A Chloroplast COR413 Protein From Physcomitrella patens Is Required for Growth Regulation Under High Light and ABA Responses.

COR413 genes belong to a poorly characterized group of plant-specific cold-regulated genes initially identified as part of the transcriptional activation machinery of plants during cold acclimation. They encode multispanning transmembrane proteins predicted to target the plasma membrane or the chloroplast inner membrane. Despite being ubiquitous throughout the plant kingdom, little is known about their biological function. In this study, we used reverse genetics to investigate the relevance of a predicted chloroplast localized COR413 protein (PpCOR413im) from the moss Physcomitrella patens in developmental and abiotic stress responses. Expression of PpCOR413im was strongly induced by abscisic acid (ABA) and by various environmental stimuli, including low temperature, hyperosmosis, salinity and high light. In vivo subcellular localization of PpCOR413im-GFP fusion protein revealed that this protein is localized in chloroplasts, confirming the in silico predictions. Loss-of-function mutants of PpCOR413im exhibited growth and developmental alterations such as growth retardation, reduced caulonema formation and hypersensitivity to ABA. Mutants also displayed altered photochemistry under various abiotic stresses, including dehydration and low temperature, and exhibited a dramatic growth inhibition upon exposure to high light. Disruption of PpCOR413im also caused altered chloroplast ultrastructure, increased ROS accumulation, and enhanced starch and sucrose levels under high light or after ABA treatment. In addition, loss of PpCOR413im affected both nuclear and chloroplast gene expression in response to ABA and high light, suggesting a role for this gene downstream of ABA in the regulation of growth and environmental stress responses. Developmental alterations exhibited by PpCOR413im knockout mutants had remarkable similarities to those exhibited by hxk1, a mutant lacking a major chloroplastic hexokinase, an enzyme involved in energy homeostasis. Based on these findings, we propose that PpCOR413im is involved in coordinating energy metabolism with ABA-mediated growth and developmental responses.

A Dehydration-Induced Eukaryotic Translation Initiation Factor iso4G Identified in a Slow Wilting Soybean Cultivar Enhances Abiotic Stress Tolerance in Arabidopsis.

Water is usually the main limiting factor for soybean productivity worldwide and yet advances in genetic improvement for drought resistance in this crop are still limited. In the present study, we investigated the physiological and molecular responses to drought in two soybean contrasting genotypes, a slow wilting N7001 and a drought sensitive TJS2049 cultivars. Measurements of stomatal conductance, carbon isotope ratios and accumulated dry matter showed that N7001 responds to drought by employing mechanisms resulting in a more efficient water use than TJS2049. To provide an insight into the molecular mechanisms that these cultivars employ to deal with water stress, their early and late transcriptional responses to drought were analyzed by suppression subtractive hybridization. A number of differentially regulated genes from N7001 were identified and their expression pattern was compared between in this genotype and TJS2049. Overall, the data set indicated that N7001 responds to drought earlier than TJ2049 by up-regulating a larger number of genes, most of them encoding proteins with regulatory and signaling functions. The data supports the idea that at least some of the phenotypic differences between slow wilting and drought sensitive plants may rely on the regulation of the level and timing of expression of specific genes. One of the genes that exhibited a marked N7001-specific drought induction profile encoded a eukaryotic translation initiation factor iso4G (GmeIFiso4G-1a). GmeIFiso4G-1a is one of four members of this protein family in soybean, all of them sharing high sequence identity with each other. In silico analysis of GmeIFiso4G-1 promoter sequences suggested a possible functional specialization between distinct family members, which can attain differences at the transcriptional level. Conditional overexpression of GmeIFiso4G-1a in Arabidopsis conferred the transgenic plants increased tolerance to osmotic, salt, drought and low temperature stress, providing a strong experimental evidence for a direct association between a protein of this class and general abiotic stress tolerance mechanisms. Moreover, the results of this work reinforce the importance of the control of protein synthesis as a central mechanism of stress adaptation and opens up for new strategies for improving crop performance under stress.

Recovery from heat, salt and osmotic stress in Physcomitrella patens requires a functional small heat shock protein PpHsp16.4.

BACKGROUND: Plant small heat shock proteins (sHsps) accumulate in response to various environmental stresses, including heat, drought, salt and oxidative stress. Numerous studies suggest a role for these proteins in stress tolerance by preventing stress-induced protein aggregation as well as by facilitating protein refolding by other chaperones. However, in vivo evidence for the involvement of sHsps in tolerance to different stress factors is still missing, mainly due to the lack of appropriate mutants in specific sHsp genes. RESULTS: In this study we characterized the function of a sHsp in abiotic stress tolerance in the moss Physcomitrella patens, a model for primitive land plants. Using suppression subtractive hybridization, we isolated an abscisic acid-upregulated gene from P. patens encoding a 16.4 kDa cytosolic class II sHsp. PpHsp16.4 was also induced by salicylic acid, dithiothreitol (DTT) and by exposure to various stimuli, including osmotic and salt stress, but not by oxidative stress-inducing compounds. Expression of the gene was maintained upon stress relief, suggesting a role for this protein in the recovery stage. PpHsp16.4 is encoded by two identical genes arranged in tandem in the genome. Targeted disruption of both genes resulted in the inability of plants to recover from heat, salt and osmotic stress. In vivo localization studies revealed that PpHsp16.4 localized in cytosolic granules in the vicinity of chloroplasts under non stress conditions, suggesting possible distinct roles for this protein under stress and optimal growth. CONCLUSIONS: We identified a member of the class II sHsp family that showed hormonal and abiotic stress gene regulation. Induction of the gene by DTT treatment suggests that damaged proteins may act as signals for the stress-induction of PpHsp16.4. The product of this gene was shown to localize in cytosolic granules near the chloroplasts, suggesting a role for the protein in association with these organelles. Our study provides the first direct genetic evidence for a role of a sHsp in osmotic and salt stress tolerance, and supports a function for this protein particularly during the stress recovery stage of P. patens.

