DEFENSE EVOLUTION IN THE GRACILARIACEAE (RHODOPHYTA): SUBSTRATE‐REGULATED OXIDATION OF AGAR OLIGOSACCHARIDES IS MORE ANCIENT THAN THE OLIGOAGAR‐ACTIVATED OXIDATIVE BURST1

Combined phylogenetic, physiological, and biochemical approaches revealed that differences in defense‐related responses among 17 species belonging to the Gracilariaceae were consistent with their evolutionary history. An oxidative burst response resulting from activation of NADPH oxidase was always observed in two of the subgenera of Gracilaria sensu lato (Gracilaria, Hydropuntia), but not in Gracilariopsis and in species related to Gracilaria chilensis (“chilensis” clade). On the other hand, all species examined except Gracilaria tenuistipitata var. liui and Gracilariopsis longissima responded with up‐regulation of agar oligosaccharide oxidase to an challenge with agar oligosaccharides. As indicated by pharmacological experiments conducted with Gracilaria chilensis and Gracilaria sp. “dura,” the up‐regulation of agar oligosaccharide oxidase involved an NAD(P)H‐dependent signaling pathway, but not kinase activity. By contrast, the activation of NADPH oxidase requires protein phosphorylation. Both responses are therefore independent, and the agar oligosaccharide‐activated oxidative burst evolved after the capacity to oxidize agar oligosaccharide, probably providing additional defensive capacity to the most recently differentiated clades of Gracilariaceae. As demonstrated with Gracilaria gracilis, Gracilaria dura, and Gracilariopsis longissima, the different responses to agar oligosaccharides allow for a fast and nondestructive distinction among different clades of gracilarioids that are morphologically convergent. Based upon sequences of the chloroplast‐encoded rbcL gene, this study suggests that at least some of the samples from NW America recorded as Gs. lemanaeiformis are probably Gs. chorda. Moreover, previous records of Gracilaria conferta from Israel are shown to be based upon misidentification of Gracilaria sp. “dura,” a species that belongs to the Hydropuntia subgenus.     

Effects of Zn and P addition on the microbial biomass in a Zn deficient calcareous soil amended with glucose

A 35-day laboratory incubation experiment at 25°C was carried out to investigate the effects of Zn and P addition on microbial biomass C, N, and P in a Zn deficient calcareous soil, sampled at 15–40 cm depth in Central Anatolia, Turkey, amended with glucose. The underlying hypothesis was that P, but also Zn addition leads to a decrease in the microbial biomass C/N ratio. In the glucose-amended soil, the microbial biomass C/N ratio was not affected by the addition of P at day 5. At day 35 in this treatment, the significant P addition × day interaction revealed a significant decrease in the microbial biomass C/N ratio from 11.3 to 8.9. In the glucose-amended soil, Zn addition also had generally significant negative effects on microbial biomass C in comparison with the pure glucose treatment. A similar tendency was observed for microbial biomass N and consequently the microbial biomass C/N ratio remained unaffected. No evidence was found in the present incubation experiment that the microbial community suffered from Zn deficiency.     

Inhibitory effects of sub-optimal root zone temperature on leaf bioactive components, photosystem II (PS II) and minerals uptake in Trichosanthes cucumerina L. Cucurbitaceae

We hypothesized that sub-optimal root zone temperature (RZT) will cause a reduction in the bioactive components contents, adversely affect PS II and hinder uptake/partitioning of mineral elements in the “Light Green Variant” of the African snake tomato (Trichosanthes cucumerina L.). Three RZT temperatures (20, 25 and 30°C) were evaluated in a digitally controlled growth chamber. Results showed that for all the mineral nutrients analyzed (Ca, Mg, P, K, Fe and Mn), the amounts absorbed by the plant increase as RZT increases with each nutrient displaying different characteristics with respect to the quantity partitioned into root, stem and leaf at the different RZT. At sub-optimal RZT (20°C), significantly higher amounts of Ca and K were found in the root, whereas at normal RZT (25 and 30°C) higher amounts of Ca were recorded in the stem, and about 50% of the amounts in the stem were found in the leaves. For all the RZT, the amounts of Mg in the leaves were significantly higher than in the root and stem, while the amounts in the stems were also significantly higher than the amounts in the roots. At normal RZT (25 and 30°C) almost equal amounts of P were present in the root, stem and leaf. The amounts of phenolics, ascorbic acid, chlorophyll a and b and total chlorophyll increase as the RZT increases. Photosystem analyses showed that at 30°C the F _v/F _m (relative photochemical efficiency) was 0.76, while at 20 and 25°C the values were 0.35 and 0.60, respectively. The F _v/F _m value (0.35) obtained at 20°C confirmed the adverse effects of sub-optimal RZT on the photosystem II (PS II). Photosynthetic measurements showed that as the RZT increased, A (net photosynthetic efficiency), E (transpiration rate), C _i (intercellular carbon dioxide concentration) and g ₁ (stomata conductance) also increased. We postulate that the higher E and g ₁ at high RZT have a great physiological implication on plant performance, because transpiration cooling would be improved, especially during the summer; but the lost water must be complemented by adequate irrigation. The totality of the results confirm our hypothesis that sub-optimal RZT will cause a reduction in the bioactive components contents, adversely affect PS II and hinder uptake/partitioning of mineral elements in T. cucumerina.     

Vegetative performance,leaf water potential,and partitioning of minerals and soluble sugars: Traits for ranking the NaCl-tolerance of tomato genotypes?

