Chirp-controlled high-harmonic and attosecond-pulse generation via coherent-wake plasma emission driven by mid-infrared laser pulses.

Coherent-wake plasma emission induced by ultrashort mid-infrared laser pulses on a solid target is shown to give rise to high-brightness, high-order harmonic radiation, offering a promising source of attosecond pulses and a probe for ultrafast subrelativistic plasma dynamics. With 80-fs, 0.2-TW pulses of 3.9-µm radiation used as a driver, optical harmonics up to the 34th order are detected, with their spectra stretching from the mid-infrared region to the extreme ultraviolet region. The harmonic spectrum is found to be highly sensitive to the chirp of the driver. Particle-in-cell analysis of this effect suggests, in agreement with the generic scenario of coherent-wake emission, that optical harmonics are radiated as trains of extremely short, attosecond ultraviolet pulses with a pulse-to-pulse interval varying over the pulse train. A positive chirp of the driver pulse can partially compensate for this variation in the interpulse separation, allowing harmonics of the highest orders to be generated in the plasma emission spectrum.

Seed Storage Globulins: Origin and Evolution of Primary and Higher Order Structures.

Legumin and vicilin are two-domain seed storage globulins similar in primary and higher order structures of their domains to single-domain plant germins as well as to the domains of two-domain and single-domain bacterial oxalate decarboxylases. Independent evolutionary pathways have been shown for the descent of the storage globulins and germins from two-domain and single-domain bacterial oxalate decarboxylases, respectively. As compared to vicilins, the primary and tertiary structures of legumins were found to most closely reflect the ancient features characteristic of a common precursor of storage globulins. During the evolution of the storage globulins, a mechanism specifically controlling their degradation has been formed. We found that limited proteolysis of soybean legumin and kidney bean vicilin in germinating seeds and in vitro leads to their regular changes, which initiate an extensive cleavage of storage globulin molecules by the one-by-one mechanism. As also shown, limited proteolysis of soybean legumin loosens the intersubunit interactions in its oligomeric molecule. Based on these data, we hypothesize that the deep one-by-one degradation of soybean legumin is triggered by its dissociation, which bares peptide bonds potentially susceptible to proteolytic attack but are masked in the oligomer.

11S Storage globulin from pumpkin seeds: regularities of proteolysis by papain.

Limited proteolysis of the α- and ß-chains and deep cleavage of the αß-subunits by the cooperative (one-by-one) mechanism was observed in the course of papain hydrolysis of cucurbitin, an 11S storage globulin from seeds of the pumpkin Cucurbita maxima. An independent analysis of the kinetics of the limited and cooperative proteolyses revealed that the reaction occurs in two successive steps. In the first step, limited proteolysis consisting of detachments of short terminal peptides from the α- and ß-chains was observed. The cooperative proteolysis, which occurs as a pseudo-first order reaction, started at the second step. Therefore, the limited proteolysis at the first step plays a regulatory role, impacting the rate of deep degradation of cucurbitin molecules by the cooperative mechanism. Structural alterations of cucurbitin induced by limited proteolysis are suggested to generate its susceptibility to cooperative proteolysis. These alterations are tentatively discussed on the basis of the tertiary structure of the cucurbitin subunit pdb|2EVX in comparison with previously obtained data on features of degradation of soybean 11S globulin hydrolyzed by papain.

[Evolution of seed storage globulins and cupin superfamily].

An extensive superfamily of cupins (clan cl09118) currently combines thousands of functionally and structurally diverse prokaryote and eukaryote proteins, which contain a beta-barrel of antiparallel beta-strands (cupin module). Possible ways of the formation of the cupin superfamily were suggested based on the comparison of primary and tertiary structures of proteins from several conserved families of cupins including seed storage globulins and plant oxalate oxydases (germins), and bacterial oxalate decarboxylases, gentisate dioxygenases and epimerases. The origin of two-domain structure of seed storage globulins from cyanobacterial two-domain oxalate decarboxylases has been deduced. The evolutionary pathway of single-domain germins previously suggested to be immediate progenitors of storage globulins was traced back. Common evolutionary roots of germins and oxalate decarboxylases descend from recent bacterial and archaebacterial proteins whose primitive structure is restricted to the cupin module. These root proteins reflect the hypothetical structure of a pro-cupin that probably gave rise to at least a part of the total diversity of members of the cupin superfamily (for instance, to the cupin module of gentisate dioxygenases). The major dilemma for the description of the cupin superfamily is distinguishing evolutionary divergence from convergence. The structural convergence can be exemplified by formation of a beta-barrel inside extremely conserved structures of the otherwise unrelated epimerases from Archaea and bacteria.

