Soybean Vegetative Storage Protein Research Articles

A single amino acid substitution in soybean VSPalpha increases its acid phosphatase activity nearly 20-fold.

Soybean [Glycine max (L.) Merr.] contains two proteins called vegetative storage proteins (VSPs) that function as temporary storage reserves, but are also closely related to plant acid phosphatases of the haloacid dehalogenase (HAD) superfamily. This study examined the biochemical basis for the relatively low catalytic activity previously reported for these VSPs. The specific activity of purified recombinant VSPalpha on GMP was about 40-fold lower than for a related soybean root nodule acid phosphatase (APase), which had a specific activity of 845 U mg(-1) protein. Conversion of Ser106 to Asp increased VSPalpha activity about 20-fold. This Asp residue is present in nodule APase and is a highly conserved nucleophile in the HAD superfamily. Related VSPs from cultivated soybean and from three wild perennial soybeans, as well as a pod storage protein (PSP) from Phaseolus vulgaris L. all lack the catalytic Asp, suggesting they too are catalytically inefficient. Phylogenetic analysis showed the VSPs and PSP are more closely related to each other than to 21 other VSP-like proteins from several plant species, all of which have the nucleophilic Asp. This study suggests that loss of catalytic activity may be a requirement for the VSPs and PSP to function as storage proteins in legumes.

Coexpression of the soybean vegetative storage protein beta subunit (S-VSPbeta) either with the bacterial feedback-insensitive dihydrodipicolinate synthase or with S-VSPalpha stabilizes the S-VSPbeta transgene protein and enhances lysine production in transgenic tobacco plants.

Soybean vegetative storage proteins (S-VSPs) are lysine-rich leaf proteins, originally found to accumulate to high levels in depodded soybean plants. In the present study, we overexpressed S-VSPbeta, the ruminant stable subunit of the S-VSP genes, in transgenic tobacco plants. The S-VSPbeta protein accumulated in all organs studied, but its level declined drastically with leaf age. This instability of S-VSPbeta could be overcome either by elevating free lysine levels or by coexpressing S-VSPbeta with S-VSPbeta. High levels of rumen-stable, lysine-rich proteins is expected to improve absorption of lysine by ruminants. Furthermore, the expression of S-VSPs in heterologous plants led to a significant increase in total soluble lysine, suggesting that these proteins may also permit better assimilation of lysine by humans and monogastric animals.

Combined expression of S-VSPalpha in two different organelles enhances its accumulation and total lysine production in leaves of transgenic tobacco plants.

Soybean vegetative storage proteins (S-VSPs) accumulate to high levels in vacuoles of both wild types and heterologous plants. Here it is shown that directing S-VSPalpha to two different organelles-chloroplasts and vacuoles-in a single transgenic plant significantly increased its accumulation. Accumulation of S-VSPalpha in heterologous plants correlated with total soluble lysine. Using this approach with essential amino-acid-rich transgene proteins may lead to a breakthrough in improving plant nutritional quality.

Resistance of soybean vegetative storage proteins (S-VSPs) to proteolysis by rumen microorganisms.

Soybean vegetative storage proteins (S-VSPs) are lysine-rich and, hence, are potentially of high nutritive value for high productive ruminants. Using S-VSPs from wild-type soybean and from transgenic tobacco plants expressing either one of the two S-VSPs subunits (S-VSP alpha or S-VSP beta) or both, we tested their stability in cow rumen fluid under in situ conditions, using SDS-polyacrylamide gel electrophoresis. Proteolysis and degradation pattern of S-VSPs from transgenic tobacco leaves occurred relatively fast compared with that of wild-type (WT) soybean plants. Comparing the two S-VSPs subunits expressed in transgenic plants, we found that S-VSP alpha was degraded much faster than S-VSP beta. The degradation pattern of S-VSPs in transgenic tobacco plants expressing both subunits resembled that of WT soybean. In contrast, the degradation pattern of transgenic tobacco plants expressing a single subunit was different. These finding suggest that the quaternary structure of S-VSPs may be an important factor determining their resistance to rumen degradation. Our results also suggest that the stability to rumen proteolysis of a given protein, when expressed in a transgenic plant, may not always be predictable and has to be verified.

