Vegetative Storage Proteins Research Articles

The Role of Jasmonic Acid and Related Compounds in the Regulation of Plant Development

Publisher Summary This chapter reviews the roles of jasmonic acid (JA) and related compounds in the regulation of plant development. JA-related compounds are widely distributed among higher plants and are potent inducers of expression of several genes, such as the genes of proteinase inhibitor in tomato plants and the genes of vegetative storage proteins in soybean plants. JA is synthesized from linolenic acid by a series of enzymes. The initial reaction in JA synthesis is catalyzed by lipoxygenase, which is a common enzyme in plant tissues. JA-related compounds are easily extracted from plant materials by methanol, ethanol, or acetone. The material should be homogenized immediately after harvest with sufficient solvent to prevent the enzymatic changes of the compounds. JA has four stereoisomers because of the presence of two chiral carbons at the C-1 and C-2 positions of a cyclopentane ring. Maslenkova et al. examined the effect of JA on the oxygen-evolving activity of chloroplasts isolated from barley leaves. The kinetics characteristics of oxygen evolution indicated that JA decreased the value of the total number of oxygen-evolving centers. JA affects the degree of structuring of the granal region or the structural integrity of the electron transport chain in barley chloroplasts.

Methyl Jasmonate Treatment Eliminates Cell-Specific Expression of Vegetative Storage Protein Genes in Soybean Leaves

Soybean (Glycine max) plants accumulate a vacuolar glycoprotein in the parenchymal cells of leaves, petioles, stems, seed pods, and germinating cotyledons that acts in temporary nitrogen storage during vegetative growth. In situ immunolocalization of this vegetative storage protein (VSP) revealed that it accumulates in those parenchymal cells in close proximity to existing and developing vasculature, as well as in epidermal and cortical cells. The protein was more prevalent in younger, nitrogen-importing tissues before pod and seed development. Removal of actively growing seed pods greatly enhanced VSP accumulation, primarily in bundle sheath and paraveinal mesophyll cells. In situ hybridization of a VSP RNA probe to mRNA in leaf sections demonstrated that cell-specific mRNA accumulation corresponded with the pattern of protein localization. Treatment of leaf explants with 50 micromolar methyl jasmonate resulted in accumulation of VSP mRNA and protein in all cell types.

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.

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.

Isolation and Characterization of a Tomato Acid Phosphatase Complementary DNA Associated with Nematode Resistance

The tomato (Lycopersicon esculentum) acid phosphatase-1 (Apase-1(1), EC 3.1.3.2) isozyme variant, genetically linked to the root-knot nematode resistance locus (Mi) on chromosome 6, has been purified by a rapid procedure from tomato cell suspension cultures. Peptide fragments of the purified enzyme were generated from trypsin and Lys-C endoprotease digests and separated by reverse-phase high-performance liquid chromatography. Amino acid sequences derived from the purified peptide fragments represented >50% of the total amino acid content of the protein and enabled the construction of degenerate oligonucleotide probes that were used to screen a tomato cell culture complementary DNA library. Clones corresponding to full-length coding sequences for Apase-1 have been isolated and sequenced. Southern blot analysis of DNA isolated from a number of tomato cultivars shows that the Apase-1(1) gene (aps1) is present at one copy per genome and that genotypes containing the aps1(1) allele have restriction fragment length polymorphisms that distinguish them from cultivars having the aps1(+) allele. Segregation analysis demonstrates that the restriction fragment length polymorphisms are associated with the aps1 locus. Tomato Apase-1(1) is also found to have significant homology at the amino acid sequence level to a class of vegetative storage proteins characterized in soybean.

