The varied ability of grains to synthesize and catabolize ABA is one of the factors affecting dormancy and its release by after-ripening in imbibed triticale grains of cultivars with different pre-harvest sprouting susceptibilities.

Abscisic acid (ABA) is a phytohormone involved in the acquisition of primary dormancy during seeds maturation as well as dormancy maintenance in imbibed seeds. After imbibition, the ABA content decreased to a much lower level in embryos of freshly harvested triticale grains of the Leontino cultivar, which is more susceptible to pre-harvest sprouting (PHS) than embryos of the Fredro cultivar. Lower ABA content in the Leontino cultivar resulted from increased expression of TsABA8'OH1 and TsABA8'OH2, which encode ABA 8'-hydroxylase and are involved in ABA catabolism.

Structural and functional characterization of the triticale (x Triticosecale Wittm.) phytocystatin TrcC-8 and its dimerization-dependent inhibitory activity.

Phytocystatins are a group of proteins with significant potential to regulate activities of cysteine proteinases of native and pest/pathogen origins. The two-domain triticale (x Triticosecale Wittm.) phytocystatin TrcC-8 was characterized in this study.

The roles of cysteine proteases and phytocystatins in development and germination of cereal seeds.

Proteolysis is an important process for development and germination of cereal seeds. Among the many types of proteases identified in plants are the cysteine proteases (CPs) of the papain and legumain families, which play a crucial role in hydrolysing storage proteins during seed germination as well as in processing the precursors of these proteins and the inactive forms of other proteases. Moreover, all of the tissues of cereal seeds undergo progressive degradation via programed cell death, which is integral to their growth.

Abscisic acid content and the expression of genes related to its metabolism during maturation of triticale grains of cultivars differing in pre-harvest sprouting susceptibility.

Abscisic acid (ABA) is a plant hormone that plays a predominant role in the onset and maintenance of primary dormancy. Peak ABA accumulation in embryos of triticale grains was observed before any significant loss of water and was higher in Fredro, a cultivar less susceptible to pre-harvest sprouting (PHS), than in Leontino, a cultivar more sensitive to PHS. At full maturity, embryonic ABA content in Fredro was twice as high as in Leontino.

Analysis of expression and inhibitory activity of a TrcC-6 phytocystatin present in developing and germinating seeds of triticale (×Triticosecale Wittm.).

Storage proteins of cereal seeds are processed during accumulation and degraded during germination primarily by cysteine proteinases. One of the mechanisms controlling the activity of these enzymes is the synthesis of specific inhibitors named phytocystatins. Here we present the complete gene sequence of a triticale ( × Triticosecale Wittm.

A triticale water-deficit-inducible phytocystatin inhibits endogenous cysteine proteinases in vitro.

Water-deficit is accompanied by an increase in proteolysis. Phytocystatins are plant inhibitors of cysteine proteinases that belong to the papain and legumain family. A cDNA encoding the protein inhibitor TrcC-8 was identified in the vegetative organs of triticale.

tyrB-2 and phhC genes of Pseudomonas putida encode aromatic amino acid aminotransferase isozymes: evidence at the protein level.

Two Pseudomonas putida aminotransferases (ArAT I and ArAT II) that exhibit activity toward L-tryptophan were purified 104- and 395-fold using a six-stage purification procedure involving ammonium sulfate fractionation and chromatographic separation on phenyl-Sepharose, Sephadex G-100 superfine, DEAE-cellulose and Protein-Pack Q8 HR columns. Mass spectrometry analysis resulted in the identification of 27 and 20 % of the total ArAT I and ArAT II amino acid sequences. In addition, N-terminal sequence fragments of ArAT I and ArAT II were determined using the Edman degradation method.

A simple method for simultaneous RP-HPLC determination of indolic compounds related to bacterial biosynthesis of indole-3-acetic acid.

In this short technical report, we present a fast and simple procedure for sample preparation and a single-run Reversed Phase High Performance Liquid Chromatography (RP-HPLC) determination of seven indoles (indole-3-acetic acid, indole-3-acetamide, indole-3-acetonitrile, indole-3-ethanol, indole-3-lactic acid, tryptamine and tryptophan) in bacterial culture supernatants. The separation of the analytes, after a single centrifugal filtration clean-up step, was performed using a gradient elution on a symmetry C8 column followed by fluorimetric detection (λ(ex) = 280/λ(em) = 350 nm). The calibration curves were linear for all of the studied compounds over the concentration range of 0.