Subtractive immunization: a tool for the generation of discriminatory antibodies to proteins of similar sequence

Antibodies specific for a protein of interest are invaluable tools for monitoring the protein's structure, location and activity. Due to the tendency of an immune system to mount a response toward the abundant, immunodominant epitopes in a protein mixture, difficulties are inherent in the isolation of antibodies specific for proteins that are rare or poorly immunogenic. Likewise, isolation of antibodies specific for a protein with significant sequence similarity to other proteins, such as those derived from protein engineering, may be challenging. Subtractive immunization is a technique proven to facilitate efforts to produce monoclonal antibodies specific for antigens that are present in low abundance in a protein mixture, poorly immunogenic and/or similar in sequence or structure to other proteins. This protocol provides a detailed, stepwise procedure for the isolation of antibodies specific for a protein with sequence similarity to other proteins. As an example, we describe methods established to isolate antibodies specific to a methionine-enriched variant of soybean vegetative storage protein β (VSPβ-Met) that shares 91.8% amino acid sequence identity to the wild-type protein (VSPβ-WT). These methods include cyclophosphamide-induced immunosuppression of mice for the wild-type protein followed by immunization with VSPβ-Met. As a result of this procedure, mouse polyclonal antibodies that exhibited 10-fold greater reactivity with VSPβ-Met than VSPβ-WT in an ELISA were generated. It is anticipated that this strategy will have utility for generating antibodies specific to protein variants derived from protein engineering.

Strategies to generate antibodies capable of distinguishing between proteins with >90% amino acid identity

Protein engineering is a common strategy for the generation of protein variants with new properties. The engineered variants often have a high degree of similarity with the wild-type progenitor protein, necessitating a tool (e.g., antibody) to distinguish the wild-type and variant protein forms. As part of an overall effort to understand the process of incorporation of amino acids into storage proteins during seed fill in soybean, we have engineered a variant of soybean vegetative storage protein β (VSPβ) that is 91.8% identical in amino acid sequence to the wild-type protein, but contains 10% methionine (VSPβ-Met, unpublished results). Thus, it would be desirable to have antibodies that specifically recognize VSPβ-Met over the endogenously expressed wild-type protein in transgenic plants. To this end, we compared three strategies for the isolation of VSPβ-Met-specific antibodies: (1) hybridoma production using VSPβ-Met protein as the antigen, (2) polyclonal antibody production in rabbits using a peptide antigen corresponding to a methionine-rich region of VSPβ-Met, and (3) subtractive immunization in mice using VSPβ-WT as the tolerogen, cyclophosphamide for immunosuppression and VSPβ-Met as the immunogen. While the first strategy generated antibodies cross-reactive to both antigens, the second strategy generated polyclonal antibodies that preferentially recognized the variant protein in immunoblots. However, using subtractive immunization, we were able to generate mouse polyclonal antibodies that exhibited 10-fold greater reactivity with VSPβ-Met than VSPβ-WT in an ELISA.

A soybean vegetative storage protein accumulates to high levels in various organs of transgenic tobacco plants

Soybean vegetative storage proteins (S-VSPs) are a group of high-lysine proteins. These proteins can accumulate up to 15% of the soluble leaf proteins in young shoots, as well as in shoots of depodded mature plants. Closely related proteins are found in forage crops, such as alfalfa. We have expressed the S-VSPα gene, fused to the strong constitutive cauliflower mosaic virus 35S promoter, in transgenic tobacco as a model plant to study the potential of producing high levels of S-VSPs for the nutritional improvement of forage crops. S-VSPα was detected in the soluble (albumin) fraction of the transgenic plants. The transgenic protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis similarly to the natural S-VSPα of soybean, suggesting that it was correctly processed in tobacco and accumulated in the vacuoles. The S-VSPα ranged between 2 and 6% of the soluble proteins in leaves of the transgenic plants and was present in various organs and in mature leaves. Our results suggest that S-VSPs can serve as an excellent protein source for improving the nutritional quality of crop plants, particularly cereal forage and grains, which contain limiting levels of lysine.

