Breaking bad news : dynamic molecular mechanisms of wound response in plants

Recognition and repair of damaged tissue are an integral part of life. The failure of cells and tissues to appropriately respond to damage can lead to severe dysfunction and disease. Therefore, it is essential that we understand the molecular pathways of wound recognition and response. In this review, we aim to provide a broad overview of the molecular mechanisms underlying the fate of damaged cells and damage recognition in plants. Damaged cells release the so-called damage associated molecular patterns to warn the surrounding tissue. Local signaling through calcium (Ca2+), reactive oxygen species (ROS), and hormones, such as jasmonic acid, activates defense gene expression and local reinforcement of cell walls to seal off the wound and prevent evaporation and pathogen colonization. Depending on the severity of damage, Ca2+, ROS, and electrical signals can also spread throughout the plant to elicit a systemic defense response. Special emphasis is placed on the spatiotemporal dimension in order to obtain a mechanistic understanding of wound signaling in plants

The transcriptional repressor complex FRS7-FRS12 regulates flowering time and growth in Arabidopsis

Most living organisms developed systems to efficiently time environmental changes. The plant-clock acts in coordination with external signals to generate output responses determining seasonal growth and flowering time. Here, we show that two Arabidopsis thaliana transcription factors, FAR1 RELATED SEQUENCE 7 (FRS7) and FRS12, act as negative regulators of these processes. These proteins accumulate particularly in short-day conditions and interact to form a complex. Loss-of-function of FRS7 and FRS12 results in early flowering plants with overly elongated hypocotyls mainly in short days. We demonstrate by molecular analysis that FRS7 and FRS12 affect these developmental processes in part by binding to the promoters and repressing the expression of GIGANTEA and PHYTOCHROME INTERACTING FACTOR 4 as well as several of their downstream signalling targets. Our data reveal a molecular machinery that controls the photoperiodic regulation of flowering and growth and offer insight into how plants adapt to seasonal changes

Glutaredoxin GRXS17 associates with the cytosolic iron-sulfur cluster assembly pathway

Cytosolic monothiol glutaredoxins (GRXs) are required in iron-sulfur (Fe-S) cluster delivery and iron sensing in yeast and mammals. In plants, it is unclear whether they have similar functions. Arabidopsis (Arabidopsis thaliana) has a sole class II cytosolic monothiol GRX encoded by GRXS17. Here, we used tandem affinity purification to establish that Arabidopsis GRXS17 associates with most known cytosolic Fe-S assembly (CIA) components. Similar to mutant plants with defective CIA components, grxs17 loss-of-function mutants showed some degree of hypersensitivity to DNA damage and elevated expression of DNA damage marker genes. We also found that several putative Fe-S client proteins directly bind to GRXS17, such as XANTHINE DEHYDROGENASE1 (XDH1), involved in the purine salvage pathway, and CYTOSOLIC THIOURIDYLASE SUBUNIT1 and CYTOSOLIC THIOURIDYLASE SUBUNIT2, both essential for the 2-thiolation step of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification of tRNAs. Correspondingly, profiling of the grxs17-1 mutant pointed to a perturbed flux through the purine degradation pathway and revealed that it phenocopied mutants in the elongator subunit ELO3, essential for the mcm5 tRNA modification step, although we did not find XDH1 activity or tRNA thiolation to be markedly reduced in the grxs17-1 mutant. Taken together, our data suggest that plant cytosolic monothiol GRXs associate with the CIA complex, as in other eukaryotes, and contribute to, but are not essential for, the correct functioning of client Fe-S proteins in unchallenged conditions

