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.     

Functional and biochemical analysis of the N-terminal domain of phytochrome A

Phytochrome A (phyA) is a versatile plant photoreceptor that mediates responses to brief light exposures (very low fluence responses, VLFR) as well as to prolonged irradiation (high irradiance responses, HIR). We identified the phyA-303 mutant allele of Arabidopsis thaliana bearing an R384K substitution in the GAF subdomain of the N-terminal half of phyA. phyA-303 showed reduced phyA spectral activity, almost normal VLFR, and severely impaired HIR. Recombinant N-terminal half oat of PHYA bearing the phyA-303 mutation showed poor incorporation of chromophore in vitro, despite the predicted relatively long distance (>13 A) between the mutation and the closest ring of the chromophore. Fusion proteins bearing the N-terminal domain of oat phyA, beta-glucuronidase, green fluorescent protein, and a nuclear localization signal showed physiological activity in darkness and mediated VLFR but not HIR. At equal protein levels, the phyA-303 mutation caused slightly less activity than the fusions containing the wild-type sequence. Taken together, these studies highlight the role of the N-terminal domain of phyA in signaling and of distant residues of the GAF subdomain in the regulation of phytochrome bilin-lyase activity.     

Epithelial cadherin is present in bovine oviduct epithelial cells and gametes,and is involved in fertilization-related events

Fertilization is a calcium-dependent process that involves sequential cell–cell adhesion events of spermatozoa with oviduct epithelial cells (OECs) and with cumulus-oocyte complexes (COCs). Epithelial cadherin (E-cadherin) participates in calcium-dependent somatic cell adhesion; the adaptor protein β-catenin binds to the E-cadherin cytoplasmic domain and links the adhesion protein to the cytoskeleton. The study was conducted to immunodetect E-cadherin and β-catenin in bovine gametes and oviduct (tissue sections and OEC monolayers), and to assess E-cadherin participation in fertilization-related events. Epithelial cadherin was found in spermatozoa, oocytes, cumulus cells, and OEC. In acrosome-intact noncapacitated spermatozoa, E-cadherin was mainly localized in the apical ridge and acrosomal cap (E1-pattern; 84 ± 9%; mean ± standard deviation of the mean). After sperm treatment with heparin to promote capacitation, the percentage of cells with E1-pattern (56 ± 12%) significantly decreased; concomitantly, the percentage of spermatozoa depicting an E-cadherin staining pattern similar to E1-pattern but showing a signal loss in the acrosomal cap (E2-pattern: 40 ± 11%) increased. After l-α-lysophosphatidylcholine–induced acrosome reaction, E-cadherin signal was mainly localized in the inner acrosomal membrane (E3-pattern: 67 ± 22%). In IVM COC, E-cadherin was immunodetected in the plasma membrane of cumulus cells and oocytes, but was absent in the polar body. The 120 KDa mature protein form was found in protein extracts from spermatozoa, oocytes, cumulus cells, and OEC. β-Catenin distribution followed E-cadherin's in all cells evaluated. Epithelial cadherin participation in cell–cell interaction was evaluated using specific blocking monoclonal antibody DECMA-1. Sperm incubation with DECMA-1 impaired sperm–OEC binding (the number of sperm bound to OEC: DECMA-1 = 6.7 ± 6.1 vs. control = 29.6 ± 20.1; P < 0.001), fertilization with COC (% fertilized COC: DECMA-1 = 68.8 ± 10.4 vs. control = 90.7 ± 3.1; P < 0.05) or denuded oocytes (% fertilized oocytes: DECMA-1 = 57.0 ± 15.2 vs. control = 89.2 ± 9.8; P < 0.05) and binding to the oolemma (the number of sperm bound to oolemma: DECMA-1 = 2.2 ± 1.1 vs. control = 11.1 ± 4.8; P < 0.05). This study describes, for the first time, the presence of E-cadherin in bovine spermatozoa, COC, and OEC, and shows evidence of its participation in sperm interaction with the oviduct and the oocyte during fertilization.     

