Howard D. Grimes

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.

A 62-kD sucrose binding protein is expressed and localized in tissues actively engaged in sucrose transport.

Sucrose transport from the apoplasm, across the plasma membrane, and into the symplast is critical for growth and development in most plant species. Phloem loading, the process of transporting sucrose against a concentration gradient into the phloem, is an essential first step in long-distance transport of sucrose and carbon partitioning. We report here that a soybean 62-kD sucrose binding protein is associated with the plasma membrane of several cell types engaged in sucrose transport, including the mesophyll cells of young sink leaves, the companion cells of mature phloem, and the cells of the developing cotyledons. Furthermore, the temporal expression of the gene and the accumulation pattern of the protein closely parallel the rate of sucrose uptake in the cotyledon. Molecular cloning and sequence analysis of a full-length cDNA for this 62-kD sucrose binding protein indicated that the protein is not an invertase, contains a 29-amino acid leader peptide that is absent from the mature protein, and is not an integral membrane protein. We conclude that the 62-kD sucrose binding protein is involved in sucrose transport, but is not performing this function independently.

Sink limitation induces the expression of multiple soybean vegetative lipoxygenase mRNAs while the endogenous jasmonic acid level remains low.

The response of individual members of the lipoxygenase multigene family in soybeans to sink deprivation was analyzed. RNase protection assays indicated that a novel vegetative lipoxygenase gene, vlxC, and three other vegetative lipoxygenase mRNAs accumulated in mature leaves in response to a variety of sink limitations. These data suggest that several members of the lipoxygenase multigene family are involved in assimilate partitioning. The possible involvement of jasmonic acid as a signaling molecule regulating assimilate partitioning into the vegetative storage proteins and lipoxygenases was directly assessed by determining the endogenous level of jasmonic acid in leaves from plants with their pods removed. There was no rise in the level of endogenous jasmonic acid coincident with the strong increase in both vlxC and vegetative storage protein VspB transcripts in response to sink limitation. Thus, expression of the vegetative lipoxygenases and vegetative storage proteins is not regulated by jasmonic acid in sink-limited leaves.

A soybean sucrose binding protein independently mediates nonsaturable sucrose uptake in yeast.

Heterologous expression of a cDNA encoding a 62-kD soybean sucrose binding protein in yeast demonstrates that this protein, independent of other plant proteins, mediates sucrose uptake across the plasma membrane. Sucrose binding protein-mediated sucrose uptake is nonsaturable up to 30 mM sucrose, is specific for sucrose, and is relatively insensitive to treatment with sulfhydryl-modifying reagents. Alteration of the external pH or pretreatment of the yeast cells with protonophores did not significantly affect the rate of 14C-sucrose uptake. This demonstrates that sucrose binding protein-mediated sucrose uptake is not dependent on H+ movement and delineates it from other plant sucrose transporters. Physiological characterization of sucrose uptake into higher plant cells has shown the presence of both saturable and nonsaturable uptake components. The nonsaturable mechanism is relatively insensitive to external pH, pretreatment with protonophores, and treatment with sulfhydryl-modifying reagents. Sucrose binding protein-mediated sucrose uptake in yeast mimics this physiologically described, but mechanistically undefined, nonsaturable sucrose uptake mechanism in higher plants. Functional characterization of the sucrose binding protein thus defines both a novel component of sucrose uptake and provides important insight into this nonsaturable sucrose uptake mechanism, which has remained enigmatic since its physiological description.

Regeneration of Indica Rice (Oryza sativa L.) from Primary Callus Derived from Immature Embryos

Summary To identify important parameters controlling the regeneration of indica rice (Oryza sativa L.) callus, immature embryos of IR54 were plated onto callus induction medium consisting of either MS or N6 basal salts. Various medium supplements were subsequently compared for ability to induce callus growth and plant regeneration. It was found that callus growth and plant regeneration were influenced by organic supplements to the basal media, medium solidifying agents, the number of embryo-derived calli per plate during plant regeneration, and phytohormone concentration during callus induction. However, these factors often influenced regeneration in contrasting ways depending on the basal medium used. For optimum plant regeneration N6 callus induction medium was supplemented with 1 g 1 -1 casein hydrolysate, 0.2 % (w/v) Gelrite, and 0.5 mg -1 -1 2,4-D. Regeneration was accomplished by subculturing one callus per plate to identical medium lacking 2,4-D.

Crystal structures of vegetative soybean lipoxygenase VLX-B and VLX-D, and comparisons with seed isoforms LOX-1 and LOX-3.

The lipoxygenase family of lipid‐peroxidizing, nonheme iron dioxygenases form products that are precursors for diverse physiological processes in both plants and animals. In soybean (Glycine max), five vegetative isoforms, VLX‐A, VLX‐B, VLX‐C, VLX‐D, VLX‐E, and four seed isoforms LOX‐1, LOX‐2, LOX‐3a, LOX‐3b have been identified. In this study, we determined the crystal structures of the substrate‐free forms of two major vegetative isoforms, with distinct enzymatic characteristics, VLX‐B and VLX‐D. Their structures are similar to the two seed isoforms, LOX‐1 and LOX‐3, having two domains with similar secondary structural elements: a β‐barrel N‐terminal domain containing highly flexible loops and an α‐helix‐rich C‐terminal catalytic domain. Detailed comparison of the structures of these two vegetative isoforms with the structures of LOX‐1 and LOX‐3 reveals important differences that help explain distinct aspects of the activity and positional specificity of these enzymes. In particular, the shape of the three branches of the internal subcavity, corresponding to substrate‐binding and O2 access, differs among the isoforms in a manner that reflects the differences in positional specificities. Proteins 2006.

