Loss of Starch Granule Initiation Has a Deleterious Effect on the Growth of Arabidopsis Plants Due to an Accumulation of ADP-Glucose

STARCH SYNTHASE4 (SS4) is required for proper starch granule initiation in Arabidopsis (Arabidopsis thaliana), although SS3 can partially replace its function. Unlike other starch-deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under long-day conditions. They have less chlorophyll and carotenoids than the wild type and lower maximal rates of photosynthesis. There is evidence of photooxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose (Glc) than wild-type plants. Restricting ADP-Glc synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1) or the small subunit of ADP-Glc pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4 and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-Glc in the ss3/ss4 mutant sequesters a large part of the plastidial pools of adenine nucleotides, which limits photophosphorylation, leading to photooxidative stress, causing the chlorotic and stunted growth phenotypes of the plants.The metabolism of starch plays an essential role in the physiology of plants. Starch breakdown provides the plant with carbon skeletons and energy when the photosynthetic machinery is inactive (transitory starch) or in the processes of germination and sprouting (storage starch). Deficiencies in the accumulation of transitory starch in Arabidopsis (Arabidopsis thaliana) have been described previously, specifically in mutants affected in the plastidial phosphoglucomutase (PGM1) or the small subunit (APS1) of the ADP-Glc pyrophosphorylase (AGPase). While they are described as “starchless,” they actually contain small amounts of starch (1%–2% of the wild-type levels; Streb et al., 2009) and share similar phenotypic alterations, such as growth retardation when cultivated under a short-day photoregime and increased levels of soluble sugars during the light phase and reduced levels during the night (Caspar et al., 1985; Lin et al., 1988b; Schulze et al., 1991). Carbon partitioning is altered in these plants. As photosynthate cannot be accumulated as starch, it is diverted via hexose phosphates in the cytosol to the synthesis of Suc, which accumulates together with the hexose sugars, Glc and Fru (Caspar et al., 1985). In Arabidopsis, there are five starch synthase isoforms: one granule-bound starch synthase and four soluble starch synthases: SS1, SS2, SS3, and SS4. We have described previously an Arabidopsis mutant plant lacking SS3 and SS4 that is also severely affected in the accumulation of starch (Szydlowski et al., 2009). SS4 is involved in the initiation of the starch granule and controls the number of granules per chloroplast (Roldán et al., 2007). The elimination of SS3 in an ss4 background leads to an absence of starch in most of the chloroplasts, despite the fact that SS1 and SS2 are still present and total starch synthase activity is only reduced by 35% (Szydlowski et al., 2009). However, a very small proportion of chloroplasts of this mutant plant contain a single huge starch granule, which is also a characteristic of chloroplasts in the ss4 single mutant (D’Hulst and Mérida, 2012). Thus, like aps1 and pgm1, ss3/ss4 plants contain only small amounts of starch. However, unlike aps1 or pgm1 plants, most of the cells of this mutant have empty chloroplasts, without starch (Szydlowski et al., 2009).In this work, we have analyzed the phenotypic effects of the impaired starch accumulation of ss3/ss4 plants. We show that this mutant displays phenotypic changes that are not found in other mutants with very low levels of starch, such as aps1 or pgm1 plants. We provide evidence that extremely high levels of ADP-Glc accumulate in the ss3/ss4 plants. Using reverse genetics to block the pathway of starch synthesis upstream of the starch synthases reduced the level of ADP-Glc in ss3/ss4 plants and reverted the other phenotypic traits. This suggests that ADP-Glc accumulation is the causal factor behind the chlorotic and stunted growth phenotypes of the ss3/ss4 mutant.     