The RING E3 ligase KEEP ON GOING modulates JASMONATE ZIM-DOMAIN12 stability

Jasmonate (JA) signaling in plants is mediated by the JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the activity of several transcription factors regulating JA-inducible gene expression. The hormone JA-isoleucine triggers the interaction of JAZ repressor proteins with the F-box protein CORONATINE INSENSITIVE1 (COI1), part of an S-phase kinase-associated protein1/Cullin1/F-box protein COI1 (SCFCOI1) E3 ubiquitin ligase complex, and their degradation by the 26S proteasome. In Arabidopsis (Arabidopsis thaliana), the JAZ family consists of 13 members. The level of redundancy or specificity among these members is currently not well understood. Here, we characterized JAZ12, encoded by a highly expressed JAZ gene. JAZ12 interacted with the transcription factors MYC2, MYC3, and MYC4 in vivo and repressed MYC2 activity. Using tandem affinity purification, we found JAZ12 to interact with SCFCOI1 components, matching with observed in vivo ubiquitination and with rapid degradation after treatment with JA. In contrast to the other JAZ proteins, JAZ12 also interacted directly with the E3 RING ligase KEEP ON GOING (KEG), a known repressor of the ABSCISIC ACID INSENSITIVE5 transcription factor in abscisic acid signaling. To study the functional role of this interaction, we circumvented the lethality of keg loss-of-function mutants by silencing KEG using an artificial microRNA approach. Abscisic acid treatment promoted JAZ12 degradation, and KEG knockdown led to a decrease in JAZ12 protein levels. Correspondingly, KEG overexpression was capable of partially inhibiting COI1-mediated JAZ12 degradation. Our results provide additional evidence for KEG as an important factor in plant hormone signaling and a positive regulator of JAZ12 stability

Approches intégrés des mécanismes moléculaires de la tolérance au cuivre chez les algues brunes