Transformation of peanut with a soybean vspB promoter-uidA chimeric gene. I. Optimization of a transformation system and analysis of GUS expression in primary transgenic tissues and plants.

Direct DNA delivery via microprojectile bombardment has become an established approach for gene transfer into peanut (Arachis hypogaea L.). To optimize our transformation protocol and to simultaneously explore the function of a heterologous promoter whose activity is developmentally regulated, embryogenic cultures from three peanut cultivars were bombarded with two plasmid constructs containing a uidA gene controlled by either a soybean vegetative storage protein gene promoter or a cauliflower mosaic virus 35S promoter. We found that GUS transient expression was useful to predict stable transformation and confirmed that image analysis could provide a quick and efficient method for semi-quantitation of transient expression. One hundred and sixty hygromycin-resistant cell lines were recovered from and maintained on selective medium, and those tested by Southern blot analysis showed integration of the foreign gene. Over 200 transgenic plants were regenerated from 38 cell lines. More than 100 plants from 32 cell lines flowered and 79 plants from 19 cell lines produced pods. Over 1000 R1 seeds were harvested. Analysis of expression in primary transgenic plants showed that GUS expression driven by the vspB promoter was modulated by chemical and positional information.

A 28-kilodalton pod storage protein of French bean plants. Purification, characterization, and primary structure.

When French bean (Phaseolus vulgaris) plants were depodded in the early stages of fruit development, relative levels of a specific protein with a relative molecular weight of 28,000 were enhanced in the young pods that formed later. The protein, designated pod storage protein (PSP), was purified from extracts of newly formed pods from plants that had been previously depodded four times at intervals of 2 weeks. Two-dimensional polyacrylamide gel electrophoresis showed the presence of three forms (designated A, B, and C) of PSP with identical electrophoretic mobilities but different charges. The molecular mass of native PSP was estimated by gel filtration to be 67 kD; therefore, the protein was most likely present as a dimer. The antisera raised against forms A and C were crossreactive with each other. Form B lacked the N-terminal alanine of forms A and C. An expression library from French bean pods was screened using the antiserum against form A, and a full-length cDNA clone was isolated. The cDNA insert included 765 bp potentially encoding a polypeptide with 255 amino acid residues (and a calculated molecular mass of 28,854 D). The amino acid sequence deduced from the PSP cDNA had 65 to 71% identity with soybean (Glycine max) vegetative storage protein sequences (P.E. Staswick [1988] Plant Physiol 87: 250-254; and Correction [1989] Plant Physiol 89: 717). Genomic Southern blot analysis suggested that PSP is derived from a single-copy gene.

Characterization of a New Lectin of Soybean Vegetative Tissues

Lectins are carbohydrate-binding proteins that occur widely among plants. Lectins of plant vegetative tissues are less well characterized than those of seeds. Previously, a protein of soybean (Glycine max [L.] Merr.) leaves was shown to possess properties similar to the seed lectin. Here we show that the N-terminal amino acid sequence of this protein shares 63% identity with the seed lectin. Immunoblot analysis indicated that the protein occurs in leaves, petioles, stems, and cotyledons of seedlings but not in seeds. These observations prompted designation of the protein as a soybean vegetative lectin (SVL). Immunohistochemical localization in leaves indicated that SVL was localized to the vacuoles of bundle-sheath and paraveinal mesophyll cells. Removal of sink tissues or exposure to atmospheric methyl jasmonate caused increased levels of SVL in leaves and cotyledons. Co-precipitation of SVL and the soybean vegetative storage protein (VSP) during purification suggested an interaction between these proteins. SVL-horseradish peroxidase conjugate bound to dot blots of VSP or SVL, and binding was inhibited by porcine stomach mucin and heparin but not simple carbohydrates. Binding between SVL and VSP and similarities in localization and regulation support a possible in vivo interaction between these proteins.

Purification and Properties of a Unique Nucleotide Pyrophosphatase/Phosphodiesterase I That Accumulates in Soybean Leaves in Response to Fruit Removal.

