Auxin - oxylipin crosstalk in stress responses of plant roots

Phytohormones regulate a wide array of developmental processes throughout the life cycle of plants. Over recent years, mounting evidence led to the widely accepted concept that plant hormone action is not the read-out of linear pathways, but determined by the extensive combinatorial activity of the signaling molecules and the integration of their signaling pathways, both in terms of regulating growth and development and in adapting to external stimuli. Recent work is beginning to shed light on the crosstalk of both nominally synergistically and antagonistically acting plant hormones such as, for example, auxins with oxylipins. Here, we report that oxylipins directly contribute to the regulation of the expression of two Arabidopsis YUCCA (YUC) genes, YUC8 and YUC9. Similar to previously characterized YUC family members, we identify both YUC8 and YUC9 as involved in local auxin biosynthesis, as demonstrated by the altered auxin contents and auxin-dependent phenotypes displayed by loss-of function mutants and transgenic overexpressing lines. Gene expression data obtained by qPCR analysis and microscopic examination of promoter-reporter lines reveal an oxylipin-mediated regulation of YUC9 expression that is dependent on the COI1 signal transduction pathway. The microscopic data indicate a functional overlap of the two analyzed auxin biosynthesis genes, but also point out specific functions for YUC8 and YUC9, which are in part related to different spatio-temporal expression pattern. In support of these findings, the analyzed yuc knockout mutants had lower free auxin contents and displayed a reduced response to oxylipins. This work provides evidence of a molecular mechanism that links oxylipin signaling with auxin homeostasis.

A role of Toc33 in the protochlorophyllide-dependent plastid import pathway of NADPH:proto��chlorophyllide oxidoreductase (POR) A

NADPH: protochlorophyllide oxido reductase (POR) A is a key enzyme of chlorophyll biosynthesis in angiosperms. It is nucleus-encoded, synthesized as a larger precursor in the cytosol and imported into the plastids in a substrate-dependent manner. Plastid envelope membrane proteins, called protochlorophyllide dependent translocon proteins, Ptcs, have been identi���ed that interact with pPORA during import. Amongthem are a 16-kDa ortholog of the previously characterized outer envelope protein Oep16 (named Ptc16) and a33-kDa protein (Ptc33) related to the GTP-binding proteins Toc33 and Toc34 of Arabidopsis. In the present work, we studied the interactions and roles of Ptc16 and Ptc33 during pPORA import. Radio labeled Ptc16/Oep16 was synthesized from a corresponding cDNA and imported into isolated Arabidopsis plastids. Crosslinking experiments revealed that import of35S-Oep16/Ptc16 is stimulated by GTP.35S-Oep16/Ptc16forms larger complexes with Toc33 but not Toc34. Plastids of the ppi1 mutant of Arabidopsis lacking Toc33, were unable to import pPORA in darkness but imported the small subunit precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSSU), precursor ferredoxin (pFd) as well as pPORB which is a close relative of pPORA. In white light, partial suppressions of pSSU, pFd and pPORB import were observed. Our results unveil a hitherto unrecognized role of Toc33 in pPORA import and suggest photo oxidative membrane damage, induced by excess Pchlide accumulating in ppi1 chloroplasts because of the lack of pPORA import, to be the cause of the general drop of protein import.

Estructura de una red esencial de se��alizaci��n que regula la homeostasis i��nica en plantas

