Legumin Storage Proteins Research Articles

Mass Spectrometry Characterization of the SDS-PAGE Protein Profile of Legumins and Vicilins from Chickpea Seed.

Chickpea (Cicer arietinum L.) seed proteins show a lot of functional properties leading this legume to be an interesting component for the development of protein-enriched foods. However, both the in-depth proteomic investigation and structural characterization of chickpea seed proteins are still lacking. In this paper a detailed characterization of chickpea seed protein fraction by means of SDS-PAGE, in-gel protein digestion, high-resolution mass spectrometry, and database searching is reported. Through this approach, twenty SDS gel bands were cut and analyzed. While the majority of the bands and the identified peptides were related to vicilin and legumin storage proteins, metabolic functional proteins were also detected. Legumins, as expected, were revealed at 45-65 kDa, as whole subunits with the α- and β-chains linked together by a disulphide bond, but also at lower mass ranges (α- and β-chains migrating alone). Similarly, but not expected, the vicilins were also spread along the mass region between 65 and 23 kDa, with some of them being identified in several bands. An MS structural characterization allowed to determine that, although chickpea vicilins were always described as proteins lacking cysteine residues, they contain this amino acid residue. Moreover, similar to legumins, these storage proteins are firstly synthesized as pre-propolypeptides (Mr 50-80 kDa) that may undergo proteolytic steps that not only cut the signal peptides but also produce different subunits with lower molecular masses. Overall, about 360 different proteins specific of the Cicer arietinum L. species were identified and characterized, a result that, up to the current date, represents the most detailed description of the seed proteome of this legume.

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.

Proteomic Analysis of Near-Isogenic Sunflower Varieties Differing in Seed Oil Traits

Near-isogenic sunflower lines containing 25% (inbred RHA280) and 48% (RHA801) oil by seed dry mass were comparatively analyzed in biological triplicate at 18 days after flowering using two-dimensional (both pI 3-10 and 4-7) Difference Gel Electrophoresis. Additionally, two inbred lines varying in oleic acid content, HA89 (18% oleic) and HA341 (89% oleic), were also analyzed in the same manner. Statistical analyses of these sunflower lines was performed beginning with fitting a mixed effects linear model to the log-transformed optical volume of each spot to account for gel variation, followed by testing the significance between varieties for mean transformed optical spot volumes. The p-values from the spot analysis procedures were then used to find the cutoff point for differential expression using a 10% false-discovery rate (FDR). Comparison of the oil content and oleic acid composition lines revealed 77 and 42 protein spots below the 10% FDR cutoff, respectively, and were therefore declared differentially expressed. Liquid chromatography-tandem mass spectrometry analysis of each of these protein spots resulted in assignments for 44 and 17 spots, respectively. Fructokinase, plastid phosphoglycerate kinase, and enolase proteins were determined to be up-regulated in the high oil line, while phosphofructokinase, cytosolic phosphoglucomutase, and cytsolic phosphoglycerate kinase were up-regulated in the low oil variety. Additionally, four activities involved in amino acid synthesis were up-regulated in the low oil variety in addition to 12S storage proteins and a protein similar to legumin storage protein. Interestingly, two 2-DE spots identified as 14-3-3 proteins were found to be up-regulated in high oleic acid variety. Alteration of glycolytic and amino acid biosynthetic enzymes, as well as storage protein levels, suggests seed oil content is tightly linked to carbohydrate metabolism and protein synthesis in a complex manner.

Physiological and morphological characteristics during development of pedunculate oak (Quercus robur L.) zygotic embryos

The developmental stages of oak zygotic embryos (ZEs) are characterized here according to morphological and physiological features. Seeds were harvested from June to September in 1-week intervals. Excised embryos were classified into four stages of development by using growth parameters. For physiological characterization, endogenous levels of abscisic acid (ABA), indole-3-acetic acid (IAA), l-proline, starch content and water status were determined. The expression of the oak legumin storage protein gene was tested in immature cotyledonary ZEs before and after ABA treatment. The ABA levels of the embryos showed a significant peak during the intermediate stage of maturation (stage III) and then decreased again at the end of the late maturation phase (stage IV). Concomitant with ABA, the moisture content declined with the maximum embryo size. High IAA levels were found at the beginning of embryo enlargement as exponential growth occurred (stage II) but decreased during further development. Starch accumulated gradually in the course of maturation, whereas significant values were found in stage IV ZEs near shedding. Proline, on fresh weight basis, was high during stages I and II. Osmotic potential increased when, by rapid dry matter accumulation, stage II ZEs reached their maximum size during early intermediate development. Expression of precocious germination was higher on hormone-free medium, in particular, among stage II and stage III ZEs. Variations in phytohormone levels in combination with changes in tissue water status seem to be important factors for oak ZE development.

High accumulation of legumin and Lea-like mRNAs during maturation is associated with increased conversion frequency of somatic embryos from pedunculate oak (Quercus robur L.).

