Alternative oxidase impacts the plant response to biotic stress by influencing the mitochondrial generation of reactive oxygen species

Previously, we showed that inoculation of tobacco with Pseudomonas syringae incompatible pv. maculicola results in a rapid and persistent burst of superoxide (O₂^‐) from mitochondria, no change in amount of mitochondrial alternative oxidase (AOX) and induction of the hypersensitive response (HR). However, inoculation with incompatible pv. phaseolicola resulted in increased AOX, no O₂^‐ burst and no HR. Here, we show that in transgenic plants unable to induce AOX in response to pv. phaseolicola, there is now a strong mitochondrial O₂^‐ burst, similar to that normally seen only with pv. maculicola. This interaction did not however result in a HR. This indicates that AOX amount is a key determinant of the mitochondrial O₂^‐ burst but also that the burst itself is not sufficient to induce the HR. Surprisingly, the O₂^‐ burst normally seen towards pv. maculicola is delayed in plants lacking AOX. This delay is associated with a delayed HR, suggesting that the burst does promote the HR. A O₂^‐ burst can also be induced by the complex III inhibitor antimycin A (AA), but is again delayed in plants lacking AOX. The similar mitochondrial response induced by pv. maculicola and AA suggests that electron transport is a target during HR‐inducing biotic interactions.     

Autoradiographic localization of angiotensin II receptors in developing rat cerebellum and brainstem

The role of Angiotensin II (Ang II) as a growth promoting or modulating factor has recently become a field of intensive research. A central issue in developmental neurobiology is the understanding of mechanisms governing the formation of spatially ordered connections. In this study, we show the localization of Ang II receptor subtypes by autoradiography in 2-week-old rat hindbrains confronting these data with membrane binding assays. Competition studies done on membrane preparations evidence no major changes on the relative affinities for both receptor subtypes between 2-week-old and adult rat tissues. By autoradiography, we found that all the areas (1-10) of the 2-week-old cerebellum showed both receptor subtypes present in complementary adjacent layers. Areas expressing a high level of AT2 receptors follow: inferior colicullus (IC), dorso tegmental nucleus, central (DTgC), subcoeruleus, alpha, sensory root of the trigeminal nerve, principal sensory root trigeminal nucleus (Pr5, Pr5VL) supragenual nucleus, genu facial nerve, facial nucleus, cerebellar peduncles, vestibular and lateral nuclei. Spinal trigeminal, (oral) and Raphe nuclei express AT1 receptor subtype. The high level of Ang II AT2 receptors present in the cerebellar peduncles might have a meaning on the establishment of the olivo-cerebellar connection. The high expression of Ang II AT2 receptors on 2-week-old rat hindbrains, a critical age on development, as well as its disappearance in the adult, strongly suggests a probable role of these receptors in cell migration and neuronal synaptogenesis.     

Effects of maternal captopril treatment during late pregnancy on neonatal lung development in rats

The renin-angiotensin system (RAS) has been implicated in pulmonary hypertension and pulmonary fibrosis. In the present study, we examined the effects of maternal exposure to captopril (2.85 mg/kg/day) during late pregnancy (G13-G21) on postnatal rat lung development. Treatment with captopril during late pregnancy caused a significant decrease in ACE activity in P0 rats. Body weight decreased at P0 (p<0.001), P8 and P15 (p<0.01) in captopril-treated rats. Lung weight of P0 and P8 pups was lower in treated-animals (p<0.05). Lungs from captopril-treated animals showed impaired alveolar formation, with enlarged distal airway spaces at P8, P15 and P30. Interalveolar wall distance measured by mean linear intercept increased in treated vs. age-matched animals at P8, P15 (p<0.001) and P30 (p<0.05) resembling new bronchopulmonary dysplasia. In control animals, the proliferating cell nuclear antigen (PCNA) marker was higher at P0 and then drops gradually, while in captopril-treated animals PCNA marker remains higher at all stages studied. α-Smooth muscle actin (α-SMA), a marker of fibroblast differentiation into myofibroblasts, was higher at the tips of developing secondary septa in captopril-treated lungs at P8 and P15. The increased expression of PCNA and α-SMA in treated pups suggest that beyond the effect caused by captopril, the developing lungs have the capacity to recover once the treatment was stopped. Taking together the low weight, histomorphological changes and increased expression of cellular markers caused by ACE inhibition during late pregnancy, it appears that the RAS could be an intrinsic factor involved in secondary septa formation during lung development.     

