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     

Arabidopsis thaliana vegetative storage protein (VSP) genes: gene organization and tissue-specific expression

We have previously identified two cDNAs encoding vegetative storage proteins (VSPs) in Arabidopsis thaliana. Unlike soybean in which VSPs accumulate at high levels in leaves, A. thaliana VSP mRNAs are abundant in flowers. To understand tissue-specific expression and possible roles of VSPs on reproductive organ development, genes corresponding to VSPs (Vsp1 and Vsp2) and their putative promoters were characterized in this study. Genomic sequence analysis revealed that Vsp1 and Vsp2 resemble each other except in their introns, and that these two genes were organized in a tandem array with an interval of 6 kb in a region. The expression patterns of Vsp1 and Vsp2 were examined using transgenic A. thaliana plants carrying a promoter from Vsp1 or Vsp2 fused to a bacterial -glucuronidase (GUS) reporter gene. The promoter from Vsp1 expressed its effect in gynoecia, especially in styles, the basal and distal ends of ovaries and in siliques, whereas the promoter from Vsp2 showed its activity in vegetative shoots, petioles, peduncles and receptacles of floral organs. These results suggest that expression of Vsp1 and Vsp2 may be developmentally regulated in A. thaliana. In the transgenic plants, the GUS activity was induced by wounding in an area around the mid-rib of leaves. Therefore, Vsp1 and Vsp2 promoters appear to have elements required for both tissue specificity and wounding.     

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.     

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 NH₄NO₃ 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.     

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.     

Expression of a vegetative-storage-protein gene from Arabidopsis is regulated by copper, senescence and ozone

Emerging data suggest that the mechanisms regulating plant copper homeostasis could be implicated in stress and senescence signal transduction pathways. To gain insight into copper-modulated patterns of gene expression, copper-treated Arabidopsis thaliana (L.) Heynh. plants were analysed by mRNA differential display. The experimental conditions were selected using aggregation of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) as a molecular sensor to monitor copper-induced oxidative stress. Two copper-induced messengers encoding a vegetative storage protein (VSP2) were isolated by this technique. Both clones differed in the length of their 3'-untranslated region according to the presence of two polyadenylation signals in this region. VSP2 expression was further studied under natural senescence and various conditions causing oxidative stress, such as ozone exposure, paraquat and H2O2 treatments. The expression of other messengers related to copper homeostasis and detoxification processes was followed in parallel to that of VSP2. Here, we describe specific gene-expression responses to copper treatment, and present arguments connecting copper homeostasis, senescence and antioxidative responses in plants. Our results are consistent with the role of VSPs as temporary nitrogen-storage proteins which accumulate if nutrients are abundant, either in developing organs or in cotyledons and mature leaves subjected to generalized protein mobilization, such as those conditions created under severe oxidative stress.     

Stable expression of silencing‐suppressor protein enhances the performance and longevity of an engineered metabolic pathway

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA‐guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co‐evolved viral silencing‐suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA‐regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three‐step transgenic pathway for the synthesis of arachidonic acid (AA), an ω‐6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP‐AA‐transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.     

Isolation and characterization of cDNA clones corresponding to the genes expressed preferentially in floral organs of Arabidopsis thaliana

Seventeen cDNA clones of genes corresponding to mRNAs expressed preferentially in floral organs of Arabidopsis thaliana were obtained by differential screening of a flower bud cDNA library, and classified into five groups (1A, 17A, 1B, 4B and 5B) by cross-hybridization and restriction analysis. Sequence analysis revealed that the 1A-1 and 17A-1 clones encode vegetative storage proteins (VSPs). The VSP mRNAs were detected in a small amount in leaves and increased to a limited level by wounding. Both 1B-1 and 5B-1 clones were homologous to transmembrane protein cDNAs. The protein encoded by 4B-1 clone contained a proline-rich region, but no homologous proteins were found in databases.     

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 and VSP, 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 and Vsp, 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.     

