Protein compositions of mesophyll and paraveinal mesophyll of soybean leaves at various developmental stages

Mesophyll and paraveinal mesophyll protoplasts (PVMP) were isolated from leaves of soybean (Glycine max) at various stages of physiological development, and protein compositions of the two protoplast types were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Polypeptides of 27, 29 (previously shown to be storage proteins), and 94 kilodaltons were found to be PVMP-specific proteins and were present in both nodulated and nonnodulated plants. The 27 and 94 kilodalton polypeptides were major PVMP constituents. All three polypeptides accumulate as early as one-quarter leaf expansion. Immunoblotting and immunocytochemical studies using antibodies against the 27/29 kilodalton proteins confirmed that they are specific to the paraveinal mesophyll (PVM) and that they are localized in the PVM vacuole. The 27 kilodalton polypeptide increased significantly by two weeks depodding, and this accumulation was restricted to the PVM vacuole. Radiolabeling experiments showed that the difference in relative amounts of the 27 and 29 kilodalton polypeptides was due to a greater rate of synthesis of the 27 kilodalton polypeptide. The 94 kilodalton polypeptide accumulated to a maximum at anthesis, but was absent at 2 weeks postanthesis in both depodded and podded nodulated plants, probably because they were nitrogen limited. In nonnodulated plants, it was present through 2 weeks postanthesis. The results confirm that the 27 and 29 kilodalton proteins of soybean leaf are stored in the PVM vacuole and show that they are accumulated early during leaf development while they are still strong sinks for nitrogen. The 94 kilodalton protein, previously found to accumulate in leaves after depodding, is also a PVM protein and is likely a third vegetative storage protein, although its accumulation appears to be more dependent on excess nitrogen availability. The results further support the hypothesis that the PVM is a specialized leaf tissue that functions in synthesis and compartmentation of storage proteins.     

Mechanism of transport of vegetative storage proteins to the vacuole of the paraveinal mesophyll of soybean leaf

Summary Microscopy techniques were used to identify the pathway of transport of soybean leaf vegetative storage proteins (VSP/ and VSP94) to the vacuoles of a specialized cell type, the paraveinal mesophyll (PVM), where they accumulate. PVM cells are enriched in endoplasmic reticulum and Golgi bodies relative to surrounding mesophyll cells. The margins of medial and trans Golgi cisternae had attached or closely associated noncoated vesicles with densely staining membranes and lumenal contents of the same appearance as material that accumulated in the vacuole. These vesicles appeared to be transported preferentially to the tonoplast, where fusion with the membrane released the granular contents into the vacuole. Cytochemical staining with phosphotungstic acid and silver methenamine supported this interpretation as both the Golgi vesicles and the tonoplast stained intensely with these reagents, unlike the tonoplast of mesophyll cells which do not accumulate VSP. Immunocytochemical localization for VSP/ labeled the Golgi bodies and associated vesicles, and vacuolar material in PVM cells, but not in mesophyll. Similar labeling was seen in PVM of another legume species previously found to accumulate antigenically similar VSPs. Immunolocalization for VSP94, a lipoxygenase, labeled the PVM cytosol and material in the PVM vacuole, but not the Golgi or vesicles. The results of this study demonstrate that the Golgi pathway is utilized for transport of VSP/ in the PVM, which follows the mechanism of deposition demonstrated for certain seed storage proteins. VSP94 appeared to follow a separate path for accumulation in PVM vacuoles.Abbreviations LOX lipoxygenase - PVM paraveinal mesophyll - RER rough endoplasmic reticulum - TEM transmission electron     

Allantoinase activity and ureide content of mesophyll and paraveinal mesophyll of soybean leaves

Paraveinal mesophyll (PVM) is a specialized soybean (Glycine max Merr.) leaf tissue which represents a significant biochemical compartment. Stereological measurements showed that PVM makes up 23% of the mesophyll volume in nodulated soybean. To get an indication of the extent of involvement of PVM in ureide metabolism, physical characteristics, distribution of allantoinase activity and ureide content were determined in isolated PVM protoplasts (PVMP) and mesophyll protoplasts (MP). PVMP were larger and contained less chlorophyll and protein than MP. PVMP had twice as much allantoinase activity per protoplast but only half as much allantoinase activity when expressed on a volume basis as compared to MP. Total leaf ureide concentration was high and nearly equally distributed between MP and PVMP. PVMP had a higher ureide content per protoplast, a higher concentration of allantoic acid and a lower ratio of allantoin to allantoic acid. These results suggest that both tissues have the capacity to assimilate allantoin in vivo. The data are discussed with reference to the relative access of the two mesophyll tissues to incoming ureides.     