Several unique proteins accumulate in soybean (Glycine max) leaves when the developing fruits are removed. In the present study, elevated levels of nucleotide pyrophosphatase and phosphodiesterase I activities were present in leaves of defruited soybean plants. The soluble enzyme catalyzing these reactions was purified nearly 1000-fold, producing a preparation that contained a single 72-kD polypeptide. The molecular mass of the holoenzyme was approximately 560 kD, indicating that the native enzyme was likely octameric. The purified enzyme hydrolyzed nucleotide-sugars, nucleotide di- and triphosphates, thymidine monophosphate p-nitrophenol, and inorganic pyrophosphate but not nucleotide monophosphates, sugar mono- and bisphosphates, or NADH. The subunit and holoenzyme molecular masses and the preference for substrates distinguish the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I from other plant nucleotide pyrophosphatase/phosphodiesterase I enzymes. Also, the N-terminal sequence of the soybean leaf enzyme exhibited no similarity to the mammalian nucleotide pyrophosphatase/phosphodiesterase I, soybean vegetative storage proteins, or other entries in the data bank. Thus, the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I appears to be a heretofore undescribed protein that is physically and enzymatically distinct from nucleotide pyrophosphatase/phosphodiesterase I from other sources, as well as from other phosphohydrolytic enzymes that accumulate in soybean leaves in response to fruit removal.

Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB, genes encoding vegetative storage protein acid phosphatases, and is regulated similarly by methyl jasmonate, wounding, sugars, light and phosphate

The soybean vegetative storage proteins, VSP alpha and VSP beta, are acid phosphatases that accumulate to very high levels in hypocotyls, young leaves and flowers and pods. The genes encoding the soybean VSP are activated by jasmonate, wounding, sugars and light and down regulated by phosphate and auxin. In this study, expression of an Arabidopsis thaliana gene (Atvsp) encoding a protein homologous to soybean Vsp alpha and Vsp beta, was examined and compared to expression of the soybean Vsp genes. Atvsp mRNA was present at high levels in flowers and buds and at low levels in roots, stems, leaves and siliques. Expression of Atvsp in leaves could be induced by wounding or by treatment of illuminated plants with methyl jasmonate and sucrose. Roots of plants with wounded leaves also accumulated Atvsp mRNA indicating that this gene can be regulated by a transmissible wound signal. Phosphate partially inhibited expression of Atvsp. Arabidopsis proteins of 29 and 30 kDa crossreacted with antibodies against soybean VSP. These proteins were very abundant in flowers and the proteins accumulated in leaves and roots of plants treated with methyl jasmonate. The level of these proteins in flowers was similar to the levels of soybean VSP in young soybean leaves. Overall, these data indicate that Arabidopsis Atvsp and soybean VspA/B genes are regulated similarly and that in both plants, the gene products can accumulate to high levels. This suggests that genes homologous to VspA/B may be of greater general significance than previously recognized.

Phosphate Modulates Transcription of Soybean VspB and Other Sugar-Inducible Genes.

The soybean vegetative storage protein genes VspA and VspB encode vacuolar glycoprotein acid phosphatases. Transcription of the Vsp is synergistically activated by jasmonic acid or methyl jasmonate (MeJA) and soluble sugars. The action of these modulators is mediated by two different DNA domains in the VspB promoter. In this study, we present new data regarding VspB regulation by sucrose and inorganic phosphate, which suggest a common mechanism of transcriptional control for Vsp and other sugar-inducible genes. We found that the sugar-mediated activation of VspB expression was inhibited by phosphate. Deletion analysis and transient assays in tobacco protoplasts identified a 130-bp DNA domain in the VspB promoter that mediates both sucrose induction and phosphate inhibition. Transcription mediated by this DNA domain was induced by phosphate elimination from the protoplast incubation medium, even in the absence of sucrose. The effect of sucrose and phosphate on VspB expression was studied in vivo in several ways. Depletion of phosphate from soybean cell cultures by the addition of mannose stimulated VspB expression, even in the absence of sucrose or MeJA. In illuminated soybean leaves treated with MeJA, inhibition of photosynthetic electron transport by DCMU decreased VspB expression. In contrast, VspB expression in soybean leaves stimulated by phosphate depletion was not influenced by DCMU. Moreover, sucrose-stimulated expression of the sugar-responsive genes lipoxygenase A and chalcone synthase of soybean and proteinase inhibitor II and class I patatin of potato was inhibited by phosphate. Like VspB, these genes were stimulated by phosphate depletion in the absence of exogenous sucrose. We propose that sugar-responsive genes are activated, in part, by accumulation of sugar-phosphates and concomitant reduction of cellular phosphate levels. These data may help explain recruitment of the Vsp, which encode acid phosphatases, as vegetative storage proteins.

