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. Higher ABA content and maintenance of dormancy in Fredro grains were correlated with intensified ABA biosynthesis, which resulted from higher expression of TsNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase. These results suggest that grains of triticale cultivars with different resistance to PHS vary in their ability to metabolize ABA after imbibition. After-ripening did not affect the ABA content in embryos of dry grains of either triticale cultivar. However, after-ripening caused dormancy release in Fredro grains and significantly affected the ABA content and the rate of its metabolism after imbibition. A more rapid decline in ABA content in imbibed Fredro grains was accompanied by decreased transcript levels of TsNCED1 as well as increased expression of TsABA8'OH1 and TsABA8'OH2. Thus, after-ripening may affect dormancy of grains through reduction of the ABA biosynthesis rate and intensified ABA catabolism. Overexpression of TsNCED1 in tobacco increases ABA content and delays germination, while overexpression of TsABA8'OH2 decreases ABA content, accelerates germination, and reduces the sensitivity to ABA of transgenic seeds compared to seeds of wild-type plants. Therefore, these genes might play an important role in the regulation of triticale grain dormancy, thus affecting susceptibility to PHS.

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. This protein belongs to the second group of phytocystatins and contains all the conserved sequences and motifs as well as both N-terminal (CY) and C-terminal (CY-L) domains that are characteristic of phytocystatins with the C-terminal extension. We demonstrated that TrcC-8 forms stable dimers with a significantly reduced inhibitory activity against papain compared to the activity of monomers, indicating the regulatory nature of the oligomerization. Moreover, according to our research, only the N-terminal domain possesses the ability to form dimers, indicating that this part of TrcC-8 is involved in the dimerization of the full-length protein. Homology modelling of TrcC-8 strongly suggests distinct specificities for the CY and CY-L domains, confirmed in experiments with inhibition of the papain. Our results suggest that the CY domain of TrcC-8 may, although markedly weakly and suboptimally, interact with papain in an analogous mode to tarocystatin, while the CY-L domain of TrcC-8 has distinct specificity than tarocystatin.

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. Two full-length cDNAs of 9-cis-epoxycarotenoid dioxygenase (TsNCED1, TsNCED2), an enzyme involved in ABA biosynthesis, and two full-length cDNAs of ABA 8'-hydroxylase (TsABA8'OH1 and TsABA8'OH2), an enzyme involved in ABA catabolism, were identified in triticale grains and characterized. The maximum transcript level of both TsNCED1 and TsNCED2 preceded the peak of ABA accumulation, suggesting that both TsNCEDs contribute to reach this peak, although the expression of TsNCED1 was significantly higher in Fredro than in Leontino. High expression of TsABA8'OH2 and TsABA8'OH1 was observed long before and at the end of the ABA accumulation peak, respectively, but no differences were observed between cultivars. The obtained results suggest that mainly TsNCED1 might be related to the higher ABA content and higher resistance of Fredro to PHS. However, Fredro embryos not only have higher ABA content, but also exhibit greater sensitivity to ABA, which may also have a significant effect on grain dormancy and lower susceptibility to PHS for grains of this cultivar.

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. In view of the important roles played by proteases, their uncontrolled activity could be harmful to the development of seeds and young seedlings. Thus, the activities of these enzymes are regulated by intracellular inhibitors called phytocystatins (PhyCys). The phytocystatins inhibit the activity of proteases of the papain family, and the presence of an additional motif in their C-termini allows them to also regulate the activity of members of the legumain family. A balance between the levels of cysteine proteases and phytocystatins is necessary for proper cereal seed development, and this is maintained through the antagonistic activities of gibberellins (GAs) and abscisic acid (ABA), which regulate the expression of the corresponding genes. Transcriptional regulation of cysteine proteases and phytocystatins is determined by cis-acting elements located in the promoters of these genes and by the expression of their corresponding transcription factors (TFs) and the interactions between different TFs.

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.) phytocystatin, TrcC-6, which encodes a 152-amino acid protein with a putative 25-amino acid signal peptide. This protein has a calculated molecular mass of 16.2 kDa, and was assigned to phylogenetic group B of phytocystatins. Because TrcC-6 transcripts are present in triticale seeds, we hypothesized that this phytocystatin regulates storage protein accumulation and degradation. Therefore, changes in gene expression during the entire period of seed development and germination were examined. TrcC-6 transcripts and TrcC-6 protein levels increased during the maturation of seeds and remained high during the first hours of germination. This enabled us to conclude that TrcC-6 likely regulates seed germination by the regulation of storage protein hydrolysis. For the analysis of TrcC-6 inhibitory activity, recombinant protein was expressed in Escherichia coli BL21 (DE3) and purified. Recombinant TrcC-6 proved to be a potent inhibitor of cysteine proteinases. It inhibited the in vitro activity of papain (EC 3.4.22.2) and ficin (EC 3.4.22.3). Furthermore, native PAGE analysis revealed that recombinant TrcC-6 inhibits the activity of endogenous cysteine proteinases present in germinating seeds of triticale. Based on these results, TrcC-6 is likely one of the important factors that regulate cysteine proteinase activity during the accumulation and mobilization of storage proteins.

