Control lumínico de la respuesta al ácido jasmónico (JA) en plantas de Arabidopsis thaliana

Al monitorear los cambios en el ambiente lumínico, las plantas pueden obtener información acerca de la proximidad de las plantas vecinas. Los jasmonatos (JAs) son reguladores lipídicos que juegan un papel central en el control de las respuestas de defensa frente a patógenos y plagas, así como también en la regulación de los procesos de crecimiento y desarrollo. Una disminución en la relación rojo: rojo lejano (R:RL) de la luz representa una señal de competencia para las plantas terrestres. Esta señal es detectada por el fotorreceptor fitocromo B (phyB) y, entre otras cosas, induce la aceleración del crecimiento y elongación y reprime la expresión de defensas de las plantas. Los efectos de las bajas relaciones R:RL sobre el sistema de defensas están mediados, al menos en parte, a través de una reducción en la señalización de los JAs. En esta tesis doctoral se pretende avanzar en la comprensión de los mecanismos que controlan el efecto de R:RL y phyB en las respuestas a JA en Arabidopsis thaliana. Se postulan posibles mecanismos mediante los cuales la inactivación del phyB por bajas relaciones R:RL produce la disminución en la sensibilidad de la vía de los JAs. Para este fin, he combinado un enfoque genético con el uso de herramientas fisiológicas, bioquímicas y moleculares para estudiar los efectos de la calidad de la luz sobre los componentes críticos de la vía de señalización de JA. Los resultados presentados en esta tesis demuestran que el efecto de las bajas relaciones R:RL, provocando una disminución de la sensibilidad a JA, requiere de la proteína JA-ZIM domain 10(JAZ10). También demostramos que la degradación de las proteínas DELLA (mediada por el aumento en la actividad de giberelinas (GAs) ), es necesaria para que se manifieste la represión de la vía de JA en condiciones de bajas relaciones R:RL. Por último, los resultados sugieren que, además de este efecto bien caracterizado de las GAs sobre la señalización de JA, mediado por la degradación de las proteínas DELLAs, GA reprime las respuestas de defensa por un nuevo mecanismo. En este mecanismo, GA aumenta la estabilidad de la JAZ10 y retarda la degradación de esta proteína inducida por JA.

Control lumínico de la respuesta al ácido jasmónico (JA) en plantas de Arabidopsis thaliana

Al monitorear los cambios en el ambiente lumínico, las plantas pueden obtener información acerca de la proximidad de las plantas vecinas. Los jasmonatos (JAs) son reguladores lipídicos que juegan un papel central en el control de las respuestas de defensa frente a patógenos y plagas, así como también en la regulación de los procesos de crecimiento y desarrollo. Una disminución en la relación rojo: rojo lejano (R:RL) de la luz representa una señal de competencia para las plantas terrestres. Esta señal es detectada por el fotorreceptor fitocromo B (phyB) y, entre otras cosas, induce la aceleración del crecimiento y elongación y reprime la expresión de defensas de las plantas. Los efectos de las bajas relaciones R:RL sobre el sistema de defensas están mediados, al menos en parte, a través de una reducción en la señalización de los JAs. En esta tesis doctoral se pretende avanzar en la comprensión de los mecanismos que controlan el efecto de R:RL y phyB en las respuestas a JA en Arabidopsis thaliana. Se postulan posibles mecanismos mediante los cuales la inactivación del phyB por bajas relaciones R:RL produce la disminución en la sensibilidad de la vía de los JAs. Para este fin, he combinado un enfoque genético con el uso de herramientas fisiológicas, bioquímicas y moleculares para estudiar los efectos de la calidad de la luz sobre los componentes críticos de la vía de señalización de JA. Los resultados presentados en esta tesis demuestran que el efecto de las bajas relaciones R:RL, provocando una disminución de la sensibilidad a JA, requiere de la proteína JA-ZIM domain 10(JAZ10). También demostramos que la degradación de las proteínas DELLA (mediada por el aumento en la actividad de giberelinas (GAs) ), es necesaria para que se manifieste la represión de la vía de JA en condiciones de bajas relaciones R:RL. Por último, los resultados sugieren que, además de este efecto bien caracterizado de las GAs sobre la señalización de JA, mediado por la degradación de las proteínas DELLAs, GA reprime las respuestas de defensa por un nuevo mecanismo. En este mecanismo, GA aumenta la estabilidad de la JAZ10 y retarda la degradación de esta proteína inducida por JA.

Estudo da associação dos genes GGR, GGPPS2 e GGPPS6 em Arabidopsis thaliana L. (Brassicaceae) a compostos alelopáticos

Dissertação (mestrado)—Universidade de Brasília, Departamento de Botânica, Programa de Pós-Graduação em Botânica, 2016.

Superexpressão do gene dehidrina de Arachis duranensis em plantas transgênicas de Arabidopsis thaliana

Dissertação (mestrado)—Universidade de Brasília, Departamento de Botânica, Programa de Pós-Graduação em Botânica, 2016.

Functional analysis of Arabidopsis thaliana lesion mimic mutant cfs1 in ESCRT complex-related protein trafficking

The Endosomal Sorting Complex Required for Transport (ESCRT)-complex is composed of four complexes, ESCRT-0-III. They sequentially act on a late endosome to sort mono-ubiquitinated transmembrane proteins into the intralumenal vesicle, forming of a multivesicular body(MVB) that is delivered to vacuole for degradation. In Arabidopsis thaliana, the loss of an ESCRT-I component, elch displays a cytokinesis defect; while a dominant negative expression of an ESCRT-III component results in cell death due to vacuolar loss. In this work, the function of a plant-specific ELCH-interactor, CELL DEATH RELATED FYVE/SYLF DOMAIN CONTAINING 1 (CFS1) and its influences on the ESCRT-complex function are investigated. CFS1 is a phosphatidylinositol-3-phosphate- and actin-binding protein. The cfs1 mutants mimic lesions in the first eldest leaf that propagate to the next eldest one. Genetic analyses have demonstrated that cell death in cfs1 does not require a functional ESCRT-I component; nevertheless, the loss of CFS1 alleviates elchcytokinesis defect, suggesting its influence on the ESCRT-I function. Further analyses reveal that cfs1 accumulates autophagosomes throughout its lifespan due to a decrease in autophagosome degradation, suggesting that as the plant ages, the cumulated autophagosomes falsely trigger effectors-triggered immunity that executes cell death in cfs1. As the ESCRT-complex has been demonstrated to be involved in the delivery of autophagosomes to vacuole and CFS1 homolog, CFS2 reportedly interacts with ATG8, it can be postulated from the results of this work that CFS1 alone or together with CFS2 function in sequestering mature autophagosomes onto MVBs. At the MVBs, the ESCRT-complex then mediates the fusion of autophagosome and MVB for subsequent delivery to vacuole.

The role of FMT and FIS1A in mitochondrial morphology and salt stress in Arabidopsis thaliana

Salt stress is known to have severe effects on plant health and fecundity, and mitochondria are known to be an essential part of the plant salt stress response. Arabidopsis thaliana serves as an excellent model to study the effects of salt stress as well as mitochondrial morphology. Arabidopsis contains several homologues to known mitochondrial proteins, including the fission protein FIS1A, and FMT, a homologue of the CLU subfamily. We sought to examine the effects of salt stress on knockout lines of FIS1A and FMT, as well as a transgenic line overexpressing FMT (FMT-OE) in columella cells in the root cap of Arabidopsis. fmt mutants displayed defects in both root and leaf growth, as well as a delay in flowering time. These mutants also showed a pronounced increase in mitochondrial clustering and number. FMT-OE mutants displayed severe defects in germination, including a decrease in total germination, and an increase in the number of days to germination. fis1A mutants exhibited shorter roots and slightly shorter leaves, as well as a tendency towards random mitochondrial clustering in root cells. Salt stress was shown to affect various mitochondrial parameters, including an increase in mitochondrial number and clustering, as well as a decrease in mitochondrial area. These results reveal a previously unknown role for FMT in germination and flowering in Arabidopsis, as well as insight into the effects of salt stress on mitochondrial morphology. FMT, along with FIS1A, may also help to regulate mitochondrial number and clustering, as well as root and leaf growth, under both control and salt-stressed conditions. This has implications for both FMT and FIS1A in whole-plant morphology as well as the plant salt stress response.

An egg apparatus-specific enhancer of Arabidopsis, identified by enhancer detection

Despite a central role in angiosperm reproduction, few gametophyte-specific genes and promoters have been isolated, particularly for the inaccessible female gametophyte (embryo sac). Using the Ds-based enhancer-detector line ET253, we have cloned an egg apparatus-specific enhancer (EASE) from Arabidopsis (Arabidopsis thaliana). The genomic region flanking the Ds insertion site was further analyzed by examining its capability to control gusA and GFP reporter gene expression in the embryo sac in a transgenic context. Through analysis of a 5' and 3' deletion series in transgenic Arabidopsis, the sequence responsible for egg apparatus-specific expression was delineated to 77 bp. Our data showed that this enhancer is unique in the Arabidopsis genome, is conserved among different accessions, and shows an unusual pattern of sequence variation. This EASE works independently of position and orientation in Arabidopsis but is probably not associated with any nearby gene, suggesting either that it acts over a large distance or that a cryptic element was detected. Embryo-specific ablation in Arabidopsis was achieved by transactivation of a diphtheria toxin gene under the control of the EASE. The potential application of the EASE element and similar control elements as part of an open-source biotechnology toolkit for apomixis is discussed.

Gene Silencing in Arabidopsis Spreads from the Root to the Shoot, through a Gating Barrier, by Template-Dependent, Nonvascular, Cell-to-Cell Movement

Upward long-distance mobile silencing has been shown to be phloem mediated in several different solanaceous species. We show that the Arabidopsis (Arabidopsis thaliana) seedling grafting system and a counterpart inducible system generate upwardly spreading long-distance silencing that travels not in the phloem but by template-dependent reiterated short-distance cell-to-cell spread through the cells of the central stele. Examining the movement of the silencing front revealed a largely unrecognized zone of tissue, below the apical meristem, that is resistant to the silencing signal and that may provide a gating or protective barrier against small RNA signals. Using a range of auxin and actin transport inhibitors revealed that, in this zone, alteration of vesicular transport together with cytoskeleton dynamics prevented or retarded the spread of the silencing signal. This suggests that small RNAs are transported from cell to cell via plasmodesmata rather than diffusing from their source in the phloem.

