The effect of intercropping leguminous green manure on theanine accumulation in the tea plant: A metagenomic analysis.

Intercropping is a widely recognised technique that contributes to agricultural sustainability. While intercropping leguminous green manure offers advantages for soil health and tea plants growth, the impact on the accumulation of theanine and soil nitrogen cycle are largely unknown. The levels of theanine, epigallocatechin gallate and soluble sugar in tea leaves increased by 52.87% and 40.98%, 22.80% and 6.17%, 22.22% and 29.04% in intercropping with soybean-Chinese milk vetch rotation and soybean alone, respectively. Additionally, intercropping significantly increased soil amino acidnitrogen content, enhanced extracellular enzyme activities, particularly ß-glucosidase and N-acetyl-glucosaminidase, as well as soil multifunctionality. Metagenomics analysis revealed that intercropping positively influenced the relative abundances of several potentially beneficial microorganisms, including Burkholderia, Mycolicibacterium and Paraburkholderia. Intercropping resulted in lower expression levels of nitrification genes, reducing soil mineral nitrogen loss and N2 O emissions. The expression of nrfA/H significantly increased in intercropping with soybean-Chinese milk vetch rotation. Structural equation model analysis demonstrated that the accumulation of theanine in tea leaves was directly influenced by the number of intercropping leguminous green manure species, soil ammonium nitrogen and amino acid nitrogen. In summary, the intercropping strategy, particularly intercropping with soybean-Chinese milk vetch rotation, could be a novel way for theanine accumulation.

Vicilin and legumin storage proteins are abundant in water and alkali soluble protein fractions of glandless cottonseed.

In this work, we sequentially extracted water (CSPw)- and alkali (CSPa)-soluble protein fractions from glandless cottonseed. SDS-Gel electrophoresis separated CSPw and CSPa to 8 and 14 dominant polypeptide bands (110-10 kDa), respectively. Liquid chromatography-electrospray ionization-tandem mass spectrometry identified peptide fragments from 336 proteins. While the majority of peptides were identified as belonging to vicilin and legumin storage proteins, peptides from other functional and uncharacterized proteins were also detected. Based on the types (unique peptide count) and relative abundance (normalized total ion current) of the polypeptides detected by mass spectrometry, we found lower levels (abundance) and types of legumin isoforms, but higher levels and more fragments of vicilin-like antimicrobial peptides in glandless samples, compared to glanded samples. Differences in peptide fragment patterns of 2S albumin and oleosin were also observed between glandless and glanded protein samples. These differences might be due to the higher extraction recovery of proteins from glandless cottonseed as proteins from glanded cottonseed tend to be associated with gossypol, reducing extraction efficiency. This work enriches the fundamental knowledge of glandless cottonseed protein composition. For practical considerations, this peptide information will be helpful to allow better understanding of the functional and physicochemical properties of glandless cottonseed protein, and improving the potential for food or feed applications.

Plant protein-based hydrophobic fine and ultrafine carrier particles in drug delivery systems.

For thousands of years, plants and their products have been used as the mainstay of medicinal therapy. In recent years, besides attempts to isolate the active ingredients of medicinal plants, other new applications of plant products, such as their use to prepare drug delivery vehicles, have been discovered. Nanobiotechnology is a branch of pharmacology that can provide new approaches for drug delivery by the preparation of biocompatible carrier nanoparticles (NPs). In this article, we review recent studies with four important plant proteins that have been used as carriers for targeted delivery of drugs and genes. Zein is a water-insoluble protein from maize; Gliadin is a 70% alcohol-soluble protein from wheat and corn; legumin is a casein-like protein from leguminous seeds such as peas; lectins are glycoproteins naturally occurring in many plants that recognize specific carbohydrate residues. NPs formed from these proteins show good biocompatibility, possess the ability to enhance solubility, and provide sustained release of drugs and reduce their toxicity and side effects. The effects of preparation methods on the size and loading capacity of these NPs are also described in this review.

Accumulation of PrLeg, a Perilla legumin protein in potato tuber results in enhanced level of sulphur-containing amino acids.

Potato is the fourth staple food in the world, following rice, wheat, and maize, whereas tubers contain high quality of starch, relatively high amounts of vitamin C and many other important substances. It also contains relatively good quality of protein (about 3 to 6% of the dried weight) and patatin, and 11S globulin is a major storage protein with high level of lysine. However, tuber protein contains relatively low amounts of sulphur-containing amino acids, which may result in low nutritional value. Recently, we cloned a gene encoding PrLeg polypeptide, a seed storage protein from Perilla, which contains relatively higher levels of sulphur-containing amino acids. We transformed PrLeg cDNA into a potato plant to over-express under the direction of the tuber-specific promoter, patatin. Most of the transgenic lines identified through PCR and RT-PCR analyses were able to accumulate high amount of prLeg transcript in their tuber tissue, while very little or no transcript that were detected in their leaf tissues. The level of methionine content was elevated up to three-fold compared to non-transgenic parental line, without any significant changes in other amino acids, suggesting that further research is required to get a deeper insight into their nutritional value.

