Eliot M. Herman

Protein Storage Bodies and Vacuoles

Plants store proteins in embryo and vegetative cells to provide carbon, nitrogen, and sulfur resources for subsequent growth and development. The storage and mobilization cycles of amino acids that compose these proteins are critical to the life cycle of plants. Mechanisms for protein storage and

Genetic Modification Removes an Immunodominant Allergen from Soybean

The increasing use of soybean (Glycine max) products in processed foods poses a potential threat to soybean-sensitive food-allergic individuals. In vitro assays on soybean seed proteins with sera from soybean-sensitive individuals have immunoglobulin E reactivity to abundant storage proteins and a few less-abundant seed proteins. One of these low abundance proteins, Gly m Bd 30 K, also referred to as P34, is in fact a major (i.e. immunodominant) soybean allergen. Although a member of the papain protease superfamily, Gly m Bd 30 K has a glycine in the conserved catalytic cysteine position found in all other cysteine proteases. Transgene-induced gene silencing was used to prevent the accumulation of Gly m Bd 30 K protein in soybean seeds. The Gly m Bd 30 K-silenced plants and their seeds lacked any compositional, developmental, structural, or ultrastructural phenotypic differences when compared with control plants. Proteomic analysis of extracts from transgenic seed detected the suppression of Gly m Bd 30 K-related peptides but no other significant changes in polypeptide pattern. The lack of a collateral alteration of any other seed protein in the Gly m Bd 30 K-silenced seeds supports the presumption that the protein does not have a role in seed protein processing and maturation. These data provide evidence for substantial equivalence of composition of transgenic and non-transgenic seed eliminating one of the dominant allergens of soybean seeds.

The Role of Aquaporins and Membrane Damage in Chilling and Hydrogen Peroxide Induced Changes in the Hydraulic Conductance of Maize Roots

When chilling-sensitive plants are chilled, root hydraulic conductance (Lo) declines precipitously; Lo also declines in chilling-tolerant plants, but it subsequently recovers, whereas in chilling-sensitive plants it does not. As a result, the chilling-sensitive plants dry out and may die. Using a chilling-sensitive and a chilling-tolerant maize genotype we investigated the effect of chilling on Lo, and its relationship to osmotic water permeability of isolated root cortex protoplasts, aquaporin gene expression, aquaporin abundance, and aquaporin phosphorylation, hydrogen peroxide (H2O2) accumulation in the roots and electrolyte leakage from the roots. Because chilling can cause H2O2 accumulation we also determined the effects of a short H2O2 treatment of the roots and examined the same parameters. We conclude from these studies that the recovery of Lo during chilling in the chilling-tolerant genotype is made possible by avoiding or repairing membrane damage and by a greater abundance and/or activity of aquaporins. The same changes in aquaporins take place in the chilling-sensitive genotype, but we postulate that membrane damage prevents the Lo recovery. It appears that the aquaporin response is necessary but not sufficient to respond to chilling injury. The plant must also be able to avoid the oxidative damage that accompanies chilling.

KDEL-Containing Auxin-Binding Protein Is Secreted to the Plasma Membrane and Cell Wall.

The auxin-binding protein ABP1 has been postulated to mediate auxin-induced cellular changes associated with cell expansion. This protein contains the endoplasmic reticulum (ER) retention signal, the tetrapeptide lysine-aspartic acid-glutamic acid-leucine (KDEL), at its carboxy terminus, consistent with previous subcellular fractionation data that indicated an ER location for ABP1. We used electron microscopic immunocytochemistry to identify the subcellular localization of ABP1. Using maize (Zea mays) coleoptile tissue and a black Mexican sweet (BMS) maize cell line, we found that ABP1 is located in the ER as expected, but is also on or closely associated with the plasma membrane and within the cell wall. Labeling of the Golgi apparatus suggests that the transport of ABP1 to the cell wall occurs via the secretory system. Inhibition of secretion of an ABP homolog into the medium of BMS cell cultures by brefeldin A, a drug that specifically blocks secretion, is consistent with this secretion pathway. The secreted protein was recognized by an anti-KDEL peptide antibody, strongly supporting the interpretation that movement of this protein out of the ER does not involve loss of the carboxy-terminal signal. Cells starved for 2,4-dichlorophenoxyacetic acid for 72 h retained less ABP in the cell and secreted more of it into the medium. The significance of our observations is 2-fold. We have identified a KDEL-containing protein that specifically escapes the ER retention system, and we provide an explanation for the apparent discrepancy that most of the ABP is located in the ER, whereas ABP and auxin act at the plasma membrane.

