Autophagy is activated and involved in cell death with participation of cathepsins during stress-induced microspore embryogenesis in barley

Microspores are reprogrammed towards embryogenesis by stress. Many microspores die after this stress, limiting the efficiency of microspore embryogenesis. Autophagy is a degradation pathway that plays critical roles in stress response and cell death. In animals, cathepsins have an integral role in autophagy by degrading autophagic material; less is known in plants. Plant cathepsins are papain-like C1A cysteine proteases involved in many physiological processes, including programmed cell death. We have analysed the involvement of autophagy in cell death, in relation to cathepsin activation, during stress-induced microspore embryogenesis in Hordeum vulgare. After stress, reactive oxygen species (ROS) and cell death increased and autophagy was activated, including HvATG5 and HvATG6 up-regulation and increase of ATG5, ATG8, and autophagosomes. Concomitantly, cathepsin L/F-, B-, and H-like activities were induced, cathepsin-like genes HvPap-1 and HvPap-6 were up-regulated, and HvPap-1, HvPap-6, and HvPap-19 proteins increased and localized in the cytoplasm, resembling autophagy structures. Inhibitors of autophagy and cysteine proteases reduced cell death and promoted embryogenesis. The findings reveal a role for autophagy in stress-induced cell death during microspore embryogenesis, and the participation of cathepsins. Similar patterns of activation, expression, and localization suggest a possible connection between cathepsins and autophagy. The results open up new possibilities to enhance microspore embryogenesis efficiency with autophagy and/or cysteine protease modulators.España, MINECO AGL2014-52028-R and AGL2017-82447-

Pectin De-methylesterification and AGP Increase Promote Cell Wall Remodeling and Are Required During Somatic Embryogenesis of Quercus suber

Somatic embryogenesis is a reliable system for in vitro plant regeneration, with biotechnological applications in trees, but the regulating mechanisms are largely unknown. Changes in cell wall mechanics controlled by methylesterification of pectins, mediated by pectin methylesterases (PMEs) and pectin methyl esterase inhibitors (PMEIs) underlie many developmental processes. Arabinogalactan proteins (AGPs) are highly glycosylated proteins located at the surface of plasma membranes, in cell walls, and in extracellular secretions, with key roles in a range of different processes. In this study, we have investigated changes in two cell wall components, pectins and AGPs, during somatic embryogenesis in Quercus suber, a forest tree of high economic and ecologic value. At early embryogenesis stages, cells of proembryogenic masses showed high levels of esterified pectins and expression of QsPME and QsPMEI genes encoding a PME and a putative PMEI, respectively. At advanced stages, differentiating cells of heart, torpedo and cotyledonary embryos exhibited walls rich in de-esterified pectins, while QsPME gene expression and PME activity progressively increased. AGPs were detected in cell walls of proembryogenic masses and somatic embryos. QsLys-rich-AGP18, QsLys-rich-AGP17, and QsAGP16L1 gene expression increased with embryogenesis progression, as did the level of total AGPs, detected by dot blot with β-glucosyl Yariv reagent. Immuno dot blot, immunofluorescence assays and confocal analysis using monoclonal antibodies to high- (JIM7, LM20) and low- (JIM5, LM19) methylesterified pectins, and to certain AGP epitopes (LM6, LM2) showed changes in the amount and distribution pattern of esterified/de-esterified pectins and AGP epitopes, that were associated with proliferation and differentiation and correlated with expression of the PME and AGP genes analyzed. Pharmacological treatments with catechin, an inhibitor of PME activity, and Yariv reagent, which blocks AGPs, impaired the progression of embryogenesis, with pectin de-esterification and an increase in AGP levels being necessary for embryo development. Findings indicate a role for pectins and AGPs during somatic embryogenesis of cork oak, promoting the cell wall remodeling during the process. They also provide new insights into the regulating mechanisms of somatic embryogenesis in woody species, for which information is still scarce, opening up new possibilities to improve in vitro embryo production in tree breeding

Dual targeting to mitochondria and plastids of AtBT1 and ZmBT1, two members of the mitochondrial carrier family

