Structure of Haze Forming Proteins in White Wines: Vitis vinifera Thaumatin-Like Proteins

Grape thaumatin-like proteins (TLPs) play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62°C), with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions.     

Functional molecular aspects of the NADH dehydrogenases of plant mitochondria

There are multiple routes of NAD(P)H oxidation associated with the inner membrane of plant mitochondria. These are the phosphorylating NADH dehydrogenase, otherwise known as Complex I, and at least four other nonphosphorylating NAD(P)H dehydrogenases. Complex I has been isolated from beetroot, broad bean, and potato mitochondria. It has at least 32 polypeptides associated with it, contains FMN as its prosthetic group, and the purified enzyme is sensitive to inhibition by rotenone. In terms of subunit complexity it appears similar to the mammalian and fungal enzymes. Some polypeptides display antigenic similarity to subunits fromNeurospora crassa but little cross-reactivity to antisera raised against some beef heart complex I subunits. Plant complex I contains eight mitochondrial encoded subunits with the remainder being nuclear-encoded. Two of these mitochondrial-encoded subunits, nad7 and nad9, show homology to corresponding nuclear-encoded subunits inNeurospora crassa (49 and 30 kDa, respectively) and beef heart CI (49 and 31 kDa, respectively), suggesting a marked difference between the assembly of CI from plants and the fungal and mammalian enzymes. As well as complex I, plant mitochondria contain several type-II NAD(P)H dehydrogenases which mediate rotenone-insensitive oxidation of cytosolic and matrix NADH. We have isolated three of these dehydrogenases from beetroot mitochondria which are similar to enzymes isolated from potato mitochondria. Two of these enzymes are single polypeptides (32 and 55 kDa) and appear similar to those found in maize mitochondria, which have been localized to the outside of the inner membrane. The third enzyme appears to be a dimer comprised of two identical 43-kDa subunits. It is this enzyme that we believe contributes to rotenone-insensitive oxidation of matrix NADH. In addition to this type-II dehydrogenases, several observations suggest the presence of a smaller form of CI present in plant mitochondria which is insensitive to rotenone inhibition. We propose that this represents the peripheral arm of CI in plant mitochondria and may participate in nonphosphorylating matrix NADH oxidation.     

Effect of Lincocin Treatment on the Greening Process in Bean (Phaseolus vulgaris) Leaves

The effect of lincocin (a plastid protein synthesis inhibitor) treatment on the greening process of bean (Phaseolus vulgaris L.) leaves have been studied. In comparison with control leaves treated ones had a decreased rate of chloroplast development. They had a marked chlorophyll deficiency and a decreased chlorophyll a/b ratio. Some long and short wavelength forms of chlorophyll a were lacking as evidenced from the absorption spectra at 25°C and the fluorescence spectra at 77°K. The –¹⁴CO₂ fixation was inhibited by 80–90% in treated leaves. The fluorescence induced by the measuring light was greater in the treated leaves than in the control ones, and the kinetics of the decline of the relative fluorescence intensity were also different. Electron microscopic studies showed macrogranum-like structures and incomplete membrane vesicles in the treated plastids. After longer treatment a destruction of membranes was observed. The results indicate some structural and functional membrane deficiencies and instability of the membranes.     

Effect of cryoprotective agents on rat cutaneous nerves.

Desheathed rat cutaneous nerves were exposed to various concentrations of ethylene glycol (EG), glycerol and dimethyl sulfoxide (DMSO) at temperatures of 1, 24, and 38 °C for periods of time ranging from 5 to 60 min. Measurements of the percent recovery of the original action potential (AP) were determined after removal of the cryoprotective agent (CPA) under various conditions, i.e., temperature, time and sequence of rinsing. A comparison of the results obtained after the nerves were exposed directly to a 15% concentration of the three CPAs at 1 °C for a 15-min period showed that the percentage of recovery of the AP was 90, 69, and 36% of the original values when treated with DMSO, EG, or glycerol, respectively. In all three groups, the nerves were rinsed at 1 °C for 15 min. If the exposure to glycerol at 1 °C was carried out in a gradual stepwise manner, the recovery of the AP in 10 and 15% solutions ranged from 58 to 64%. If the temperatures of the exposure and rinse were increased to 24 and 38 °C, glycerol produced some toxicity within 10 min and after 25 min no recovery of AP was obtained. The results of a 10-min direct exposure to EG at 1 °C showed a moderate decrease in recovery of the AP as the concentration was increased from 10 to 15–20%. Increasing the exposure time to 15 and 30 min at 1 °C also contributed to further reduction in recovery. DMSO, however, in concentrations of 10, 15, and 20% produced only a slight decline of AP after a 5–15 min exposure at 1 °C. Recovery ranged from 96% after 10 min in a 10% solution to 88% after 15 min in a 20% solution. Toxicity became more apparent with DMSO when nerves were exposed to 30% concentrations for 5–10 min; the latter time resulted in a 49% recovery of the AP. Exposure of nerves to a CPA solution containing isotonic concentrations of electrolytes resulted in a 10–30% improvement in recovery when compared with specimens treated with lower levels of salt. The effect of raising the temperature of the rinse to 38 °C and increasing the wash time to 20 min was studied in a few selected experiments. After a direct 15-min exposure to a 15% solution of a CPA at 1 °C the recovery in the case of glycerol was significantly increased with such treatment whereas with EG and DMSO it remained unchanged. There was no evidence of thermal or cold shock in this work.     