Dinoflagellate Expressed Sequence Tag Data Indicate Massive Transfer of Chloroplast Genes to the Nuclear Genome

The peridinin-pigmented plastids of dinoflagellates are very poorly understood, in part because of the paucity of molecular data available from these endosymbiotic organelles. To identify additional gene sequences that would carry information about the biology of the peridinin-type dinoflagellate plastid and its evolutionary history, an analysis was undertaken of arbitrarily selected sequences from cDNA libraries constructed from Lingulodinium polyedrum (1012 non-redundant sequences) and Amphidinium carterae (2143). Among the two libraries 118 unique plastid-associated sequences were identified, including 30 (most from A. carterae) that are encoded in the plastid genome of the red alga Porphyra. These sequences probably represent bona fide nuclear genes, and suggest that there has been massive transfer of genes from the plastid to the nuclear genome in dinoflagellates. These data support the hypothesis that the peridinin-type plastid has a minimal genome, and provide data that contradict the hypothesis that there is an unidentified canonical genome in the peridinin-type plastid. Sequences were also identified that were probably transferred directly from the nuclear genome of the red algal endosymbiont, as well as others that are distinctive to the Alveolata. A preliminary report of these data was presented at the Botany 2002 meeting in Madison, WI.

Synthesis and protein body deposition of maize 15-kd zein in transgenic tobacco seeds.

The maize 15‐Kd zein structural gene was placed under the regulation of French bean β‐phaseolin gene flanking regions. Agrobacterium tumefaciens‐mediated transformation was used to insert the chimeric phaseolin–zein gene into the tobacco genome. Transgenic plants synthesized zein in a tissue‐specific manner during the latter half of seed development. Transcription of the chimeric gene was initiated in phaseolin‐derived sequences, and was terminated within the phaseolin gene 3′ flanking region. Both zein‐ and phaseolin‐derived polyadenylation signals were used in the processing of zein RNA in transgenic plant seeds. Zein accumulation, though subject to an 80‐fold variation among 19 plants tested, could reach as much as 1.6% of the total seed protein in several plants. In developing tobacco seeds, zein was correctly processed by the removal of a 20‐amino‐acid signal peptide. Electron microscope immunogold localization of the zein expressed in embryo and endosperm tissue indicates that the monocot protein accumulates in the crystalloid component of vacuolar protein bodies. The density of gold label over the protein bodies is several fold greater in the embryo than the endosperm. Zein is found in roots, hypocotyls and cotyledons of germinating transgenic tobacco seeds.

Cosuppression of the α Subunits of β-Conglycinin in Transgenic Soybean Seeds Induces the Formation of Endoplasmic Reticulum–Derived Protein Bodies

The expression of the α and α′ subunits of β-conglycinin was suppressed by sequence-mediated gene silencing in transgenic soybean seed. The resulting seeds had similar total oil and protein content and ratio compared with the parent line. The decrease in β-conglycinin protein was apparently compensated by an increased accumulation of glycinin. In addition, proglycinin, the precursor of glycinin, was detected as a prominent polypeptide band in the protein profile of the transgenic seed extract. Electron microscopic analysis and immunocytochemistry of maturing transgenic soybean seeds indicated that the process of storage protein accumulation was altered in the transgenic line. In normal soybeans, the storage proteins are deposited in pre-existing vacuoles by Golgi-derived vesicles. In contrast, in transgenic seed with reduced β-conglycinin levels, endoplasmic reticulum (ER)–derived vesicles were observed that resembled precursor accumulating–vesicles of pumpkin seeds and the protein bodies accumulated by cereal seeds. Their ER–derived membrane of the novel vesicles did not contain the protein storage vacuole tonoplast-specific protein α-TIP, and the sequestered polypeptides did not contain complex glycans, indicating a preGolgi and nonvacuolar nature. Glycinin was identified as a major component of these novel protein bodies and its diversion from normal storage protein trafficking appears to be related to the proglycinin buildup in the transgenic seed. The stable accumulation of proteins in a protein body compartment instead of vacuolar accumulation of proteins may provide an alternative intracellular site to sequester proteins when soybeans are used as protein factories.

