Teodoro Coba de la Peña

The natural compound benzoxazolin-2(3H)-one selectively retards cell cycle in lettuce root meristems

Benzoxazolin-2(3H)-one (BOA) is a natural plant product that is phytotoxic to target plant species, inhibiting germination and growth and causing oxidative damage. We investigated its effects on the root meristems of seedlings of lettuce (Lactuca sativa) by means of light and transmission electron microscopy, flow cytometry, and conventional determination of mitotic index. Flow cytometry analyses and mitotic index showed a retard of cell cycle in BOA-treated meristems with selective activity at G2/M checkpoint.

Legumes in the reclamation of marginal soils, from cultivar and inoculant selection to transgenic approaches

Mineral nitrogen deficiency is a frequent characteristic of arid and semi-arid soils. Biological nitrogen fixation by legumes is a sustainable and environmental-friendly alternative to chemical fertilization. Therefore, legumes have a high potential for the reclamation of marginal soils. Such issue is becoming more urgent due to the ever-rising requirement for food and feed, and the increasing extension of salinized and degraded lands, both as a consequence of global change and irrigation practices. This manuscript reviews current research on physiological and molecular mechanisms involved in the response and tolerance to environmental stresses of the Rhizobium–legume symbiosis. We report in particular recent advances on the isolation, characterization, and selection of tolerant rhizobial strains and legume varieties, both by traditional methods and through biotechnological approaches. The major points are the following. (1) Understanding mechanisms involved in stress tolerance is advancing fast, thus providing a solid basis for the selection and engineering of rhizobia and legumes with enhanced tolerance to environmental constraints. (2) The considerable efforts to select locally adapted legume varieties and rhizobial inocula that can fix nitrogen under conditions of drought or salinity are generating competitive crop yields in affected soils. (3) Biotechnological approaches are used to obtain improved legumes and rhizobia with enhanced tolerance to abiotic stresses, paying particular attention to the sensitive nitrogen-fixing activity. Those biotechnologies are yielding transgenic crops and inocula with unquestionable potential. In conclusion, the role of legumes in sustainable agriculture, and particularly, their use in the reclamation of marginal lands, certainly has a very promising future.

Flavodoxin overexpression reduces cadmium-induced damage in alfalfa root nodules.

Flavodoxins are electron carrier flavoproteins that are involved in the response to oxidative stress in bacteria and cyanobacteria. Recently, we obtained Sinorhizobium meliloti bacteria that overexpressed a flavodoxin from the cyanobacterium Anabaena variabilis [Redondo et al. (2009) Plant Physiology 149:1166–1178]. In the present work, tolerance to cadmium was evaluated in free-living transformed S. meliloti and in alfalfa plants nodulated by the flavodoxin-overexpressing rhizobia, in comparison with plants nodulated by wild-type bacteria. Overexpression of flavodoxin protected free-living S. meliloti from cadmium toxicity and had a positive effect on nitrogen fixation of alfalfa plants subjected to cadmium stress. Flavodoxin notably reduced cadmium-induced structural and ultrastructural alterations in alfalfa nodules. Putative protection mechanisms in flavodoxin-overexpressing nodules are discussed. Flavodoxin could have applications as a biotechnological tool to improve the symbiotic performance of alfalfa and other legumes in cadmium polluted soils.

Nitrogen fixation persists under conditions of salt stress in transgenic Medicago truncatula plants expressing a cyanobacterial flavodoxin.

Several recent studies have demonstrated that the expression of a cyanobacterial flavodoxin in plants can provide tolerance to a wide range of environmental stresses. Indeed, this strategy has been proposed as a potentially powerful biotechnological tool to generate multiple-tolerant crops. To determine whether flavodoxin expression specifically increased tolerance to salt stress and whether it might also preserve legume nitrogen fixation under saline conditions, the flavodoxin gene was introduced into the model legume Medicago truncatula. Expression of flavodoxin did not confer saline tolerance to the whole plant, although the sensitive nitrogen-fixing activity was maintained under salt stress in flavodoxin-expressing plants. Our results indicate that flavodoxin induced small but significant changes in the enzymatic activities involved in the nodule redox balance that might be responsible for the positive effect on nitrogen fixation. Expression of flavodoxin can be regarded as a potential tool to improve legume symbiotic performance under salt stress, and possibly other environmental stresses.

A salt stress-responsive cytokinin receptor homologue isolated from Medicago sativa nodules

A new cytokinin receptor homologue, MsHK1, was isolated from Medicago sativa root nodules. MsHK1 expression was induced in alfalfa seedlings by exogenous application of the cytokinin trans-zeatin. Transcript accumulation was detected in different plant organs. MsHK1 expression was induced by salt stress in alfalfa roots, leaves and nodules, and transcript accumulation in the vascular bundles pointed to a putative role in osmosensing for MsHK1 and/or other close cytokinin receptor homologues. Expression in the meristem and the invasion zone of the nodule suggest a role for cytokinin receptors in cytokinin sensing during nodule cell division and differentiation.