Morphological plasticity associated with environmental hypoxia in characiform fishes from neotropical floodplain lakes

The ability to develop reversible dermal extensions on the lower jaw in some South American characiform fishes has been proposed as a way to optimize the performance of aquatic surface respiration (ASR) during hypoxic conditions. These structures are formed by edema of the hypodermal tissues and can develop in a large proportion of individuals inhabiting a lake within a few hours following daily changes in dissolved oxygen. Our study report the development of dermal lip protuberances in eleven species of characiform fishes associated with periods of strong environmental hypoxia in floodplain lakes of Salado River, Argentina. Protuberances occurred in different basic forms in fishes with divergent head morphology (genera Astyanax, Ctenobrycon, Aphyocharax, Brycon, Mylossoma, Triportheus, Oligosarcus and Acestrorhynchus). The discovery of dermal projections on the anterior border of maxillary bone extends the known range of structures affected by lip protuberances. Dermal structures began to develop simultaneously in both jaws below dissolved oxygen (DO) concentrations of 1.20–1.75 mgl⁻¹ and rose in a steep manner as oxygen level decreased. The degree of morphological plasticity differed among traits and species. The shape of response of morphology to DO was similar to that previously reported on ASR, providing additional evidence about the functional link between these traits. Our results suggests that dermal lip protuberances are widely spread among characiform fishes, affecting several mouth structures. The different types of protuberances would make up for the limitations imposed by body size and mouth shape and position on the performance of ASR in fishes with divergent morphology.     

Cysteine-rich secretory proteins (CRISP) and their role in mammalian fertilization

Epididymal protein CRISPI is a member of the CRISP (Cysteine-RIch Secretory proteins) family and is involved in sperm-egg fusion through its interaction with complementary sites on the egg surface. Results from our laboratory have shown that this binding ability resides in a 12-amino-acid region corresponding to a highly conserved motif of the CRISP family, named Signature 2 (S2). In addition to this, our results revealed that CRISP1 could also be involved in the previous step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. As another approach to elucidate the participation of CRISP1 in fertilization, a mouse line containing a targeted disruption of CRISP1 was generated. Although CRISP1-deficient mice exhibited normal fertility, CRISP1-defficient sperm presented a decreased level of protein tyrosine phosphorylation during capacitation, and an impaired ability to fertilize both zona-intact and zona-free eggs in vitro, confirming the proposed roles for the protein in fertilization. Evidence obtained in our laboratory indicated that testicular CRISP2 would also be involved in sperm-egg fusion. Competition assays between CRISP1 and CRISP2, as well as the comparison of their corresponding S2 regions, suggest that both proteins bind to common complementary sites in the egg. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization.     

Environmental context shapes the bacterial community structure associated to Peltigera cyanolichens growing in Tierra del Fuego,Chile

The structure of the associated bacterial community of bipartite cyanolichens of the genus Peltigera from three different environmental contexts in the Karukinka Natural Park, Tierra del Fuego, Chile, was assessed. The sampling sites represent different habitat contexts: mature native forest, young native forest and grassland. Recently it has been determined that the bacterial community associated to lichens could be highly structured according to the mycobiont or photobiont identities, to the environmental context and/or to the geographic scale. However, there are some inconsistencies in defining which of these factors would be the most significant on determining the structure of the microbial communities associated with lichens, mainly because most studies compare the bacterial communities between different lichen species and/or with different photobiont types (algae vs. cyanobacteria). In this work bipartite lichens belonging to the same genus (Peltigera) symbiotically associated with cyanobacteria (Nostoc) were analyzed by TRFLP to determine the structure of the bacterial community intimately associated with the lichen thalli and the one present in the substrate where they grow. The results indicate that the bacterial community intimately associated differs from the one of the substrate, being the former more influenced by the environmental context where the lichen grows.     