Characterization of soybean vegetative storage proteins and genes

Summary Soybean vegetative storage proteins (VSPs) were purified and characterized. Anion exchange HPLC resolved partially purified VSPs into fractions containing 27-kD/27-kD and 29-kD/29-kD homodimers and 27-kD/29-kD heterodimers. Reversed-phase HPLC resolved partially purified VSPs into three fractions. One fraction contained only 27-kD VSP and the other two contained 29-kD VSP. The two 29-kD VSP fractions differed with respect to their cyanogen bromide cleavage patterns, an observation that indicated the 29-kD VSPs were heterogeneous. Genomic clones that contained 29-kD VSP genes were also isolated and characterized. One genomic clone contained a complete 29-kD VSP gene and was sequenced. The coding region in the clone contained two introns whose borders had regulatory sequences typical of other eukaryotic genes. Putative polyadenlyation signals were present in the 3-flanking region of the gene, while putative TATA, CAAT, and enhancer core sequences were found in the 5-flanking regions. A second genomic clone that was studied contained the 5 regions of two partial 29-kD VSP genes in an inverted linkage. Genomic DNA gel blots showed that the two genes were organized in the same arrangement in the soybean genome.Cooperative research between USDA/Agricultural Research Service and the Indiana Agricultural Experiment Station. Journal Paper No. 12,192 from the Indiana Agricultural Experiment Station     

Local and differential control of vegetative storage protein expression in response to herbivore damage in Arabidopsis thaliana

Vegetative storage proteins (VSPs) are thought to fulfil important nutritional roles during plant development and stress adaptation. Plant responses to mechanical wounding and herbivore damage include an activation of VSP expression. It was recently suggested that vsp is part of the systemic response of Arabidopsis to wounding. To test this proposal, we monitored the spatial regulation of vsp mRNAs and VSP proteins. Arabidopsis contains two vsp genes and real-time quantitative PCR allowed us to characterize their differential expression. The ratio of vsp1 to vsp2 mRNA abundance increased when plants were challenged with diamondback moth larvae or Egyptian cotton worms, but not when they were mechanically wounded. We observed a dramatic increase of vsp1 and vsp2 mRNA as well as VSP protein levels in leaves that experienced herbivore damage. By contrast, there was a relatively minor increase of vsp mRNA and VSP protein levels in undamaged leaves of infested plants. These results clearly demonstrate that VSPs are part of the local plant response to herbivore attack. To obtain additional information on vsp regulation, we analysed a fusion of a soybean vspB promoter fragment to the β-glucuronidase gene in transgenic Arabidopsis plants. The vspB promoter responded to both jasmonate and herbivore treatments, suggesting that similar signals regulate its expression in both plant species.     

The soybean 94-kilodalton vegetative storage protein is a lipoxygenase that is localized in paraveinal mesophyll cell vacuoles.

Soybean leaves contain three proteins (the vegetative storage proteins or VSPs) that respond to nitrogen status and are believed to be involved in the temporary storage of nitrogen. One of these proteins, with a molecular mass of 94 kD and termed vsp94, was microsequenced. Partial amino acid sequence indicated that vsp94 was highly homologous to the lipoxygenase protein family. Further evidence that vsp94 is a lipoxygenase was obtained by demonstrating that vsp94 cross-reacted with a lipoxygenase antibody. Also, a lipoxygenase cDNA coding region was able to detect changes in an mRNA that closely parallel changes in vsp94 protein levels resulting from alteration of nitrogen sinks. Extensive immunocytochemical data indicate that this vsp94/lipoxygenase is primarily expressed in the paraveinal mesophyll cells and is subcellularly localized in the vacuole. These observations are significant in that they suggest that plant lipoxygenases may be bifunctional proteins able to function enzymatically in the hydroperoxidation of lipids and also to serve a role in the temporary storage of nitrogen during vegetative growth.     

The occurrence and gene expression of vegetative storage proteins and a Rubisco Complex Protein in several perennial soybean species

Vegetative storage proteins (VSPs) have been extensively studiedin Glycine max, but not in perennial relatives of the cultivatedsoybean. The occurrence and gene expression of VSPs and a RubiscoComplex Protein (RCP) in several Glycine species was investigatedby mRNA blot hybridization and protein immunoblotting. RCP hada developmental pattern of gene expression that closely paralleledthat of VSP. The RCP gene was also induced by depodding, methyljasmonatetreatment, wounding, and to a lesser extent by nitrogen fertilization,as was previously found for the VSPs. VSP in leaves of 13 perennialsoybeans was heterogeneous in apparent size and number of bandsdetected by immunoblotting following SDS-PAGE. In contrast,RCP was detected as a single band of nearly identical mobilityin all species. Both proteins were most abundant in young leavesof the perennials, and methyljasmonate and wounding inducedboth VSP and RCP gene expression in perennial soybeans. Theseresults suggest that the VSPs in perennial soybeans functionas storage reserves, as they do in G. max. Key words: Soybean, methyljasmonate, perennial, storage     

Tissue- and Cell-Specific Distribution of Connexin 32-and Connexin 26-related Proteins from Vicia faba L.

Different tissues (leaf lamina and midveins, epidermis, primary roots, etiolated shoots, hypocotyl, petioles) and protoplasts from mesophyll, guard cells, and petioles of Vicia faba L., obtained from defined developmental stages were analyzed with polyclonal antibodies raised against mouse liver connexins 26 and 32. Immunostaining after treatment with CX 26 antibodies showed two polypeptides of 21 and 16 kD in all tissues examined except for hypocotyl and epidermis. Additionally, a stronger immunoresponse at 40 kD occurred in extracts from leaf material, mesophyll protoplasts, and roots. Incubation with the CX 32 antibodies resulted in an immunoreactive band at 29 kD in mesophyll protoplast samples, and, a very weak band in extracts from leaf material. A 36 kD polypeptide, present in samples prepared from root, etiolated shoots, petioles, and midveins, did not appear in leaf laminas and mesophyll protoplasts. Extracts from guard cells did not show any immunoreactive band. The tissue- and cell-specific distribution of CX 23- and CX 26-related proteins is supposed to reflect diversities in physiological functions together with alterations in plasmodesmatal ultrastructures during development.     

Efficient Down-Regulation of the Major Vegetative Storage Protein Genes in Transgenic Soybean Does Not Compromise Plant Productivity

Soybean (Glycine max L. Merr.) contains two related and abundant proteins, VSP alpha and VSP beta, that have been called vegetative storage proteins (VSP) based on their pattern of accumulation, degradation, tissue localization, and other characteristics. To determine whether these proteins play a critical role in sequestering N and other nutrients during early plant development, a VspA antisense gene construct was used to create transgenic plants in which VSP expression was suppressed in leaves, flowers, and seed pods. Total VSP was reduced at least 50-fold due to a 100-fold reduction in VSP alpha and a 10-fold reduction in VSP beta. Transgenic lines were grown in replicated yield trials in the field in Nebraska during the summer of 1999 and seed harvested from the lines was analyzed for yield, protein, oil, and amino acid composition. No significant difference (alpha = 0.05) was found between down-regulated lines and controls for any of the traits tested. Young leaves of antisense plants grown in the greenhouse contained around 3% less soluble leaf protein than controls at the time of flowering. However, total leaf N did not vary. Withdrawing N from plants during seed fill did not alter final seed protein content of antisense lines compared with controls. These results indicate that the VSPs play little if any direct role in overall plant productivity under typical growth conditions. The lack of VSPs in antisense plants might be partially compensated for by increases in other proteins and/or non-protein N. The results also suggest that the VSPs could be genetically engineered or replaced without deleterious effects.     

15科温带树木营养贮藏蛋白质的细胞学研究

利用光学显微镜技术和组织化学方法研究１５科２９种２变种 温带树木中的营养贮藏蛋白质（ＶＳＰｓ）的分布和形态。结果表明,ＶＳＰｓ在树木中的分布分为ＶＳＰｓ丰富、贫乏和缺乏３种类型。同时,ＶＳＰｓ有多种形态,大致可分为蛋白体状、颗粒状和絮状３种,这些形态的ＶＳＰｓ存在于不同的树木中或同一树种的不同细胞中。ＶＳＰｓ的有无、多少及其形态在不同科树木之间及同一科不同属树木之间存在在较大差异,但在同一属树种之     

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.     

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.     

Paraveinal mesophyll: Review and survey of the subtribeErythrininae (Phaseoleae,Papilionoideae, Leguminosae)

Paraveinal mesophyll (PVM) is a distinctive anatomical feature of the leaf mesophyll of some plant taxa that may represent a specialized physiological compartment. A comprehensive review of the 42 published references that mention PVM or similar cell layers and a survey of 121 of the 272 species of all nine genera of thePhaseoleae subtribeErythrininae demonstrate that PVM is nearly exclusively found inLeguminosae. InLeguminosae, PVM is either rare or absent in subfamilyCaesalpinioideae, uncommon inMimosoideae, and extensively distributed amongPapilionoideae. In subtribeErythrininae, PVM is ubiquitous inErythrina, and occurs in four other genera. ThreeErythrininae genera (Apios, Mucuna, andCochlianthus) lack PVM. Unique chloroplast-poor, enlarged conical cells (pellucid palisade idioblasts) occur in 80 species ofErythrina but not in any other genus ofErythrininae.     

Vegetative storage protein in Litchi chinensis, a subtropical evergreen fruit tree, possesses trypsin inhibitor activity

BACKGROUND AND AIMS: Vegetative storage proteins (VSPs) are commonly bioactive in herbaceous plants but few VSPs with bioactivity have been identified in trees. In addition, information on the characterization of VSPs in evergreen trees is limited. The objective of this study was to characterize the VSPs with bioactivity in evergreen trees. Methods The VSP in lychee (Litchi chinensis), an evergreen fruit tree, was characterized by a combination of cytological, biochemical and molecular biological techniques. KEY RESULTS: The VSP in lychee was a 22-kDa protein. It accumulated in the large central vacuoles of protein-storing cells (PSCs) in two distinguishable forms, granular and floccular. The PSCs were of a novel type. The 22-kDa protein is distributed in mature leaves, bark tissues of branches, trunk and large roots, paralleling the distribution of PSCs. Its homologues were present in mature seed. During young shoot development and fruiting, the 22-kDa protein decreased apparently, suggesting a nitrogen-storage function. The 22-kDa protein had several isoforms encoded by a small multigene family. One gene member, LcVSP1, was cloned. The LcVSP1 had no intron and contained a 675 bp open reading frame encoding a putative protein of 225 amino acids. LcVSP1 was homologous to Kunitz trypsin inhibitors. The 22-kDa protein inhibited trypsin and chymotrypsin, but had no inhibitory effect on subtilisin. CONCLUSIONS: Lychee is rich in a 22-kDa VSP with trypsin inhibitor activity. The VSP plays an important role in nitrogen storage while its possible defensive function remains to be elucidated.     

Characterization of the Functional Domains of a Mammalian Voltage-Sensitive Phosphatase

Voltage-sensitive phosphatases (VSPs) are proteins that directly couple changes in membrane electrical potential to inositol lipid phosphatase activity. VSPs thus couple two signaling pathways that are critical for cellular functioning. Although a number of nonmammalian VSPs have been characterized biophysically, mammalian VSPs are less well understood at both the physiological and biophysical levels. In this study, we aimed to address this gap in knowledge by determining whether the VSP from mouse, Mm-VSP, is expressed in the brain and contains a functional voltage-sensing domain (VSD) and a phosphatase domain. We report that Mm-VSP is expressed in neurons and is developmentally regulated. To address whether the functions of the VSD and phosphatase domain are retained in Mm-VSP, we took advantage of the modular nature of these domains and expressed each independently as a chimeric protein in a heterologous expression system. We found that the Mm-VSP VSD, fused to a viral potassium channel, was able to drive voltage-dependent gating of the channel pore. The Mm-VSP phosphatase domain, fused to the VSD of a nonmammalian VSP, was also functional: activation resulted in PI(4,5)P₂ depletion that was sufficient to inhibit the PI(4,5)P₂-regulated KCNQ2/3 channels. While testing the functionality of the VSD and phosphatase domain, we observed slight differences between the activities of Mm-VSP-based chimeras and those of nonmammalian VSPs. Although the properties of VSP chimeras may not completely reflect the properties of native VSPs, the differences we observed in voltage-sensing and phosphatase activity provide a starting point for future experiments to investigate the function of Mm-VSP and other mammalian VSPs. In conclusion, our data reveal that both the VSD and the lipid phosphatase domain of Mm-VSP are functional, indicating that Mm-VSP likely plays an important role in mouse neurophysiology.     

Age-Dependent Modifications and Further Localization of the CX 26-like Protein from Vicia faba L.

A strong age dependency together with alterations in the cellular distribution of CX 26 immunorelated protein(s) was found for differently developed leaves of Vicia faba L. With increasing age, an immunoreactive 40 kD band was observed in the soluble and microsomal fraction. In the cell wall protein preparation of young and fully differentiated leaves the 40 kD band was the minor constituent. A 33 kD polypeptide was dominantly localized in the microsomal fractions of all developmental stages and in SDS-extracts of total cell proteins of young leaves. A 21 kD protein together with a 16 kD polypeptide was associated with the cell wall fraction. The 21 kD protein, assumed to represent a plasmodesmatal constituent, was reduced with age. In SDS extracts, prepared from the different developmental stages of the leaves and of mesophyll protoplasts, the age-dependent appearance of the several immunostained bands was most obvious. A correlation of the 16, 33, and 40 kD bands to a turnover of the 21 kD protein is suggested. The reduced amount of the 21 kD protein with increasing age may be contemplated as an indication for a relative decrease of symplastic connections between cells of maturing leaves. This is in agreement with the results obtained by immunofluorescence studies using guard cell protoplasts. Here, observations pointed also to a reduction and final loss of CX 26-related protein at the protoplast surfaces.     

The paraveinal mesophyll of soybean leaves in relation to assimilate transfer and compartmentation

Nitrogen and carbohydrate assimilates were temporally and spatially compartmented among various cell types in soybean (Glycine max L., Merr.) leaves during seed filling. The paraveinal mesophyll (PVM), a unique cell layer found in soybean, was demonstrated to function in the synthesis, compartmentation and remobilization of nitrogen reserves prior to and during the seed-filling stages. At anthesis, the PVM vacuoles contain substantial protein which completely disappears by two weeks into the seed filling. Distinct changes in the PVM cytoplasm, tonoplast and organelles were correlated with the presence or absence of the vacuolar material. Microautoradiography following the accumulation of several radiolabeled sugars and amino acids demonstrated the glycoprotein nature of the vacuolar material. Incorporation of methionine, leucine, glucose, and glucosamine resulted in heavy labelling of the PVM vacuole, in contrast to galactose, proline, and mannose which resulted in a much reduced labelling pattern. In addition, starch is unequally compartmented and degraded among the various leaf cells during seed filling. At the end of the photoperiod at the flowering stage, the highest starch accumulation was in the second palisade layer followed by the spongy mesophyll and the first (uppermost) palisade layer. Starch in the first palisade layer was completely degraded during the dark whereas the starch in the second palisade and spongy mesophyll was not remobilized to any appreciable extent. By mid-podfilling (approximately five weeks postanthesis) starch was absent in the first palisade layer at the end of the photoperiod while the second palisade and spongy mesophyll layers contained substantial starch. Starch was remobilized from these latter cells during the remainder of seed filling when current photosynthetic production is low. Structural changes associated with cell senescence first appear in the upper palisade layer and then progress (excluding the PVM) to the second palisade and spongy mesophyll layer. The PVM and phloem appear to retain their structural integrity into the leaf yellowing stage. Reducing sink capacity by pod removal resulted in a continued accumulation of vacuolar protein, an increase in cytoplasmic volume, and fragmentation of the vacuole in the PVM. Pod removal also resulted in an increased amount of accumulated starch (which did not turn over) in all mesophyll layers, and an increase in cell size and cell-wall thickness.     

Antigenic variation in Giardia lamblia and the host's immune response.

Giardia lamblia, a protozoan parasite of the small intestine of humans and other animals, undergoes surface antigenic variation. The antigens involved belong to a family of variant-specific surface proteins (VSPs), which are unique, cysteine-rich zinc finger proteins. The patterns of infection in humans and animals fail to show the expected cyclical waves of increasing and decreasing numbers of parasites expressing unique VSPs. Nevertheless, changes in VSP expression occur within the population in vivo owing to selection of VSPs by both immune and non-immune mechanisms. After inoculation of a single G. lamblia clone (able to persist in the absence of immune pressure) expressing one VSP (> or = 90%) into mice or humans, the original VSP continues to be expressed until 2 weeks post inoculation (p.i.), when many other VSPs gradually replace it. Selection by immune-mediated processes is suggested because switching occurs at the same time that humoral responses are first detected. In most mouse strains, switching also occurs at about two weeks. Almost all trophozoites are eliminated at three weeks (p.i.), but a barely detectable infection persists over months. In neonatal mice, apparent self-cure is delayed until the sixth or seventh week. Antigenic switching does not occur in adult or neonatal severe combined immunodeficiency disease (SCID) mice, but does occur in neonatal nude mice, thus implicating B-cell-mediated mechanisms in immune switching. Not all VSPs are expressed to the same degree in vivo. Some VSPs appear to be preferentially selected whereas others are eliminated on a non-immune basis. In infections in which immunity does not play a role, such as in SCID mice, and during the first week of infection in immunocompetent mice or gerbils, persisting VSPs are preferentially expressed and maintained whereas non-persisting VSPs are replaced within the first week of infection. The purpose of antigenic variation may be presentation of a wide assortment of VSPs to hosts, increasing the chance of a successful initial infection or reinfection. Immune selection of variants comes into play following biological selection.