Patterns of Starchy Endosperm Acidification and Protease Gene Expression in Wheat Grains following Germination

The family of 14-3-3 proteins is ubiquitous in eukaryotes and has been shown to exert an array of functions. We were interested in the possible role of 14-3-3 proteins in seed germination. Therefore, we studied the expression of 14-3-3 mRNA and protein in barley (Hordeum distichum L.) embryos during germination. With the use of specific cDNA probes and antibodies, we could detect individual expression of three 14-3-3 isoforms, 14-3-3A, 14-3-3B, and 14-3-3C. Each homolog was found to be expressed in barley embryos. Whereas protein levels of all three isoforms were constant during germination, mRNA expression was found to be induced upon imbibition of the grains. The induction of 14-3-3A gene expression during germination was different from that of 14-3-3B and 14-3-3C. In situ immunolocalization analysis showed similar spatial expression for 14-3-3A and 14-3-3B, while 14-3-3C expression was markedly different. Whereas 14-3-3A and 14-3-3B were expressed throughout the embryo, 14-3-3C expression was tissue specific, with the strongest expression observed in the scutellum and the L2 layer of the shoot apical meristem. These results show that 14-3-3 homologs are differently regulated in barley embryos, and provide a first step in acquiring more knowledge about the role of 14-3-3 proteins in the germination process.     

Structural requirements of carbohydrates to bind Agaricus bisporus lectin

Galbeta1-3GalNAc (T-disaccharide) and related molecules were assayed to describe the structural requirements of carbohydrates to bind Agaricus bisporus lectin (ABL). Results provide insight into the most relevant regions of T-disaccharide involved in the binding of ABL. It was found that monosaccharides bind ABL weakly indicating a more extended carbohydrate-binding site as compared to those involvedin the T- disaccharide specific lectins such as jacalin and peanut agglutinin. Lacto-N-biose (Galbeta1-3GlcNAc) unlike T-disaccharide, is unable to inhibit the ABL interaction, thus showing the great importance of the position of the axial C-4 hydroxyl group of GalNAc in T-disaccharide. This finding could explain the inhibitory ability of Galbeta1-6GlcNAc and lactose because C-4 and C-3 hydroxyl groups of reducing Glc, respectively, occupy a similar position as reported by conformational analysis. From the comparison of different glycolipids bearing terminal T-disaccharide bound to different linkages, it can be seen than ABL binding is even more impaired by an adjacent C-6 residual position than by the anomeric influence of T-disaccharide. Furthermore, the addition of beta-GlcNAc to the terminal T-disaccharide in C-3 position of Gal does not affect the ABL binding whereas if an anionic group such as glucuronic acid is added to C-3, the binding is partially affected. These findings demonstrate that ABL holds a particular binding nature different from that of other T-disaccharide specific lectins.      

Immunoreactivity of the <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> 19-kDa lipoprotein

Background  

The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed.     

Overexpression of Aquaporin-1 in lung adenocarcinomas and pleural mesotheliomas

Aquaporin-1 (AQP1) is the main water channel responsible for water transport through many epithelia and endothelia. The latest evidence pointed toward an important role of this protein also in gas permeation, angiogenesis, cell proliferation and migration. In the present work we studied the expression of AQP1 by immunohistochemical staining of 92 lung biopsies from patients diagnosed with a pleuro-pulmonary tumor (71 lung and 21 pleural neoplasms). AQP1 expression was analyzed comparing the results among the different histological patterns and against 9 control cases (5 parenchyma and 4 healthy pleura). Clear staining of AQP1 was detected in 39 of the 92 tumors analyzed. In parenchyma, AQP1 was more frequently detected in primary lung adenocarcinomas (55%, P<0.001); in contrast, small cell carcinomas were the least AQP1 expressive tumors studied (93% of negative staining, P<0.05). Carcinomas analyzed in pleura (mesotheliomas and metastatic adenocarcinomas) also revealed strong expression of AQP1. High expression of this protein was detected in small capillaries in areas near or surrounding the tumor, and novel intense AQP1 immunostaining was detected over thicker alveolar walls in alveoli inside or next to the tumoral tissue regardless of the tumor type. An important role of AQP1 in tumor angiogenesis is sustained by the abundant expression of this protein in the endothelia of tumor capillaries. Further studies are necessary to elucidate the potential pathophysiological role of this protein in pleuro-pulmonary neoplasms.     

