Soybean vegetative storage protein structure and gene expression

Depodded soybean (Glycine max [L] Merr. cv Williams) plants accumulate high levels of a glycoprotein in their leaves that has many features of a storage protein. The protein is found in all vegetative tissues which have been examined but not in the seeds. Translation in vitro indicated that elevated mRNA levels were at least partially responsible for the specific increase in vegetative storage protein. cDNA clones were isolated and sequenced, and an amino acid sequence was predicted. Although the amino acid composition is similar to that of seed storage proteins, no sequence similarity could be detected. Northern blot hybridization confirmed a large increase in vegetative storage protein mRNA in leaves of depodded plants. The vegetative storage proteins are represented by about four gene copies in the haploid genome.     

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.     

Calmodulin mRNA in Barley (Hordeum vulgare L.) : Apparent Regulation by Cell Proliferation and Light

Calmodulin is encoded by a 650-nucleotide mRNA in higher plants. This messenger was identified in barley and pea by a combination of in vitro translation and blot hybridization experiments using anti-sense RNA produced from an eel calmodulin cDNA probe. In all plant tissues tested, calmodulin mRNA represents between 0.01 and 0.1% of the total translatable mRNA population. Calmodulin mRNA levels are three- to fourfold higher in the meristematic zone of the first leaf of barley. At all other stages of leaf cell differentiation, calmodulin mRNA levels are nearly identical. During light-induced development in barley leaves, the relative proportion of translatable calmodulin mRNA declines about twofold. Cytoplasmic mRNAs that may encode calmodulin-like proteins were also detected. The levels of several of these putative Ca²⁺-binding protein mRNAs are modulated during the course of light-induced barley leaf cell development.     

Testing the optimal defense theory and the growth-differentiation balance hypothesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Two prominent theories proposed to explain patterns of chemical defense expression in plants are the optimal defense theory (ODT) and the growth-differentiation balance hypothesis (GDBH). The ODT predicts that plant parts with high fitness value will be highly defended, and the GDBH predicts that slow growing plant parts will have more resources available for defense and thus will have higher defense levels than faster growing tissues. We examined growth rate, fitness value, and defense protein levels in leaves of a wild and lab ecotype of Arabidopsis thaliana to address whether patterns of defense protein expression in this plant conform to predictions of either the ODT or the GDBH. We divided leaves of A. thaliana into six leaf classes based on three developmental stages: vegetative, bolting, and flowering; with two leaf ages at each stage: young and old. We assessed the fitness value of leaves by determining the impact of the removal of each leaf class on total seed production and germination rates. Although A. thaliana was highly tolerant to defoliation, young leaves were more valuable than old in general, and young leaves on bolting plants were the most valuable leaf class in particular. Young leaves on vegetative plants grew fastest in both ecotypes, while old leaves on bolting and flowering plants grew slowest. Finally, defense levels were assessed in each leaf class by quantifying the constitutive and inducible expression of four defense-related proteins. Expression of guaiacol peroxidase and chitinase activity conformed largely to GDBH predictions. Expression of trypsin inhibitor and polyphenoloxidase activity varied by leaf class and treatment, but conformed to neither GDBH nor ODT predictions.     

Overexpression of serine acetyltransferase in maize leaves increases seed‐specific methionine‐rich zeins

Maize kernels do not contain enough of the essential sulphur‐amino acid methionine (Met) to serve as a complete diet for animals, even though maize has the genetic capacity to store Met in kernels. Prior studies indicated that the availability of the sulphur (S)‐amino acids may limit their incorporation into seed storage proteins. Serine acetyltransferase (SAT) is a key control point for S‐assimilation leading to Cys and Met biosynthesis, and SAT overexpression is known to enhance S‐assimilation without negative impact on plant growth. Therefore, we overexpressed Arabidopsis thaliana AtSAT1 in maize under control of the leaf bundle sheath cell‐specific rbcS1 promoter to determine the impact on seed storage protein expression. The transgenic events exhibited up to 12‐fold higher SAT activity without negative impact on growth. S‐assimilation was increased in the leaves of SAT overexpressing plants, followed by higher levels of storage protein mRNA and storage proteins, particularly the 10‐kDa δ‐zein, during endosperm development. This zein is known to impact the level of Met stored in kernels. The elite event with the highest expression of AtSAT1 showed 1.40‐fold increase in kernel Met. When fed to chickens, transgenic AtSAT1 kernels significantly increased growth rate compared with the parent maize line. The result demonstrates the efficacy of increasing maize nutritional value by SAT overexpression without apparent yield loss. Maternal overexpression of SAT in vegetative tissues was necessary for high‐Met zein accumulation. Moreover, SAT overcomes the shortage of S‐amino acids that limits the expression and accumulation of high‐Met zeins during kernel development.     