A molecular cytogenetic map of sorghum chromosome 1. Fluorescence in situ hybridization analysis with mapped bacterial artificial chromosomes

We used structural genomic resources for Sorghum bicolor (L.) Moench to target and develop multiple molecular cytogenetic probes that would provide extensive coverage for a specific chromosome of sorghum. Bacterial artificial chromosome (BAC) clones containing molecular markers mapped across sorghum linkage group A were labeled as probes for fluorescence in situ hybridization (FISH). Signals from single-, dual-, and multiprobe BAC-FISH to spreads of mitotic chromosomes and pachytene bivalents were associated with the largest sorghum chromosome, which bears the nucleolus organizing region (NOR). The order of individual BAC-FISH loci along the chromosome was fully concordant to that of marker loci along the linkage map. In addition, the order of several tightly linked molecular markers was clarified by FISH analysis. The FISH results indicate that markers from the linkage map positions 0.0-81.8 cM reside in the short arm of chromosome 1 whereas markers from 81.8-242.9 cM are located in the long arm of chromosome 1. The centromere and NOR were located in a large heterochromatic region that spans approximately 60% of chromosome 1. In contrast, this region represents only 0.7% of the total genetic map distance of this chromosome. Variation in recombination frequency among euchromatic chromosomal regions also was apparent. The integrated data underscore the value of cytological data, because minor errors and uncertainties in linkage maps can involve huge physical regions. The successful development of multiprobe FISH cocktails suggests that it is feasible to develop chromosome-specific "paints" from genomic resources rather than flow sorting or microdissection and that when applied to pachytene chromatin, such cocktails provide an especially powerful framework for mapping. Such a molecular cytogenetic infrastructure would be inherently cross-linked with other genomic tools and thereby establish a cytogenomics system with extensive utility in development and application of genomic resources, cloning, transgene localization, development of plant "chromonomics," germplasm introgression, and marker-assisted breeding. In combination with previously reported work, the results indicate that a sorghum cytogenomics system would be partially applicable to other gramineous genera.     

Eosinophils express functional IL-13 in eosinophilic inflammatory diseases

IL-13 is an immunoregulatory and effector cytokine in allergic diseases such as bronchial asthma. A variety of immune and non-immune cells are known as IL-13 producers. In this study we investigated whether and under what conditions human eosinophils generate IL-13. Freshly isolated highly purified peripheral blood eosinophils from patients with several eosinophilic inflammatory diseases and from normal control individuals were investigated. We observed that blood eosinophils from patients suffering from bronchial asthma, atopic dermatitis, parasitic infections, hypereosinophilic syndrome, and idiopathic eosinophilic esophagitis expressed IL-13, as assessed by ELISA, ELISPOT assay, flow cytometry, and immunocytochemistry. By using nasal polyp tissues and immunohistochemistry, we demonstrated IL-13 expression in eosinophils under in vivo conditions. In contrast, blood eosinophils from control individuals as well as blood neutrophils from both eosinophilic and control patients did not produce detectable IL-13 levels. However, when blood eosinophils from control individuals were stimulated with GM-CSF or IL-5 in vitro, they generated IL-13 mRNA and protein, suggesting that IL-13 expression by eosinophils under inflammatory conditions is a cytokine-driven process. Stimulation of blood eosinophils containing IL-13 by eotaxin resulted in a rapid release of this cytokine. Eosinophil-derived IL-13 was functional, as it increased the surface expression of the low affinity IgE receptor (CD23) on purified B cells. In conclusion, human eosinophils are able to produce and release functional IL-13 in eosinophilic inflammatory responses.     

QTL analysis of genotype × environment interactions affecting cotton fiber quality

Cotton is unusual among major crops in that large acreages are grown under both irrigated and rainfed conditions, making genotype x environment interactions of even greater importance than usual in designing crop-improvement strategies. We describe the impact of well-watered versus water-limited growth conditions on the genetic control of fiber quality, a complex suite of traits that collectively determine the utility of cotton. Fiber length, length uniformity, elongation, strength, fineness, and color (yellowness) were influenced by 6, 7, 9, 21, 25 and 11 QTLs (respectively) that could be detected in one or more treatments. The genetic control of cotton fiber quality was markedly affected both by general differences between growing seasons ("years") and by specific differences in water management regimes. Seventeen QTLs were detected only in the water-limited treatment while only two were specific to the well-watered treatment, suggesting that improvement of fiber quality under water stress may be even more complicated than improvement of this already complex trait under well-watered conditions. In crops such as cotton with widespread use of both irrigated and rainfed production systems, the need to manipulate larger numbers of genes to confer adequate quality under both sets of conditions will reduce the expected rate of genetic gain. These difficulties may be partly ameliorated by efficiencies gained through identification and use of diagnostic DNA markers, including those identified herein.     

Pollinator rarity as a threat to a plant with a specialized pollination system

An increasing diversity of highly specialized pollination systems are being discovered, many of which are likely to be vulnerable to anthropogenic landscape modification. Here, we investigate if a specialized pollination system limits the persistence of Caladenia huegelii (Orchidaceae), an endangered species pollinated by sexual deception of thynnine wasps. Once locally common in part of its geographical range, C. huegelii is now largely restricted to small habitat remnants in urban areas. Pollinator surveys coupled with DNA barcoding detected a single pollinator taxon, a small form of Macrothynnus insignis. Phylogenetic analysis revealed that small M. insignis from within the range of C. huegelii are strongly divergent from other wasp populations, suggesting that some reproductive isolation may exist. Although common in intact landscapes outside the range of C. huegelli, small M. insignis individuals were recorded at only 4% of sites in suitable C. huegelii habitat. Accordingly, reproductive success in C. huegelii was low compared with related Caladenia spp., with 33–60% of populations failing to set fruit in any given year. As such, populations are likely to now persist primarily through individual plant longevity rather than reproduction. Due to the low reproductive success of C. huegelii, ongoing human intervention will almost certainly be needed to sustain the species. Future research will need to focus on optimizing hand pollination to maintain reproduction and high seed fitness. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 511–525.