Characterization and evolutionary relationship of methionine-rich legumin-like protein from buckwheat.

We have isolated and characterized a full-length cDNA for legumin-like storage polypeptide from buckwheat seed (Fagopyrum esculentum Moench) and compared its deduced amino acid sequence with those from different representatives of dicots, monocots and gymnosperms. The cDNA sequence was reconstructed from two overlapping clones isolated from a cDNA library made on mRNA of buckwheat seed at the mid-maturation stage of development. Analysis of the deduced amino acid sequence revealed that this specific buckwheat storage polypeptide should be classified in the methionine-rich legumin subfamily present in the lower angiosperm clades, a representative of which was first characterized in Magnolia salicifolia (clone B 14). The fact that a methionine-rich legumin coexists together with methionine-poor legumins in buckwheat should be an important element regarding the evolutionary position of buckwheat. This may also be supporting evidence that the B14 ortholog was not lost in evolution but was protected under pressure of an increased need for sulfur. Using primers designed from characterized cDNA, we also isolated its corresponding gene from buckwheat genomic DNA and analyzed the characteristic exon/intron structure. The firstly identified two-intron structure of buckwheat legumin gene is an important contribution to study of methionine-rich legumins in lower angiosperms.

Seed-specific promoters direct gene expression in non-seed tissue.

The organ specificity of four promoters that are known to direct seed-specific gene expression was tested. Whereas the phaseolin (phas)- and legumin B4 (leB4)-promoters were from genes encoding 7S and 11S globulins from Phaseolus vulgaris and Vicia faba, respectively, the usp- and the sbp-promoters were from non-storage protein genes of V. faba. The expression of different promoter-reporter gene fusions was followed either by RT-PCR or by registering the reporter enzyme activity in organs of transgenic tobacco, pea, narbon bean, or linseed. In addition to seeds, the promoters directed reporter gene expression in pollen and in seed coats. USP-, vicilin- and legumin-mRNA were detected by RT-PCR in pollen of Pisum sativum and V. faba. Expression during microsporogenesis and embryogenesis seems to be a general character of various seed protein genes.

Polypeptide components of oligomeric legumin-like thiamin-binding protein from buckwheat seeds characterized by partial amino acid sequencing and photoaffinity labeling.

Among thiamin-binding proteins that ubiquitously occur in plant seeds, that of common buckwheat became a model of extensive studies of the chemical mechanism of ligand-protein interaction. In this work, the polypeptide components of buckwheat seed thiamin-binding protein (BSTBP) are identified and characterized. We suggest that BSTBP is probably a fraction of major storage 13 S globulin (legumin), has an average molecular mass of 235 kDa and comprises hexamers of 57-kDa and 38-kDa subunits in variable combinations. Each subunit is a pair of disulfide-linked polypeptide chains, 36 kDa plus 24 kDa and two-times 22 kDa, respectively. The N-terminal sequences of 22-kDa and 24-kDa components show strict homology with those reported for "basic subunits" of buckwheat legumin. By photoaffinity labeling of BSTBP with 4-azido-2-nitrobenzoylthiamine, it is shown that the 36-kDa chain plays the major role in thiamin binding, but the other chains may also be variably involved. Putative thiamin-binding fragments are identified and sequenced.

Amino acid sequence of the 26 kDa subunit of legumin-type seed storage protein of common buckwheat (Fagopyrum esculentum Moench): molecular characterization and phylogenetic analysis.

The paper describes the amino acid sequence of a 26 kDa basic subunit of 13S globulin of common buckwheat (Fagopyrum esculentum Moench). The protein has 93 and 75% sequence homology with 11S globulin of Coffea arabica and beta subunit of 11S globulin of Cucurbita pepo respectively. The subunit has the "globally conserved" N-terminal sequence consisting of Gly-Ile-Asp-Glu and the cysteine at P7' from the proteolytic processing site. A conserved 7 residue domain of Pro-His-Trp-Asn-Ile-Asn-Ala, characteristic of basic subunits of legumins from non-leguminous angiosperms, is also present in this protein. A distinguishing features of this subunit is the relatively high level of lysine and methionine.

Stored cysteine proteinases start globulin mobilization in protein bodies of embryonic axes and cotyledons during vetch (Vicia sativa L.) seed germination.