Vegetative Storage Proteins in Poplar

Bark, wood, and root tissues of several Populus species contain a 32- and a 36-kilodalton polypeptide which undergo seasonal fluctuations and are considered to be storage proteins. These two proteins are abundant in winter and not detectable in summer as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection. An antibody raised against the 32-kilodalton storage protein of Populus trichocarpa (T. & G.) cross-reacts with the 36-kilodalton protein of this species. The synthesis of the 32- and 36-kilodalton proteins can be induced in micropropagated plants by short-day conditions in the growth chamber. These proteins are highly abundant in structural roots, bark, and wood and combined represent >25% of the total soluble proteins in these tissues. Nitrate concentration in the leaves and nitrate uptake rate decreased dramatically when LD plants were transferred to short-day conditions; the protein content in leaves was unaffected. A decrease of the 32- and 36-kilodalton polypeptides occurs after transferring induced plants back to LD conditions. Both polypeptides are glycosylated and can be efficiently purified by affinity chromatography using concanavalin A-Sepharose 4B. The 32- and the 36-kilodalton polypeptides have identical basic isoelectric points and both consist of at least three isoforms. The storage proteins show a loss in apparent molecular mass after deglycosylation with trifluoromethanesulfonic acid. It is concluded that the 32- and 36-kilodalton polypeptides are glycoforms differing only in the extent of glycosylation. The relative molecular mass of the native storage protein was estimated to be 58 kilodalton, using gel filtration. From the molecular mass and the elution pattern it is supposed that the storage protein occurs as a heterodimer composed of one 32- and one 36-kilodalton subunit. Preliminary data suggest the involvement of the phytochrome system in the induction process of the 32- and 36-kilodalton polypeptides.

The 32-Kilodalton Vegetative Storage Protein of Salix microstachya Turz

A 32-kilodalton vegetative storage protein, found in Salix microstachya Turz. bark during the overwintering period, was purified and characterized using several polyacrylamide gel electrophoretic procedures. Solubility characteristics and amino acid analyses were also performed. The protein is water soluble, is glycosylated, has no disulfide-bonded subunits, but is composed of a family of isoelectric isomers. The majority of these isomers are basic. Characteristic of storage proteins, the protein is rich in glutamine/glutamate and asparagine/aspartate (28%), the basic nature of the isomers indicating that most of these amino acid residues are in the amide form. The protein was purified using preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and antibodies raised in chickens. Immunoblot analysis suggested an annual cyclic nature of the accumulation and mobilization of this vegetative storage protein. Immunologically, it is related to a similar molecular weight protein found in the bark of Populus deltoides Marsh. but not to any overwintering storage proteins of the other hardwoods tested. Indirect immunolocalization revealed that the protein was sequestered in protein-storage vacuoles in parenchymatous cells of the inner bark tissues of Salix during the winter months.

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.

Acid Phosphatase-1 from Nematode Resistant Tomato

Aps-1 encodes acid phosphatase-1, one of the many acid phosphatases present in tomato (Lycopersicon esculentum Mill.). Aps-1 is closely linked to Mi, a gene conferring resistance against nematodes. Thus, a clone of Aps-1 would provide access to the region of the genome containing Mi. Acid phosphatase-1 was purified from tomato suspension culture cells. Fragmentary amino acid sequences were derived from the purified protein and from its proteolytic and chemical digestion products. One of these amino acid sequences was used to design an oligodeoxyribonucleotide probe expected to hybridize to acid phosphatase-1 cDNA. This probe identified, in a cDNA library, a clone encoding the carboxyl-terminal sequence of a protein that is very similar, but not identical, to acid phosphatase-1. Using this clone, we discovered a second cDNA clone that corresponds in its carboxyl terminal sequence to acid phosphatase-1 but, surprisingly, retains sequences of an Aps-1 intron. The second cDNA clone was used to detect both a cDNA clone and a genomic clone corresponding to Aps-1. The identity of these clones was confirmed by sequence analysis and by the correlation of a restriction fragment length polymorphism with two Aps-1 alleles in a segregating tomato population. The deduced amino acid sequence of the Aps-1 open reading frame predicts a hydrophobic animoterminal signal sequence and a mature protein with a molecular weight of 25,000. The amino acid sequence of this protein has a strong similarity in size and sequence to a vegetative storage protein of soybean.