Storage and mobilization as antagonistic functional constraints on seed storage globulin evolution.

When seeds germinate nearly all the proteins are degraded in senescing storage tissue cells. All these proteins act as amino acid reserves which are mobilized to nourish the seedling. Nevertheless, the major amount of the seeds' protein reserve consists of a few enzymatically inactive, abundant, genuine storage proteins. In their metabolism the conflicting processes of biosynthesis, protein turnover and breakdown, are temporally separated. No degradation of correctly formed storage proteins was observed at the time of synthesis and accumulation during seed maturation. Breakdown takes place after a (long) period of rest when seeds germinate and seedlings start growing. At that time genuine storage proteins are no longer synthesized. Genuine storage proteins have evolved structural features permitting controlled temporal patterns of protection and proteolysis. The acquisition of inserted sequence stretches as sites accessible to limited proteolysis played a key role in the evolution of this control system and happened in coevolution of genuine storage proteins with specific proteinases. This can be deduced from the results of current research on the mechanisms of limited and unlimited proteolysis of storage globulins and on storage globulin evolution. The evolved system of controlled structure-function interplay between storage globulins and proteinases is part of a syndrome that, in addition, comprises differential compartmentation and gene expression of storage proteins and proteinases for controlling the total spatial and temporal patterns of globulin storage and mobilization in maturing and germinating seeds.

Sequence peculiarity of gnetalean legumin-like seed storage proteins.

The development of seeds as a specialized organ for the nutrition, protection, and dispersal of the next generation was an important step in the evolution of land plants. Seed maturation is accompanied by massive synthesis of storage compounds such as proteins, starch, and lipids. To study the processes of seed storage protein evolution we have partially sequenced storage proteins from maturing seeds of representatives from the gymnosperm genera Gnetum, Ephedra, and Welwitschia-morphologically diverse and unusual taxa that are grouped in most formal systems into the common order Gnetales. Based on partial N-terminal amino acid sequences, oligonucleotide primers were derived and used for PCR amplification and cloning of the corresponding cDNAs. We also describe the structure of the nuclear gene for legumin of Welwitschia mirabilis. This first gnetalean nuclear gene structure contains introns in only two of the four conserved positions previously characterized in other spermatophyte legumin genes. The distinct phylogenetic status of the gnetalean taxa is also reflected in a sequence peculiarity of their legumin genes. A comparative analysis of exon/intron sequences leads to the hypothesis that legumin genes from Gnetales belong to a monophyletic evolutionary branch clearly distinct from that of legumin genes of extant Ginkgoales and Coniferales as well as from all angiosperms.

A gene encoding a vicilin-like protein is specifically expressed in fern spores. Evolutionary pathway of seed storage globulins.

The isolation and characterisation of a cDNA coding for a vicilin-like protein of the fern Matteuccia struthiopteris is described. The corresponding gene is specifically expressed during late stages of spore development. Extensive sequence comparisons suggest that the fern protein can be considered as a molecular missing link between single-domain germin/spherulin-like proteins and two-domain seed storage globulins of gymnosperms and angiosperms. Further, evidence is provided for the existence of a superfamily of structurally related, functionally different proteins which includes storage globulins of the vicilin and legumin families, a membrane-associated sucrose-binding protein of soybean, a Forssman antigen-binding lectin of velvet bean, the precursor of the vacuolar membrane bound proteins MP27/MP32 of pumpkin, the embryogenesis-specific protein Gea8 of carrot, the fern-spore-specific protein described here as well as the functionally diverse family of germins/germin-like proteins and the spherulins of myxomycetes. We propose that seed storage globulins of spermatophytes evolved from desiccation-related single-domain proteins of prokaryotes via a duplicated two-domain ancestor that is best represented by the extant fern spore-specific vicilin-like protein.