Sucrose Modulation of Soybean Vsp Gene Expression Is Inhibited by Auxin.

We have shown that auxin represses soybean (Glycine max L.) vegetative storage protein gene (Vsp) expression in suspension-cultured cells and in leaves and petioles of excised trifoliates. The auxin analog naphthyleneacetic acid (NAA) at 10 [mu]M strongly inhibited methyl jasmonate-induced Vsp expression in soybean suspension-cultured cells. Both indole-3-acetic acid and NAA inhibited methyl jasmonate- and wound-induced expression of the Vsp and LoxA excised soybean trifoliate leaves and petioles. The less active auxin analog phenylacetic acid had less effect on methyl jasmonate- and wound-induced expression of these genes. Addition of cytokinin to alter the auxin:cytokinin ratio did not reverse auxin inhibition of Vsp expression. Transcription of [beta]-glucuronidase (Gus) modulated by a methyl jasmonate-responsive domain derived from the VspB promoter was minimally influenced by auxin. In contract, sucrose-induced expression of Gus mediated by a sucrose-responsive domain of the VspB promoter was strongly inhibited by NAA. We conclude that auxin inhibits Vsp mRNA accumulation, in part, by repressing sugar-mediated activation of Vsp expression.

Purification of the Major Soybean Leaf Acid Phosphatase That Is Increased by Seed-Pod Removal.

Fruit removal for 5 weeks after flowering increased acid phosphatase activity 10-fold in soybean (Glycine max L. Merr. Var Hobbit) leaves compared with normal seed-pod-bearing plants. The major acid phosphatase activity in leaves was purified over 2700-fold, yielding a single polypeptide of 51 kD with a specific activity of 1353 units/mg protein using p-nitrophenylphosphate as the substrate. Isoelectric focusing demonstrated that the purified protein co-migrated with a majority of the activity that increased in leaves following seed-pod removal. Immunoblot analysis demonstrated that at least part of the increased activity was due to an increased abundance of the phosphatase protein. In situ enzyme activity staining localized most of the total phosphatase activity to vascular tissues, the leaf paraveinal mesophyll cell layer, and the lower epidermis. This distribution and the response to seed-pod removal paralleled previous results for soybean vegetative storage protein (VSP) [alpha] and [beta]. However, in a native polyacrylamide gel the VSP detected by immunological staining of electrophoretically transferred protein did not migrate with the majority of the phosphatase activity. Fractionation of crude leaf protein on concanavalin A-Sepharose yielded a fraction containing 97% of the total VSP but only 0.1% of the total acid phosphatase activity.

The soybean vegetative storage proteins VSP alpha and VSP beta are acid phosphatases active on polyphosphates.

The soybean vegetative storage protein genes (vspA, and vspB) are regulated in a complex manner developmentally and in response to external stimuli such as wounding and water deficit. The proteins accumulate to almost one-half the amount of soluble leaf protein when soybean plants are continually depodded and have been identified as storage proteins because of their abundance and pattern of expression in plant tissues. We have shown that purified VSP homodimers (VSP alpha and VSP beta) and heterodimers (VSP alpha/beta) possess acid phosphatase activity (alpha = 0.3-0.4 units/mg; beta = 2-4 units/mg; alpha/beta = 7-10 units/mg). Specific activities were determined by monitoring o-carboxyphenyl phosphate (0.7 mM) cleavage at pH 5.5 (VSP alpha) or pH 5.0 (VSP alpha/beta and VSP beta) in 0.15 M sodium acetate buffer at 25 degrees C. These enzymes are active over a broad pH range, maintaining greater than 40% of maximal activity from pH 4.0 to 6.5 and having maximal activity at pH 5.0-5.5. They are inactivated by sodium fluoride, sodium molybdate, and heating at 70 degrees C for 10 min. These phosphatases can liberate Pi from several different substrates, including napthyl acid phosphate, carboxyphenyl phosphate, sugar-phosphates, glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, ATP, ADP, PPi, and short chain polyphosphates. VSP alpha/beta cleaved phosphoenolpyruvate, ATP, ADP, PPi, and polyphosphates most efficiently. Apparent Km and Vmax values at 25 degrees C and pH 5.0 were 42 microM and 2.0 mumol/min/mg, 150 microM and 4.2 mumol/min/mg, and 420 microM and 4.1 mumol/min/mg, for tetrapolyphosphate, pyrophosphate, and phosphoenolpyruvate, respectively.