Carboxypeptidase I from triticale grains and the hydrolysis of salt-soluble fractions of storage proteins.

Carboxypeptidase I was purified from triticale grains (×Triticosecale Wittm.) by a 5-step purification procedure including gel filtration, cation-exchange chromatography and affinity chromatography. The enzyme was purified 595.

TsPAP1 encodes a novel plant prolyl aminopeptidase whose expression is induced in response to suboptimal growth conditions.

A triticale cDNA encoding a prolyl aminopeptidase (PAP) was obtained by RT-PCR and has been designated as TsPAP1. The cloned cDNA is 1387 bp long and encodes a protein of 390 amino acids with a calculated molecular mass of 43.9 kDa.

Molecular Cloning and Expression Analysis of Triticale Phytocystatins During Development and Germination of Seeds.

Three triticale cDNAs encoding inhibitors of cysteine endopeptidases, belonging to phytocystatins, have been identified and designated as , and . Full-length cDNAs of (617 bp) and (940 bp), as well as a fragment of cDNA (369 bp), were obtained. A high-level identity of the deduced amino acid sequence of TrcCs with other known phytocystatins, especially with wheat and barley, has been observed.

Biochemical characterisation of prolyl aminopeptidase from shoots of triticale seedlings and its activity changes in response to suboptimal growth conditions.

Prolyl aminopeptidase (PAP) was isolated from the shoots of three-day-old triticale seedlings and was purified using a five-step purification procedure (acid precipitation, gel filtration, anion-exchange chromatography, hydrophobic chromatography and rechromatography). The enzyme was purified 460-fold with a recovery of 6%. Prolyl aminopeptidase appears to be a tetramer consisting of four subunits, each with a molecular weight of approximately 54kDa.

Isolation and characterization of carboxypeptidase III from germinating triticale grains.

Carboxypeptidase III from germinating triticale grains was purified 434.2-fold with a six-step procedure including: homogenization, ammonium sulfate precipitation, cation-exchange chromatography on CM-cellulose, gel filtration chromatography on Sephadex G-150, cation-exchange chromatography on SP8HR column (HPLC), and affinity chromatography on CABSSepharose 4B. Triticale carboxypeptidase III is a monomer with a molecular weight of 45 kDa, which optimally hydrolyzes peptides at temperature 30-50 degrees C and pH 4.

[Mobilization of storage proteins in cereal grains].

Mobilization of seed reserves is a gradual process leading to the total degradation of accumulated biopolymers. In cereals cysteine endopeptidases and serine carboxypeptidases play essential role in hydrolysis of storage proteins. Peptidases of other catalytic groups seem to take part in regulatory processes or various processes that are not directly connected with storage proteins breakdown in the endosperm of germinating grains.

The properties and functions of bacterial aminopeptidases.

Aminopeptidases are enzymes that release N-terminal amino residues from oligopeptides, polypeptides and proteins. The classification of aminopeptidases has often been based on mechanism of catalysis, structure of active site, substrate specificity kinetic and molecular properties. In terms of catalytic mechanism bacterial aminopeptidases can be divided into three main catalytic groups: metallo-, cysteine- and serine aminopeptidases.

Ability of Pseudomonas sp. to synthesize aminopeptidases in the presence of various carbon and nitrogen sources.

A soil strain of Pseudomonas sp. is able to synthesize at least two aminopeptidases exhibiting high activity in the presence of Phe-beta-NA and Ala-beta-NA as substrates. Irrespective of the used substrate, total activity of studied enzymes was strongly related to concentrations of organic components (peptone, glutamic acid, glucose) in mineral media and was the higher, the higher the concentration.

Purification and properties of phenylalanyl aminopeptidase synthesised by Pseudomonas sp.

Intracellular aminopeptidase synthesized by a soil strain of Pseudomonas sp. was purified 323-fold using the following procedure: saturation with ammonium sulfate, separation by preparative electrophoresis, anion-exchange chromatography and gel filtration chromatography. Molecular weight of the enzyme determined according to the latter method was 57 kDa.