Selection of Single-Chain Antibodies against the VP8* Subunit of Rotavirus VP4 Outer Capsid Protein and Their Expression in Lactobacillus casei

Single-chain antibodies (scFv) recognizing the VP8* fraction of rotavirus outer capsid and blocking rotavirus infection in vitro were isolated by phage display. Vectors for the extracellular expression in Lactobacillus casei of one of the scFv were constructed. L. casei was able to secrete active scFv to the growth medium, showing the potential of probiotic bacteria to be engineered to express molecules suitable for in vivo antirotavirus therapies.     

Effect of the synthetic auxin 2,4-DP on fruit development of loquat

Application of the butylglycol ester of 2,4-DP increased final fruit size in loquat without causing fruit thinning. Its effectiveness depended on the concentration applied and treatment date. When applied at the onset of the cell enlargement fruit stage, 25 mg l^–1 was the most effective treatment. Fruit diameter distribution showed a significant shift to the larger size for treated trees; further, fruit colour break and maturation were encouraged and harvest time was earlier than in untreated trees.     

Tree age estimates in Fagus sylvatica and Quercus robur: testing previous and improved methods

The accuracy of direct (based on increment cores) and indirect (based on age-size relationships) methods of tree age estimation in Fagus sylvatica and Quercus robur was tested. This was done through increment cores and stem discs taken in an old-growth forest of Northern Spain. It was found that cross-dating was more precise than ring counting by up to 7 years per tree. Furthermore, cross-dating permitted the estimation of the age of trees with floating ring-width series, which were 7% of cored F. sylvatica and 40% of Q. robur ones. In partial cores with the arcs of the inner rings, the length of the missing radius was estimated with both a geometric method, based on the curvature of the arcs, and a new graphical method, based on the convergence of xylem rays at the pith. The graphical method was more accurate when the radial growth was eccentric, as happens in Q. robur, while both methods showed a similar accuracy for F. sylvatica, whose growth is relatively concentric. Empirical models of initial radial growth (IRG), built to estimate the number of missing rings, reduced the errors associated with other methods that assume constant growth rates. Age estimates obtained from the graphical method combined with the IRG models were within 4% of the actual age. This combination ensured age estimates with a mean accuracy of 8 years for 98% of the F. sylvatica trees, and 4 years for 89% of the Q. robur. In partial cores without the arcs of the inner rings, the length of the missing radius was estimated as the distance to the geometric centre of the tree. In that case, age estimates obtained by extrapolating the mean growth rate of the 20 innermost rings in the cores were from 10 to 20% of actual age, which coincided with results obtained in other tree species with this method. Finally, the age-diameter equations of the different cohorts produced better age estimates (from 8 to 14% of actual age) than equations of the population as a whole (from 20 to 40% of actual age). These results proved that the errors derived from doubtful assumptions, such as concentric radial growth, constant growth and recruitment rates, or the absence of anomalous rings, could be reduced by applying more realistic methods of tree age estimation.     

CONSTITUTIVE PHOTOMORPHOGENIC 1 promotes seed germination by destabilizing RGA-LIKE 2 in Arabidopsis

Under favorable moisture, temperature, and light conditions, gibberellin (GA) biosynthesis is induced and triggers seed germination. A major mechanism by which GA promotes seed germination is by promoting the degradation of the DELLA protein RGA-LIKE 2 (RGL2), a major repressor of germination in Arabidopsis (Arabidopsis thaliana) seeds. Analysis of seed germination phenotypes of constitutive photomorphogenic 1 (cop1) mutants and complemented COP1-OX/cop1-4 lines in response to GA and paclobutrazol (PAC) suggested a positive role for COP1 in seed germination and a relation with GA signaling. cop1-4 mutant seeds showed PAC hypersensitivity, but transformation with a COP1 overexpression construct rendered them PAC insensitive, with a phenotype similar to that of rgl2 mutant (rgl2-SK54) seeds. Furthermore, cop1-4 rgl2-SK54 double mutants showed a PAC-insensitive germination phenotype like that of rgl2-SK54, identifying COP1 as an upstream negative regulator of RGL2. COP1 interacted directly with RGL2, and in vivo this interaction was strongly enhanced by SUPPRESSOR OF PHYA-105 1. COP1 directly ubiquitinated RGL2 to promote its degradation. Moreover, GA stabilized COP1 with consequent RGL2 destabilization. By uncovering this COP1–RGL2 regulatory module, we reveal a mechanism whereby COP1 positively regulates seed germination and controls the expression of germination-promoting genes.

A master regulator of photomorphogenesis positively regulates germination in Arabidopsis seeds by directly ubiquitinating and promoting the degradation of a key repressor of seed germination.     