The Soybean 94-Kilodalton Vegetative Storage Protein Is a Lipoxygenase That Is Localized in Paraveinal Mesophyll Cell Vacuoles

The Soybean 94-Kilodalton Vegetative Storage Protein Is a Lipoxygenase That Is Localized in Paraveinal Mesophyll Cell Vacuoles

Induction of soybean vegetative storage proteins and anthocyanins by low-level atmospheric methyl jasmonate.

Soybean seedlings were exposed to atmospheric methyl jasmonate (MJ) to determine if low levels of this compound could regulate the expression and accumulation of the vegetative storage proteins (VSPs) in soybeans. Low levels of atmospheric MJ induced the accumulation of three VSPs with molecular masses of 27 kDa, 29 kDa, and 94 kDa (vsp27, vsp29, and vsp94, respectively). Atmospheric MJ caused vsp94 to be accumulated in all above-ground organs of the seedling uniformly after just 3 days of exposure. vsp27 preferentially accumulated in shoot tips and primary leaves, whereas vsp29 preferentially accumulated in the cotyledons. In addition to these effects, MJ also induced the biosynthesis of anthocyanins in light-grown seedlings but inhibited anthocyanin biosynthesis in etiolated seedlings. It is concluded that low levels of atmospheric MJ regulate anthocyanin biosynthesis and the organspecific accumulation of VSPs in developing soybean seedlings. The organ-specific differential accumulation may reflect changes in the pattern of nitrogen partitioning between various compounds and/or organs. These results lend substance to the hypothesis that volatile MJ may act as a gaseous messenger or growth regulator in plants.

Nitrogen and Methyl Jasmonate Induction of Soybean Vegetative Storage Protein Genes

Vegetative storage protein (VSP) and VSP mRNA levels in soybean (Glycine max) leaves correlated with the amount of NH(4)NO(3) provided to nonnodulated plants. The mRNA level declined as leaves matured, but high levels of N delayed the decline. This is consistent with the proposed role for VSP in the temporary storage of N. Wounding, petiole girdling, and treatment with methyljasmonate (MeJA) increased VSP mRNA in leaves 24 hours after treatment. The magnitude of the response depended on leaf age and N availability. N deficiency essentially eliminated the response to wounding and petiole girdling. MeJA was almost as effective in N-deficient plants as in those receiving abundant N. Inhibitors of lipoxygenase, the first enzyme in the jasmonic acid biosynthetic pathway, blocked induction by wounding and petiole girdling but not by MeJA. This supports a role for endogenous leaf jasmonic acid (or MeJA) in the regulation of VSP gene expression.

Low water potentials affect expression of genes encoding vegetative storage proteins and plasma membrane proton ATPase in soybean

We have examined growth, water status and gene expression in dark-grown soybean (Glycine max L. Merr.) seedlings in response to water deficit (low water potentials) during the first days following germination. The genes encoded the plasma membrane proton ATPase and two proteins of 28 kDa and 31 kDa putatively involved in vegetative storage. Water potentials of stems and roots decreased when 2-day-old seedlings were transferred to water-saturated air. Stem growth was inhibited immediately. Root growth continued at control rates for one day and then was totally inhibited when the normal root-stem water potential gradient was reversed. Expression of mRNA for the 28 kDa and 31 kDa proteins, measured independently using specific 3'-end probes, occurred about equally in stems. However, only the mRNA for the 31 kDa protein was detected in roots and at a lower abundance than in stems. Low water potentials increased the mRNA only for the 28 kDa protein in stems and the 31 kDa protein in roots. This differential expression followed the inhibition of stem growth but preceded the inhibition of root growth. The expression of the message for the ATPase, measured using a probe synthesized from a partial oat ATPase clone, was low in stems and roots but there was a 6-fold increase at low water potentials in roots. The increase followed the inhibition of root growth. This appears to be the first instance of regulation of ATPase gene expression in plants and the first demonstration of differential expression of the 28 kDa, 31 kDa, and ATPase messages. The correlation with the differential growth responses of the stems and roots raises the possibility that the differential gene expression could be involved in the growth response to low water potentials.