This study addresses the physiological response of four tomato genotypes with distinct sensitivity to high NaCl concentrations, with the aim of identifying physiological traits to rank the genotypes’ sensitivity to salt stress. The central hypothesis was that tomato genotypes grown in saline environments show a characteristic salinity-triggered absorption and translocation of ions, leading to a distinct distribution pattern of Na, K, and soluble sugars. Experiments were conducted on two commercial cultivars: one assumed to be sensitive (Solanum lycopersicum L. F1 hybrid Harzfeuer) and one known to be tolerant (S. lycopersicum L. var. edkawi) to high salt concentrations. Furthermore, two wild salt-tolerant relatives (S. pennellii and S. lycopersicum var. cerasiforme) were selected. Based on our results regarding vegetative performance and partitioning of Na, K, glucose, fructose, and sucrose, it is possible to classify the genotype S. lycopersicum F1 hybrid Harzfeuer as moderately sensitive to salt stress and the genotypes S. lycopersicum var. edkawi, S. lycopersicum var. cerasiforme, and S. pennellii as moderately resistant to 210 mM NaCl. Calculations of the percentage of modification revealed non-specific genotype responses for the amount of sodium in roots and leaves, as well as the sucrose concentration and the osmotic potential of leaves. As shown, the salt-induced changes in potassium levels in leaves, and glucose concentration in roots might be used as additional traits to discriminate genotypes regarding their salt-sensitivity. These parameters might be useful when comparisons of fruit production or vegetative performance provide no conclusive indication e.g. due to the distinct growth habits of commercial cultivars selected for fruit productivity and native genotypes having a stronger vegetative development. However, additional studies should be conducted to evaluate a large number of genotypes differing in their NaCl tolerance. Furthermore, plant responses considering the dynamic source-sink relations due to fruit load needs to be considered.     

Complementary retrieval of 16S rRNA gene sequences using broad-range primers with inosine at the 3'-terminus: implications for the study of microbial diversity

We evaluated the impact of the base analogue inosine substituted at the 3'-terminus of broad-range 16S rRNA gene primers on the recovery of microbial diversity using terminal restriction fragment length polymorphism and clonal analysis. Oral plaque biofilms from 10 individuals were tested with modified and unmodified primer pairs. Besides a core overlap of shared terminal restriction fragments (T-RFs), each primer system provided unique information on the occurrence of T-RFs, with a higher number generally displayed with inosine primers. All clones sequenced were at least 99% identical to publicly available full-length sequences. Analysis of the corresponding primer-binding sites showed that most sequence types were 100% complementary to the unmodified primers so that the characteristic of inosine to bind with all four nucleotides was not crucial for the observed increase in microbial richness. Instead, differences in community compositions were correlated with the identity of the nearest-neighbor 3' of the primer-targeting region. By influencing the thermal stability of primer hybridization, this position may play a previously unrecognized role in biased amplification of 16S rRNA gene sequences. In conclusion, the combined use of inosine and unmodified primers enables the complementary retrieval of 16S rRNA gene types, thereby expanding the observed diversity of complex microbial communities.     

Antisense tools for functional studies of human Argonaute proteins

The Argonaute proteins play essential roles in development and cellular metabolism in many organisms, including plants, flies, worms, and mammals. Whereas in organisms such as Caenorhabditis elegans and Arabidopsis thaliana, creation of Argonaute mutant strains allowed the study of their biological functions, in mammals the application of this approach is limited by its difficulty and in the specific case of Ago2 gene, by the lethality of such mutation. Hence, in human cells, functional studies of Ago proteins relied on phenotypic suppression using small interfering RNA (siRNA) which involves Ago proteins and the RNA interference mechanism. This bears the danger of undesired or unknown interference effects which may lead to misleading results. Thus, alternative methods acting by different regulatory mechanisms would be advantageous in order to exclude unspecific effects. The knockdown may be achieved by using specific antisense oligonucleotides (asONs) which act via an RNase H-dependent mechanism, not thought to interfere with processes in which Agos are involved. Different functional observations in the use of siRNA versus asONs indicate the relevance of this assumption. We developed asONs specific for the four human Agos (hAgos) and compared their activities with those obtained by siRNA. We confirm that hAgo2 is involved in microRNA (miRNA)- and in siRNA-mediated silencing pathways, while the other hAgos play a role only in miRNA-based gene regulation. Using combinations of asONs we found that the simultaneous down-regulation of hAgo1, hAgo2, and hAgo4 led to the strongest decrease in miRNA activity, indicating a main role of these proteins.     

A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor

Staphylococcus aureus and Staphylococcus epidermidis can cause serious chronic and recurrent infections that are difficult to eradicate. An important pathogenicity factor in these infections caused by S. aureus is its ability to be internalized by non‐professional phagocytes thereby evading the host immune system and antibiotic treatment. Here, we report a novel mechanism involved in staphylococcal internalization by host cells, which is mediated by the major autolysin/adhesins Atl and AtlE from S. aureus and S. epidermidis respectively. In a flow cytometric internalization assay, atl and atlE mutants are significantly reduced in their capacities to be internalized by endothelial cells. Moreover, pre‐incubation of endothelial cells with recombinant Atl dose‐dependently inhibited internalization. As putative Atl‐host cell receptor, the heat shock cognate protein Hsc70 was identified by mass spectrometry. The importance of Hsc70 in internalization was demonstrated by the inhibition of S. aureus internalization with anti‐Hsc70 antibodies. In conclusion, this novel Atl‐ or AtlE‐mediated internalization mechanism may represent a ‘back‐up’ mechanism in S. aureus internalization, while it may represent the major or even sole mechanism involved in the internalization of coagulase‐negative staphylococci and thus may play an important role in the pathogenesis of chronic and relapsing infections with these serious pathogens.