Zea mays and Arabidopsis thaliana Brittle 1 (ZmBT1 and AtBT1, respectively) are members of the mitochondrial carrier family. Although they are presumed to be exclusively localized in the envelope membranes of plastids, confocal fluorescence microscopy analyses of potato, Arabidopsis and maize plants stably expressing green fluorescent protein (GFP) fusions of ZmBT1 and AtBT1 revealed that the two proteins have dual localization to plastids and mitochondria. The patterns of GFP fluorescence distribution observed in plants stably expressing GFP fusions of ZmBT1 and AtBT1 N-terminal extensions were fully congruent with that of plants expressing a plastidial marker fused to GFP. Furthermore, the patterns of GFP fluorescence distribution and motility observed in plants expressing the mature proteins fused to GFP were identical to those observed in plants expressing a mitochondrial marker fused to GFP. Electron microscopic immunocytochemical analyses of maize endosperms using anti-ZmBT1 antibodies further confirmed that ZmBT1 occurs in both plastids and mitochondria. The overall data showed that (i) ZmBT1 and AtBT1 are dually targeted to mitochondria and plastids; (ii) AtBT1 and ZmBT1 N-terminal extensions comprise targeting sequences exclusively recognized by the plastidial compartment; and (iii) targeting sequences to mitochondria are localized within the mature part of the BT1 proteins. © 2011 The Author.This research was supported by the Comisio´n Interministerial de Ciencia y Tecnologı´a and Fondo Europeo de Desarrollo Regional (Spain) [grant BIO200763915]; Iden Biotechnology S.L.; Grant Agency APVV [grant No. APVV-0432-06 to M.O.]; Grant Academy VEGA [grant No. 2/0200/10 to M.O.]; the Spanish Ministry of Culture and Education [pre-doctoral fellowship to I.E.]; the Spanish Ministry of Science and Innovation [grants No. BFU2008-00203 and AGL2008-04255].Peer Reviewe

Reprogramación del polen y obtención de plantas doble-haploide de Capsisum annuum L.: Marcadores celulares y expresión de moléculas señalizadoras de estrés.