Genomic relationships between hexaploid Helianthus resinosus and diploid Helianthus annuus (Asteraceae)

Genus Helianthus comprises diploid and polyploid species. An autoallopolyploid origin has been proposed for hexaploid species but the genomic relationships remain unclear. Mitotic and meiotic studies in annual Helianthus annuus (2n = 2x = 34) and perennial Helianthus resinosus (2n = 6x = 102) as well as the F₁ hybrids between both species were carried out. Chromosome counting confirmed the hybrid origin of the latter plants and their tetraploid condition. Bivalents in hybrids ranged from 12 to 28 ( $ \bar{x} $  = 20.8). Univalents, trivalents and quadrivalents were also observed. Meiotic products comprised dyads, triads and normal tetrads and pollen grains were heterogeneous in size. These observations suggest the occurrence of 2n pollen in addition to the expected n. Genomic in situ hybridization (GISH) of total H. annuus DNA on H. resinosus chromosomes rendered weak but uniform signals; similar hybridization pattern was observed using three other annual species. Hybridization with H. annuus probe performed on root tip cells of F₁ H. annuus × H. resinosus hybrids revealed 17 chromosomes with a strong hybridization signal. GISH in hybrid meiocytes distinguished chromosomes from parental species and revealed autosyndetic pairing of H. resinosus chromosomes, allosyndetic pairing in bivalents, trivalents and quadrivalents, and the presence of univalents derived from parents, H. annuus and H. resinosus. Results obtained from classical and molecular cytogenetics do not support H. annuus as a direct ancestor of H. resinosus. The occurrence of allosyndetic pairing and the relatively high fertility of the F₁ hybrids point to the possibility that useful genes could be transferred from H. resinosus to cultivate sunflower, although the effective rate of recombination has not been evaluated. GISH method proved effective to recognize parental chromosomes in H. annuus × H. resinosus progeny.     

Variation in sclerophylly among Iberian populations of Quercus coccifera L. is associated with genetic differentiation across contrasting environments

Evergreen oaks are an emblematic element of the Mediterranean vegetation and have a leaf phenotype that seems to have remained unchanged since the Miocene. We hypothesise that variation of the sclerophyll phenotype among Iberian populations of Quercus coccifera is partly due to an ulterior process of ecotypic differentiation. We analysed the genetic structure of nine Iberian populations using ISSR fingerprints, and their leaf phenotypes using mean and intracanopy plasticity values of eight morphological (leaf angle, area, spinescence, lobation and specific area) and biochemical traits (VAZ pool, chlorophyll and β-carotene content). Climate and soil were also characterised at the population sites. Significant genetic and phenotypic differences were found among populations and between NE Iberia and the rest of the populations of the peninsula. Mean phenotypes showed a strong and independent correlation with both genetic and geographic distances. Northeastern plants were smaller, less plastic, with smaller, spinier and thicker leaves, a phenotype consistent with the stressful conditions that prevailed in the steppe environments of the refugia within this geographic area during glaciations. These genetic, phenotypic, geographic and environmental patterns are consistent with previously reported palaeoecological and common evidence. Such consistency leads us to conclude that there has been a Quaternary divergence within the sclerophyllous syndrome that was at least partially driven by ecological factors.     

INTRALOCUS SEXUAL CONFLICT AND ENVIRONMENTAL STRESS

Intralocus sexual conflict (IaSC) occurs when selection at a given locus favors different alleles in males and females, placing a fundamental constraint on adaptation. However, the relative impact of IaSC on adaptation may become reduced in stressful environments that expose conditionally deleterious mutations to selection. The genetic correlation for fitness between males and females (r_MF) provides a quantification of IaSC across the genome. We compared IaSC at a benign (29°C) and a stressful (36°C) temperature by estimating r_MFs in two natural populations of the seed beetle Callosobruchus maculatus using isofemale lines. In one population, we found substantial IaSC under benign conditions signified by a negative r_MF (?0.51) and, as predicted, a significant reduction of IaSC under stress signified by a reversed and positive r_MF (0.21). The other population displayed low IaSC at both temperatures (r_MF: 0.38; 0.40). In both populations, isofemale lines harboring alleles beneficial to males but detrimental to females at benign conditions tended to show overall low fitness under stress. These results offer support for low IaSC under stress and suggest that environmentally sensitive and conditionally deleterious alleles that are sexually selected in males mediate changes in IaSC. We discuss implications for adaptive evolution in sexually reproducing populations.