Las plantas son organismos s��siles que han desarrollado la capacidad para detectar variaciones sutiles en su ambiente y producir respuestas adaptativas mediante rutas de se��alizaci��n. Los est��mulos causados por el estr��s bi��tico y abi��tico son numerosos y dependiendo del tiempo de exposici��n y su intensidad, pueden reducir la tasa de crecimiento de las plantas y la producci��n. Los cambios en la concentraci��n del calcio citos��lico libre constituyen una de las primeras reacciones intracelulares a las situaciones de estr��s abi��tico. En esta situaci��n, el calcio act��a como segundo mensajero y las variaciones en su concentraci��n son descodificadas por prote��nas de uni��n a calcio. Las m��s conocidas son las manos-EF y los dominios C2. Los dominios C2 han sido descritos como dominios de uni��n a l��pidos dependientes de calcio. Estos dominios se consideran prote��nas perif��ricas solubles en agua que se asocian de manera reversible a los l��pidos de la membrana mediante una o dos regiones funcionales: el sitio de uni��n a calcio y el sitio polib��sico. A pesar de que se conoce la estructura molecular de algunos dominios C2, se desconocen aspectos relacionados como las reglas que dirigen su forma de interaccionar con los diferentes fosfol��pidos y prote��nas, la posici��n que ocupan en la bicapa lip��dica y su papel en la transmisi��n de se��ales. En esta tesis se ha estudiado una prote��na de Arabidopsis thaliana (At3g17980) representativa de una nueva familia de prote��nas con dominios C2, que consiste ��nicamente de un dominio C2. Esta prote��na, llamada AtC2.1, ha sido clonada en el vector pETM11, expresada en E. coli y purificada a homogeneidad en dos pasos cromatogr��ficos. Se obtuvieron cristales de AtC2.1 de buena calidad mediante t��cnicas de difusi��n de vapor. La prote��na fue co-cristalizada con calcio, fosfocolina (POC) y el fosfol��pido 1,2-dihexanoil-sn-glicero-3-fosfo-L-serina (PSF). Se recogieron ocho conjuntos de datos de difracci��n de rayos X empleando radiaci��n sincrotr��n. Los cristales difractaron hasta 1.6 �� de resoluci��n. Siete de ellos pertenec��an al grupo ortorr��mbico P212121, con las dimensiones de la celdilla unidad a = 35.3, b = 88.9, c = 110.6 ��, y un cristal pertenec��a al grupo espacial monocl��nico C2, con a = 124.84, b = 35.27, c = 92.32 �� y ������= 121.70��. La estructura se resolvi�� mediante la t��cnica MR-SAD utilizando el cinc como dispersor an��malo. La estructura cristalina mostr�� que la mol��cula forma un d��mero en el que cada prot��mero se pliega como un dominio C2 t��pico, con la topolog��a tipo II y presenta una inserci��n de 43 amino��cidos que la diferencia de los dominios C2 conocidos. El mapa de densidad electr��nica mostr�� dos ��tomos de calcio por prot��mero. Se resolvieron las estructuras de AtC2.1 en complejo con POC o PSF. En ambos complejos, el an��lisis cristalogr��fico detect�� m��ximos de densidad electr��nica en la regi��n correspondiente al sitio polib��sico formado por las hebras ���2, ���3 ���5 y el lazo 3. ��stos se interpretaron correctamente como dos mol��culas de POC y un ��tomo de cinc, en un complejo, y como la cabeza polar del PSF en el otro. AtC2.1 define un sitio de interacci��n con l��pidos dependiente de cinc. En conclusi��n, en este trabajo se presenta la estructura tridimensional de AtC2.1, miembro representativo de una familia de prote��nas de Arabidopsis thaliana, identificadas como prote��nas que interaccionan con los receptores de ABA. Estas prote��nas est��n constituidas ��nicamente por un dominio C2. El an��lisis conjunto de los datos biof��sicos y cristalogr��ficos muestra que AtC2.1 es un sensor de calcio que une l��pidos usando dos sitios funcionales. Estos datos sugieren un mecanismo de inserci��n en membrana dependiente de calcio que trae consigo la disociaci��n de la estructura dim��rica y, por consiguiente, un cambio en las propiedades de superficie de la mol��cula. Este mecanismo proporciona las bases del reconocimiento y transporte de los receptores de ABA y/o otras mol��culas a la membrana celular. Plants are sessile organisms that have developed the capacity to detect slight variations of their environment. They are able to perceive biotic and abiotic stress signals and to transduce them by signaling pathways in order to trigger adaptative responses. Stress factors are numerous and, depending on their exposition time and their concentration, can reduce plant growth rate, limiting the productivity of crop plants. Changes in the cytosolic free calcium concentration are observed as one of the earliest intracellular reactions to abiotic stress signals. Calcium plays a key role as a second messenger, and calcium concentration signatures, called calcium signals, are decodified by calcium binding proteins. The main calcium binding structures are the EF-hand motif and the C2 domains. C2 domain is a calcium dependent lipid-binding domain of approximately 130 amino acids. C2 domain displays two functional regions: the Ca-binding region and the polybasic cluster. Both of them can interact with the membrane phospholipids. Despite the number of C2 domain 3D structures currently available, questions about how they interact with the different target phospholipids, their precise spatial position in the lipid bilayer, interactions with other proteins and their role in transmitting signals downstream, have not yet been explored. In this work we have studied an uncharacterized protein from Arabidopsis thaliana (At3g17980) consisting of only a single C2 domain, as member of a new protein C2-domain family. This protein called AtC2.1 was cloned into the pETM11 vector and expressed in E. coli, allowing the purification to homogeneity in two chromatographic steps. Good quality diffracting crystals were obtained using vapor-diffusion techniques. Crystals were co-crystalized with calcium; phosphocholine (POC) and/or the phospholipid 1,2-dihexanoyl-sn-glycero-3-phospho-L-serine (PSF). Eight data set were collected with synchrotron radiation. Crystals diffracted up to 1.6 �� resolution and seven of them belong to the orthorhombic space group P212121, with unit-cell parameters a = 35.3, b = 88.9, c = 110.6 ��. Another crystal was monoclinic, space group C2, with a = 124.84, b = 35.27, c = 92.32 �� and ������= 121.70��. The structural model was solved by MR-SAD using Zn2+ as anomalous scatterer. The crystal structure shows that the molecule is a dimer. Each monomer was folded as a canonical C2 domain with the topology II with a 43 residues insertion. The electron density map reveals two calcium ions per molecule. Structures of AtC2.1, complexed with POC and PSF, have been solved. Well-defined extra electron densities were found, in both complexes, within the concave surface formed by strands ���2, ���3, ���5 and loop 3 of AtC2.1. These densities were clearly explained by the presence of the two POC molecules, one zinc atom and head groups of PSF, occupying the cavity of the polybasic site. AtC2.1 defines a new metal dependent lipid-binding site into the polybasic site. In conclusion, in this thesis it is presented the molecular structure of AtC2.1, a representative member of a family of Arabidopsis thaliana C2 domain proteins, of unknown function, but identified as a molecular interacting unit of the ABA receptors. The joint analyses of the biophysical and crystallographic data show that AtC2.1 is a calcium sensor that binds lipids in two sites and suggest a model of calcium-dependent membrane insertion mechanism that will involve either dimer dissociation or a strong rearrangement of the dimeric structure. This mechanism may be the basis for the recognition and delivery of ABA receptors or other protein molecules to cell membranes.

Genes implicados en la respuesta molecular al estr��s h��drico en Pinus pinaster Ait