Universidad: Complutense De Madrid Departamento: Genetica Fecha De Lectura: 11/04/2008 Dirección: Director: Maria Del Carmen Risueño Almeida Codirector: Pilar Sanchez Testillano Tribunal: Presidente: Benjamín Fernández Ruiz Secretario: Cristina Pardo Martín Vocal: María Ángeles Bueno Pérez Vocal: Óscar Visente Meana Vocal: José María Seguí SimarroEl polen es capaz de cambiar su proceso de desarrollo, reprogramarse y entrar en una nueva ruta denominada embriogénica, tras un tratamiento de estrés. Tras su reprogramación, la microspora comienza a dividirse simétricamente dando lugar a proembriones y embriones que posteriormente continúan su desarrollo Regenerando plantas haploides o doble-haploides. En este contexto, el cultivo de anteras es, hoy en día, uno de los métodos más eficientes para la obtención de plantas doble-haploides, de gran utilidad para generar nuevas líneas isogénicas y variedades, así como para Estudios genéticos y de mutagénesis. El pimiento (capsicum annuum l.) Es uno de los cultivos hortícolas más importantes desde el punto de vista Económico en españa, siendo éste el país con mayor producción de la unión europea. En esta tesis doctoral, un primer objetivo ha sido la obtención plantas doble-haploides a partir de cultivos de Anteras de capsicum annuum yolo Wonder var. B. Hemos optimizado los protocolos existentes (Mitykó et al.,1995), estudiando diversos factores tales como la correlación entre la morfología de los botones florales, el tamaño y la coloración de las anteras con antocianina y el estadio de desarrollo de las microsporas, así como diversos aspectos del cultivo in vitro y el tratamiento de estrñes inductor de la embriogénesis. Por otro lado, se ha llevado a cabo la caracterización celular de las primeras etapas de desarrollo hasta el estado de torpedo en la embriogénesis de microsporas de Capsicum annuum L. Se ha determinado el nivel de ploidía de las plantas procedentes del cultivo de microsporas, mediante citometría de flujo. Un segundop objetivo ha sido el análisis de distintos aspectos del proceso de reprogramación del polen a embriogénesis, concretamente la identificación de marcadores en etapas clave y la expresión de proteínas de estrés y señalizadoras.Un segundo objetivo ha sido el análisis de distintos aspectos del proceso de reprogramación del polen a embriogénesis, concretamente la identificación de marcadores en etapas clave y la expresión de proteínas de estrés y señalizadoras. Existe una estrecha relación entre las proteínas de choque térmico (HSPs) y el abandono de la ruta gametofítica hacia la embriogénesis. Hemos estudiado los cambios en la expresión y en la localización subcelular de dos proteínas de choque térmico, HSP70 y HSP90, en la ruta gametofítica y en varias etapas de la ruta embriogénica. Tras el tratamiento inductor se observó un gran aumento de la expresión de HSP70, y una traslocación al nucleolo de la misma; sugiriendo una función específicamente protectora de la maquinaria nuclear y reguladora de la expresión génica. Se ha realizado una caracterización celular y ultraestructural (gametofítico y embriogénico) secuencial de todas las etapas de los procesos de desarrollo, mediante el empleo de técnicas histoquímicas e inmunocitoquímicas específicas, para elegir las fases fundamentales de estudio donde aparecen las características celulares y funcionales diferentes. Ha sido objeto de especial atención la definición de marcadores celulares que caracterizan y diferencian los do programas de desarrollo del polen, el gametofítico y el embriogénico, y que indican los principales cambios de actividad metabólica que están asociados a la reprogramación del polen.Entre estos marcadores se han identificado los cambios en el tamaño y forma celular y nuclear, el patrón de condensación cromatínica, la organización funcional de la región intercromatínica y la arquitectura nucleolar, la población ribosómica y la densidad citoplásmica, la presencia y tamaño de gránulos de almidón y por último, la estructura y composición de las paredes celulares. Se ha analizado la expresión in situ de moléculas señalizadoras como las ERKs, P-MAPKs y LOX, encontrándose diferencias en su expresión y localización subcelular entre las microsporas que se reprograman hacia proliferación (ruta embriogénica) frente a las que siguen su proceso normal de diferenciación (ruta gametofítica). Asimismo se han detectado cambios en la composición de las paredes celulares en relación a los procesos de proliferación y diferenciación. Muchos de los anticuerpos empleados han resultado ser buenos marcadores que nos permiten identificar de forma temprana en el cultivo las microsporas que se reprograman e inician la proliferación (ruta embriogénica): mayor proporción de ERK y MAPK en núcleo, aumento de cuerpos de Cajal nucleares, expresi¿¿on de PCNA asociado a replicación y mayor proporción de pectinas esterificadas (marcadas por JIM7) en las paredes celulares; frente a las que siguen su proceso normal de diferenciación (ruta gametofítica): acumulación de almidón, mayor proporción de ERK y PMAPK en citoplasma, y de pectinas no esterificadas (marcadas por JIM5), xiloglucanos (XG) y ramnogalacturonano II (RGII) en las paredes celulares. Los resultados obtenidos aportan nueva información muy valiosa sobre los mecanismos que controlan el proceso de reprogramación del polen a embriogénesis en Capsicum annuum L, una especie de gran interés económico; información que puede ayudar a su explotación de forma más eficiente y a dirigir futuras estrategias en la mejora de este cultivo.Peer reviewe

Suppression of Metacaspase- and Autophagy-Dependent Cell Death Improves Stress-Induced Microspore Embryogenesis in Brassica napus

Microspore embryogenesis is a biotechnological process that allows us to rapidly obtain doubled-haploid plants for breeding programs. The process is initiated by the application of stress treatment, which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study, we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by the transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), an increase in MCA proteolytic activity and the activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programs

Microspore-derived embryogenesis in Capsicum annuum: subcellular rearrangements through development