A jasmonate-responsive element within the A. thaliana vsp1 promoter

The vsp1 gene of Arabidopsis thaliana encodes a storage protein that accumulates in vegetative organs. Transgenic plants expressing a vsp1 promoter-gus (beta-glucuronidase) gene fusion were found to contain high GUS activity when challenged with jasmonate, a volatile plant hormone. The induction of vsp1-gus expression by jasmonate could be measured in tobacco mesophyll protoplasts, after transient expression. A number of deletions were operated in the vsp1 promoter in order to locate its jasmonate-responsive element. A 41 bp sequence taken approximately 150 bp upstream of the vsp1 TATA box could confer jasmonate responsiveness upon a short CaMV 35S promoter. Whereas the deletion of a CAAT box-like element within the 41 bp sequence did not affect promoter activity, mutation of a short palindromic motif completely abolished jasmonate responsiveness. This motif shows no homology with the jasmonate-responsive elements of other promoters.     

Isolation and characterization of wound-inducible carboxypeptidase inhibitor from tomato leaves

In a previous report [Mol. Gen. Genet. 228 (1991) 281], carboxypeptidase inhibitor protein (CPI) mRNA was found to accumulate in leaves of wounded tomato plants, but CPI protein could not be detected. In contrast, we found that CPI protein does accumulate in tomato leaves in response to wounding, and also in response to treatment with either systemin, methyl jasmonate (MeJ), oligogalacturonic acid, or chitosan. Identification of CPI protein was confirmed by its inhibition of metallo-carboxypeptidase A (CPAase), which was used as an assay during purification of the inhibitor from leaves of MeJ-treated tomato plants. Amino acid sequence analysis and mass spectroscopic analyses of the pure protein confirmed its identity as CPI. The pure protein inhibited CPAase in a 1:1 stoichimetric interaction. Time course analyses of the induction of CPI mRNA in tomato leaves in response to wounding indicated that the gene is a member of the group of "late genes" that code for defensive proteins synthesized in leaves in response to herbivore attack.     

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.     

Molecular cloning of a tomato leaf cDNA encoding an aspartic protease, a systemic wound response protein

A full-length cDNA encoding an aspartic protease (LeAspP) has been cloned from a tomato leaf cDNA library. Using LeAspP cDNA as a probe in gel blots, LeAspP mRNA was shown to be systemically induced in tomato leaves by wounding. Application of methyl jasmonate to leaves of intact tomato plants, or supplying systemin to young tomato plants through their cut stems, induces synthesis of LeAspP mRNA. LeAspP message is regulated in tomato similar to several systemic wound response proteins (swrps) that are part of the defense response in tomato plants directed against herbivore attacks.     

Fluctuation of Vegetative Storage Proteins in the Seedlings of Swietenia macrophylla, Analogous to the Seasonal Changes of Those in the Shoot of the Adult Tree

In order to Identify appropriate plant materials for studying the gene expression and biological function of vegetative storage proteins （VSPs） in woody plants, the VSPs in the seedlings of Swietenla rnacrophylla King were investigated by using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western.blotting. The seed of S. macrophylla was rich in storage proteins that accumulated In the vacuoles of cotyledon parenchyma cells in appearance of compact spherical grains. The growth and development of S. macrophylla seedlings were characterized by an obvious growth rhythm. The storage proteins In seeds disappeared during seedling growth while VSPs appeared in the stem 2 weeks after seedling leaves matured. Thereafter, the VSPs In the seedling stem almost exhausted during new shoot growth, and when the leaves of new shoot Just matured, both the stem beneath the new shoot of seedlings and the stem of new shoot started to accumulate VSPs. Nitrogen application dramatically Increased the level of VSPs, but had little influence on the dynamics of VSP consumption and accumulation in seedling stem. Together with these data, the fluctuation of VSPs in seedlings was very similar to that in the branches of the adult trees. In addition, seedlings are easy to be treated due to their small size. Our results suggested that S. rnacrophylla seedlings were suitable for Investigating the biological roles of VSPs and the mechanism of nitrogen storage in trees.