La presente tesis doctoral se centra en el estudio de la respuesta molecular de las con��feras mediterr��neas al estr��s h��drico. Para ello se ha escogido como especie modelo Pinus pinaster Ait., la con��fera m��s abundante en Espa��a, y que habita un amplio rango de situaciones ecol��gicas, especialmente en lo relativo a la disponibilidad de agua. En primer lugar, se ha aplicado un estr��s h��drico controlado en cultivo hidrop��nico y se ha generando una genoteca sustractiva con objeto de identificar los genes inducidos por el estr��s, analizando su expresi��n en ra��ces, tallos y ac��culas. A continuaci��n, se ha analizado, la expresi��n de los genes anteriormente obtenidos as�� como de otros seleccionados de las bases de datos disponibles, durante una sequ��a prolongada en tierra, similar a las que las plantas deben afrontar en la naturaleza. Se ha utilizado en este caso, adem��s de P. pinaster, P. pinea, otra con��fera mediterr��nea adaptada a las sequ��as recurrentes. Este trabajo ha permitido identificar genes candidato expresionales, presumiblemente comunes en la respuesta molecular de las con��feras al d��ficit h��drico. Se han detectado diferencias notables en la expresi��n de determinados genes, que podr��an ser los responsables de las diferencias exhibidas por ambas especies en el comportamiento frente a la sequ��a. Entre los genes identificados como inducidos por el estr��s h��drico se encuentran varios miembros de la familia de las deshidrinas. Trabajos previos han utilizado deshidrinas como genes candidato; no obstante, la falta de especificidad de ciertos fragmentos y marcadores utilizados, debido a la complejidad estructural de esta familia, resta fiabilidad a algunos de los resultados publicados. Por este motivo, se ha estudiado en detalle esta familia en P. pinaster, se han identificado y caracterizado 8 miembros y se ha analizado su patr��n de expresi��n frente a sequ��a. Este estudio ha permitido describir por primera vez unos segmentos conservados en la secuencia de amino��cidos de las deshidrinas de pin��ceas, cuya presencia y n��mero de repeticiones parece estar relacionado con su especificidad. Por ��ltimo, se han escogido tres genes implicados en distintas fases de la respuesta al estr��s h��drico para su an��lisis exhaustivo: una deshidrina, una nodulina y un factor de transcripci��n tipo AP2. Se ha caracterizado su estructura ex��n/intr��n y secuenciado su regi��n promotora. Adem��s, se han obtenido l��neas transformadas que sobreexpresan estos genes tanto de forma heter��loga, en la especie modelo Arabidopsis thaliana, como en el propio P. pinaster. Este material facilitar�� la realizaci��n de futuros estudios sobre la funci��n y el mecanismo de actuaci��n de estos genes en la respuesta al estr��s h��drico. ABSTRACT This thesis focuses in the study of the molecular response to water stress in Mediterranean conifers. For this purpose, P. pinaster was selected as model species. It���s the most abundant conifer in Spain, living in a wide range of ecological conditions, especially regarding water availability. First, we have applied a controlled polyethylene glycol-induced water stress in hydroponic culture and obtained a suppression subtractive hybridization (SSH) library, with the aim of identifying genes induced by water stress, analysing their expression in roots, stems and needles. We have then analysed the expression patterns of the identified genes, together with other genes selected from public databases. This study was conducted throughout a prolonged drought stress in soil, similar to the ones plants have to face in nature. In this case not only P. pinaster was analysed but also P. pinea, another Mediterranean conifer well adapted to recurrent droughts. This work has enabled us to identify of reliable candidate genes, presumably shared with other conifers in the response to water stress. We observed remarkable differences in the expression of some genes, which could be involved in the differential behaviour that these species show in the water stress response. Within the genes induced by water stress, several members of the dehydrin gene family were identified. Due to the structural complexity of the family, certain ambiguities and inconsistencies have been detected in previous works that have used dehydrins as candidate genes. For this reason, we have analysed thoroughly this gene family in P. pinaster, and have identified and characterized eight different members, whose expression patterns during drought have also been assessed. This study has allowed us to identify for the first time novel conserved segments in the amino acids sequences of Pinaceae. The presence and number of repetitions of these segments could be associated with the functional specificity of these proteins. Finally, three genes involved in different steps of the water stress response were selected for an exhaustive analysis: a dehydrin, a nodulin and an AP2 transcription factor. For all of them, the exon/intron structure was established and their promoter region was sequenced. Also, transformed lines were obtained both in Arabidopsis thaliana and in P. pinaster for the constitutive overexpression of these genes. This material will facilitate the development of further studies to investigate the function of these genes during the water stress response

Purification and Characterization of AsES Protein A Subtilisin Secreted by Acremonium Strictum is a Novel Plant Defense Elicitor.

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2����) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

Characterization of tomato Cycling Dof Factors reveals conserved and new functions in the control of flowering time and abiotic stress responses.

DNA binding with One Finger (DOF) transcription factors are involved in multiple aspects of plant growth and development but their precise roles in abiotic stress tolerance are largely unknown. Here we report a group of five tomato DOF genes, homologous to Arabidopsis Cycling DOF Factors (CDFs), that function as transcriptional regulators involved in responses to drought and salt stress and flowering-time control in a gene-specific manner. SlCDF1?5 are nuclear proteins that display specific binding with different affinities to canonical DNA target sequences and present diverse transcriptional activation capacities in vivo. SlCDF1?5 genes exhibited distinct diurnal expression patterns and were differentially induced in response to osmotic, salt, heat, and low-temperature stresses. Arabidopsis plants overexpressing SlCDF1 or SlCDF3 showed increased drought and salt tolerance. In addition, the expression of various stress-responsive genes, such as COR15, RD29A, and RD10, were differentially activated in the overexpressing lines. Interestingly, overexpression in Arabidopsis of SlCDF3 but not SlCDF1 promotes late flowering through modulation of the expression of flowering control genes such as CO and FT. Overall, our data connect SlCDFs to undescribed functions related to abiotic stress tolerance and flowering time through the regulation of specific target genes and an increase in particular metabolites