Coregulation of Soybean Vegetative Storage Protein Gene Expression by Methyl Jasmonate and Soluble Sugars

The soybean vegetative storage protein genes vspA and vspB are highly expressed in developing leaves, stems, flowers, and pods as compared with roots, seeds, and mature leaves and stems. In this paper, we report that physiological levels of methyl jasmonate (MeJA) and soluble sugars synergistically stimulate accumulation of vsp mRNAs. Treatment of excised mature soybean (Glycine max Merr. cv Williams) leaves with 0.2 molar sucrose and 10 micromolar MeJA caused a large accumulation of vsp mRNAs, whereas little accumulation occurred when these compounds were supplied separately. In soybean cell suspension cultures, the synergistic effect of sucrose and MeJA on the accumulation of vspB mRNA was maximal at 58 millimolar sucrose and was observed with fructose or glucose substituted for sucrose. In dark-grown soybean seedlings, the highest levels of vsp mRNAs occurred in the hypocotyl hook, which also contained high levels of MeJA and soluble sugars. Lower levels of vsp mRNAs, MeJA, and soluble sugars were found in the cotyledons, roots, and nongrowing regions of the stem. Wounding of mature soybean leaves induced a large accumulation of vsp mRNAs when wounded plants were incubated in the light. Wounded plants kept in the dark or illuminated plants sprayed with dichlorophenyldimethylurea, an inhibitor of photosynthetic electron transport, showed a greatly reduced accumulation of vsp mRNAs. The time courses for the accumulation of vsp mRNAs induced by wounding or sucrose/MeJA treatment were similar. These results strongly suggest that vsp expression is coregulated by endogenous levels of MeJA (or jasmonic acid) and soluble carbohydrate during normal vegetative development and in wounded leaves.

Nucleotide Sequence of a Soybean Vegetative Storage Protein vspA Gene

Nucleotide Sequence of a Soybean Vegetative Storage Protein <i>vspA</i> Gene

Nucleotide Sequence of a Soybean Vegetative Storage Protein vspB Gene

Nucleotide Sequence of a Soybean Vegetative Storage Protein <i>vspB</i> Gene

Jasmonic acid-dependent increase in vegetative storage protein in soybean tissue cultures

Adding jasmonic acid (JA) to autotrophic, photomixotrophic, or heterotrophic suspension cultures of soybean specifically increased the level of the Mr 30,000 subunit of soybean vegetative storage protein (VSP-30) and a polypeptide at Mr 18,000 that interacted with antibody raised against VSP. Using photomixotrophic cells, the increase was observed at concentrations as low as 10 nM JA and the increase was evident within 2 h following treatment. Below 10 μM, JA did not inhibit growth of the cells but did cause browning at higher concentrations. Other plant growth regulators, including abscisic acid (ABA), gibberellic acid, and benzyl adenine, did not alter the level of VSP-30 either in the presence or absence of JA. Methyl jasmonate (JA-Me), 3-oxo-2-butyl-cyclopentane-1-acetate, and 3-oxo-2-pentyl-cyclopentane-1-acetate also increased VSP-30 but at higher concentrations than JA. Altering the level of reduced nitrogen or sucrose in the medium did not alter VSP-30 levels in the cells, but at higher sucrose concentrations, sensitivity to JA was reduced. The dramatic increase in VSP-30 elicited by JA appears to be a specific response to the phytohormone.