A comparative analysis of the NADPH thioredoxin reductase C-2-Cys peroxiredoxin system from plants and cyanobacteria

Redox regulation based on disulfide-dithiol conversion catalyzed by thioredoxins is an important component of chloroplast function. The reducing power is provided by ferredoxin reduced by the photosynthetic electron transport chain. In addition, chloroplasts are equipped with a peculiar NADPH-dependent thioredoxin reductase, termed NTRC, with a joint thioredoxin domain at the carboxyl terminus. Because NADPH can be produced by the oxidative pentose phosphate pathway during the night, NTRC is important to maintain the chloroplast redox homeostasis under light limitation. NTRC is exclusive for photosynthetic organisms such as plants, algae, and some, but not all, cyanobacteria. Phylogenetic analysis suggests that chloroplast NTRC originated from an ancestral cyanobacterial enzyme. While the biochemical properties of plant NTRC are well documented, little is known about the cyanobacterial enzyme. With the aim of comparing cyanobacterial and plant NTRCs, we have expressed the full-length enzyme from the cyanobacterium Anabaena species PCC 7120 as well as site-directed mutant variants and truncated polypeptides containing the NTR or the thioredoxin domains of the protein. Immunological and kinetic analysis showed a high similarity between NTRCs from plants and cyanobacteria. Both enzymes efficiently reduced 2-Cys peroxiredoxins from plants and from Anabaena but not from the cyanobacterium Synechocystis. Arabidopsis (Arabidopsis thaliana) NTRC knockout plants were transformed with the Anabaena NTRC gene. Despite a lower content of NTRC than in wild-type plants, the transgenic plants showed significant recovery of growth and pigmentation. Therefore, the Anabaena enzyme fulfills functions of the plant enzyme in vivo, further emphasizing the similarity between cyanobacterial and plant NTRCs.     

Plant responses to fungal volatiles involve global posttranslational thiol redox proteome changes that affect photosynthesis

Microorganisms produce volatile compounds (VCs) that promote plant growth and photosynthesis through complex mechanisms involving cytokinin (CK) and abscisic acid (ABA). We hypothesized that plants' responses to microbial VCs involve posttranslational modifications of the thiol redox proteome through action of plastidial NADPH‐dependent thioredoxin reductase C (NTRC), which regulates chloroplast redox status via its functional relationship with 2‐Cys peroxiredoxins. To test this hypothesis, we analysed developmental, metabolic, hormonal, genetic, and redox proteomic responses of wild‐type (WT) plants and a NTRC knockout mutant (ntrc) to VCs emitted by the phytopathogen Alternaria alternata. Fungal VC‐promoted growth, changes in root architecture, shifts in expression of VC‐responsive CK‐ and ABA‐regulated genes, and increases in photosynthetic capacity were substantially weaker in ntrc plants than in WT plants. As in WT plants, fungal VCs strongly promoted growth, chlorophyll accumulation, and photosynthesis in ntrc–Δ2cp plants with reduced 2‐Cys peroxiredoxin expression. OxiTRAQ‐based quantitative and site‐specific redox proteomic analyses revealed that VCs promote global reduction of the thiol redox proteome (especially of photosynthesis‐related proteins) of WT leaves but its oxidation in ntrc leaves. Our findings show that NTRC is an important mediator of plant responses to microbial VCs through mechanisms involving global thiol redox proteome changes that affect photosynthesis.