Chaperonas moleculares y tolerancia a estr��s abi��tico en especies arb��reas

Las temperaturas extremas, la sequ��a y otros estreses abi��ticos limitan la producci��n forestal de forma significativa, causando grandes p��rdidas econ��micas en el sector. Los ��rboles, al ser organismos s��siles, han desarrollado una serie de estrategias para percibir dichos factores, activando respuestas defensivas apropiadas. Entre ellas ocupa un lugar preeminente la s��ntesis de prote��nas con actividad chaperona molecular. Las chaperonas moleculares interaccionan con prote��nas desnaturalizadas total o parcialmente, promoviendo su correcto plegamiento y ensamblaje. Las chaperonas moleculares que se sintetizan de forma predominante en plantas, pero no en otros eucariotas, pertenecen a la familia sHSP (small heat-shock proteins). Se trata de una familia inusualmente compleja y heterog��nea, cuyos miembros son de peque��o tama��o (16-42 kD) y poseen un dominio ���alfa-cristalina��� muy conservado. Estas prote��nas est��n implicadas en protecci��n frente a estr��s abi��tico mediante la estabilizaci��n de prote��nas y membranas, si bien su mecanismo de acci��n se conoce de forma incompleta. A pesar del evidente potencial aplicado de las prote��nas sHSP, son muy escasos los estudios realizados hasta el momento con un enfoque netamente biotecnol��gico. Por otra parte, casi todos ellos se han llevado a cabo en especies herb��ceas de inter��s agron��mico o en especies modelo, como Arabidopsis thaliana. De ah�� que las sHSP de arb��reas hayan sido mucho menos caracterizadas estructural y funcionalmente, y ello a pesar del inter��s econ��mico y ecol��gico de los ��rboles y de su prolongada exposici��n vital a m��ltiples factores estresantes. La presente Tesis Doctoral se centra en el estudio de sHSP de varias especies arb��reas de inter��s econ��mico. El escrutinio exhaustivo de genotecas de cDNA de ��rganos vegetativos nos ha permitido identificar y caracterizar los componentes mayoritarios de tallo en dos especies productoras de madera noble: nogal y cerezo. Tambi��n hemos caracterizado la familia completa en chopo, a partir de su secuencia gen��mica completa. Mediante expresi��n heter��loga en bacterias, hemos analizado el efecto protector de estas prote��nas in vivo frente a distintos tipos de estr��s abi��tico, relevantes para el sector productivo. Los resultados demuestran que las prote��nas sHSP-CI: (i) aumentan la viabilidad celular de E.coli frente a casi todos estos factores, aplicados de forma individual o combinada; (ii) ejercen un rol estabilizador de las membranas celulares frente a condiciones adversas; (iii) sirven para mejorar la producci��n de otras prote��nas recombinantes de inter��s comercial. El efecto protector de las prote��nas sHSP-CI tambi��n ha sido analizado in planta, mediante la expresi��n ect��pica de CsHSP17.5-CI en chopos. En condiciones normales de crecimiento no se han observado diferencias fenot��picas entre las l��neas transg��nicas y los controles, lo que demuestra que se pueden sobre-expresar estas prote��nas sin efectos pleiotr��picos delet��reos. En condiciones de estr��s t��rmico, por el contrario, los chopos transg��nicos mostraron menos da��os y un mejor crecimiento neto. En l��nea con lo anterior, las actividades biol��gicas de varias enzimas resultaron m��s protegidas frente a la inactivaci��n por calor, corroborando la actividad chaperona propuesta para la familia sHSP y su conexi��n con la tolerancia al estr��s abi��tico. En lo que respecta a la multiplicaci��n y propagaci��n de chopo in vitro, una forma de cultivo que comporta estr��s para las plantas, todas las l��neas transg��nicas se comportaron mejor que los controles en t��rminos de producci��n de biomasa (callos) y regeneraci��n de brotes, incluso en ausencia de estr��s t��rmico. Tambi��n se comportaron mejor durante su cultivo ex vitro. Estos resultados tienen gran potencial aplicado, dada la recalcitrancia de muchas especies vegetales de inter��s econ��mico a la micropropagaci��n y a la manipulaci��n in vitro en general. Los resultados derivados de esta Tesis, aparte de aportar datos nuevos sobre el efecto protector de las prote��nas sHSP citos��licas mayoritarias (clase CI), demuestran por vez primera que la termotolerancia de los ��rboles puede ser manipulada racionalmente, incrementando los niveles de sHSP mediante t��cnicas de ingenier��a gen��tica. Su inter��s aplicado es evidente, especialmente en un escenario de calentamiento global. ABSTRACT Abiotic stress produces considerable economic losses in the forest sector, with extreme temperature and drought being amongst the most relevant factors. As sessile organisms, plants have acquired molecular strategies to detect and recognize stressful factors and activate appropriate responses. A wealth of evidence has correlated such responses with the massive induction of proteins belonging to the molecular chaperone family. Molecular chaperones are proteins which interact with incorrectly folded proteins to help them refold to their native state. In contrast to other eukaryotes, the most prominent stress-induced molecular chaperones of plants belong to the sHSP (small Heat Shock Protein) family. sHSPs are a widespread and diverse class of molecular chaperones that range in size from 16 to 42k Da, and whose members have a highly conserved ���alpha-crystallin��� domain. sHSP proteins play an important role in abiotic stress tolerance, membrane stabilization and developmental processes. Yet, their mechanism of action remains largely unknown. Despite the applied potential of these proteins, only a few studies have addressed so far the biotechnological implications of this protein family. Most studies have focused on herbaceous species of agronomic interest or on model species such as Arabidopsis thaliana. Hence, sHSP are poorly characterized in long-lived woody species, despite their economic and ecological relevance. This Thesis studies sHSPs from several woody species of economic interest. The most prominent components, namely cytosolic class I sHSPs, have been identified and characterized, either by cDNA library screening (walnut, cherry) or by searching the complete genomic sequence (poplar). Through heterologous bacterial expression, we analyzed the in vivo protective effects of selected components against abiotic stress. Our results demonstrate that sHSP-CI proteins: (i) protect E. coli cells against different stressful conditions, alone or combined; (ii) stabilize cell membranes; (iii) improve the production of other recombinant proteins with commercial interest. The effects of CsHSP17.5-CI overexpression have also been studied in hybrid poplar. Interestingly, the accumulation of this protein does not have any appreciable phenotypic effects under normal growth conditions. However, the transgenic poplar lines showed enhanced net growth and reduced injury under heat-stress conditions compared to vector controls. Biochemical analysis of leaf extracts revealed that important enzyme activities were more protected in such lines against heat-induced inactivation than in control lines, lending further support to the chaperone mode of action proposed for the sHSP family. All transgenic lines showed improved in vitro and ex vitro performance (calli biomass, bud induction, shoot regeneration) compared to controls, even in the absence of thermal stress. Besides providing new insights on the protective role of HSP-CI proteins, our results bolster the notion that heat stress tolerance can be readily manipulated in trees through genetic engineering. The applied value of these results is evident, especially under a global warming scenario.