Inhibition of protein synthesis by cycloheximide during vetch seed germination, did not prevent globulin breakdown as indicated by a decrease in vicilin- and legumin-specific immunosignals on Western blots. Protein bodies isolated from embryo axes and cotyledons of dry vetch (Vicia sativa L.) seeds using a non-aqueous method were found to be free of cytoplasmic and organellar contaminations. Lysates of these purified protein bodies were capable of degrading globulins; this process was blocked by the cysteine proteinase (CPR) inhibitor iodoacetic acid. Protein bodies contained the papain-like CPR2 and CPR4, and the legumain-like CPR VsPB2. In vitro assays showed that albumin extracts from protein bodies degraded oligopeptide substrates in the PepTag-Assay and degraded the legumain substrate N-benzoyl-asparaginyl-p-nitroanilide. We conclude that, during germination, globulin mobilization is initiated by stored CPRs in protein bodies of embryonic axes as well as cotyledons, and that de-novo-formed proteolytic enzymes mainly mediate bulk degradation of stored globulin in cotyledons after germination.

Mungbean [Vigna radiata (L.) Wilczek] globulins: purification and characterization.

Vicilin type (8S) and basic 7S globulins and legumin type (11S) globulins were isolated from mungbean [Vigna radiata (L.) Wilczek]. The native molecular weights of the different globulin types were 360000 for legumin, 200000 for vicilin, and 135000 for basic 7S. Some of the 8S globulin apparently complexed and coeluted with the 11S on gel filtration. On SDS-PAGE, 11S was composed of two bands of 40000 and 24000, 8S was composed of 60000, 48000, 32000, and 26000 bands, and basic 7S was composed of 28000 and 16000 bands. The percent composition of total globulins was estimated to be as follow: 8S, 89%; basic 7S, 3.4%; and 11S, 7.6%. The basic 7S and 11S but not the 8S globulins were found to have disulfide bonds. The presence of carbohydrates by conjugated peroxidase reaction was observed in all bands of 8S, the acidic polypeptide of basic 7S, and its complex but not in 11S. The 28000 basic 7S band and its 42000 complex and the first three major bands of 8S cross-reacted with antibodies to all types of soybean conglycinin subunits (alpha, alpha', and beta), whereas the fourth band cross-reacted only with the anti-beta subunit. None of the mungbean globulins cross-reacted with anti-soybean glycinin. Basic 7S was found to be easily extracted with 0.15 M NaCl, 11S was extracted with 0.35 M NaCl,and 8S was extracted over a wide range of NaCl concentrations. The N-terminal sequences of the different subunits/fragments of the globulins were determined and found to have strong homology with storage proteins of other legumes and crops.

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.).

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2-3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants.

Isolation and characterisation of legumin promoter sequence from chickpea (Cicer arietinum L.).

Seed specific promoters are useful for expression of foreign genes in the seeds. We have isolated a Cicer arietinum legumin promoter from lambda EMBL genomic library and subcloned in pBluescript II KS (-) in Eco RV and Pst I site. The 2.762 kb Hind II Pst I fragment was sequenced completely by dideoxy chain termination method by creating a set of unidirectional deletions of the inserts in pAKKIII. The insert contains mainly upstream sequence (2240 bps) and only a part of structural gene (522 bps) sequence. The 522 bps of the structural gene shows approximately 80% homology with pea legumin A and this is almost the same as chickpea legumin in its sequence. The amino acid sequence derived from the part of the structural gene was similar to the chickpea 5' part of the legumin structural gene with a few variations. A 21 amino acid signal peptide was also deduced like many other legumes. Transcription start site (CAT) was located at 55 bp upstream of the initiation codon ATG. One codon downstream to ATG codon Hind III site was present. TATA box was observed at -30 position, with a consensus of CCTATAAATAACC. The consensus CATGCAAG, a part of legumin box was noticed at -110 bp position. At -295 to -265 bp upstream AGGA box like sequences were observed and a 56 bp perfect repeat was located between -913 bp and -972 bp. Strong homology with pea promoter sequence near the CAT sequence was noticed which gradually decreased towards the upstream region. Thus the cloned fragment contains a full length promoter which can be utilised for expression of foreign genes in seeds of chickpea.

Interaction of chickpea (Cicer arietinum L.) legumin with oxidized linoleic acid.

Chickpea legumin has been purified and incubated under oxidizing conditions with linoleic acid to investigate the influence of this acid on the structure and nutritional quality of the protein. At the end of the incubation time, >30% of the linoleic acid was oxidized. The oxidized linoleic acid was highly detrimental to legumin, and the electrophoretic pattern of the protein was completely changed after the incubation period. Nevertheless, neither polymerization nor cleavage of the protein was observed as deduced from gel filtration chromatography, suggesting that the changes observed in native electrophoresis were probably due to oxidation of legumin. The incubation of legumin with linoleic acid also produced a diminution of the contents of methionine and histidine, by 81.3 and 24.3%, respectively. Finally, in vitro protein digestibility of chickpea legumin was also seriously affected by the incubation with linoleic acid, decreasing from 84.1 to 69.2%.