Wheat vegetative nitrogen compositional changes in response to reduced reproductive sink strength

N redistribution patterns and the N composition of vegetative tissues above the peduncle node of wheat (Triticum aestivum L.) plants with altered reproductive sink strength were evaluated to determine the role of vegetative storage proteins in the temporary storage of excess N destined for export. The degree of leaf senescence symptoms (loss of chlorophyll, total N, and ribulose-1,5-bisphosphate carboxylase/oxygenase) were initially reduced, but the complete senescence of vegetative tissues proceeded even for plants completely lacking reproductive sinks. Plants with 50% less sink strength than control plants with intact spikes redistributed vegetative N to the spike almost as effectively as the control plants. Plants without reproductive sinks exported less N from the flag leaf and had flag leaf blades and peduncle tissues with higher soluble protein and α-NH₂ amino acid levels than control plants. An abundant accumulation of polypeptides in the soluble protein profiles of vegetative tissues was not evident in plants with reduced sink strength. Storage of amino acids apparently accommodates any excess N accumulated by vegetative tissues during tissue reproductive growth. Any significant role of vegetative storage proteins in the N economy of wheat is unlikely.     

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.     

Binding-protein expression is subject to temporal,developmental and stress-induced regulation in terminally differentiated soybean organs

Binding protein (BiP) is a widely distributed and highly conserved endoplasmic-reticulum luminal protein that has been implicated in cotranslational folding of nascent polypeptides, and in the recognition and disposal of misfolded polypeptides. Analysis of cDNA sequences and genomic blots indicates that soybeans (Glycine max L. Merr.) possess a small gene family encoding BiP. The deduced sequence of BiP is very similar to that of other plant BiPs. We have examined the expression of BiP in several different terminally differentiated soybean organs including leaves, pods and seed cotyledons. Expression of BiP mRNA increases during leaf expansion while levels of BiP protein decrease. Leaf BiP mRNA is subject to temporal control, exhibiting a large difference in expression in a few hours between dusk and night. The expression of BiP mRNA varies in direct correlation with accumulation of seed storage proteins. The hybridization suggests that maturing-seed BiP is likely to be a different isoform from vegetative BiPs. Levels of BiP protein in maturing seeds vary with BiP mRNA. High levels of BiP mRNA are detected after 3 d of seedling growth. Little change in either BiP mRNA or protein levels was detected in maturing soybean pods, although BiP-protein levels decrease in fully mature pods. Persistent wounding of leaves by whiteflies induces massive overexpression of BiP mRNA while only slightly increasing BiP-protein levels. In contrast single-event puncture wounding only slightly induces additional BiP expression above the temporal variations. These observations indicate that BiP is not constitutively expressed in terminally differentiated plant organs. Expression of BiP is highest during the developmental stages of leaves, pods and seeds when their constituent cells are producing seed or vegetative storage proteins, and appears to be subject to complex regulation, including developmental, temporal and wounding.The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned.Abbreviations BiP binding protein The sequences reported in this paper have been submitted to Gen-Bank and are identified with the accession numbers BiP-A (U08384), BiP-B (U08383), BiP-C (U08382) and -1,3 glucanase (U08405).     

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.     

Effect of photoperiod on growth of the plants,and sesamin content and CYP81Q1 gene expression in the leaves of sesame (Sesamum indicum L.)