Characterization of four bifunctional plant IAM/PAM-amidohydrolases capable of contributing to auxin biosynthesis.

Amidases [EC 3.5.1.4] capable of converting indole-3-acetamide (IAM) into the major plant growth hormone indole-3-acetic acid (IAA) are assumed to be involved in auxin de novo biosynthesis. With the emerging amount of genomics data, it was possible to identify over forty proteins with substantial homology to the already characterized amidases from Arabidopsis and tobacco. The observed high conservation of amidase-like proteins throughout the plant kingdom may suggest an important role of theses enzymes in plant development. Here, we report cloning and functional analysis of four, thus far, uncharacterized plant amidases from Oryza sativa, Sorghum bicolor, Medicago truncatula, and Populus trichocarpa. Intriguingly, we were able to demonstrate that the examined amidases are also capable of converting phenyl-2-acetamide (PAM) into phenyl-2-acetic acid (PAA), an auxin endogenous to several plant species including Arabidopsis. Furthermore, we compared the subcellular localization of the enzymes to that of Arabidopsis AMI1, providing further evidence for similar enzymatic functions. Our results point to the presence of a presumably conserved pathway of auxin biosynthesis via IAM, as amidases, both of monocot, and dicot origins, were analyzed.

YUCCA8 and YUCCA9 overexpression reveals a link between auxin signaling and lignification through the induction of ethylene biosynthesis.

Auxin is associated with the regulation of virtually every aspect of plant growth and development. Many previous genetic and biochemical studies revealed that, among the proposed routes for the production of auxin, the so-called indole-3-pyruvic acid (IPA) pathway is the main source for indole-3-acetic acid (IAA) in plants. The IPA pathway involves the action of 2 classes of enzymes, tryptophan-pyruvate aminotransferases (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1(TAA1)/TRYPTOPHAN AMINOTRANSFERASE RELATED (TAR)) and flavin monooxygenases (YUCCA). Both enzyme classes appear to be encoded by small gene families in Arabidopsis consisting of 5 and 11 members, respectively. We recently showed that it is possible to induce transcript accumulation of 2 YUCCA genes, YUC8 and YUC9, by methyl jasmonate treatment. Both gene products were demonstrated to contribute to auxin biosynthesis in planta.1 Here we report that the overexpression of YUC8 as well as YUC9 led to strong lignification of plant aerial tissues. Furthermore, new evidence indicates that this abnormally strong secondary growth is linked to increased levels of ethylene production.

Jasmonate-dependent modifications of the pectin matrix during potato development function as a defense mechanism targeted by Dickeya dadantii virulence factors

The plant cell wall constitutes an essential protection barrier against pathogen attack. In addition, cell-wall disruption leads to accumulation of jasmonates (JAs), which are key signaling molecules for activation of plant inducible defense responses. However, whether JAs in return modulate the cell-wall composition to reinforce this defensive barrier remains unknown. The enzyme 13-allene oxide synthase (13-AOS) catalyzes the first committed step towards biosynthesis of JAs. In potato (Solanum tuberosum), there are two putative St13-AOS genes, which we show here to be differentially induced upon wounding. We also determine that both genes complement an Arabidopsis aos null mutant, indicating that they encode functional 13-AOS enzymes. Indeed, transgenic potato plants lacking both St13-AOS genes (CoAOS1/2 lines) exhibited a significant reduction of JAs, a concomitant decrease in wound-responsive gene activation, and an increased severity of soft rot disease symptoms caused by Dickeya dadantii. Intriguingly, a hypovirulent D. dadantii pel strain lacking the five major pectate lyases, which causes limited tissue maceration on wild-type plants, regained infectivity in CoAOS1/2 plants. In line with this, we found differences in pectin methyl esterase activity and cell-wall pectin composition between wild-type and CoAOS1/2 plants. Importantly, wild-type plants had pectins with a lower degree of methyl esterification, which are the substrates of the pectate lyases mutated in the pel strain. These results suggest that, during development of potato plants, JAs mediate modification of the pectin matrix to form a defensive barrier that is counteracted by pectinolytic virulence factors from D. dadantii.

Large-scale identification of gibberellin-related transcription factor defines group VII ERFs as functional of DELLA partners

DELLA proteins are the master negative regulators in gibberellin (GA) signaling acting in the nucleus as transcriptional regulators. The current view of DELLA action indicates that their activity relies on the physical interaction with transcription factors (TFs). Therefore, the identification of TFs through which DELLAs regulate GA responses is key to understanding these responses from a mechanistic point of view. Here, we have determined the TF interactome of the Arabidopsis (Arabidopsis thaliana) DELLA protein GIBBERELLIN INSENSITIVE and screened a collection of conditional TF overexpressors in search of those that alter GA sensitivity. As a result, we have found RELATED TO APETALA2.3, an ethylene-induced TF belonging to the group VII ETHYLENE RESPONSE FACTOR of the APETALA2/ethylene responsive element binding protein superfamily, as a DELLA interactor with physiological relevance in the context of apical hook development. The combination of transactivation assays and chromatin immunoprecipitation indicates that the interaction with GIBBERELLIN INSENSITIVE impairs the activity of RELATED TO APETALA2.3 on the target promoters. This mechanism represents a unique node in the cross regulation between the GA and ethylene signaling pathways controlling differential growth during apical hook development.