Immunogold-localization and synthesis of an oil-body membrane protein in developing soybean seeds

The synthesis of a major oil-body membrane brotein was studied in maturing soybean (Glycine max (L.) Merr.) cotyledons. The membrane contained four abundant proteins with apparent molecular mass (Mr) of 34000, 24000, 18000 and 17000. The Mr=24000 protein (mP 24) was selected for more detailed analysis. The protein was purified to apparent homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isolated from the gel by electroelution or chemical hydrolysis of gel crosslinks. It was then used to elicit rabbit antibodies which were judged to be specific when assayed by SDS-PAGE-immunoblot procedures. The mP 24 was localized in immature soybean cotyledon cells by indirect immunogold procedures on thin sections of Lowicryl- and LR-White-embedded tissue. Indirect labeling with the primary antiserum followed by colloidal gold-protein A showed specific labeling of the oil-body membrane and an absence of label on the other subcellular organelles including the endoplasmic reticulum (ER). Parallel tissue samples were studied by conventional transmission electron microscopy. Although segments of the ER were observed to be closely juxtaposed to the oil bodies, continuity between the two organelles was not observed. The synthesis of mP 24 was studied by in-vitro translation and in-vivo labeling with [3H]leucine followed by indirect immunoaffinity isolation of the labeled products. The SDS-PAGE fluorography results indicated that the primary translation product and the in-vivo synthesized protein have the same Mr, and this is also the same Mr as the protein in the mature membrane.

Vacuolar-type H+-ATPases are associated with the endoplasmic reticulum and provacuoles of root tip cells

To understand the origin of vacuolar H+ -ATPases (V-ATPases) and their cellular functions, the subcellular location of V-H+ -ATPases was examined immunologically in root cells of oat seedlings. A V-ATPase complex from oat roots consists of a large peripheral sector (V1) that includes the 70-kD (A) catalytic and the 60-kD (B) regulatory subunits. The soluble V1 complex, thought to be synthesized in the cytoplasm, is assembled with the membrane integral sector (V0) at a yet undefined location. In mature cells, V-ATPase subunits A and B, detected in immunoblots with monoclonal antibodies (Mab) (7A5 and 2E7), were associated mainly with vacuolar membranes (20–22% sucrose) fractionated with an isopycnic sucrose gradient. However, in immature root tip cells, which lack large vacuoles, most of the V-ATPase was localized with the endoplasmic reticulum (ER) at 28 to 31% sucrose where a major ER-resident binding protein equilibrated. The peripheral subunits were also associated with membranes at 22% sucrose, at 31 to 34% sucrose (Golgi), and in plasma membranes at 38% sucrose. Immunogold labeling of root tip cells with Mab 2E7 against subunit B showed gold particles decorating the ER as well as numerous small vesicles (0.1–0.3 [mu]m diameter), presumably pro-vacuoles. The immunological detection of the peripheral subunit B on the ER supports a model in which the V1 sector is assembled with the V0 on the ER. These results support the model in which the central vacuolar membrane originates ultimately from the ER. The presence of V-ATPases on several endomembranes indicates that this pump could participate in diverse functional roles.

Suppression of Soybean Oleosin Produces Micro-Oil Bodies that Aggregate into Oil Body/ER Complexes

Using RNAi, the seed oil body protein 24-kDa oleosin has been suppressed in transgenic soybeans. The endoplasmic reticulum (ER) forms micro-oil bodies about 50 nm in diameter that coalesce with adjacent oil bodies forming a hierarchy of oil body sizes. The oil bodies in the oleosin knockdown form large oil body-ER complexes with the interior dominated by micro-oil bodies and intermediate-sized oil bodies, while the peripheral areas of the complex are dominated by large oil bodies. The complex merges to form giant oil bodies with onset of seed dormancy that disrupts cell structure. The transcriptome of the oleosin knockdown shows few changes compared to wild-type. Proteomic analysis of the isolated oil bodies of the 24-kDa oleosin knockdown shows the absence of the 24-kDa oleosin and the presence of abundant caleosin and lipoxygenase. The formation of the micro-oil bodies in the oleosin knockdown is interpreted to indicate a function of the oleosin as a surfactant.