Characterization and expression analyses of the H⁺-pyrophosphatase gene in rye.

The H⁺-pyrophosphatase (H⁺-PPase) gene plays an important role in maintaining intracellular proton gradients. Here, we characterized the full-length complementary DNA (cDNA) and DNA of the H⁺-PPase gene ScHP1 in rye (Secale cereale L. 'Qinling'). We determined the subcellular localization of this gene and predicted the corresponding protein structure. We analysed the evolutionary relationship between ScHP1 and H⁺-PPase genes in other species, and did real-time quantitative polymerase chain reaction to explore the expression patterns of ScHP1 in rye plants subjected to N, P and K deprivation and to cold, high-salt and drought stresses. ScHP1 cDNA included a 2289 bp open reading frame (ORF) encoding 762 amino acid residues with 14 transmembrane domains. The genomic ScHP1 DNA was 4354 bp and contained eight exons and seven introns. ScHP1 was highly homologous with other members of the H⁺-PPase gene family. When the full-length ORF was inserted into the expression vector pA7-YFP, the fluorescent microscopy revealed that ScHP1-YFP fusion protein was located in the plasma membrane. Rye plants that were subjected to N deprivation, cold and high-salt stresses, ScHP1 expression was higher in the leaves than roots. Conversely, plants subjected to P and K deprivation and drought stress, ScHP1 expression was higher in the roots than leaves. Under all the investigated stress conditions, expression of ScHP1 was lower in the stem than in the leaves and roots. Our results imply that ScHP1 functions under abiotic stress response.

Oat (Avena sativa) seed extract as an antifungal food preservative through the catalytic activity of a highly abundant class I chitinase.

Extracts from different higher plants were screened for the ability to inhibit the growth of Penicillium roqueforti, a major contaminating species in industrial food processing. Oat (Avena sativa) seed extracts exhibited a high degree of antifungal activity and could be used directly on rye bread to prevent the formation of P. roqueforti colonies. Proteins in the oat seed extracts were fractionated by column chromatography and proteins in fractions containing antifungal activity were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database searches. Identified antifungal candidates included thaumatin-like proteins, 1,3-beta-glucanase, permatin precursor, pathogenesis-related protein type 1, and chitinases of class I and II. Class I chitinase could be specifically removed from the extracts and was found to be indispensable for 50% of the P. roqueforti inhibiting activity. The purified class I chitinase has a molecular weight of approximately 34 kDa, optimal chitinase activity at pH 7, and exists as at least two basic isoforms (pI values of 7.6 and 8.0). Partial sequencing of the class I chitinase isoforms by LC-MS/MS revealed a primary structure with high similarity to class I chitinases of wheat (Triticum aestivum), barley (Hordeum vulgare), and rye (Secale cereale). Oat, wheat, barley, and rye seed extracts were compared with respect to the abundance of the class I chitinase and decrease in antifungal activity when class I chitinase is removed. We found that the oat seed class I chitinase is at least ten times more abundant than the wheat, barley, and rye homologs and that oat seed extracts are highly active toward P. roqueforti as opposed to extracts of other cereal seeds.

Genetic analysis of a cross-pollinated species, Secale cereale L., for the character with polymorphic genetic basis.

Theoretical basis and an algorithm of calculations for frequencies of genes and genotypes in the population of species with obligate cross-pollination under assessment for the character of polymorphic genetic basis are proposed. Use of four rye populations and the F1 and F2 from their crossings allows to determine genetic control of beta-amylase spectrum through identification of two genes, establishment of the allele number in each gene, and showing the linkage between beta-Amy-1 and the gene for self-incompatibility S2.

Determination of phytase activity in cereal grains by direct incubation.

In contrast to supplemented microbial phytases, considerably lower cereal phytase activities were found after application of an enzyme solution extracted in citrate/NaOH buffer (pH 5.5) for 1 h as compared with the direct incubation of the plant material. Differences between both methods obtained were 70% for wheat and spelt and 50% for barley and rye. The determination of phytase activity of a wheat sample by direct incubation was affected by the sample size and amount of substrate (Na-phytate) added. Optimal conditions are a maximal level of phytase activity of < or = 0.05 U incubated for 60 min in a buffered solution (citrate/NaOH, pH 5.5) with 10 ml of 32.6 mmol/l Na-phytate at 37 composite function C. Possible reasons for the differences between both methods and for the factors affecting phytase activity by direct incubation are discussed.

Nutritional significance of phytic acid and phytase.

In the nutrition of monogastric animals phytate-P represents a poorly available source of phosphorus, especially in the case of diets low in phytase activity. Similarly the bioavailability of different minerals and trace elements is considerably reduced by phytate complexes. High concentrations of Ca increase the anti-nutritive effect of phytic acid on mineral and trace element bioavailability and thus impede the action of phytase. This effect can in part be compensated by an increased supply of vitamin D. There is also evidence for protective functions of phytic acid such as the prevention of the formation of free radicals, the delaying of post prandial glucose absorption, the decrease in plasma cholesterol and triglycerides as well as a change in the carry over of heavy metals. The basic mechanisms by which phytic acid may exert these effects are still not clear. In several studies reported in the literature, evidence for the nutritional significance and ecological importance of microbial phytase for pigs and poultry has been given. As the monogastric organism contains no or only negligible amounts of endogenous phytase in the stomach and small intestine, it is therefore dependent on plant or microbial phytase. Plant phytase, e.g. from rye, triticale, wheat or, in smaller amounts from barley, and supplemented Aspergillus-phytase display cumulative effects.

Chromosomal location of three wheat sequences with homology to pollen allergen encoding, DNA replication regulating, and DNA (cytosine-5)-methyltransferase genes in wheat and rye.

Three wheat sequences, shown to be homologous to pollen allergen encoding, DNA replication regulating, and DNA (cytosine-5)-methyltransferase genes were localized on chromosomes using nullisomic-tetrasomic wheat ('Chinese Spring') and wheat-rye ('Chinese Spring'/'Imperial') addition lines. Whereas the loci for the pollen allergen encoding sequence (Tri a III) were shown to be located on homoeologous group 4, the DNA replication regulating (Rep) and DNA (cytosine-5)-methyltransferase (Mtase) genes were located to homoeologous groups 1 and 7, respectively, of Triticeae. Chromosomal rearrangements in wheat and rye relative to each other are discussed.

PCR and sequencing from a single pollen grain.

In order to eliminate the laborious step of DNA extraction preceding all studies within the field of plant molecular biology we attempted to do PCR amplifications directly on pollen grains. Successful PCR amplification was obtained in reactions including a single pollen grain from Hordeum vulgare or Secale strictum. Both the plastid gene encoding ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) and the nuclear-encoded internal transcribed spacer regions (ITS) and the 5.8S rDNA region were amplified and sequenced to verify PCR amplification.