Short Exogenous Peptides Regulate Expression of CLE, KNOX1, and GRF Family Genes in Nicotiana tabacum.

Exogenous short biologically active peptides epitalon (Ala-Glu-Asp-Gly), bronchogen (Ala-Glu-Asp-Leu), and vilon (Lys-Glu) at concentrations 10-7-10-9 M significantly influence growth, development, and differentiation of tobacco (Nicotiana tabacum) callus cultures. Epitalon and bronchogen, in particular, both increase growth of calluses and stimulate formation and growth of leaves in plant regenerants. Because the regulatory activity of the short peptides appears at low peptide concentrations, their action to some extent is like that of the activity of phytohormones, and it seems to have signaling character and epigenetic nature. The investigated peptides modulate in tobacco cells the expression of genes including genes responsible for tissue formation and cell differentiation. These peptides differently modulate expression of CLE family genes coding for known endogenous regulatory peptides, the KNOX1 genes (transcription factor genes) and GRF (growth regulatory factor) genes coding for respective DNA-binding proteins such as topoisomerases, nucleases, and others. Thus, at the level of transcription, plants have a system of short peptide regulation of formation of long-known peptide regulators of growth and development. The peptides studied here may be related to a new generation of plant growth regulators. They can be used in the experimental botany, plant molecular biology, biotechnology, and practical agronomy.

Mechanisms of catalytic action and chemical modifications of endonucleases WEN1 and WEN2 from wheat seedlings.

Hydrolysis of DNA catalyzed by wheat endonucleases WEN1 and WEN2 is pronouncedly processive. A correlation has been revealed between appearance of new products of DNA hydrolysis with different length and conformational changes in the enzymes. The first conformational conversion of the endonucleases is associated with appearance of large fragments of DNA hydrolysis with length longer than 500 bp, and the second conversion is associated with formation of oligonucleotides with length of 120-140 bp, and the third conversion is associated with formation of short oligonucleotides and mononucleotides. Formation of the DNA-enzyme complex is accompanied by appearance of fluorescence at λ = 410-440 nm. The intensity, positions, and numbers of maximums of the fluorescence spectra of DNA-WEN1 and DNA-WEN2 complexes are different and depend on the methylation status of the DNA and on the presence of Mg2+. The endonucleases hydrolyze DNA by two mechanisms: one is metal-independent, and the other depends on one or two Mg2+ ions. One Mg2+ ion is located inside the catalytic center of WEN1, whereas the WEN2 center contains two Mg2+ ions. The first (site-specific) stage of DNA hydrolysis does not depend on Mg2+. Mg2+ ions evoke changes in the site specificity of the endonuclease action (WEN1) and abolish their ability to recognize the methylation status of DNA. Products of DNA hydrolysis by endonucleases WEN1 and WEN2 in the presence of Mg2+ are similar in length (120-140 bp). The endonucleases have at least two centers (domains) - catalytic and substrate-binding. Two histidine and apparently two lysine plus two dicarboxylic amino acid residues are present inside the catalytic center of WEN1. The catalytic center of WEN2 contains at least one histidine residue and apparently two residues of aspartic or glutamic acid, which are involved in coordination of the metal ions. The catalytic centers of WEN1 and WEN2 seem to be formed, respectively, by HD/E(D/EK)KH and HD/ED/E amino acid residues. The site-specificity of the endonuclease action is due to the DNA-binding domain. This domain contains dicarboxylic amino acid residues, which seem to be responsible for sensitivity of the enzymes to the methylation status of DNA. The hydroxyl groups of tyrosine residues in the enzymes also seem to contribute to recognizing methylated bases in DNA.

Modulation of action of wheat seedling endonucleases WEN1 and WEN2 by histones.