New insights into the transciptional regulation of the mithocondrial AtTRXO1 and its role under salinity

Utilizando una genoteca ordenada de aproximadamente 1200 factores trancripcionales (TFs) de Arabidopsis thaliana en levadura, y utilizando como cebo un motivo conservado en los promotores de los genes de las Brassicaceae ort��logos al AtTrxo1, se han localizado una decena de posibles TFs que regulan el gen AtTrxo1 que codifica una tioredoxina o mitocondrial. La selecci��n del m��s probable TF se realiza por an��lisis de RTqPCR e hibridaci��n in situ de mRNAs.

Signwalling: signals derived from arabiopsis cell wall activate specific resistance to pathogens

The cell wall is a dynamic structure that regulates both constitutive and inducible plant defence responses. Different molecules o DAMPs (damage-associated molecular patterns) can be released from plant cell walls upon pathogen infection or wounding and can trigger immune responses. To further characterize the function of cell wall on the regulation of these immune responses, we have performed a biased resistance screening of putative/well-characterized primary/secondary Arabidopsis thaliana cell wall mutants (cwm). In this screening we have identified more than 20 cwm mutants with altered susceptibility/resistance to at least one of the following pathogens: the necrotrophic fungi Plectosphaerella cucumerina, the vascular bacterium Ralstonia solanacearum, the biotrophic oomycete Hyaloperonospora arabidopsidis and the powdery mildew fungus Erisyphe cruciferarum. We found that cell wall extracts from some of these cwm plants contain novel DAMPs that activate immune responses and conferred enhanced resistance to particular pathogens when they were applied to wild-type plants. Using glycomic profiling we have performed an initial characterization of the active carbohydrate structures present in these cwm wall fractions, and we have determined the signalling pathways regulated by thesse fractions. . The data generated with this collection of wall mutants support the existence of specific correlations between cell wall structure/composition, resistance to particular type of pathogens and plant fitness. Remarkably, we have identified specific cwm mutations that uncoupled resistance to pathogens from plant trade-offs, further indicating the plasticity of wall structures in the regulation of plant immune responses.

New insights into the regulation of NADPH oxidase dependent reactive oxygen species signaling during the plant immune response

Las NADPH oxidasas de plantas, denominadas ���respiratory burst oxidase homologues��� (RBOHs), producen especies reactivas del ox��geno (ROS) que median un amplio rango de funciones. En la c��lula vegetal, el ajuste preciso de la producci��n de ROS aporta la especificidad de se��al para generar una respuesta apropiada ante las amenazas ambientales. RbohD y RbohF, dos de los diez genes Rboh de Arabidopsis, son pleiotr��picos y median diversos procesos fisiol��gicos en respuesta a pat��genos. El control espacio-temporal de la expresi��n de los genes RbohD y RbohF podr��a ser un aspecto cr��tico para determinar la multiplicidad de funciones de estas oxidasas. Por ello, generamos l��neas transg��nicas de Arabidopsis con fusiones de los promoters de RbohD y RbohF a los genes delatores de la B-glucuronidasa y la luciferasa. Estas l��neas fueron empleadas para revelar el patr��n de expresi��n diferencial de RbohD y RbohF durante la respuesta inmune de Arabidopsis a la bacteria pat��gena Pseudomonas syringae pv. tomato DC3000, el hongo necr��trofo Plectosphaerella cucumerina y en respuesta a se��ales relacionadas con la respuesta inmune. Nuestros experimentos revelan un patr��n de expresi��n diferencial de los promotores de RbohD y RbohF durante el desarrollo de la planta y en la respuesta inmune de Arabidopsis. Adem��s hemos puesto de manifiesto que existe una correlaci��n entre el nivel de actividad de los promotores de RbohD y RbohF con la acumulaci��n de ROS y el nivel de muerte celular en respuesta a pat��genos. La expression de RbohD y RbohF tambi��n es modulada de manera diferencial en respuesta a patrones moleculares asociados a pat��genos (PAMPs) y por ��cido absc��sico (ABA). Cabe destacar que, mediante una estrategia de intercambio de promotores, hemos revelado que la regi��n promotora de RbohD, es necesaria para dirigir la producci��n de ROS en respuesta a P. cucumerina. Adicionalmente, la activaci��n del promotor de RbohD en respuesta al aislado de P. cucumerina no adaptado a Arabidopsis 2127, nos llev�� a realizar ensayos de susceptibilidad con el doble mutante rbohD rbohF que han revelado un papel desconocido de estas oxidasas en resistencia no-huesped. La interacci��n entre la se��alizaci��n dependiente de las RBOHs y otros componentes de la respuesta inmune de plantas podr��a explicar tambi��n las distintas funciones que median estas oxidasas en relaci��n con la respuesta inmune. Entre la gran cantidad de se��ales coordinadas con la actividad de las RBOHs, existen evidencias gen��ticas y