Fungal biomass and decomposition in Spartina maritima leaves in the Mondego salt marsh (Portugal)

Spartina maritima (Curtis) Fernald is a dominant species in the Mondego salt marsh on the western coast of Portugal, and it plays a significant role in estuarine productivity. In this work, leaf litter production dynamics and fungal importance for leaf decomposition processes in Spartina maritima were studied. Leaf fall was highly seasonal, being significantly higher during dry months. It ranged from 42 g m^-2 in June to less than 6 g m^-2 during the winter. Fungal biomass, measured as ergosterol content, did not differ significantly between standing-decaying leaves and naturally detached leaves. Fungal biomass increased in wet months, with a maximum of 614 g g^-1 of ergosterol in January in standing-decaying leaves, and 1077 g g^-1 in December, in naturally detached leaves, decreasing greatly in summer. Seasonal pattern of fungal colonization was similar in leaves placed in litterbags on the marsh-sediment surface. However, ergosterol concentrations associated with standing-decaying and naturally detached leaves were always much higher than in litterbagged leaves, suggesting that fungal activity was more important before leaf fall. Dry mass of litterbagged leaves declined rapidly after 1 month (about 50%), mostly due to leaching of soluble organic compounds. After 13 months, Spartina leaves had lost 88% of their original dry weight. The decomposition rate constant (k) for Spartina maritima leaves was 0.151 month^-1.     

The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.     

Distinctive patterns of DNA methylation associated with Parkinson disease

Parkinson disease (PD) is a multifactorial neurodegenerative disorder with high incidence in the elderly, where environmental and genetic factors are involved in etiology. In addition, epigenetic mechanisms, including deregulation of DNA methylation have been recently associated to PD. As accurate diagnosis cannot be achieved pre-mortem, identification of early pathological changes is crucial to enable therapeutic interventions before major neuropathological damage occurs. Here we investigated genome-wide DNA methylation in brain and blood samples from PD patients and observed a distinctive pattern of methylation involving many genes previously associated to PD, therefore supporting the role of epigenetic alterations as a molecular mechanism in neurodegeneration. Importantly, we identified concordant methylation alterations in brain and blood, suggesting that blood might hold promise as a surrogate for brain tissue to detect DNA methylation in PD and as a source for biomarker discovery.     

Quantitative evaluation of siRNA delivery in vivo

Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.     

Cell-free propagation of prion strains

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). Disease is transmitted by the autocatalytic propagation of PrP(Sc) misfolding at the expense of the normal prion protein. The biggest challenge of the prion hypothesis has been to explain the molecular mechanism by which prions can exist as different strains, producing diseases with distinguishable characteristics. Here, we show that PrP(Sc) generated in vitro by protein misfolding cyclic amplification from five different mouse prion strains maintains the strain-specific properties. Inoculation of wild-type mice with in vitro-generated PrP(Sc) caused a disease with indistinguishable incubation times as well as neuropathological and biochemical characteristics as the parental strains. Biochemical features were also maintained upon replication of four human prion strains. These results provide additional support for the prion hypothesis and indicate that strain characteristics can be faithfully propagated in the absence of living cells, suggesting that strain variation is dependent on PrP(Sc) properties.     

A population genomics insight into the Mediterranean origins of wine yeast domestication

The domestication of the wine yeast Saccharomyces cerevisiae is thought to be contemporary with the development and expansion of viticulture along the Mediterranean basin. Until now, the unavailability of wild lineages prevented the identification of the closest wild relatives of wine yeasts. Here, we enlarge the collection of natural lineages and employ whole‐genome data of oak‐associated wild isolates to study a balanced number of anthropic and natural S. cerevisiae strains. We identified industrial variants and new geographically delimited populations, including a novel Mediterranean oak population. This population is the closest relative of the wine lineage as shown by a weak population structure and further supported by genomewide population analyses. A coalescent model considering partial isolation with asymmetrical migration, mostly from the wild group into the Wine group, and population growth, was found to be best supported by the data. Importantly, divergence time estimates between the two populations agree with historical evidence for winemaking. We show that three horizontally transmitted regions, previously described to contain genes relevant to wine fermentation, are present in the Wine group but not in the Mediterranean oak group. This represents a major discontinuity between the two populations and is likely to denote a domestication fingerprint in wine yeasts. Taken together, these results indicate that Mediterranean oaks harbour the wild genetic stock of domesticated wine yeasts.