Wheat core histones and various subfractions of histone H1 modulate differently the action of endonucleases WEN1 and WEN2 from wheat seedlings. The character of this modulation depends on the nature of the histone and the methylation status of the substrate DNA. The modulation of enzyme action occurs at different stages of processive DNA hydrolysis and is accompanied by changes in the site specificity of the enzyme action. It seems that endonuclease WEN1 prefers to bind with protein-free DNA stretches in histone H1-DNA complex. The endonuclease WEN1 does not compete with histone H1/6 for DNA binding sites, but it does compete with histone H1/1, probably for binding with methylated sites of DNA. Unlike histone H1, the core histone H2b binds with endonuclease WEN1 and significantly increases its action. This is associated with changes in the site specificity of the enzyme action that is manifested by a significant increase in the amount of low molecular weight oligonucleotides and mononucleotides produced as a result of hydrolysis of DNA fragments with 120-140-bp length. The WEN2 endonuclease binds with histone-DNA complexes only through histones. The action of WEN2 is increased or decreased depending on the nature of the histone. Histone H1/1 stimulated the exonuclease activity of WEN2. It is supposed that endonucleases WEN1 and WEN2, in addition to the catalytic domain, should have a regulatory domain that is involved in binding of histones. As histone H1 is mainly located in the linker chromatin areas, it is suggested that WEN2 should attack DNA just in the chromatin linker zones. As differentiated from WEN2, DNA hydrolysis with endonuclease WEN1 is increased in the presence of core histones and, in particular, of H2b. Endonuclease WEN1 initially attacks different DNA sites in chromatin than WEN2. Endonuclease WEN2 activity can be increased or diminished depending on presence of histone H1 subfractions. It seems that just different fractions of the histone H1 are responsible for regulation of the stepwise DNA degradation by endonuclease WEN2 during apoptosis. Modulation of the action of the endonucleases by histones can play a significant role in the epigenetic regulation of various genetic processes and functional activity of genes.

Interaction of short peptides with FITC-labeled wheat histones and their complexes with deoxyribooligonucleotides.

Judging from fluorescence modulation (quenching), short peptides (Ala-Glu-Asp-Gly, Glu-Asp-Arg, Ala-Glu-Asp-Leu, Lys-Glu-Asp-Gly, Ala-Glu-Asp-Arg, and Lys-Glu-Asp-Trp) bind with FITC-labeled wheat histones H1, H2B, H3, and H4. This results from the interaction of the peptides with the N-terminal histone regions that contain respective and seemingly homologous peptide-binding motifs. Because homologous amino acid sequences in wheat core histones were not found, the peptides seem to bind with some core histone regions having specific conformational structure. Peptide binding with histones and histone-deoxyribooligonucleotide complexes depends on the nature of the histone and the primary structures of the peptides and oligonucleotides; thus, it is site specific. Histones H1 bind preferentially with single-stranded oligonucleotides by homologous sites in the C-terminal region of the protein. Unlike histone H1, the core histones bind predominantly with double-stranded methylated oligonucleotides and methylated DNA. Stern-Volmer constants of interaction of histone H1 and core histones with double-stranded hemimethylated oligonucleotides are higher compared with that of binding with unmethylated ones. DNA or deoxyribooligonucleotides in a complex with histones can enhance or inhibit peptide binding. It is suggested that site-specific interactions of short biologically active peptides with histone tails can serve in chromatin as control epigenetic mechanisms of regulation of gene activity and cellular differentiation.

Processing character of the action of wheat endonucleases WEN1 and WEN2. Kinetic parameters.

The wheat seedling endonucleases WEN1 and WEN2 dependent on Mg(2+), Ca(2+), and S-adenosyl-L-methionine (SAM) and sensitive to the substrate DNA methylation status have an expressed processing action. The enzymes hydrolyze DNA at a few subsequent stages: first, they split λ phage DNA specifically at CNG-sites (WEN1) with liberation of large fragments; second, they hydrolyze these fragments to 120-140 bp oligonucleotides that finally are hydrolyzed to very short fragments and mononucleotides. Initial stages of DNA hydrolysis may proceed in the absence of Mg(2+), but subsequent hydrolysis stages are very strongly stimulated by Mg(2+). It cannot be ruled out that modulation of enzymatic activity with Mg(2+) and probably with DNA fragments formed is associated with reorganization of the structure of eukaryotic (wheat) endonucleases with respective changes in their catalytic properties and site specificity of action. Michaelis constant value for WEN1 endonuclease on hydrolysis of methylated λ phage DNA containing Cm(5)CWGG and Gm(6)ATC sites is four-fold lower compared with that observed on hydrolysis of unmethylated λ phage DNA. This may indicate that affinity of WEN1 enzyme to methylated DNA is higher than that to unmethylated DNA. In the presence of SAM, the Michaelis constant for WEN2 on the DNA hydrolysis stage characterized by formation of 120-140 bp fragments is decreased, but for WEN1 it is increased by 1.5-2.0-fold. This means that SAM inhibits WEN1 but stimulates WEN2. Thus, wheat endonucleases WEN1 and WEN2 differ significantly in affinities to substrate DNAs with different methylation status, in velocities of DNA hydrolysis, and time of production of DNA fragments of similar length. It seems that the investigated plant endonucleases can hydrolyze DNA in the nucleus as well to both large and very short fragments including mononucleotides, that is, in particular, essential for utilization of cell nucleic acid material during apoptosis.