Estudio comparativo entre soluciones conservadoras con y sin formol en placenta humana / Comparative study between conservative solutions with and without formaldehyde in human placenta

Las técnicas de fijación y conservación anatómica son realizadas habitualmente con soluciones que contienen formol, dado su bajo costo. Estas tienen varias desventajas como el olor irritante, rigidez, cambios de coloración de las estructuras, así como toxicidad con potencial cancerígeno, teratogénico y mutagénico para quien lo manipula. Por esto, es deseable utilizar soluciones sin formol. El objetivo de este trabajo fue comparar 2 métodos de conservación cadavérica, uno con formol (solución de Montevideo) y otro sin formol (método de Prives) utilizando la placenta humana como órgano experimental, evaluando sus parámetros macroscópicos. Se utilizaron 46 placentas humanas de partos normales y gestación a término. Las placentas fueron separadas en dos grupos (n=22 y n=24 respectivamente). El primer grupo de placentas fue perfundido con una solución con formol y el segundo grupo en una solución sin formol. Luego ambos grupos fueron sumergidos y mantenidos en sus soluciones respectivas por dos semanas y posteriormente retiradas dejándolas al aire a temperatura ambiente por 2 semanas más. Se analizaron las variables cuantitativas de peso y diámetro en cada una de las piezas, así como las variables cualitativas de consistencia, color, olor y crecimiento de micro/macro organismos. La recopilación de datos fue realizada previo al lavado, a los 14, 21 y 28 días. Los resultados mostraron que las placentas conservadas con el método de Prives presentaron mejor conservación en relación a su diámetro, consistencia, color y menor olor irritante en relación a las placentas tratadas con solución con formol. En ningún caso hubo crecimiento de micro o macroorganismos. En conclusión, emplear soluciones alternativas que sustituyan ventajosamente al formol como la fórmula de Prives conservan mejor las características macroscópicas de las placentas sin generar un olor irritante, deteniendo el proceso de descomposición.

The fixation and conservation techniques of anatomic material are commonly performed with solutions containing formaldehyde, given its low cost. These have several disadvantages such as the irritating odor, stiffness, discoloration of the structures, toxicity, carcinogenic, teratogenic and mutagenic risk for those who are exposed. Therefore it is desirable to use solutions without formaldehyde. The aim of this study was to compare 2 methods of anatomical conservation, one with formalin (Montevideo's solution) and one without formalin (Prives method) using the human placenta as an experimental organ model evaluating its macroscopic parameters. We used 46 human placentas from normal deliveries and term pregnancy. The placentas were separated into two groups (n=22 and n=24 respectively). The first group of placentas was perfused with formaldehyde solution and the second group in a solution without formaldehyde. Then both groups were immersed and maintained in their respective solutions for two weeks and then withdrawn leaving the air at room temperature for 2 weeks. Quantitative variables were analyzed for weight and diameter of each piece, and qualitative variables as consistency, color, odor and growth of micro/macro organisms were evaluated. Data collection was performed prior to washing at 14, 21 and 28 days. The results showed that conserved placentas with Prives method showed better conservation in relation to its diameter, consistency, color and less irritating odor in relation to placentas treated with formalin solution. In no case was growth of micro or macro organism. In conclusion, using advantageously at alternative solutions to formaldehyde, as the formula of Prives method, better preserved macroscopic characteristics of placentas without generating an irritating smell, stopping the decomposition process.

Differential contribution of individual dehydrin genes from Physcomitrella patens to salt and osmotic stress tolerance.

The moss Physcomitrella patens can withstand extreme environmental conditions including drought and salt stress. Tolerance to dehydration in mosses is thought to rely on efficient limitation of stress-induced cell damage and repair of cell injury upon stress relief. Dehydrin proteins (DHNs) are part of a conserved cell protecting mechanism in plants although their role in stress tolerance is not well understood. Four DHNs and two DHN-like proteins were identified in the predicted proteome of P. patens. Expression of PpDHNA and PpDHNB was induced by salt and osmotic stress and controlled by abscisic acid. Subcellular localization of the encoded proteins suggested that these dehydrins are localized in cytosol and accumulate near membranes during stress. Comparative analysis of dhnA and dhnB targeted knockout mutants of P. patens revealed that both genes play a role in cellular protection during salt and osmotic stress, although PpDHNA has a higher contribution to stress tolerance. Overexpression of PpDHNA and PpDHNB genes in transgenic Arabidopsis improved rosette and root growth in stress conditions, although PpDHNA was more efficient in this role. These results suggest that specific DHNs contribute considerably to the high stress tolerance of mosses and offer novel tools for genetic engineering stress tolerance of higher plants.