14 páginas, 7 figuras -- PAGS nros. 709-722Background information. In vitro-cultured microspores, after an appropriate stress treatment, can switch towards an embryogenic pathway. This process, known as microspore embryogenesis, is an important tool in plant breeding. Basic studies on this process in economically interesting crops, especially in recalcitrant plants, are very limited and the sequence of events is poorly understood. In situ studies are very convenient for an appropriate dissection of microspore embryogenesis, a process in which a mixture of different cell populations (induced and non-induced) develop asynchronically. Results. In the present study, the occurrence of defined subcellular rearrangements has been investigated during early microspore embryogenesis in pepper, an horticultural crop of agronomic interest, in relation to proliferation and differentiation events. Haploid plants of Capsicum annuum L. (var. Yolo Wonder B) have been regenerated from in vitro anther cultures by a heat treatment at 35°C for 8 days. Morphogenesis of microspore-derived embryos has been analysed, at both light and electron microscopy levels, using low-temperature-processed, well-preserved specimens. The comparison with the normal gametophytic development revealed changes in cell organization after embryogenesis induction, and permitted the characterization of the time sequence of a set of structural events, not previously defined in pepper, related to the activation of proliferative activity and differentiation. These changes mainly affected the plastids, the vacuolar compartment, the cell wall and the nucleus. Further differentiation processes mimicked that of the zygotic development. Conclusions. The reported changes can be considered as markers of the microspore embryogenesis. They have increased the understanding of the mechanisms controlling the switch and progression of the microspore embryogenesis, which could help to improve its efficiency and to direct strategies, especially in agronomically interesting cropsThis work was supported by grants from the Spanish MCyT (BOS2002-03572) and Comunidad de Madrid (CM 07G/0026/2003Peer reviewe

Nuclear bodies domain changes with microspore reprogramming to embryogenesis

10 pages, 5 figures.-- PMID: 16584983 [PubMed].We analysed the presence of nuclear bodies and particularly Cajal bodies during representative stages of gametophytic and haploid embryogenic development in isolated microspore and anther cultures of a model system (Brassica napus cv. Topas) and a recalcitrant species (Capsicum annuum L. var. Yolo Wonder B). The nuclear bodies domain is involved on several important roles on nuclear metabolism, and Cajal bodies are specifically involved on the storage and maturation of both snRNPs and snoRNPs, as well as other splicing factors, necessary for mRNA and pre-rRNA processing, but not directly on the transcription. In this study, immunofluorescence and immunogold labelling with anti-trimethylguanosine antibodies against the specific cap of snRNAs, ultrastructural and cytochemical analysis were performed on cryoprocessed samples at confocal and electron microscopy respectively. Results showed that Cajal bodies increase during the early stages of microspore embryogenic development (young pro-embryos), compared to microspore and pollen development. Our results suggest that Cajal bodies may have a role in the transcriptionally active, proliferative stages that characterise early microspore embryogenic development.This work was supported by projects granted by Spanish Ministry of Education and Science (MEC) BOS2002-03572, AGL2005-05104, BFU2005-01094, and Comunidad de Madrid, CM 07G/0026/2003.Peer reviewe

Microspore reprogramming to embryogenesis induces changes in cell wall and starch accumulation dynamics associated with proliferation and differentiation events

Short Communication.-- PMID: 20383055 [PubMed]Plant cell wall polymers are regulated during development, but the specific roles of their different molecular components and the functional meaning of cell wall changes in different cell types and cell processes are still unclear. In the present work the presence and distribution of different cell wall components in Capsicum annuum L. pollen have been analyzed in situ in order to monitor how they change during two developmental programs. These programs are: pollen development, which is a differentiation process, and stress-induced pollen reprogramming to embryogenesis, which involves proliferation followed later by differentiation processes. Specific antibodies recognizing the major cell wall polymers, the major hemicellulose, xyloglucan (XG), the rhamnogalacturonan II (RGII) pectin domain, and high- and low-methyl-esterified pectins were used for both dot-blot and immunolocalization assays at light and electron microscopy levels during defined developmental stages. For comparison purposes, a similar approach was also used in zygotic embryogenesis and root apical tip growth. Results showed differences in the distribution pattern of these molecular complexes, in the proportion of esterified and de-esterified pectins in the two pollen developmental pathways, and defined wall changes during microspore reprogramming. These changes were associated with proliferation and differentiation events where highly esterified pectins were characteristic of proliferation, while de-esterified pectins, XG and RGII were abundant in walls of differentiating cells. Starch deposits were also studied and the results revealed changes in starch synthesis dynamics after switching the pollen embryogenic developmental program. These changes occurred together with modifications in the distribution patterns of cell wall polymers, starch accumulation being associated with cell differentiation. As in the case of proliferating cells, esterified pectins were also abundant in the apertures of developing microspores, regions of new cell wall formation. The different distribution patterns of cell wall polymers were common for proliferating cells and differentiating cells in all the plant systems analyzed, including zygotic embryos and root tip cells, suggesting that these patterns are markers of proliferation and differentiation events as well as markers of pollen reprogramming to embryogenesis.Peer reviewe