Proteinase A, a storage-globulin-degrading endopeptidase of vetch (Vicia sativa L.) seeds, is not involved in early steps of storage-protein mobilization.

Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia sativa L.) which was assumed to initiate storage-globulin breakdown just after the onset of seed germination. This enzyme was purified from cotyledons of vetch seedlings. On gelatin-containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDS/PAGE was 29 kDa. Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein-body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor have been obtained by PCR. The precursor is composed of an N-terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C-terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25,244 Da does not have the KDEL sequence. The derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydryl-endopeptidase (SH-EP), a cysteine endopeptidase from germinating Vigna mungo seedlings. Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of 1-day-germinated vetch seeds, where its amount increases up to day 6. No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised against the purified and against recombinantly produced proteinase A, the 29-kDa bands of mature proteinase A were detected in cotyledon extracts of 6-day-germinated seeds when globulin degradation has already far proceeded. The reported data do not agree with the proposed triggering role of proteinase A in storage-globulin breakdown during germination.

Limited proteolysis of beta-conglycinin and glycinin, the 7S and 11S storage globulins from soybean [Glycine max (L.) Merr.]. Structural and evolutionary implications.

The G2 (A2B1a) glycinin subunit from soybean (Glycine max L. Merr.) was purified and renatured to the homohexameric holoprotein. This protein along with purified beta-conglycinin were subjected to limited proteolysis by trypsin. The generated polypeptide fragments were separated via SDS/PAGE and the amino acid sequence of the N-terminals was determined. Four cleavage points were detected in the alpha-chain A2 of glycinin as well as in the alpha'-chain of beta-conglycinin. From the known three-dimensional structure of 7S globulin and the hypothetical model of 7S globulin-like 11S globulin structure, it was possible to draw the conclusion that two distinct types of susceptible sites for proteolytic cleavage are characteristic of the subunits of both globulins. The first includes the sequences linking N- and C-terminal domains of both globulins and the sequence of N-terminal extensions of 70-kDa subunits from the vicilin-like 7S globulins. The second type includes the loop between beta-strands E and F of the N-terminal domain of 11S globulins and of the C-terminal domain of 7S globulins. A statistically significant similarity was found between the N-terminal extension of the alpha'-chain of beta-conglycinin and the interdomain linker regions of soybean glycinin and pea legumin. It is proposed that the three sequence regions which form the first type of susceptible sites are of similar structural function and might have evolved from the N-terminal segment of a putative single-domain ancestor.

A vicilin-like seed protein of cycads: similarity to sucrose-binding proteins.

Seed storage globulins of the 7S and 11S type are synthesized in the seeds of angiosperms and gymnosperms. We have isolated and characterized a vicilin-like gene expressed in the cycad Zamia furfuraceae. Sequence comparisons reveal clear similarities to a sucrose-binding protein isolated from soybean. We suggest the existence of a superfamily of related genes including both vicilin-like and legumin-like seed globulin genes as well as genes coding for spherulins, germins and sucrose-binding-proteins.

Legumin-like and vicilin-like seed storage proteins: evidence for a common single-domain ancestral gene.

Legumin-like 11S and vicilin-like 7S globulins are the main storage proteins of most angiosperms and gymnosperms. The subunits of the hexameric legumin are synthesized as a precursor comprising a N-terminal acidic alpha- and a C-terminal basic beta-chain. The trimeric vicilin molecule consists of subunits composed of two symmetrical N- and C-terminal structural domains. In a multiple alignment we have compared the N-terminal and C-terminal domains of 11 legumins and seven vicilins of several dicot, monocot, and gymnosperm species. The comparisons using all six possible pairwise combinations reveal that the N-terminal and C-terminal domains of both protein families are similar to each other. These results together with data on the distribution of variable and conserved regions, on the positions of susceptible sites for proteolytic attack, as well as on the published 7S protein tertiary structure suggest that both protein families share a common single-domain ancestor molecule and lead to the hypothesis that a triplication event has occurred during the evolution of a putative legumin/vicilin ancestor gene. Moreover, the comparison of the intron/exon pattern reveals that at least three out of five intron positions are precisely conserved between the genes of both protein families, further supporting the idea of a common evolutionary origin of recent legumin and vicilin encoding genes.