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. In response to water-deficit, increases in the mRNA levels of TrcC-8 were observed in leaf and root tissues. Immunoblot analysis indicated that accumulation of the TrcC-8 protein occurred after 72h of water-deficit in the seedlings. Using recombinant protein, inhibitory activity of TrcC-8 against cysteine proteases from triticale and wheat tissues was analyzed. Under water-deficit conditions, there are increases in cysteine proteinase activities in both plant tissues. The cysteine proteinase activities were inhibited by addition of the recombinant TrcC-8 protein. These results suggest a potential role for the triticale phytocystatin in modulating cysteine proteinase activities during water-deficit conditions.

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. Based on the analyses performed, the studied proteins were identified as products of the tyrB-2 and phhC genes, and the presence of these genes in the investigated bacterial strain was confirmed using molecular biology methods. Extensive analysis of the substrate specificities of ArAT I and ArAT II revealed that both enzymes most efficiently catalyzed reactions involving aromatic amino acids and 2-oxoacids followed by dicarboxylic compounds. The best substrates for ArAT I and ArAT II were L-phenylalanine and phenylpyruvate. Based on these results, the studied proteins were classified as aromatic amino acid aminotransferase isozymes.

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 0.0625-125 µg mL(-1) (r ( 2 ) ≥ 0.998) and the limits of detection were below 0.015 µg mL(-1). The applicability of the method was confirmed by analysis of Pseudomonas putida culture supernatants.

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 595.9 fold with a 1.58% recovery. Triticale carboxypeptidase I is a homodimer with a molecular weight of 124.2 kDa and a subunit weight of 55.2 kDa. Each subunit is composed of two polypeptide chains (33.4 and 21.3 kDa). Serine was found in the active site of triticale carboxypeptidase I; DFP (diisopropylflourophosphate) and other applied inhibitors of serine proteases inhibited the enzyme activity. Triticale carboxypeptidase I hydrolyzes N-CBZ-dipeptide (N-carbobenzoxy-dipeptide) substrates at low pH. N-CBZ-Phe-Ala, N-CBZ-Phe-Leu and N-CBZ-Ala-Met were hydrolyzed with the highest rates. The lowest K(m) value and the highest k(cat)/K(m) ratio were observed for hydrolysis of N-CBZ-Phe-Ala. Studies on the amino acid sequence revealed that the purified enzyme is homologous to carboxypeptidase I from barley. Analyses of conserved regions in the sequence of triticale carboxypeptidase I revealed the presence of Ser, Asp and His that compose the catalytic triad. Intact storage proteins were poor substrates for carboxypeptidases. Carboxypeptidase I together with carboxypeptidase III effectively degraded albumins proteolytically modified by endopeptidase EP8. Modified globulins were degraded at a slower rate, and all three carboxypeptidases were required for a significantly increased activity. Studies of the expression of the carboxypeptidase I gene revealed that the synthesis of the enzyme occurs mainly in the scutellum of the grain. The enzyme is also expressed in the aleurone layer of the grains, although its function in this tissue is unknown.

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. The deduced TsPAP1 protein exhibits a considerable sequence identity with the biochemically characterized bacterial and fungal PAP proteins of small molecular masses (∼35 kDa). Moreover, the presence of conserved regions that are characteristic for bacterial monomeric PAP enzymes (the GGSWG motif, the localization of the catalytic triad residues and the segment involved in substrate binding) has also been noted. Primary structure analysis and phylogenetic analysis revealed that TsPAP1 encodes a novel plant PAP protein that is distinct from the multimeric proteins that have thus far been characterized in plants and whose counterparts have been recognized only in bacteria and fungi. A significant increase in the TsPAP1 transcript level in the shoots of triticale plants was observed under drought and saline conditions as well as in the presence of cadmium and aluminium ions in the nutrient medium. This paper is the first report describing changes in the transcript levels of any plant PAP in response to suboptimal growth conditions.

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 TrcC-1, TrcC-4 and TrcC-5. Full-length cDNAs of TrcC-1 (617 bp) and TrcC-4 (940 bp), as well as a fragment of TrcC-5 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. Moreover, the presence of conserved domain, containing the G and W residues, the sequence of QxVxG and the sequence of LARFAV, characteristic for plant cysteine endopeptidase inhibitors, has been noted. The profiles of TrcC-1 and TrcC-5 mRNA levels in the developing seeds of two triticale cultivars that differ in their resistance to preharvest sprouting (Zorro and Disco) were similar. However, the expression of TrcC-4 was, higher in the developing seeds, and in the scutellum of germinating seeds of a cultivar more resistant to preharvest sprouting (Zorro) than in the less resistant (Disco). Additionally, the expression of TrcC-4 remained longer in developing seeds of Zorro as compared to Disco. The performed studies suggest that TrcC-4 might have an influence on the higher resistance of Zorro cultivar to preharvest sprouting.