Assessing the biosynthetic capabilities of secretory glands in Citrus peel.

Epithelial cells (ECs) lining the secretory cavities of Citrus peel have been hypothesized to be responsible for the synthesis of essential oil, but direct evidence for such a role is currently sparse. We used laser-capture microdissection and pressure catapulting to isolate ECs and parenchyma cells (as controls not synthesizing oil) from the peel of young grapefruit (Citrus × paradisi ‘Duncan’), isolated RNA, and evaluated transcript patterns based on oligonucleotide microarrays. A Gene Ontology analysis of these data sets indicated an enrichment of genes involved in the biosynthesis of volatile terpenoids and nonvolatile phenylpropanoids in ECs (when compared with parenchyma cells), thus indicating a significant metabolic specialization in this cell type. The gene expression patterns in ECs were consistent with the accumulation of the major essential oil constituents (monoterpenes, prenylated coumarins, and polymethoxylated flavonoids). Morphometric analyses demonstrated that secretory cavities are formed early during fruit development, whereas the expansion of cavities, and thus oil accumulation, correlates with later stages of fruit expansion. Our studies have laid the methodological and experimental groundwork for a vastly improved knowledge of the as yet poorly understood processes controlling essential oil biosynthesis in Citrus peel.

Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways

Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 alpha-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lipoxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in Escherichia coli, and then analyzed for lipoxygenase activity using linoleic, alpha-linolenic and arachidonic acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five preferred alpha-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lipoxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses indicated that the two arachidonic acid lipoxygenases form (12S)-hydroperoxy eicosatetraenoic acid as the main product, while the other five lipoxygenases produce mainly (13S)-hydroperoxy octadecatrienoic acid from alpha-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin pathways.

A Plasma Membrane Sucrose-binding Protein That Mediates Sucrose Uptake Shares Structural and Sequence Similarity with Seed Storage Proteins but Remains Functionally Distinct

Photoaffinity labeling of a soybean cotyledon membrane fraction identified a sucrose-binding protein (SBP). Subsequent studies have shown that the SBP is a unique plasma membrane protein that mediates the linear uptake of sucrose in the presence of up to 30 mm external sucrose when ectopically expressed in yeast. Analysis of the SBP-deduced amino acid sequence indicates it lacks sequence similarity with other known transport proteins. Data presented here, however, indicate that the SBP shares significant sequence and structural homology with the vicilin-like seed storage proteins that organize into homotrimers. These similarities include a repeated sequence that forms the basis of the reiterated domain structure characteristic of the vicilin-like protein family. In addition, analytical ultracentrifugation and nonreducing SDS-polyacrylamide gel electrophoresis demonstrate that the SBP appears to be organized into oligomeric complexes with aM r indicative of the existence of SBP homotrimers and homodimers. The structural similarity shared by the SBP and vicilin-like proteins provides a novel framework to explore the mechanistic basis of SBP-mediated sucrose uptake. Expression of the maize Glb protein (a vicilin-like protein closely related to the SBP) in yeast demonstrates that a closely related vicilin-like protein is unable to mediate sucrose uptake. Thus, despite sequence and structural similarities shared by the SBP and the vicilin-like protein family, the SBP is functionally divergent from other members of this group.

The Effects of High Salinity, Water-Deficit, and Abscisic Acid on Phosphoeno/pyruvate Carboxylase Activity and Proline Accumulation in Mesembryanthemum crystallinum Cell Cultures

Summary It has been established from previous studies in the halophyte Mesembryanthemum crystallinum that saline conditions induce the synthesis of phospho enol pyruvate carboxylase (PEPC), the performance of Crassulacean acid metabolism (CAM), and the production of compatible solutes such as proline. The objective of the present study was to determine whether factors causing the induction of these processes in leaves were also effective in tissue cultures of M. crystallinum grown under different conditions. In heterotrophically (sucrose)-grown calli of M. crystallinum , low levels of NaCl (10 to 25 mM) were required for optimum growth, while increasing concentrations of salt up to 200 mM caused a progressive decline in growth. Addition of salt to the culture medium caused a progressive increase in proline accumulation (13 fold increase in calli grown in 200 mM NaCl compared with calli grown without salt) but had little affect on PEPC activity in calli grown in sucrose media in the dark or light. Addition of 10 -7 M abscisic acid in the presence of 200 mM NaCl doubled the proline content in light-grown calli, while abscisic acid alone had no affect. However, growth with 10 -7 to 10 -5 M abscisic acid had no affect on levels of PEPC in dark or light-grown calli, in contrast to the previously reported induction of this enzyme and CAM by provision of abscisic acid to whole plants. Water stress (imposed by addition of polyethylene glycol) and low levels of NaCl (10 mM) in combination caused a large increase in proline content in heterotrophically cultured cells, whereas neither were effective alone. In photomixotrophic cell cultures, obtained by replacement of sucrose by soluble starch as the external carbon supply, there was a much higher chlorophyll content, and a limited salt induced increase in PEPC activity (4 fold enhancement during a 12-d, 200-mM, salt-shock period).