Seed storage proteins of spermatophytes share a common ancestor with desiccation proteins of fungi.

The legumin- and vicilin-like seed storage globulins of spermatophytes are specifically accumulated during embryogenesis and seed development. Previous studies have shown that a precursor common to both legumin and vicilin genes might have evolved by duplication from a single-domain ancestral gene. We here report that amino acid sequences of legumin and vicilin domains share statistically significant similarity to the germination-specific germins of wheat as well as to the spherulation-specific spherulins of myxomycetes. This conclusion is further supported by the derived intron-exon structure of a spherulin gene. Spherulins are thought to be involved in tissue desiccation or hydration. It is suggested that the present-day seed globulins of spermatophytes have evolved from a group of ancient proteins functional in cellular desiccation/hydration processes.

Purification, cDNA cloning and characterization of proteinase B, an asparagine-specific endopeptidase from germinating vetch (Vicia sativa L.) seeds.

Proteinase B, an asparagine-specific endopeptidase, has been purified from germinating vetch (Vicia sativa L.) seeds. The final preparation consists of two enzymically active proteins with molecular masses of approximately 39 kDa and 37 kDa. Synthetic substrates were used to confirm cleavage specificity of the proteinase B preparation. As expected, the enzyme cleaves the substrates at the C-terminal side of Asn residues. The octapeptide ETRNGVEE was digested most efficiently. When Gly was replaced by Ile or Glu, cleavage took place with lower efficiency. Polyclonal antibodies displayed both proteins in cotyledon extracts of germinated vetch seeds. In addition, a strong cross-reacting protein band was found in cotyledon extracts of developing seeds, indicating the presence of a very similar enzyme during seed development. cDNA clones encoding proteinase B precursor have been obtained on the basis of the N-terminal amino acid sequence DDDFEGTRWAILLAGS, by means of the polymerase chain reaction. The cDNA clones contain an open reading frame of 1479 bp encoding a polypeptide of 493 amino acids. The precursor displayed 59% sequence identity to the cDNA-derived amino acid sequence of a vacuolar Asn-specific enzyme from the developing castor beam endosperm which is thought to catalyze the post-translational processing of pro-proteins into the mature forms. Proteinase B is synthesized de novo during seed germination. The results of Southern-blot analyses suggested that there are at least two genes for proteinase B.

Action of trypsin on glycinin. Mixed-type proteolysis and its kinetics; molecular mass of glycinin-T.

The formation of a relatively stable high-molecular-mass product on trypsin hydrolysis of glycinin (glycinin-T) is interpreted to be a result of 'zipper' proteolysis. Evidence of parallel one-by-one degradation of glycinin occurring after the formation of glycinin-T is presented. At a relatively low concentration of the substrate, the one-by-one proteolysis proceeds as a first-order reaction. A method of determination of the changes in the molecular mass of a protein during the mixed-type proteolysis and some other parameters of this process is developed on the basis of the analysis of the proteolysis kinetics. The value of the molecular mass of glycinin-T calculated by means of this method makes up 70% of the initial molecular mass and coincides with the result of direct determination by gradient gel electrophoresis.

[Participation of phenylalanine-p-nitroanilidase in the decomposition of reserve proteins of germinating vetch seeds]. / Ob uchastii fenilalanin-p-nitroanilidazy v raspade zapasnykh belkov prorastaiushchikh semian viki.