A molecular phylogenetic appraisal of the systematics of the Aglaopheniidae (Cnidaria: Hydrozoa,Leptothecata) from the north‐east Atlantic and west Mediterranean

The hydrozoan family Aglaopheniidae (Cnidaria) is widespread worldwide and contains some of the most easily recognizable hydroids because of their large colony size and characteristic microscopic structure. The systematics of the group has, however, been controversial and dedicated molecular analyses are lacking. We therefore analysed existing and new 16S rRNA sequences of Aglaopheniidae, in a total of 98 16S sequences corresponding to 25 putative species (25 nominal and three undescribed) from seven genera. The monophyly of the subfamilies Gymnangiinae and Aglaopheniinae, and tribes Aglaopheniini and Cladocarpini were not verified with 16S sequence data. The genera Gymnangium and Aglaophenia can only be considered valid if both Gymnangium gracicaule and Aglaophenia latecarinata are removed from their respective genera. The phenotypically similar Cladocarpus and Streptocaulus are probably monophyletic and clearly distinct genetically. The genus Lytocarpia may be polyphyletic. The nominal species Aglaophenia pluma, Aglaophenia tubiformis, and Aglaophenia octodonta are probably conspecific, as are also the species Aglaophenia acacia and Aglaophenia elongata. The 16S data revealed the existence of two potentially unnamed species of Aglaophenia respectively from the Azores and Madeira. The phylogeographical structure of the taxa with the greatest representation of haplotypes from the north‐east Atlantic and Mediterranean, revealed the influence of Mediterranean waters in Madeira and the Azores, and gene flow between deep waters of the Mediterranean and Atlantic. The last glaciations in Europe may have caused genetic bottlenecks but also high intraspecific haplotype diversity. Finally, Macrorhynchia philippina was detected in samples from Madeira and possibly represents an invasive species. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 164 , 717–727.     

Consequences of grazing on the vertical transmission of a fungal Neotyphodium symbiont in an annual grass population

Symbiosis between cool‐season grasses and vertically transmitted fungal endophytes are common and significantly impact on ecosystem function. This makes the understanding of the underlying mechanisms to symbiotic individuals frequency in local populations much more interesting. Most studies have been focused on the differential fitness between symbiotic and non‐symbiotic counterparts (relative fitness), barely considering other mechanisms. We performed a microcosms experiment to evaluate whether grazing alters the dynamics of the endophyte Neotyphodium occultans in the annual grass Lolium multiflorum by simultaneously modifying the relative fitness and the endophyte efficiency to be transmitted from host plants to seeds. Grazing was simulated by means of clipping and trampling on symbiotic and non‐symbiotic plants growing separately, in soils obtained from paddocks, differing in their agronomic management history (natural grassland vs. ryegrass promotion). Seed production showed a complex pattern as it depended on the symbiotic status of the plants, the level of grazing and the agro‐ecological context. Grazed plants produced three times fewer seeds than ungrazed plants only in microcosms with endophyte‐symbiotic plants in soils from ryegrass promotion. Endophyte benefits on seed production were exclusively observed in ungrazed plants in the same soil. Symbiotic plants produced symbiotic and non‐symbiotic seeds in all the treatments. While the production of non‐symbiotic seeds by these plants was not affected by grazing and the soil, grazing reduced the production of symbiotic seeds in both contexts. Grazing negative effect on the density of fully infected spikes determined a significant increment in the transmission failures which were not modified by agro‐ecological contexts. Therefore, grazing can modulate symbiosis dynamics through reducing seed production and endophyte transmission efficiency. Transmission has been disregarded, but it is a context‐dependent process that could lead to a gradual reduction in the symbiotic plants frequency in a population if the mutualism effectiveness does not outweigh transmission failures.     

The Torradovirus‐specific RNA2‐ORF1 protein is necessary for plant systemic infection

Tomato apex necrosis virus (ToANV, species Tomato marchitez virus, genus Torradovirus, family Secoviridae) causes a severe tomato disease in Mexico. One distinctive feature of torradoviruses compared with other members of the family Secoviridae is the presence of an additional open reading frame (ORF) in genomic RNA2 (denominated RNA2‐ORF1), located upstream of ORF2. RNA2‐ORF2 encodes a polyprotein that is processed into a putative movement protein and three capsid proteins (CPs). The RNA2‐ORF1 protein has homologues only amongst other torradoviruses and, so far, no function has been associated with it. We used recombinant and mutant ToANV clones to investigate the role of the RNA2‐ORF1 protein in various aspects of the virus infection cycle. The lack of a functional RNA2‐ORF1 resulted in an inability to systemically infect Nicotiana benthamiana and tomato plants, but both positive‐ and negative‐strand RNA1 and RNA2 accumulated locally in agroinfiltrated areas in N. benthamiana plants, indicating that the RNA2‐ORF1 mutants were replication competent. Furthermore, a mutant with a deletion in RNA2‐ORF1 was competent for virion formation and cell‐to‐cell movement in the cells immediately surrounding the initial infection site. However, immunological detection of the ToANV CPs in the agroinfiltrated areas showed that this mutant was not detected in the sieve elements even if the surrounding parenchymatic cells were ToANV positive, suggesting a role for the RNA2‐ORF1 protein in processes occurring prior to phloem uploading, including efficient spread in inoculated leaves.     

Involvement of c-Src tyrosine kinase in SHP-1 phosphatase activation by Ang II AT2 receptors in rat fetal tissues

Angiotensin II (Ang II) AT(2) receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10(-7) M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT(2) antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT(2) receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT(2) or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT(2) or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT(2) receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT(2) receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT(2) subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes.