas Modificadas Genéticamente , Nicotiana , Azul de TripanoRESUMEN
We infected a transgenic Arabidopsis line (GxA), containing an amplicon-silenced 35S : : GFP transgene, with cauliflower mosaic virus (CaMV), a plant pararetrovirus with a DNA genome. Systemically infected leaves showed strong GFP fluorescence and amplicon transcripts were detectable in Northern blots, indicating that silencing of GFP had been suppressed during CaMV-infection. Transgenic Arabidopsis lines expressing CaMV protein P6, the major genetic determinant of symptom severity, were crossed with GxA. Progeny showed strong GFP fluorescence throughout and amplicon transcripts were detectable in Northern blots, indicating that P6 was suppressing local and systemic silencing. However, levels of 21 nt siRNAs derived from the GFP transgene were not reduced. In CaMV-infected plants, the P6 transgene did not reduce levels of CaMV leader-derived 21 and 24 nt siRNAs relative to levels of CaMV 35S RNA. These results demonstrate that CaMV can efficiently suppress silencing of a GFP transgene, and that P6 acts as a silencing suppressor.
Asunto(s)
Arabidopsis/virología , Caulimovirus/química , Enfermedades de las Plantas/virología , Interferencia de ARN/fisiología , Proteínas Virales/fisiología , Arabidopsis/inmunología , Arabidopsis/metabolismo , Caulimovirus/patogenicidad , Regulación hacia Abajo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Enfermedades de las Plantas/inmunología , Hojas de la Planta/inmunología , Hojas de la Planta/metabolismo , Hojas de la Planta/virología , Plantas Modificadas Genéticamente/metabolismo , ARN Interferente Pequeño/metabolismo , VirulenciaRESUMEN
Long-distance virus transport takes place through the vascular system and is dependent on the movement of photoassimilates. Here, patterns of symptom development, virus movement and gene expression were analysed in Arabidopsis following inoculation with Cauliflower mosaic virus (CaMV) on a single leaf. Virus accumulation and expression of markers for the salicylic acid (SA) and ethylene/jasmonate (Et/JA) defence pathways, PR-1 and PDF1.2, were analysed on a leaf-by-leaf basis by real-time reverse transcription polymerase chain reaction (qRT-PCR). Virus spread followed a strictly defined pattern identical to that of a source-sink relationship. This was exploited to study differences between local and systemic defence responses in a developmental and spatial manner. In infected plants, PR-1 transcripts accumulated primarily but not exclusively in leaves with a direct vascular connection to the inoculated leaf. Abundances fell significantly as virus accumulated. By contrast, PDF1.2 transcripts were significantly lower than in controls in all leaves at early stages of infection, but recovered as virus accumulated. Virus and PR-1 transcript abundances are negatively correlated, and SA- and Et/JA-mediated signalling of gene expression occurs independently of the presence of virus. Although SA-dependent signalling responses were mainly linked to the orthostichy, Et/JA-dependent responses were independent of vascular connections.
Asunto(s)
Arabidopsis/metabolismo , Arabidopsis/virología , Caulimovirus/fisiología , Hojas de la Planta/anatomía & histología , Hojas de la Planta/virología , Arabidopsis/anatomía & histología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Defensinas/genética , Defensinas/metabolismo , Regulación de la Expresión Génica de las Plantas , Regulación Viral de la Expresión Génica , Hojas de la Planta/metabolismoRESUMEN
PR-1 has been extensively used as a marker for salicylic acid (SA)-mediated defence and systemic and local acquired resistance. The Arabidopsis Genome Project annotates At2g19990 as PR-1. This gene is also identified as PR-1 in two "full genome" Arabidopsis microarrays, and TAIR cites approximately 60 articles to describe its patterns of expression. However, most of these citations are incorrect; the probes used were not At2g19990, but a homologous gene At2g14610, which is annotated as "PR-1-like". Because of the potential for confusion, we analyzed the expression of both genes in Arabidopsis thaliana (L.) Heynh. At2g14610 (PR-1-like) showed the archetypal patterns of SA-responsive expression: mRNA levels increased following SA-treatment, inoculation with an avirulent (but not a virulent) strain of Pseudomonas syringae, and in wild-type (but not NahG) Arabidopsis infected with cauliflower mosaic virus (CaMV). In cpr5 mutants it was expressed constitutively. In contrast, expression of At2g19990 (annotated as PR-1) was detectable in neither SA-treated Col-0 nor in cpr5. Infection by virulent and avirulent isolates of P. syringae up-regulated expression, but to a similar level, and infection by CaMV induced a modest increase in expression in both the wild type and NahG. At2g19990, although pathogen responsive, does not show the SA-dependent patterns of expression expected from a member of the PR-1 regulon, and its annotation as " PR-1" is inappropriate. The annotations should identify At2g14610 as the authentic PR-1.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/biosíntesis , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas , Hojas de la Planta/metabolismo , Pseudomonas syringae , ARN Mensajero/metabolismo , ARN de Planta/metabolismoRESUMEN
Protein P6 is the main symptom determinant of cauliflower mosaic virus (CaMV), and transgene-mediated expression in Arabidopsis induces a symptom-like phenotype in the absence of infection. Seeds of a P6-transgenic line, A7, were mutagenized by gamma-irradiation and M2 seedlings were screened for mutants that suppressed the phenotype of chlorosis and stunting. We identified four mutants that were larger and less chlorotic than the A7 parent but which contained an intact and transcriptionally active transgene. The two mutants with the strongest suppression phenotype, were recessive and allelic. The transgene was eliminated by back-crossing with wild-type Arabidopsis. In progeny lines that were homozygous for the putative suppressor mutation the proportion of plants becoming infected following inoculation with CaMV was 40% that of wild-type, although in plants that did become infected, levels of virus DNA in mutants and wild-type did not differ significantly. Symptoms in the mutants were milder and delayed although this was somewhat dependent on the virus isolate. This phenotype was inherited stably. Both mutant alleles showed a partially ethylene-insensitive phenotype in an ethylene triple response assay. P6-transgenic plants were also almost completely insensitive to ethylene in the triple response assay. We suggest that the chlorosis and stunting in P6-transgenic and CaMV-infected plants are dependent on interactions between P6 and components involved in ethylene signalling, and that the suppressor gene product may function to augment these interactions.