Drought-induced increase in water-use efficiency reduces secondary tree growth and tracheid wall thickness in a Mediterranean conifer

In order to understand the impact of drought and intrinsic water-use efficiency (iWUE) on tree growth, we evaluated the relative importance of direct and indirect effects of water availability on secondary growth and xylem anatomy of Juniperus thurifera, a Mediterranean anisohydric conifer. Dendrochronological techniques, quantitative xylem anatomy, and ¹³C/¹²C isotopic ratio were combined to develop standardized chronologies for iWUE, BAI (basal area increment), and anatomical variables on a 40-year-long annually resolved series for 20 trees. We tested the relationship between iWUE and secondary growth at short-term (annual) and long-term (decadal) temporal scales to evaluate whether gains in iWUE may lead to increases in secondary growth. We obtained a positive long-term correlation between iWUE and BAI, simultaneously with a negative short-term correlation between them. Furthermore, BAI and iWUE were correlated with anatomical traits related to carbon sink or storage (tracheid wall thickness and ray parenchyma amount), but no significant correlation with conductive traits (tracheid lumen) was found. Water availability during the growing season significantly modulated tree growth at the xylem level, where growth rates and wood anatomical traits were affected by June precipitation. Our results are consistent with a drought-induced limitation of tree growth response to rising CO₂, despite the trend of rising iWUE being maintained. We also remark the usefulness of exploring this relationship at different temporal scales to fully understand the actual links between iWUE and secondary growth dynamics.     

Proteome analysis of primary neurons and astrocytes from rat cerebellum

Neurons and astrocytes are predominant cell types in brain and have distinguished morphological and functional features. Although several proteomics studies were carried out on the brain, work on individual brain cells is limited. Generating individual proteomes of neurons and astrocytes, however, is mandatory to assign protein expression to cell types rather than to tissues. We aimed to provide maps of rat primary neurons and astrocytes using two-dimensional gel electrophoresis with subsequent in-gel digestion, followed by MALDI-TOF/TOF. 428 protein spots corresponding to 226 individual proteins in neurons and 406 protein spots representing 228 proteins in astrocytes were unambiguously identified. Proteome data include proteins from several cascades differentially expressed in neurons and astrocytes, and specific expressional patterns of antioxidant, signaling, chaperone, cytoskeleton, nucleic acid binding, proteasomal, and metabolic proteins are demonstrated. We herein present a reference database of primary rat primary neuron and astrocyte proteomes and provide an analytical tool for these structures. The concomitant expressional patterns of several protein classes are given and potential neuronal and astrocytic marker candidates are presented.     

Pleistocene paleoenvironmental evolution at continental middle latitude inferred from carbon and oxygen stable isotope analysis of ostracodes from the Guadix-Baza Basin (Granada, SE Spain)

A representative paleoenvironmental reconstruction of continental middle latitude from ca. 2 my to the upper part of Middle Pleistocene (279 ± 77 ky) was obtained from the carbon and oxygen stable isotopes analyzed in ostracode shells (Cyprideis torosa) recovered in the Guadix-Baza Basin (SE Spain), an intramontaneous closed depression filled by alluvial and lacustrine sediments. This study was performed along a 356-m-thick composite section, dated previously by paleomagnetism and the amino acid racemization method. δ¹³C and δ¹⁸O profiles reflected changes in temperature, the evaporation/infill ratio in the water bodies and the amount of rain. δ¹³C is also affected by changes in plant biomass: periods with high δ¹³C and δ¹⁸O values are associated with warm and dry regimes, and with less vegetation, which, in some cases, coincide with the development of displacive gypsum crystals, whereas low δ¹³C and δ¹⁸O values correlate with cold and humid episodes, which cause more vegetation biomass and, therefore, increasing the input of isotopically light carbon. Intermediate δ¹⁸O values are linked to temperate dry or humid episodes when they coincide with high or low δ¹³C values, respectively. 86 paleoclimatic events were distinguished in the Pleistocene record from the δ¹³C and δ¹⁸O profiles. From both the statistical analysis of the geochemical data and the geological observations, four Cold and Humid Long Periods (low δ¹⁸O) and four Warm and Dry Long Periods (high δ¹⁸O) were defined. This differs with respect to the paleoclimatological behavior established for the Northern Hemisphere where during cold periods (glacial), no water was available while permafrost conditions persisted, whereas in warm episodes (interglacial), higher precipitation rates occurred. Good correspondences between the Guadix-Baza Basin paleoclimatic record and a marine oxygen-isotope sequence, two continental cores and other long Mediterranean paleoenvironmental records (pollen sequences from Israel) were found, which suggested that climate changes in the Guadix-Baza Basin were in tune with global climatic changes.     

Isolation and selection of plant growth-promoting rhizobacteria as inducers of systemic resistance in melon

Backgroud and aims

Powdery mildew elicited by Podosphaera fusca is an important threat to cucurbits. In order to find alternatives to the current use of chemicals, we examined the potential use of plant growth-promoting rhizobacteria (PGPR) for controlling the disease by induction of systemic resistance in the host plant.

Methods

A collection of Bacillus and Pseudomonas strains from different origins was studied, including strains isolated from roots of disease-free melon plants obtained from a greenhouse plagued by powdery mildew. The selection of best candidates was based on the evaluation of different traits commonly associated with PGPR, such as antifungal and siderophore production, swimming and swarming motilities, biofilm formation, auxin production and promotion of root development.

Results

Three Bacillus strains, B. subtilis UMAF6614 and UMAF6639 and B. cereus UMAF8564, and two Pseudomonas fluorescens strains, UMAF6031 and UMAF6033, were selected after ranking the strains using a nonparametric statistics test. Applied to melon seedlings, the selected strains were able to promote plant growth, increasing fresh weight up to 30%. Furthermore, these strains provided protection against powdery mildew and also against angular leaf spot caused by Pseudomonas syringae pv. lachrymans, with disease reductions of up to 60%.

Conclusions

These results suggest that the use of ISR-promoting PGPR could be a promising strategy for the integrated control of cucurbit powdery mildew and other cucurbit diseases.