Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial compounds and associate with the nucleus through tubular extensions called stromules. Although it is well established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1)-mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector-mediated suppression of BAK1-mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant–pathogen interactions.     

Immunohistochemical detection of proliferating cells in vivo

Incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrUdR) into newly synthesized DNA provides the basis of a simple technique for identifying proliferating cells. BrUdR was administered to C57BL/6 mice by continuous infusion for 1-7 days, or by intraperitoneal injection for shorter intervals. Various tissue types, including gut, kidney, and liver, were excised, fixed in neutral buffered formalin, and paraffin-embedded for sectioning. De-paraffinized 4-micron tissue sections and bone marrow samples were incubated with an anti-BrUdR antibody and cells that had traversed S-phase during the BrUdR exposure period were identified immunohistochemically. Proliferation and migration of intestinal epithelial cells were identified by antibody staining after continuous in vivo exposure to BrUdR for 1-4 days, and BrUdR incorporation into proliferating marrow cells was detected within 30 min. Tissues such as normal liver, known to have low levels of proliferation, remained unstained after 3 days' exposure to BrUdR. After we established that normal proliferating cells could be identified using this technique, BrUdR was administered to mice bearing B16 melanomas. Again, proliferating tumor cells were clearly identified in histological sections. The nuclei from these paraffin-embedded tumors were also collected for flow cytometric analysis after de-waxing, rehydration, and pepsin treatment. This combination of techniques made possible the comparison in adjacent tissue sections of labeling index, obtained from stained sections, with percentage S-phase, measured using DNA flow cytometry. The % S-phase was consistently higher than the labeling index obtained with immunocytochemistry, and two-parameter DNA vs BrUdR flow cytometry showed that this difference could be accounted for by a population of unlabeled cells with an S-phase DNA content.     

Carbon isotope effect on the enzymatic decarboxylation of pyruvic acid

The decarboxylation of pyruvic acid by the thiamine pyrophosphate dependent pyruvate decarboxylase from brewer's yeast is accompanied by a carboxyl carbon isotope effect $\text{k}{}^{12}\text{k}{}^{13}\text{= 1.0083±0.0003}$ at 25°, pH 6.8. The small size of the isotope effect indicates that decarboxylation is not rate-determining in the overall reaction. The rate constant for decarboxylation of the enzyme-bound pyruvate-thiamine pyrophosphate complex is greater by about a factor of five than the rate constant for dissociation of this complex to form free pyruvate and the enzyme-thiamine pyrophosphate complex.     

Indirect consequences of fishing: reduction of coralline algae suppresses juvenile coral abundance

Removing predatory fishes has effects that cascade through ecosystems via interactions between species and functional groups. In Kenyan reef lagoons, fishing-induced trophic cascades produce sea urchin-dominated grazing communities that greatly reduce the overall cover of crustose coralline algae (CCA). Certain species of CCA enhance coral recruitment by chemically inducing coral settlement. If sea urchin grazing reduces cover of settlement-inducing CCA, coral recruitment and hence juvenile coral abundance may also decline on fished reefs. To determine whether fishing-induced changes in CCA influence coral recruitment and abundance, we compared (1) CCA taxonomic compositions and (2) taxon-specific associations between CCA and juvenile corals under three fisheries management systems: closed, gear-restricted, and open-access. On fished reefs (gear-restricted and open-access), abundances of two species of settlement-inducing CCA, Hydrolithon reinboldii and H. onkodes, were half those on closed reefs. On both closed and fished reefs, juveniles of four common coral families (Poritidae, Pocilloporidae, Agariciidae, and Faviidae) were more abundant on Hydrolithon than on any other settlement substrate. Coral densities were positively correlated with Hydrolithon spp. cover and were significantly lower on fished than on closed reefs, suggesting that fishing indirectly reduces coral recruitment or juvenile success over large spatial scales via reduction in settlement-inducing CCA. Therefore, managing reefs for higher cover of settlement-inducing CCA may enhance coral recruitment or juvenile survival and help to maintain the ecological and structural stability of reefs.