Copper is an essential cofactor for metalloproteins involved in many physiological processes including photosynthesis, respiration. However, this metal is extremely toxic at high concentrations. Over 60 years of mine activities in northern Chile have led to a major decrease in biodiversity along coastal areas polluted by mine wastes allowing few brown algal species to establish such as Ectocarpus sp. Therefore our aim was to investigate the molecular mechanisms leading to copper tolerance in brown algae. Copper excess in Laminaria digitata, triggered, rapid responses involved in the formation of C6 and C9 aldehydes. These compounds are thought to activate rapid general stress responses. Later, long chain oxylipins are produced including C18 and C20 cyclopentenones such as 12-OPDA and prostaglandins as well as unique compounds such as the 18-hydroxy-17-oxo-eicosatetraenoic acid. These mechanisms were correlated with the up-regulation of stress-responsive genes. Our results suggest that copper-induced lipid peroxidation may regulate protective mechanisms employing plant-like octadecanoid signals but also eicosanoid oxylipins which are absent in vascular plants.Furthermore, we have characterized through physiological approaches two strains of E. siliculosus isolated from an uncontaminated coast in southern Peru (strain Es32) and from a copper-polluted coast in northern Chile (strain Es524). Analysis of copper toxicity in both strains revealed high-tolerance in Es524, contrasting to Es32. In order to analyze global soluble proteomes from these strains by 2-DE analysis, a reliable protein extraction method was developed. Differential soluble proteome profiling between control and copper stress conditions for each strain allowed to identify the induction of proteins involved in stress responses related to processes such as energy production, glutathione metabolism, phenolics biosynthesis or Heat Shock Protein accumulation. In addition, the comparison of stress-related proteomes between strains led to features related to copper tolerance in Es524. This strain presented marked expression of the copper stress targeted proteins OEC33 and fucoxanthine chlorophyll a-cbinding protein, which are important components of the photosynthetic apparatus. In addition, Es524 expressed specific stress-related enzymes such as RNA helicases from the DEAD box families involved in RNA folding and maturation and a vanadium-dependent bromoperoxidase. Therefore, two different phenotypes at the proteomic level strongly suggest that persistent copper stress may have driven selective forces leading to the development of ecotypes genetically adapted to copper-contaminated sites. In addition the exploration of the recently sequenced Ectocarpus genomes allowed the identification of conserved features of copper homeostasis and detoxification and to initiate preliminary characterization of some important metal-chelating mechanisms using functional approaches.El cobre es un cofactor esencial para metaloproteínas, por lo que es fundamental para procesos biológicos tales como la fotosíntesis o la respiración celular. Sin embargo, una acumulación incontrolada de este metal puede acarrear efectos nefastos a nivel celular. Más de 60 años de actividad minera en el norte de Chile ha causado un gran impacto en la biodiversidad de las zonas costeras contaminadas por cobre. En aquellas zonas, solamente algunas especies de algas pardas (Phaeophyceae) han logrado establecerse tales como Ectocarpus sp. Tomando en cuenta la poca información bioquímica y molecular acerca de estos organismos, mi trabajo de tesis consistió en estudiar estos mecanismos.El estrés por cobre en Laminaria digitata acarrea respuestas rápidas asociadas a la aparición de oxilipinas volátiles de tipo aldeídos en C6 y C9. Estos derivados de ácidos grasos podrían activar mecanismos generales de respuesta al estrés. En un mayor lapso de tiempo (24h) un segundo tipo de oxilipinas en C18 y C20 fueron detectadas. Estos compuestos incluyen la presencia de cliclopentenonas de carácter enzimatico como el ácido 12-oxo-fitodienoico, característico de plantas o prostaglandinas, característico de animales. Además se descubrieron compuestos únicos en algas pardas como el ácido 18-hidroxi-17-oxo–eicosatetraenoico. La biosíntesis de estos compuestos se relaciona con la regulación de genes implicados en mecanismos de desintoxicación celular. En conclusión, pese a que la peroxidación lipídica constituye uno de los efectos nocivos del exceso de cobre, algunos de estos compuestos octadecanoicos y eicosanoicos son producidos de forma enzimática los que podrían relacionarse con la activación de mecanismos de tolerancia al cobre. Por otro lado las respuestas fisiológicas al estrés por cobre fueron evaluadas en aislados de E. siliculosus provenientes de una zona sin registros de contaminacion por cobre (Es32) en comparación con otro aislado (Es524) proveniente de áreas contaminadas por cobre en la bahía de Chañaral, Chile. Los resultados muestran la clara tolerancia al cobre en el aislado Es524 comparado a Es32. Para llevar a cabo un análisis global de la expresión protéica por electroforesis bidimensional, fue previamente necesario crear un protocolo adaptado a esta especie. Los patrones de expresión protéicos en ambos aislados estresados por cobre, mostraron rasgos comunes tales como la aumentación de procesos energéticos, la activación del metabolismo del glutatión y de compuestos fenólicos o la accumulación de HSP (Heat Shock Proteins). Por otro lado la comparación de patrones entre ambos aislados mostró la aumentación marcada en Es524 de proteínas preferencialmente degradadas por cobre tales como componentes estructurales fotosintéticos. Además, este aislado expresa proteínas importantes para la desintoxicación como por ejemplo DEADbox helicasas o Bromoperoxidasas a vanadio. Estas observaciones nos han llevado a proponer la hipótesis que la tolerancia al cobre en Es524 es la resultante de un proceso adaptativo. Por otro lado la participación al proceso de anotación del genoma de E. siliculosus permitió identificar por homología a otros organismos componentes moleculares adicionales que participarían en la homeostasis y desintoxicación de cobre.Les algues brunes (Phaeophyceae) sont des organismes photosynthétiques macroscopiques fixés qui représentent une biomasse considérable dans les écosystèmes rocheux des côtes tempérés et froides de tous les océans en zone intertidale et subtidale. Par conséquent elles doivent faire face à des agressions constantes d’origine naturelle ou anthropique. Le déversement des métaux lourds comme le cuivre provenant des activités minières, portuaires et agricoles constitue une importante source de pollution dans l'environnement marin.Dans ce contexte, l’objectif de ce travail visait à apporter des réponses concernant le maintien d’une biodiversité algale très réduite dans les zones impactées par les rejets des mines de cuivre sur les côtes du nord du Chili par de nouvelles approches intégratives pour comprendre l’adaptation dans des environnements pollués. Au cours de mes travaux, j’ai notamment développé une approche de profilage métabolique des acides gras oxydés chez Laminaria digitata. Ces résultats ont permis d’appréhender la nature des lipoperoxydes produits suite au stress par le cuivre tels que des prostaglandines ou le 12-oxo-PDA, mais egalement des composés originaux comme l’acide 18-hydroxy-17-oxoeicosatetraènoique. Ces travaux ont permis d’atribuer à ces composés des fonctions potentielles notamment dans la signalisation cellulaire qui contrôle les mécanismes de tolérance au cuivre. D'autre part une approche protéomique globale par électrophorèse bidimensionnelle (2-DE) a permis d'idéntifier des proteines impliqués dans la tolérance au cuivre chez Ectocarpus siliculosus

Study of the molecular mechanisms involved in the copper tolerance of brown algae by integrative approaches

Les algues brunes (Phaeophyceae) sont des organismes photosynthétiques macroscopiques fixés qui représentent une biomasse considérable dans les écosystèmes rocheux des côtes tempérés et froides de tous les océans en zone intertidale et subtidale. Par conséquent elles doivent faire face à des agressions constantes d origine naturelle ou anthropique. Le déversement des métaux lourds comme le cuivre provenant des activités minières, portuaires et agricoles constitue une importante source de pollution dans l'environnement marin.Dans ce contexte, l objectif de ce travail visait à apporter des réponses concernant le maintien d une biodiversité algale très réduite dans les zones impactées par les rejets des mines de cuivre sur les côtes du nord du Chili par de nouvelles approches intégratives pour comprendre l adaptation dans des environnements pollués. Au cours de mes travaux, j ai notamment développé une approche de profilage métabolique des acides gras oxydés chez Laminaria digitata. Ces résultats ont permis d appréhender la nature des lipoperoxydes produits suite au stress par le cuivre tels que des prostaglandines ou le 12-oxo-PDA, mais egalement des composés originaux comme l acide 18-hydroxy-17-oxoeicosatetraènoique. Ces travaux ont permis d atribuer à ces composés des fonctions potentielles notamment dans la signalisation cellulaire qui contrôle les mécanismes de tolérance au cuivre. D'autre part une approche protéomique globale par électrophorèse bidimensionnelle (2-DE) a permis d'idéntifier des proteines impliqués dans la tolérance au cuivre chez Ectocarpus siliculosus.PARIS-BIUSJ-Physique recherche (751052113) / SudocROSCOFF-Observ.Océanol. (292393008) / SudocSudocFranceF

The Arabidopsis iron-sulfur protein GRXS17 is a target of the ubiquitin E3 ligases RGLG3 and RGLG4

The stability of signaling proteins in eukaryotes is often controlled by post-translational modifiers. For polyubiquitination, specificity is assured by E3 ubiquitin ligases. Although plant genomes encode hundreds of E3 ligases, only few targets are known, even in the model Arabidopsis thaliana. Here, we identified the monothiol glutaredoxin GRXS17 as a substrate of the Arabidopsis E3 ubiquitin ligases RING DOMAIN LIGASE 3 (RGLG3) and RGLG4 using a substrate trapping approach involving tandem affinity purification of RING-dead versions. Simultaneously, we used a ubiquitin-conjugating enzym (UBC) panel screen to pinpoint UBC30 as a cognate E2 UBC capable of interacting with RGLG3 and RGLG4 and mediating auto-ubiquitination of RGLG3 and ubiquitination of GRXS17 in vitro. Accordingly, GRXS17 is ubiquitinated and degraded in an RGLG3-and RGLG4-dependent manner in planta. The truncated hemoglobin GLB3 also interacted with RGLG3 and RGLG4 but appeared to obstruct RGLG3 ubiquitination activity rather than being its substrate. Our results suggest that the RGLG family is intimately linked to the essential element iron

Modulation of Arabidopsis root growth by specialized triterpenes

Plant roots are specialized belowground organs that spatiotemporally shape their development in function of varying soil conditions. This root plasticity relies on intricate molecular networks driven by phytohormones, such as auxin and jasmonate (JA). Loss-of-function of the NOVEL INTERACTOR OF JAZ (NINJA), a core component of the JA signaling pathway, leads to enhanced triterpene biosynthesis, in particular of the thalianol gene cluster, in Arabidopsis thaliana roots. We have investigated the biological role of thalianol and its derivatives by focusing on Thalianol Synthase (THAS) and Thalianol Acyltransferase 2 (THAA2), two thalianol cluster genes that are upregulated in the roots of ninja mutant plants. THAS and THAA2 activity was investigated in yeast, and metabolite and phenotype profiling of thas and thaa2 loss-of-function plants was carried out. THAA2 was shown to be responsible for the acetylation of thalianol and its derivatives, both in yeast and in planta. In addition, THAS and THAA2 activity was shown to modulate root development. Our results indicate that the thalianol pathway is not only controlled by phytohormonal cues, but also may modulate phytohormonal action itself, thereby affecting root development and interaction with the environment