The lysine and methionine rich basic subunit of buckwheat grain legumin: some results of a structural study.

The 26 kD basic subunit of 280 kD buckwheat grain legumin has been partially characterized by measurement of its fluorescence and CD spectra. The protein has 22% alpha-helix, 36% beta-sheet, 12% beta-turn and 30% random coil secondary structure. In comparison with the basic subunits of other legumin-type proteins, the buckwheat legumin subunit has a high content of lysine and methionine. The protein also has higher ratios of lysine to arginine and methionine to arginine.

Structural and functional changes of faba bean legumin during super-limited tryptic hydrolysis.

The influence of a super-limited tryptic hydrolysis on physicochemical and surface functional properties of faba bean legumin has been studied using size-exclusion HPLC, SDS-PAGE, UV and fluorescence spectroscopy, fluorescence probe techniques, surface tension measurements as well as determination of emulsifying activity index (EAI) and emulsion droplets diameter (D). The extent of legumin hydrolysis comprised the range between about 14 and 60 split peptide bonds per molecule resulting in a stepwise decrease of legumin molecular weight to 240 kDa (legumin-T) via discrete intermediates with characteristic subunit patterns. These changes are accompanied by an increase in the surface hydrophobicity and the exposure of aromatic chromophores. No differences were found in the surface tension between the variously hydrolyzed legumin samples. Best emulsifying properties (highest EAI and lowest D values) were attained after a rather low tryptic hydrolysis (about 30 split peptide bonds per mol). Further hydrolysis impaired the emulsifying parameter which were, however, higher (EAI) or lower (D) than those for native legumin.

The evolution of angiosperm seed proteins: a methionine-rich legumin subfamily present in lower angiosperm clades.

Analysis of legumin-encoding cDNAs from Dioscorea caucasica Lipsky (Dioscoreaceae) and from Asarum europaeum L. (Aristolochiaceae) shows that there is an especially methionine-rich legumin subfamily present in the lower angiosperm clades including the Monocotyledoneae. It is characterized by a methionine content of 3-4 mol% which is roughly triple the methionine proportion of most other legumins. These "MetR" legumins, if present, still have to be detected in the higher angiosperms including the important seed crops. Evolutionary analysis suggests that the MetR legumins are the result of a gene duplication allowing the differentiation of legumin genes according to their sulfur content. The duplication event must have taken place before the split into mono- and dicotyledonous plants but probably after the separation of angiosperms and gymnosperms.

Use of polymerase chain reaction in analysing recombinant cDNA clones encoding storage proteins of chickpea Cicer arietinum L.

A simple and reliable method was undertaken for the use of polymerase chain reaction in analyzing cDNA clones. Amplification was done of the inserts from positive legumin clones isolated from a cDNA library constructed from developing chickpea cotyledons in the expression vector, gt11. Amplification was made simple by using oligonucleotide primers which allowed convenient sizing, subcloning and sequencing of inserts by di-deoxy chain termination method. This simple method may provide opportunity to isolate large number of agronomically important genes from gene libraries.

Construction and screening of subgenomic library of chickpea (Cicer arietinum L.) for legumin genes and their analysis.

Legumin storage protein genes of chickpea were identified by hybridizing restricted chickpea genomic DNA transferred on Southern blot with heterologous pea legumin cDNA probes. Southern hybridization of restricted chickpea genomic DNA with pea legumin pDUB6 and pDUB8 cDNA probes indicated presence of pea legumin homologous sequences in chickpea. About 4-5 kb EcoRI fragment was hybridized with both the probes used. DNA corresponding to this size was isolated from the gel and used for construction of subgenomic library in lambda ZAPII vector. After amplification, library was screened for presence of legumin genes in chickpea. Out of clones screened (0.1 x 10(6)), three positive clones were obtained. Restriction analysis and hybridization studies showed presence of complete structural gene in each of the identified positive clones.

Physico-chemical characterization of legumin-T from faba bean (Vicia faba L).

Legumin-T, the high-molecular mass product of limited tryptic hydrolysis of faba bean legumin, was investigated using hydrodynamic methods, static light scattering, fluorescence and ultraviolet spectroscopy. The following physico-chemical parameters were determined in a high-ionic strength buffer system: molecular mass, 2.4 x 10(5) g/mol; sedimentation coefficient, SO20 = 10.8 x 10(-13)Si; diffusion coefficient, DO20 = 4.1 x 10(-7) cm2 s-1; intrinsic viscosity, [eta] = 3.51 mL/g; partial specific volume, v = 0.719 mL/g; frictional ratio, f/f0 = 1.22; shape factor, beta = 2.17 x 10(6). Conformational changes during the formation of legumin-T can be deduced from the fluorescence emission and UV spectra.