CNG site-specific and methyl-sensitive endonuclease WEN1 from wheat seedlings.

Endonuclease WEN1 with apparent molecular mass about 27 kDa isolated from cytoplasmic vesicular fraction of aging coleoptiles of wheat seedlings has expressed site specificity action. This is a first detection and isolation of a site-specific endonuclease from higher eukaryotes, in general, and higher plants, in particular. The enzyme hydrolyzes deoxyribooligonucleotides of different composition on CNG (N is G, A, C, or T) sites by splitting the phosphodiester bond between C and N nucleotide residues in CNG sequence independent from neighbor nucleotide context except for CCCG. WEN1 prefers to hydrolyze methylated λ phage DNA and double-stranded deoxyribooligonucleotides containing 5-methylcytosine sites (m(5)CAG, m(5)CTG) compared with unmethylated substrates. The enzyme is also able to hydrolyze single-stranded substrates, but in this case it splits unmethylated substrates predominantly. Detection in wheat seedlings of WEN1 endonuclease that is site specific, sensitive to the substrate methylation status, and modulated with S-adenosyl-L-methionine indicates that in higher plants restriction--modification systems or some of their elements, at least, may exist.

Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA.

Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

Site-specific binding of short peptides with DNA modulated eukaryotic endonuclease activity.

Short peptides (2-4 amino acid residues) inhibit or stimulate hydrolysis of λ phage DNA by eukaryotic endonucleases WEN1 and WEN2 depending on DNA methylation status. Peptide modulation of endonucleases activity most likely appears as a result of their binding to DNA. Peptides discriminate (recognize) not only certain DNA sequences, but also their methylation status. Apart from intact DNA, the test peptides bind to single-stranded DNA structures (oligonucleotides) containing NG- and CG-sites methylated in eukaryotes. Peptides affect the set of hydrolyzed sites during endonuclease hydrolysis of double-stranded structures. The effects of peptides with different primary structure on DNA hydrolysis by endonucleases are different and are modulated by histones (histone H1). Site-specific peptide interactions with DNA may epigenetically control genetic functions of the cell. These interactions probably played an important role at the very early stages of evolution.

H1 histone modulates DNA hydrolysis with WEN1 and WEN2 endonucleases from wheat coleoptiles.

We show that total H1 histone from wheat seedlings or rat liver enhances hydrolysis of lambda phage DNA with plant endonucleases WEN1 and WEN2 isolated from wheat coleoptiles. Optimal DNA/protein weight ratio in the hydrolysis reaction is 1 : 1. The action of fractions I and IV (obtained from total wheat H1 histone by electrophoresis) on DNA hydrolysis with WEN1 and WEN2 enzymes depends on the DNA methylation status. Fraction IV of wheat histone H1 stimulates hydrolysis of unmethylated lambda phage DNA with WEN1 and WEN2 enzymes. Hydrolysis of methylated lambda phage DNA (it contains 5-methylcytosine in Cm(5)CWGG sequences and N(6)-methyladenine in Gm(6)ATC sites) with WEN1 is inhibited with fractions I and IV of wheat H1 histone. Fractions II and III of wheat H1 histone do not influence DNA hydrolysis with WEN1 and WEN2. S-Adenosyl-L-methionine (SAM) stimulates activity of these plant enzymes. But in the presence of H1 histone, SAM does not add to the ability of the enzyme to hydrolyze more DNA compared with that induced with H1 histone itself. Therefore, the stimulating effects of SAM and H1 histone on DNA hydrolysis with plant endonucleases may be similar. It could be suggested that SAM and H1 histone can induce more or less analogous allosteric transformations in the structure of the investigated plant endonucleases. Thus, DNA hydrolysis with plant endonucleases is modulated with total H1 histone. H1 histone fractions affect DNA hydrolysis in a different fashion; they enhance or inhibit hydrolysis depending on the DNA methylation status. We suggest that H1 histone changes site specificity of endonucleases or it might be responsible for formation of new or masking of old sites available for these enzymes due to changes in DNA structure induced in a DNA-histone complex.

Wheat coleoptile endonuclease Wen2 is dependent on S-adenosyl-L-methionine and sensitive to DNA methylation status.