farmacol��gicas que indican que las prote��nas G heterotrim��ricas est��n implicadas en algunas de las rutas de se��alizaci��n mediadas por ROS derivadas de los RBOHs en respuesta a se��ales ambientales. Por ello hemos estudiado la relaci��n entre estas RBOH-NADPH oxidasas y AGB1, la subunidad �� de las prote��nas G heterotrim��ricas en la respuesta inmune de Arabidopsis. An��lisis de epistasis indican que las prote��nas G heterotrim��ricas est��n implicadas en distintas rutas de se��alizaci��n en defensa mediadas por las RBOHs. Nuestros resultados ilustran la relaci��n compleja entre la se��alizaci��n mediada por las RBOHs y las prote��nas G heterotrim��ricas, que var��a en funci��n de la interacci��n planta-pat��geno analizada. Adem��s, hemos explorado la posible asociaci��n entre AGB1 con RBOHD y RBOHF en eventos tempranos de la respuesta immune. Cabe se��alar que experimentos de co��mmunoprecipitaci��n apuntan a una posible asociaci��n entre AGB1 y la kinasa citoplasm��tica reguladora de RBOHD, BIK1. Esto indica un posible mecanismo de control de la funci��n de esta NADPH oxidase por AGB1. En conjunto, estos datos aportan nuevas perspectivas sobre c��mo, a trav��s del control transcripcional o mediante la interacci��n con las prote��nas G heterotrim��ricas, las NADPH oxidases de plantas median la producci��n de ROS y la se��alizaci��n por ROS en la respuesta inmune. Nuestro trabajo ejemplifica c��mo la regulaci��n diferencial de dos miembros de una familia multig��nica, les permite realizar distintas funciones fisiol��gicas especializadas usando un mismo mecanismo enzim��tico. ABSTRACT The plant NADPH oxidases, termed respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) which mediate a wide range of functions. Fine tuning this ROS production provides the signaling specificity to the plant cell to produce the appropriate response to environmental threats. RbohD and RbohF, two of the ten Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes in response to pathogens. One aspect that may prove critical to determine the multiplicity of functions of RbohD and RbohF is the spatio-temporal control of their gene expression. Thus, we generated Arabidopsis transgenic lines with RbohD- and RbohF-promoter fusions to the ��-glucuronidase and the luciferase reporter genes. These transgenics were employed to reveal RbohD and RbohF promoter activity during Arabidopsis immune response to the pathogenic bacterium Pseudomonas syringae pv tomato DC3000, the necrotrophic fungus Plectosphaerella cucumerina and in response to immunity-related cues. Our experiments revealed a differential expression pattern of RbohD and RbohF throughout plant development and during Arabidopsis immune response. Moreover, we observed a correlation between the level of RbohD and RbohF promoter activity, the accumulation of ROS and the amount of cell death in response to pathogens. RbohD and RbohF gene expression was also differentially modulated by pathogen associated molecular patterns and abscisic acid. Interestingly, a promoter-swap strategy revealed the requirement for the promoter region of RbohD to drive the production of ROS in response to P. cucumerina. Additionally, since the RbohD promoter was activated during Arabidopsis interaction with a non-adapted P. cucumerina isolate 2127, we performed susceptibility tests to this fungal isolate that uncovered a new role of these oxidases on non-host resistance. The interplay between RBOH-dependent signaling with other components of the plant immune response might also explain the different immunity-related functions mediated by these oxidases. Among the plethora of signals coordinated with RBOH activity, pharmacological and genetic evidence indicates that heterotrimeric G proteins are involved in some of the signaling pathways mediated by RBOH���derived ROS in response to environmental cues. Therefore, we analysed the interplay between these RBOH-NADPH oxidases and AGB1, the Arabidopsis ��-subunit of heterotrimeric G proteins during Arabidopsis immune response. We carried out epistasis studies that allowed us to test the implication of AGB1 in different RBOH-mediated defense signaling pathways. Our results illustrate the complex relationship between RBOH and heterotrimeric G proteins signaling, that varies depending on the type of plant-pathogen interaction. Furthermore, we tested the potential association between AGB1 with RBOHD and RBOHF during early immunity. Interestingly, our co-immunoprecipitation experiments point towards an association of AGB1 and the RBOHD regulatory kinase BIK1, thus providing a putative mechanism in the control of the NADPH oxidase function by AGB1. Taken all together, these studies provide further insights into the role that transcriptional control or the interaction with heterotrimeric G-proteins have on RBOH-NADPH oxidase-dependent ROS production and signaling in immunity. Our work exemplifies how, through a differential regulation, two members of a multigenic family achieve specialized physiological functions using a common enzymatic mechanism.