A 22-nt artificial microRNA mediates widespread RNA silencing in Arabidopsis

It is known that 22-nucleotide (nt) microRNAs (miRNAs) derived from asymmetric duplexes trigger phased small-interfering RNA (phasiRNA) production from complementary targets. Here we investigate the efficacy of 22-nt artificial miRNA (amiRNA)-mediated RNA silencing relative to conventional hairpin RNA (hpRNA) and 21-nt amiRNA-mediated RNA silencing. CHALCONE SYNTHASE (CHS) was selected as a target in Arabidopsis thaliana due to the obvious and non-lethal loss of anthocyanin accumulation upon widespread RNA silencing. Over-expression of CHS in the pap1-D background facilitated visual detection of both local and systemic RNA silencing. RNA silencing was initiated in leaf tissues from hpRNA and amiRNA plant expression vectors under the control of an Arabidopsis RuBisCo small subunit 1A promoter (SSU). In this system, hpRNA expression triggered CHS silencing in most leaf tissues but not in roots or seed coats. Similarly, 21-nt amiRNA expression from symmetric miRNA/miRNA* duplexes triggered CHS silencing in all leaf tissues but not in roots or seed coats. However, 22-nt amiRNA expression from an asymmetric duplex triggered CHS silencing in all tissues, including roots and seed coats, in the majority of plant lines. This widespread CHS silencing required RNA-DEPENDENT RNA POLYMERASE6-mediated accumulation of phasiRNAs from the endogenous CHS transcript. These results demonstrate the efficacy of asymmetric 22-nt amiRNA-directed RNA silencing and associated phasiRNA production and activity, in mediating widespread RNA silencing of an endogenous target gene. Asymmetric 22-nt amiRNA-directed RNA silencing requires little modification of existing amiRNA technology and is expected to be effective in suppressing other genes and/or members of gene families.

Interaction between Arabidopsis Brca2 and its partners Rad51, Dmc1, and Dss1

The Arabidopsis (Arabidopsis thaliana) orthologs of Brca2, a protein whose mutations are involved in breast cancer in humans, were previously shown to be essential at meiosis. In an attempt to better understand the Brca2-interacting properties, we examined four partners of the two isoforms of Brca2 identified in Arabidopsis (AtRad51, AtDmc1, and two AtDss1 isoforms). The two Brca2 and the two Dss1 isoforms are named AtBrca2(IV), AtBrca2(V), AtDss1(I), and AtDss1(V) after their chromosomal localization. We first show that both AtBrca2 proteins can interact with either AtRad51 or AtDmc1 in vitro, and that the N-terminal region of AtBrca2 is responsible for these interactions. More specifically, the BRC motifs (so called because iterated in the Brca2 protein) in Brca2 are involved in these interactions: BRC motif number 2 (BRC2) alone can interact with AtDmc1, whereas BRC motif number 4 (BRC4) recognizes AtRad51. The human Rad51 and Dmc1 proteins themselves can interact with either the complete (HsRad51) or a shorter version of AtBrca2 (HsRad51 or HsDmc1) that comprises all four BRC motifs. We also identified two Arabidopsis isoforms of Dss1, another known partner of Brca2 in other organisms. Although all four Brca2 and Dss1 proteins are much conserved, AtBrca2(IV) interacts with only one of these AtDss1 proteins, whereas AtBrca2(V) interacts with both of them. Finally, we show for the first time that an AtBrca2 protein could bind two different partners at the same time: AtRad51 and AtDss1(I), or AtDmc1 and AtDss1(I).

An upstream open reading frame is essential for feedback regulation of ascorbate biosynthesis in arabidopsis

Ascorbate (vitamin C) is an essential antioxidant and enzyme cofactor in both plants and animals. Ascorbate concentration is tightly regulated in plants, partly to respond to stress. Here, we demonstrate that ascorbate concentrations are determined via the posttranscriptional repression of GDP-l-galactose phosphorylase (GGP), a major control enzyme in the ascorbate biosynthesis pathway. This regulation requires a cis-acting upstream open reading frame (uORF) that represses the translation of the downstream GGP open reading frame under high ascorbate concentration. Disruption of this uORF stops the ascorbate feedback regulation of translation and results in increased ascorbate concentrations in leaves. The uORF is predicted to initiate at a noncanonical codon (ACG rather than AUG) and encode a 60- to 65-residue peptide. Analysis of ribosome protection data from Arabidopsis thaliana showed colocation of high levels of ribosomes with both the uORF and the main coding sequence of GGP. Together, our data indicate that the noncanonical uORF is translated and encodes a peptide that functions in the ascorbate inhibition of translation. This posttranslational regulation of ascorbate is likely an ancient mechanism of control as the uORF is conserved in GGP genes from mosses to angiosperms.