Endonuclease WEN2 with an apparent molecular mass 21.5 kD was isolated from subcellular vesicular fraction obtained from aging apoptotic coleoptiles of 8-day-old etiolated wheat seedlings and partially characterized. Similar to wheat endonuclease WEN1 of the same origin described earlier, the WEN2 enzyme is a neutral Ca2+,Mg2+,Mn2+-dependent endonuclease. Both enzymatic activities were found also in nuclei from the same cells. Mn2+ activates WEN2 more efficiently than Mg2+ or Ca2+. High ionic strength, Zn2+, and EDTA inhibit the enzyme completely. In the presence of Mg2+, elevated WEN2 activity was observed at pH between 5.5 and 7.7 and at 37 degrees C, and without Mg2+ added it was observed in narrower pH range (from pH 6.8 to pH 7.7). The enzyme is active even at high temperature (65 degrees C). WEN2 splits preferentially unmethylated, but WEN1 - methylated lambda phage DNA. Double-stranded DNA is a preferential substrate to be hydrolyzed with WEN2. S-Adenosyl-L-methionine (SAM) significantly activates endonuclease WEN2 (the optimal SAM concentration is 0.3 mM). Contrary to strong stimulating action on WEN1, the competitive inhibitors of the DNA methylation reaction (SAM analogs S-adenosyl-L-homocysteine and S-isobutyladenosine) at concentration 0.3 mM increase WEN2 activity slightly. It is suggested that WEN2 may take part in apoptotic DNA degradation. Thus, in plants there are endonucleases that recognize methylation status of substrate DNAs and are modulated by the methyl group donor, SAM, in different fashions. Therefore, all this may indicate the presence of a restriction-modification (R-M) system in higher plants.

Primary structures of two ribonucleases from ginseng calluses. New members of the PR-10 family of intracellular pathogenesis-related plant proteins.

The amino acid sequences of two ribonucleases from a callus cell culture of Panax ginseng were determined. The two sequences differ at 26% of the amino acid positions. Homology was found with a large family of intracellular pathogenesis-related proteins, food allergens and tree pollen allergens from both dicotyledonous and monocotyledonous plant species. There is about 30% sequence difference with proteins from species belonging to the same plant order (Apiales: parsley and celery), 60% with those from four other dicotyledonous plant orders and about 70% from that of the monocotyledonous asparagus. More thorough evolutionary analyses of sequences lead to the conclusion that the general biological function of members of this protein family may be closely related to the ability to cleave intracellular RNA and that they have an important role in cell metabolism. As the three-dimensional structure of one of the members of this protein family has been determined recently [Gajhede et al., Nature Struct Biol 3 (1996) 1040-1045], it may be possible to assign active-site residues in the enzyme molecule and make hypotheses about its mode of action. Structural features in addition to the cellular site of biosynthesis indicate that this family of ribonucleases is very different from previously investigated ones.

High sequence similarity between a ribonuclease from ginseng calluses and fungus-elicited proteins from parsley indicates that intracellular pathogenesis-related proteins are ribonucleases.

A ribonuclease from a callus cell culture of Panax ginseng C.A. Mey strain R1 was isolated. A pure protein with an apparent molecular mass of 18 kDa was obtained. The N-terminal sequences of the protein and of the C-terminal CNBr peptide were determined. No homology with other ribonucleases was found, but there was 60-70% sequence identity with two intracellular pathogenesis-related (IPR) proteins from parsley, indicating that not only these two proteins, but also homologous IPR proteins identified in other plant species are ribonucleases.

Effect of ethylene producer ethrel and antioxidant ionol (BHT) on the proteolytic apparatus in coleoptiles of wheat seedlings during apoptosis.

It was established that total proteolytic activity in etiolated wheat seedlings changes in ontogenesis in cycles: peaks of proteolytic activity correspond to the 3rd, 5th, and 8th days of seedling growth, respectively. The maximum of proteolytic activity preceded the maximum of nuclease activity, which may be due to activation of nucleases by proteolytic enzymes. According to inhibitory analysis the cysteine and serine proteases play the main role in apoptosis in wheat coleoptiles. Growing of seedlings in the presence of ethrel stimulated apoptosis in the coleoptile, and it increased (almost 6-fold) the proteolytic activity in its cells. On the other hand, the antioxidant ionol (BHT) suppressed the induction of proteases, particularly at the second stage of coleoptile development, and it slowed down the increase in the nuclease activity after 6th day of the seedling life. It is suggested that phytohormones and antioxidants participate in regulation of apoptosis in the ageing coleoptile, directly or indirectly effecting the proteolytic apparatus in the coleoptile cells.