Proteins in the roots of the perennial weeds chicory (Cichorium intybus L.) and dandelion (Taraxacum officinale Weber) are associated with overwintering

Roots are the overwintering structures of herbaceous perennial weeds growing in temperate climates. During the fall they accumulated reserves which are remobilized when growth resumes in the spring. An 18kDa (kilodalton) protein increases in both chicory and dandelion roots during the fall months. The proteins in both species are antigenically similar, and are recognized also by an antibody to a storage-protein deposited in Jerusalem artichoke (Helianthus tuberosus) tubers. In chicory, the protein is root-specific, but in dandelion it is detectable in the flowers, vestigial stem and the seed. Electrophoretic characterization of the 18-kDa protein shows that it is a single polypeptide, without subunits, with charge isomers of pI values close to pH 6.5. The major protein present in chicory and dandelion roots is unlike the vegetative storage proteins recently found in soybean or the storage proteins in the bark of trees.

Characterization of soybean vegetative storage proteins and genes

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.

Expression of Two Soybean Vegetative Storage Protein Genes during Development and in Response to Water Deficit, Wounding, and Jasmonic Acid

Expression of Two Soybean Vegetative Storage Protein Genes during Development and in Response to Water Deficit, Wounding, and Jasmonic Acid

Expression of two soybean vegetative storage protein genes during development and in response to water deficit, wounding, and jasmonic acid.

The expression of vspA and vspB genes encoding soybean vegetative storage proteins was studied during seedling development and in response to water deficit, tissue wounding, and jasmonic acid treatment. vspA and vspB encode VSP-alpha and VSP-beta, 28-kilodalton and 31-kilodalton vacuole-localized polypeptides that are 80% homologous. vspA and vspB mRNAs could be distinguished on RNA blots using 3'-end probes. vspA mRNA was threefold to sevenfold more abundant than vspB mRNA in leaves, about equal expression was observed in stems, and vspB mRNA exceeded vspA in roots. Transcripts were not detected in dry seeds but appeared in intact or excised seedling axes between 12 hr and 24 hr after initiation of imbibition. Both transcripts were highly abundant in the meristematic region of seedling stems and in developing leaves but were rare in mature stems, leaves, and roots. In situ localization showed that vsp transcripts were found throughout the hypocotyl hook but were concentrated in cells associated with the epidermis and vascular bundles. Water deficit caused increased vsp mRNA levels in leaves and stems, which suggests that inhibition of growth necessitates temporary storage of amino acids. Wounding induced primarily vspB mRNA in etiolated seedlings, whereas both vspA and vspB mRNA levels increased in wounded leaves. Jasmonic acid and methyl jasmonate were potent inducers of vsp gene expression in cell cultures, developing axes, leaves, and roots. We hypothesize that jasmonic acid levels modulate vsp mRNA abundance in vivo.

Novel Regulation of Vegetative Storage Protein Genes.

Novel Regulation of Vegetative Storage Protein Genes.

Novel Regulation of Vegetative Storage Protein Genes

Novel Regulation of Vegetative Storage Protein Genes

Proteins as a potential nitrogen storage compound in bark and leaves of several softwoods

The occurrence of vegetative storage proteins in the leaf and bark tissues of several softwood species during overwintering was investigated by sodium dodecyl sulphate polyacrylamide electrophoresis. Monthly protein profiles from leaves and bark of six evergreen softwood species (Pinus strobus, P. sylvestris, Picea abies, P. glauca, Abies balsamea, and Thuja occidentalis) and the bark of one deciduous softwood species (Larix decidua) suggest that storage proteins are present in bark tissues of L. decidua, Pinus sylvestris, and P. strobus. The remaining species did not show similar specific proteins. However, the total soluble protein content which was determined during active growth and during overwintering in the same tissues indicated that protein levels were higher in the winter compared to the summer in the bark of all species and in the leaves of Pinus spp. and T. occidentalis. While vegetative storage proteins do not appear prevalent in all softwood species, proteins may constitute a major form of overwintering nitrogen storage for many species.