L-phenylalanine-p-nitroanilidase (PPA-ase) from vetch cotyledons was purified 1600-fold with a 6.7% recovery. Data from gel electrophoresis suggest that the preparation obtained (specific activity 232 U/mg) contains only a small admixture of inactive protein. PPA-ase splits off more than 14% of peptide bonds of di- and oligopeptides formed by reserve protein hydrolysis with endogenous protease A. The cotyledon PPA-ase is located outside the protein bodies and differs from seedling enzymes hydrolyzing PPA by its chromatographic behaviour. The results obtained suggest that PPA-ase takes part in the hydrolysis of short peptides produced during the reserve protein degradation and transported from protein bodies in the cytoplasm.

[Purification and partial characterization of protease B from germinating vetch seeds]. / Ochistka i chastichnaia kharakteristika proteazy B iz prorastaiushchikh semian viki.

The sulfhydryl protease B was isolated from the cotyledons of 8-day old vetch seedlings and purified 1580-fold with a 38% recovery. The preparation obtained proved to be homogeneous by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis. The molecular weight of the enzyme as shown by Na-DS gel electrophoresis is 38 000. Protease B hydrolyzes the peptide bonds involving the carboxyl groups of asparagine in insulin chains A and B. Since protease B fails to attack the native reserve proteins the high molecular weight products of the initial hydrolysis of reserve proteins by the earlier discovered protease A seem to be the most probable substrates of protease B. A considerable part of these substrates is split by protease B to form large peptides. The role of protease B in reserve proteins degradation is discussed.

[Modification of vetch seed reserve proteins during germination and limited proteolysis]. / Issledovanie modifikatsii zapasnykh belkov semian viki pro prorastanii i ogranichennom proteolize.

The split-off 1-2 short peptides is the first stop in the endogenous protease A effect on the vetch legumin, which results in a step-wise rise of its hydrolyzability by two other endogenous proteases (B and C). Short neutral and basic peptides are consecutively split off from the acid subunits in the course of subsequent hydrolysis by protease A, while the breakdown of these subunits into larger fragments, which are retained in the legumin molecule by non-covalent bonds, occurs later. Similar results were obtained in experiments on trypsin action of legumin. Thus the initial course of legumin hydrolysis is largely determined by its structure. The changes of legumin during germination are similar to those occurring upon limited proteolysis by protease A. However, some differences are indicative of the existence of other factors responsible for the modification of this protein during germination. The modification of vicilin during germination and limited proteolysis occurs apparently in a similar way.

The action of pepsin on the reserve proteins of some leguminous seeds.

The action of pepsin on the 11-S and 7-S proteins of vetch, 11-S protein of soybean and 7-S protein of Phaseolus vulgaris was investigated. The first three proteins are hydrolyzed almost completely, the rate of hydrolysis being close to that of hemoglobin, while the hydrolysis of Ph. vulgaris 7-S protein stops after the cleavage of only 2,4% of peptide bonds. The nonhydrolyzable high molecular weight core makes up to 87% of the initial protein and differs from the latter in its electrophoretic mobility and sedimentation coefficient. The action of pepsin does not increase the digestibility of Ph. vulgaris 7-S protein by trypsin. After the consecutive action of these enzymes about two thirds of the protein remain unhydrolyzed. The digestion of Ph. vulgaris 7-S protein by pepsin is completed only after its denaturation by heat treatment or by the action of 6 M guanidine hydrochloride.

[Partial purification and characterization of protease A of germinating vetch seeds, hydrolyzing native reserve proteins]. / Chastichnaia ochistka i kharakteristika proteazy A prorostaiushchikh semian viki, gidrolizuiushchei nativnye zapasnye belki.

Protease A is 870-fold purified by means of isoelectric precipitation, DEAE-cellulose chromatography and gel filtration through Sephadex G-50, the yield of the enzyme being 28%. The purified preparation is free of contaminant proteolytic activity and is almost homogenous chromatographically, but it produces a complex pattern under electrophoresis in 30% polyacrylamide gel, which is probably due to enzyme autolysis. As evidenced from the effect of protease A on A and B chains of insulin, the enzyme has a wide substrate specificity. It hydrolyses native vetch legumin and vicilin up to peptides having on average 9 and 16 amino acid residues respectively. No free amino acids were found in hydrolysates of both vetch proteins. Thus, protease A is an endopeptidase, which probably plays the main role in the process of reserve proteins degradation.