Nuevos mecanismos moleculares de tolerancia a sequ��a y otros tipos de estr��s abi��tico en especies arb��reas de inter��s econ��mico

Las masas forestales tienen una importancia colosal para nuestra sociedad y el conjunto de la biosfera. Estudios recientes a escala mundial indican que la sequ��a es el factor abi��tico que m��s afecta a su crecimiento y supervivencia, seguida por las temperaturas extremas y la salinidad. Aunque comprender los mecanismos con que las especies arb��reas toleran estas formas de estr��s tiene un inter��s aplicado evidente, dichos mecanismos se han estudiado mucho m��s en especies herb��ceas modelo o de inter��s agron��mico. Existen sin embargo diferencias notables entre ellas, como se demuestra en esta tesis y en otros trabajos recientes. Nuestro estudio se centra concretamente en la respuesta molecular del chopo ���el sistema modelo forestal m��s desarrollado��� al estr��s abi��tico, con particular ��nfasis en la sequ��a. Utilizando una estrategia prote��mica y tratamientos controlados, hemos identificado componentes mayoritarios de dicha respuesta. Su participaci��n en la misma se ha validado mediante an��lisis transcripcionales detallados utilizando tecnolog��a qRT-PCR (PCR cuantitativa en tiempo real). Hemos identificado prote��nas cuyo nexo funcional con mecanismos de tolerancia ya era conocido, como chaperonas moleculares sHSP o enzimas que aten��an el estr��s oxidativo, pero tambi��n prote��nas cuya relaci��n funcional con el estr��s es menos clara o incluso novedosa, como polifenol oxidasas (PPO), deshidrogenasas/reductasas de cadena corta (SDR), o bicupinas (BIC), entre otras. El cuerpo central de la tesis consiste en la caracterizaci��n detallada de una PPO inusual, cuya inducci��n por estr��s h��drico se describe por vez primera. Estas enzimas est��n ampliamente distribuidas en plantas, si bien su n��mero es muy variable de unas especies a otras. Algunas, como nogal, tienen un ��nico gen, mientras que Arabidopsis no tiene ninguno. En la ��ltima versi��n del genoma de chopo hemos identificado un total de 12 miembros bona fide, corrigiendo trabajos previos, y hemos caracterizado su expresi��n individual ante diferentes situaciones de estr��s controlado y tratamientos hormonales. La isoforma antedicha es el ��nico miembro de la familia que responde claramente a la deshidrataci��n. Tambi��n responde a salinidad y a la mayor parte de tratamientos hormonales ensayados, pero no a da��o mec��nico o tratamientos con metil jasmonato. Esto la diferencia de enzimas hom��logas presentes en otras especies de plantas, que se han relacionado experimentalmente con estr��s bi��tico. Los patrones de acumulaci��n de transcritos en ��rboles adultos son compatibles con un papel protector frente a la sequ��a. La integraci��n de nuestros estudios funcionales y filogen��ticos sugiere que la familia ha sufrido un proceso reciente de diversificaci��n y neofuncionalizaci��n, siendo la protecci��n frente a deshidrataci��n su papel primigenio. Aunque se conoce la actividad bioqu��mica in vitro de este tipo de enzimas, sus sustratos naturales son esencialmente una inc��gnita. Mediante expresi��n heter��loga en Escherichia coli BL21(DE3) hemos detectado que la enzima de chopo es capaz de oxidar L-DOPA a dopaquinona, siendo menos activa frente a otros sustratos. Por otra parte, hemos demostrado su localizaci��n cloropl��stica mediante transformaci��n transitoria de protoplastos con fusiones a la prote��na fluorescente YFP. Mediante la obtenci��n de plantas transg��nicas de A. thaliana hemos demostrado que la enzima de chopo aumenta considerablemente la tolerancia in vivo frente a la deshidrataci��n y al estr��s salino. El an��lisis fenot��pico detallado de las l��neas transg��nicas, combinando m��ltiples metodolog��as, nos ha permitido sustanciar que la tolerancia tiene una base compleja. Esta incluye una mayor protecci��n del sistema fotosint��tico, una capacidad antioxidante muy incrementada y la acumulaci��n de solutos osmoprotectores como la prolina. Los an��lisis metabol��micos nos han permitido asociar la expresi��n de la prote��na a la s��ntesis de un flavano no descrito previamente en A. thaliana, vinculando la enzima de chopo con la s��ntesis de fenilpropanoides. Tambi��n hemos observado alteraciones en los niveles hormonales que podr��an subyacer a efectos pleiotr��picos con inter��s aplicado, como un aumento consistente del tama��o de la planta o el acortamiento del ciclo de crecimiento. Adem��s de aportar datos novedosos sobre la funcionalidad in vivo de esta familia de oxidasas, los resultados de esta tesis demuestran que los ��rboles son sistemas de estudio interesantes para caracterizar nuevas estrategias de tolerancia al estr��s abi��tico con potencial aplicado. ABSTRACT Forests masses have an extraordinary importance for our society and the biosphere. Recent worldwide studies indicate that drought is the abiotic factor that affects more their growing and survival, followed by extreme temperatures and salinity. The understanding of how the arboreal species tolerate the stress has an evident practical interest, but their mechanisms have been studied much more in herbaceous species or with agronomic interest. However, considerable differences exist between them, as this thesis and recent studies show. Our study is focused on the molecular response of the poplar ���the more developed forestry model system- to abiotic stress, specifically focused in the drought. Using a proteomic strategy and controlled treatments, we have identified main components in such response. Its participation has been validated through transcriptional analysis using qRT-PCR technology. We have identified proteins whose functional connection with tolerance mechanisms were already known, as molecular chaperones sHSP or enzymes that attenuate the oxidative stress, but also some proteins whose functional relationship with the stress is less clear or even novel, as polifenol oxidases (PPO), short chain deshidrogenases/reductases (SDR), or bicupines (BIC), among others. The central body of the thesis consists of the detailed characterization of an unsual PPO, whose induction due to drought stress is first described. These enzymes are thoroughly distributed in plants, but their number of members is very variable among species. Some of them, as the walnut tree, have a single gene, while Arabidopsis has none. We have identified a total of 12 members in the last version of the poplar genome, correcting previous works, and have characterized their individual expression against different situations of controlled stress and hormone treatments. The aforementioned isoform is the only member of the family that responds clearly to the drought. It also reacts to salinity and the majority of hormonal treatments tested, but it does not respond to mechanical damage or treatments with methyl jasmonate. This is the difference with homologue enzymes present in other plant species, which have been related experimentally with abiotic stress. The accumulation patterns of transcripts in adult trees are compatible with a protector role against drought. The integration of our functional and phylogenetic studies suggests that the family has suffered a recent process of diversification and neofunctionalization, being the protection against drought their original role. Although the in vitro biochemistry activity of this kind of enzymes is already known, their natural substracts are essentially a mystery. By means of heterologous expression of Escherichia coli BL21(DE3) we have detected that the enzyme of poplar is able to oxidize L-DOPA to dopaquinone, being less active against other substrates. Additionally, we have proven its chloroplastic location with transitory transformation of protoplasts with YFP protein fusion. By means of getting transgenic plants of A. thaliana, we have demonstrated that the poplar enzyme increases notably the in vivo tolerance against the drought and salinity stresses. The phenotypic analysis of the transgenic lines, and the use of multiple methodologies, allowed us to test the complexity of the tolerance. This includes a major protection of the photosynthetic system, a very increased antioxidant capacity and the accumulation of osmoprotectant solutes as the proline. The metabolic analysis has allowed to associate the protein expression with the synthesis of a Flavan non described previously in A. thalaiana, linking the enzyme of poplar with the synthesis of phenylpropanoids. We have observed alterations in the hormonal levels that could underlie pleiotropic effects with applied interest, as a consistent increase of the size of the plant and the reduction of the growth cycle. The results of this thesis, in addition to provide novel data about the in vivo functionality of the oxidase family, demonstrate that the trees are interesting systems of study to characterize new strategies of tolerance against abiotic stress with applied potential.