Alfalfa nodules elicited by a flavodoxin-overexpressing Ensifer meliloti strain display nitrogen-fixing activity with enhanced tolerance to salinity stress

Nitrogen fixation by legumes is very sensitive to salinity stress, which can severely reduce the productivity of legume crops and their soil-enriching capacity. Salinity is known to cause oxidative stress in the nodule by generating reactive oxygen species (ROS). Flavodoxins are involved in the response to oxidative stress in bacteria and cyanobacteria. Prevention of ROS production by flavodoxin overexpression in bacteroids might lead to a protective effect on nodule functioning under salinity stress. Tolerance to salinity stress was evaluated in alfalfa nodules elicited by an Ensifer meliloti strain that overexpressed a cyanobacterial flavodoxin compared with nodules produced by the wild-type bacteria. Nitrogen fixation, antioxidant and carbon metabolism enzyme activities were determined. The decline in nitrogenase activity associated to salinity stress was significantly less in flavodoxin-expressing than in wild-type nodules. We detected small but significant changes in nodule antioxidant metabolism involving the ascorbate–glutathione cycle enzymes and metabolites, as well as differences in activity of the carbon metabolism enzyme sucrose synthase, and an atypical starch accumulation pattern in flavodoxin-containing nodules. Salt-induced structural and ultrastructural alterations were examined in detail in alfalfa wild-type nodules by light and electron microscopy and compared to flavodoxin-containing nodules. Flavodoxin reduced salt-induced structural damage, which primarily affected young infected tissues and not fully differentiated bacteroids. The results indicate that overexpression of flavodoxin in bacteroids has a protective effect on the function and structure of alfalfa nodules subjected to salinity stress conditions. Putative protection mechanisms are discussed.

Multiple roles for cytokinin receptors and cross-talk of signaling pathways

Cytokinin receptors (CRs) are hybrid-type histidine kinases, membrane proteins with a cytokinin-binding extracellular domain. CRs initiate and propagate cytokinin signaling by means of phosphorylation and phosphotransfer to downstream proteins. In legumes, some members of the CR multigenic family are essential for nodulation. In two recent works, we investigated the involvement of two new CRs, MsHK1 from Medicago sativa, and LaHK1 from Lupinus albus, in nodule morphogenesis, senescence and stress response. LaHK1 expression increased during the first stages of lupin nodule development, while MsHK1 expression was localized in the meristem and the invasion zone of alfalfa nodules pointing to a role for CRs in nodule cell proliferation and differentiation. Both CRs were also induced during nodule senescence. MsHK1 expression increased under osmotic stress and both genes were induced following dark stress, indicating that CRs are also likely to play a significant role in the response to stress. We propose multiple roles for CRs which, when analyzed jointly with recent results from other authors, suggest coordinated cross-talk of different signaling systems. Addendum to: Coba de la Peña T, Cárcamo CB, Almonacid L, Zaballos A, Lucas MM, Balomenos D, Pueyo JJ. A salt stress-responsive cytokinin receptor homologue isolated from Medicago sativa nodules. Planta 2008; 227:769-79. and Coba de la Peña T, Cárcamo CB, Almonacid L, Zaballos A, Lucas MM, Balomenos D, Pueyo JJ. A cytokinin receptor homologue is induced during root nodule organogenesis and senescence in Lupinus albus L. Plant Physiol Biochem 2008; 46:219-25.

Cell cycle analyses for understanding growth inhibition

Cell division appears in the organisms to be an answer to the necessity of cell growth, and this uniform division of the all cell components allows an equilibrated growth of the organism. This process underlies growth and development and is central to the heredity and evolution of all life forms. Therefore, information about the mode of action to alter growth is central in the research of stress conditions and, of course, in allelopathy (Einhellig, 1994). At present, it is well known that allelochemical compounds can interfere with many vital processes of organisms. Some works have been performed using the light and electron microscopy to study the effects of

Flow Cytometry: Cell Cycle

Quantitative analyses of cell cycle can give essential information about the response of plants to short- or long-term abiotic or biotic stress, as most species alter leaf expansion or root growth as one of the first responses to cope with adverse environmental conditions (Boyer 1982). Tardieu and Granier (2000) observed a reduction of leaf area under water and light deficits due to partial blockage of nuclei in G1, which increased cell cycle duration and decreased final cell number. This effect can be detected shortly after the application of the stress and, sometimes, does not alter the photosynthetic rate, as is independent of carbon metabolism.

Molecular characterization and protein localization of the antimicrobial peptide big defensin from the scallop Argopecten purpuratus after Vibrio splendidus challenge

Abstract Big defensins are antimicrobial peptides (AMPs) that are proposed as important effectors of the immune response in mollusks, chelicerates and chordates. At present, only two members of the big defensin family have been identified in scallop. In the present work, a cDNA sequence encoding a new big defensin homologue was characterized from the scallop Argopecten purpuratus, namely ApBD1. ApBD1 cDNA sequence comprised 585 nucleotides, with an open reading frame of 375 bp and 5’‐ and 3′‐UTRs of 41 and 167 bp, respectively. The deduced protein sequence contains 124 amino acids with a molecular weight of 13.5 kDa, showing characteristic motifs of the big defensin family and presenting 76% identity with the big defensin from the scallop A. irradians. Phylogenetic analysis revealed that ApBD1 is included into the cluster of big defensins from mollusks. Tissue‐specific transcript expression analysis by RT‐qPCR showed that ApBD1 was present in all tissues tested from non‐immune challenged scallops but it was most strongly expressed in the mantle. The transcript levels of ApBD1 were significantly up‐regulated in gills at 24 and 48 h post‐injection with the heat‐attenuated bacteria Vibrio splendidus. Additionally, immunofluorescence analysis using a polyclonal anti‐ApBD1 antibody showed that this protein was abundantly located in epithelial linings of gills and mantle; and also in digestive gland showing ApBD1‐infiltrating hemocytes from immune challenged scallops. This is the first time that a big defensin is detected and located at the protein level in a mollusk. These results suggest an important role of ApBD1 in the mucosal immune response of A. purpuratus. HighlightsA new big defensin homologue, ApBD1, was characterized in Argopecten purpuratus.ApBD1 mRNA is constitutively present in all tissues and strongly expressed in mantle.ApBD1 mRNA is upregulated in gills of scallops challenged with Vibrio splendidus.ApBD1 protein is located in gills and mantle epithelia, digestive gland and infiltrating hemocytes.Results suggest an important role of ApBD1 in the mucosal immune response of A. purpuratus.