The expression patterns of the storage protein legumin gene and Em- and dehydrin-like homologues were investigated in somatic embryos from Quercus robur L. The effect of different maturation treatments (1% agar, 6% sorbitol, or 5% polyethylene glycol) and partial desiccation on transcript accumulations as well as conversion capacity of somatic embryos was also investigated. Differential expression of putative Em- and two dehydrin-like homologues (designated as Dhn1a and Dhn1b) was detected in somatic oak embryos with heterologous probes. Low expression levels of legumin, Em- and dehydrin-like mRNAs were detected in somatic embryos prior to maturation treatment. A high accumulation of these transcripts was found in embryos that had been cultured on media supplemented with 6% sorbitol or 1% agar. These embryos also showed a high conversion frequency into plantlets. In contrast, no improvement in plant conversion as well as a low accumulation of legumin, Em-like and Dhn1b-like transcripts was observed in embryos that were matured on polyethylene glycol medium. Partial-desiccation treatment significantly enhanced the plant conversion. Nevertheless, a decline in expression of legumin, Em-like and Dhn1a-like homologues was detected upon dehydration. In contrast, Dhn1b and oak homologues to peach dehydrin were also strongly expressed in desiccated embryos. In addition, the treatment of embryos with abscisic acid promoted the accumulation of all investigated transcripts. These results suggest that the regulation of the legumin storage protein gene and Lea-like homologues in somatic oak embryos is under developmental control and that their regulation can be influenced by manipulating the culture conditions.

Cloning and Characterization of Legumin Storage Protein Gene of Chickpea (Cicer arietinum L)

Genomic DNA clones coding for legumin storage protein were isolated from chickpea (Cicer arietinum L.) subgenomic library using pea legumin cDNAs as a probe. The complete nucleotide sequence of the legumin gene (leg3) was determined. Sequence analysis indicated that leg3 contains 2022 bp long structural gene interrupted by three small introns. The gene encodes a polypeptide of 496 amino acids including a signal peptide of 21 amino acid residues and a-b subunit cleavage site. Leg3 polypeptide is rich in amide amino acids and contains 5 methionine and 6 cysteine amino acids. DNA sequence comparison showed about 70–80% identity with legumin genes from pea, soybean and broad bean. The 5’ flanking region contains regulatory elements such as TATA and legumin box and 3’ region contains a stop codon and two polyadenylation signals. Southern hybridisation indicated the presence of multiple copies of /eg3in chickpea and estimated about 10–12 copies per haploid genome. Northern blot hybridisation showed that leg3 expresses in developing seed and not in other tissues of chickpea indicating temporal and tissue-specific expression of this gene.

Cotton Mat5-A (C164) Gene and Mat5-D cDNAs Encoding Methionine-Rich 2S Albumin Storage Proteins

Several abundant mRNAs are coordinately expressed specifically during the maturation stage of embryogenesis (6, 7). The cotton Mat mRNAs include those of the major vicilin and legumin storage protein genes (6), and representatives of these genes have been sequenced (2, 3). To help define the temporal and spatial regulation of the maturation program of gene expression, we isolated an additional cotton Mat gene, MatS, which encodes mRNAs represented by cDNA clone C164. The mRNAs from the two Mat5 alloalleles in the allotetraploid genome of Gossypium hirsutum make up 2% of the mRNA in maturation stage embryos (6), and the sequences show that they encode methionine-rich 2S albumin storage proteins. Three genomic fragments were recovered, but restriction site analysis and sequencing showed that they contain the same alloallele, Mat5-A, present in the A genome. One was extensively sequenced, as was all of cDNA C 164 and extensive portions of five other cDNA clones (Table I). Figure 1 shows the sequence of clone GC 1 64-24RC of MatS-A. All six cDNAs are identical with each other in the regions sequenced, but they differ significantly from the sequence of Mat5-A. The gene and cDNA sequences are colinear, with eight nucleotide differences, until MatS-A nucleotide 3661 and C164 nucleotide 514. There is no obvious similarity for the next 62 nucleotides until the site of polyadenylation of the cDNAs (Fig. 1). Because there are only two alloallelic genes in G. hirustum (6), we presume that all the sequenced cDNAs are transcribed from the other alloallele, MatS-D. The two MatS alloalleles encode proteins that differ in only three of 139 amino acid residues. Their sequences are very similar to those of the 2S albumin storage protein family (1, 8, 9), and an alignment of the cotton sequences with other 2S albumin preproproteins is unambiguous in its important features (Fig. 2). There is complete identity at the cysteine and leucine residues that are diagnostic of the family and about 44% of the cotton residues are identical with those of Bertholletia (1) and Arabidopsis (9) 2S albumins. The predicted mature form (9; Fig. 2) of the cotton 2S albumin shares with that of Bertholletia a high methionine content. It is 10% in cotton and 19% in Bertholletia, compared with 2 to 3% in other 2S albumins (1, 8, 9). Four of the 10 methionine residues in the cotton protein are at novel positions that may be sites at which other 2S albumins could be engineered for higher methionine content. Because none of the six cDNAs that were examined are transcribed from MatS-A, it is possible that this gene is not active. However, it is more likely that the apparent absence of MatS-A cDNAs is due to failure to process or polyadenylate its transcripts. In the sequence in Figure 1 and in overlapping phage isolate GC164-27 (Table I), there is a class I-like retrotransposon (10) in reverse orientation to the Mat5-A transcription unit (data not shown). The retrotransposon's putative internal domain/3'-long terminal repeat boundary is at MatS-A nucleotide 3791/92, and the 130 nucleotides between this boundary and the Mat5-A/Mat5-D cDNA divergence point can contain only a highly truncated long terminal repeat, if any part of it. We predict that the element, or element-induced rearrangements of Mat5-A sequence, interferes with the processing of the 3' end of MatS-A transcripts. Rearrangement of MatS-A in this region is suggested by the presence of long direct repeats, part of one of which is present in Mat5-D cDNAs (Fig. 1). A sequence very similar to MatS-A nucleotides 907 to 1765 has been found in reverse orientation in the upstream region of an unrelated cotton Lea4 gene (4). Portions of this element are also duplicated at Mat5-A nucleotides 2289 to 2416 (Fig. 1, repeats 1 and 2). Consequently, the sequences that are responsible for transcription of MatS-A are probably between nucleotide 2615 and the transcription start at nucleotide 3146. In this region the sequence of MatS-A has some similarities with those of other 2S albumin storage proteins, in particular two elements (double underlined in Fig. 1) present in similar locations in many of these genes (8, 9). Other elements suggested to be important in various storage protein genes (5) are not obvious in MatS-A. Finally, MatS-A has been compared with two other cotton Mat genes, vicilin (2) and legumin A (3), with which Mat5-A is coordinately expressed in cotton (6, 7), but no similarities are compelling in any sequence pair. ' Supported by a grant from the National Institutes of Health.