Sesamin is a major lignan constituent of sesame (Sesamum indicum) seed and considered responsible for a number of beneficial human health effects. We previously reported that sesamin is present in sesame leaves, and proposed use of sesame leaves as a sesamin-containing material. This study focused on the possibility that both leaf yield and sesamin content would be increased with increasing photoperiod. Additionally, it was hypothesized that sesamin content would be affected by photoperiod in relation to CYP81Q1 gene expression. We thus investigated the effect of photoperiod on growth and leaf sesamin content in relation to CYP81Q1 gene expression to confirm our hypothesis. Under short-day (SD) condition, increase of leaf area was suppressed due to the phase transition from vegetative to reproductive growth, which resulted in reduction of leaf yield. Under long-day (LD) conditions, vegetative growth was continued, and both leaf area and yield increased as photoperiod increased up to 24 h (continuous light). Sesamin accumulated particularly in the leaves of plants grown under a 24-h photoperiod for 4 weeks. High expression level of the CYP81Q1 gene in those plants indicates that photoperiod-dependent differences in leaf sesamin content correlate with differences in CYP81Q1 gene expression levels. We conclude that cultivation under continuous light enables high-yield production of sesame leaves containing distinctively high levels of sesamin.     

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.     

Hpa1 harpin needs nitroxyl terminus to promote vegetative growth and leaf photosynthesis in Arabidopsis

Hpa1 is a harpin protein produced by Xanthomonas oryzae, an important bacterial pathogen of rice, and has the growth-promoting activity in plants. To understand the molecular basis for the function of Hpa1, we generated an inactive variant protein, Hpa1ΔNT, by deleting the nitroxyl-terminal region of the Hpa1 sequence and compared Hpa1ΔNT with the full-length protein in terms of the effects on vegetative growth and related physiological responses in Arabidopsis. When Hpa1 was applied to plants, it acted to enhance the vegetative growth but did not affect the floral development. Enhanced plant growth was accompanied by induced expression of growth-promoting genes in plant leaves. The growth-promoting activity of Hpa1 was further correlated with a physiological consequence shown as promoted leaf photosynthesis as a result of facilitated CO₂ conduction through leaf stomata and mesophyll cells. On the contrary, plant growth, growth-promoting gene expression, and the physiological consequence changed little in response to the Hpa1ΔNT treatment. These analyses suggest that Hpa1 requires the nitroxyl-terminus to facilitate CO₂ transport inside leaf cells and promote leaf photosynthesis and vegetative growth of the plant.     

Overexpression of the enzyme p-hydroxyphenolpyruvate dioxygenase in Arabidopsis and its relation to tocopherol biosynthesis

The enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of p-hydroxyphenylpyruvate to homogentisic acid (HGA), the aromatic precursor for the biosynthesis of vitamin E (α-tocopherol) and plastoquinone. In order to determine if increased HPPD activity could positively impact tocopherol yields, transgenic plants were generated that overexpressed the gene encoding Arabidopsis HPPD. Transgenic plants exhibiting high levels of HPPD expression were identified by increased tolerance to a competitive inhibitor of HPPD, the herbicide sulcotrione. HPPD gene expression in these transgenic lines, as determined at the RNA, protein and activity levels, was at least 10-fold higher than that of wild-type plants. Subsequent tocopherol analysis of leaf and seed material revealed that the increased HPPD expression resulted in up to a 37% increase in leaf tocopherol levels and a 28% increase in seed tocopherol levels relative to control plants. These results demonstrate that HPPD activity, and likely HGA levels, are at least one factor limiting the production of tocopherols in photosynthetic and non-photosynthetic plant tissues.     

The Arabidopsis homeobox gene, ATHB16, regulates leaf development and the sensitivity to photoperiod in Arabidopsis

This report describes the characterisation of ATHB16, a novel Arabidopsis thaliana homeobox gene, which encodes a homeodomain-leucine zipper class I (HDZip I) protein. We demonstrate that ATHB16 functions as a growth regulator, potentially as a component in the light-sensing mechanism of the plant. Endogenous ATHB16 mRNA was detected in all organs of Arabidopsis, at highest abundance in rosette leaves. Reduced levels of ATHB16 expression in transgenic Arabidopsis plants caused an increase in leaf cell expansion and consequently an increased size of the leaves, whereas leaf shape was unaffected. Transgenic plants with increased ATHB16 mRNA levels developed leaves that were smaller than wild-type leaves. Therefore, we suggest ATHB16 to act as a negative regulator of leaf cell expansion. Furthermore, the flowering time response to photoperiod was increased in plants with reduced ATHB16 levels but reduced in plants with elevated ATHB16 levels, indicating that ATHB16 has an additional role as a suppressor of the flowering time sensitivity to photoperiod in wild-type Arabidopsis. As deduced from the response of transgenic plants with altered levels of ATHB16 expression in hypocotyl elongation assays, the gene may act to regulate plant development as a mediator of a blue light response.