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. Its pH and temperature optimum are pH 7.5 and 37°C, respectively. The enzyme prefers substrates with Pro and Hyp at the N-terminus, but is also capable of hydrolysing ß-naphthylamides (ß-NA) of Ala, Phe, and Leu. The K(m) value of PAP against Pro-ß-NA was the lowest among the substrates tested and it was 1.47×10(-5)M. The activity of PAP was not inhibited by EDTA, 1,10-phenantroline, or pepstatin A. The most effective inhibitors were DFP, Pefabloc, and PMSF, which are serine protease inhibitors. However, significant inhibition was also observed in the presence of E-64, which modifies sulfhydryl groups. A significant increase of the aminopeptidase activity against Pro-ß-NA was observed in shoots of triticale plants grown under salinity, drought stress, and in the presence of cadmium and aluminium ions in the nutrient solution.

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 4.6. N-CBZ-Ala-Phe, N-CBZ-Ala-Leu, and N-CBZ-Ala-Met are hydrolyzed at the highest rates. Amino acids with aromatic or large aliphatic side chains are preferred in position P1', whereas the presence of these types of groups in position P1 of the substrate results in a lower rate of hydrolysis. Peptides containing glutamic acid in positions P1 are poor substrates for the enzyme. This phenomenon suggests the hydrophobic substrate- binding sites S1 and S1'. The active site contains serine since diisopropylfluorophosphate and phenylmethanesulfonyl fluoride reduce the activity by 89.9% and 81.5%, respectively. Moreover, the activity of triticale carboxypeptidase III is reduced by mercury ions and organomercurial compounds, which suggests the presence of a sulfhydryl group adjacent to the active site of the enzyme. Identification of purified enzyme by mass spectrometry method demonstrated that the enzyme is a homolog of barley carboxypeptidase III.

[Mobilization of storage proteins in cereal grains]. / Mobilizacja bialek zapasowych ziarniaków zbóz.

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 rate of the hydrolysis depends on the presence of biologically active gibberellins in the grain tissues. The presence of gibberellins determines peptidases synthesis in the aleurone layer, and acidification of starchy endosperm where the process occurs. Although the researches are highly advanced the functions of many peptidases in the storage proteins degradation are still not identified.

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. According to their substrate specificity the enzymes can be ordered into two sub-groups: having broad or narrow specificity. Almost half of the characterized aminopeptidases show a subunit structure. Enzymes having a quaternary structure are most often built of a combination of 2, 4, 6 subunits. Bacterial aminopeptidases may be localised in the cytoplasm, on membranes, associated with the envelope or secreted into the extracellular media. Regulation of the synthesis of aminopeptidases is assumed to take place mainly at the level of transcription. Most genes encoding the enzymes are monocistronic and contain a promotor characteristic for the genes transcribed by RNA polymerase associated with the factor sigma70. Aminopeptidases play an important role in the initial and final steps of protein turnover and they are involved in several specific regulatory functions.

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. Aminopeptidase showed a high substrate specificity and affinity to Phe-beta-naphtylamide (Phe-beta-NA) as a substrate. A considerable inhibition of the enzymatic activity by iodoacetamide and p-chloromercuribenzoate (p-CMB) led to the conclusion that it was a cysteine aminopeptidase. Hydrosulphide compounds markedly stabilised the enzyme. Ethylenediaminetetra-acetic acid (EDTA), a metalloenzyme inhibitor, caused a double increase in the phenylalanyl aminopeptidase activity.( )Mg(2+) ions activated the enzyme to a negligible extent, whereas Co(2+), Cu(2+), Cd(2+) and Pb(2+) ions contributed to its inhibition. The highest enzymatic activity was observed at 37 degrees C and pH 7.0.

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. Tendency of changes in total activity was similar for alanyl- and phenylalanylaminopeptidase though their response to different concentrations of organic components was different. Specific activity measured in the presence of Phe-beta-NA and Ala-beta-NA as the substrates was not strictly dependent on increasing concentrations of organic components in the media. The highest specific activity of aminopeptidase was obtained in the presence of Phe-beta-NA as a substrate on the fifth day of culture in medium containing 1% glucose. The obtained results seem to indicate the inductive character of the studied aminopeptidases. On the other hand, however, they do not exclude other regulatory mechanisms of their synthesis, including catabolic repression.