The legumin gene family: structure and evolutionary implications of Vicia faba B-type genes and pseudogenes

We have characterized several Vicia faba genes encoding methionine residue-free group B subunits of the 11S or legumin storage proteins. The respective gene subfamily consists of 10 to 15 members, six of them having been studied by DNA sequence analysis. Four functional genes (LeB2, LeB4, LeB6, LeB7) are highly homologous in their coding region and 0.3 kb of their 3' flanking sequences. On the other hand, two pseudogenes (psi LeB1, psi LeB5) have accumulated a large number of mutations including an identical 0.7 kb internal deletion; they are both flanked by a repetitive element. Analysis of sequence changes show that transitions are nearly double as frequent as transversions. CpG is the most infrequent dinucleotide whereas TpA is significantly underrepresented in exon sequences. End points of deletions are correlated with short direct repeats and preferentially found in the two introns. Our studies indicate that the Vicia faba legumin B gene subfamily contains a group of expressed, highly homologous genes as well as more diverged pseudogenes.

Correlated in situ hybridisation and immunochemical studies of legumin storage protein deposition in pea (Pisum sativum L.)

Legumin and vicilin are the major globulin seed storage proteins of pea. They are synthesised predominantly in the cotyledons where they are sequestered within membrane-bounded vacuolar protein bodies. In situ hybridisation histochemistry, with both biotinylated and 35S-labelled cDNA probes, has been used to visualise the temporal and spatial patterns of distribution of legumin and vicilin mRNAs during seed development. These patterns have been compared with those of storage protein deposition which have been determined by immunocytochemistry. Results indicate that within the cotyledons high levels of legumin and civilin mRNAs are restricted to the storage parenchyma tissues, whilst the epidermal cells and vascular parenchyma do not show such accumulation. The tissues of the embryo axis also show differential levels of expression, although where present the levels of mRNAs appear much lower than in the cotyledons. Throughout the embryo the patterns shown by in situ hybridisation are similar to those shown by immunocytochemistry, although the transient endosperm of early seed development does not show such a correlation.

A transposon-like structure in the 5' flanking sequence of a legumin gene from Pisum sativum.

The legumin storage proteins of Pisum sativum are coded for by a multigene family. An insertion element (Pis1) has been found integrated into the 5' flanking sequence of the legC legumin seed storage protein gene. This element contains all the sequence features of the CACTA family of insertion elements, has perfect 12 bp inverted repeats at its termini, and generates a target host site duplication upon integration. An 8 bp sequence within the left arm of the insertion element shows perfect homology to a sequence in the legC flanking region. Three stem-loop structures which can be formed within the element have the same stem sequence.

Tissue-specific expression of a pea legumin gene in seeds of Nicotiana plumbaginifolia

A 3.4-kilobase genomic DNA fragment from Pisum sativum L. containing the LegA gene, which encodes a major legumin storage protein, was transferred to Nicotiana plumbaginifolia using an Agrobacterium tumefaciens strain containing the Bin 19 binary vector system. Northern hybridisation analysis of legA-transformed plants demonstrated that legumin-specific RNA was present in developing seeds but not in developing leaves. Legumin protein was immunologically detected in the mature seeds of legA-transformed plants, and was present as the correct-size protein composed of disulphide-bonded polypeptides. It is concluded that the transferred pea genomic fragment contains all the information necessary for seed-specific expression of the legA gene, and for correct processing of the primary transcript and the precursor legumin protein.