Transient N-acetylglucosamine in the biosynthesis of phytohemagglutinin: attachment in the Golgi apparatus and removal in protein bodies.

Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the lectin phytohemagglutinin (PHA) during seed development. The polypeptides of PHA are synthesized by endoplasmic reticulum-bound polysomes and co-translationally glycosylated, pass through the Golgi complex, and accumulate in protein bodies, which constitute the lysosomal compartment in these cells. Some of the high-mannose sidechains of PHA are modified in the Golgi complex, and in mature PHA they contain N-acetylglucosamine, mannose, fucose, and xylose in the molar ratios 2, 3.8, 0.6, and 0.5. The results reported here show that the Golgi complex is also the site of additional N-acetylglucosamine incorporation into the modified sidechains. When developing cotyledons are labeled with [3H]glucosamine and glycopeptides of PHA present in the Golgi complex isolated, the radioactivity can be released as [3H]N-acetylglucosamine by digestion of the glycopeptides with beta-N-acetylglucosaminidase, indicating that the residues are in a terminal position. Arrival of PHA in the protein bodies is followed by the slow removal of these terminal N-acetylglucosamine residues, resulting in a decrease in the Mr of the modified sidechains. The biosynthetic intermediates of the glycoproteins destined for the lysosomal compartments of animal cells contain high-mannose sidechains modified by phosphate groups covered by N-acetylglucosamine that is labile to mild acid treatment. When cotyledons are labeled with [32P]orthophosphate, there is no radioactivity in PHA obtained from any of the subcellular fractions. There is also no release of radioactivity when [3H]glucosamine-labeled glycopeptides obtained from PHA in the Golgi complex are subjected to mild acid hydrolysis. These results indicate that the sorting-signals and posttranslational processing steps for proteins that are transported to the lysosomal compartment are different in plant cells and animal cells.

The plant vacuolar protein, phytohemagglutinin, is transported to the vacuole of transgenic yeast.

Phytohemagglutinin (PHA), the major seed lectin of the common bean, Phaseolus vulgaris, accumulates in the parenchyma cells of the cotyledons. It has been previously shown that PHA is cotranslationally inserted into the endoplasmic reticulum with cleavage of the NH2-terminal signal peptide. Two N-linked oligosaccharide side chains are added, one of which is modified to a complex type in the Golgi apparatus. PHA is then deposited in membrane-bound protein storage vacuoles which are biochemically and functionally equivalent to the vacuoles of yeast cells and the lysosomes of animal cells. We wished to determine whether yeast cells would recognize the vacuolar sorting determinant of PHA and target the protein to the yeast vacuole. We have expressed the gene for leukoagglutinating PHA (PHA-L) in yeast under control of the yeast acid phosphatase (PHO5) promoter. Under control of this promoter, PHA-L accumulates to 0.1% of the total yeast protein. PHA-L produced in yeast is glycosylated as expected for a yeast vacuolar glycoprotein. Cell fractionation studies show that PHA-L is efficiently transported to the yeast vacuole. This is the first demonstration that vacuolar targeting information is recognized between two highly divergent species. A small proportion of yeast PHA-L is secreted which may be due to inefficient recognition of the vacuolar sorting signal because of the presence of an uncleaved signal peptide on a subset of the PHA-L polypeptides. This system can now be used to identify the vacuolar sorting determinant of a plant vacuolar protein.

In vivo and in vitro processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps.

Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the reserve protein phaseolin. The polypeptides are synthesized on membrane-bound polysomes, pass through the endoplasmic reticulum (ER) and accumulate in protein bodies. For a study of the biosynthesis and processing of phaseolin, developing cotyledons were labeled with radioactive amino acids, glucosamine and mannose, and isolated fractions (polysomal RNA, polysomes, and rough ER) were used for in vitro protein synthesis. Newly synthesized phaseolin present in the ER of developing cotyledons can be fractioned into four glycopolypeptides by SDS PAGE. In vitro synthesis with polysomal RNA results in the formation of two polypeptides by polysome run-off shows that glycosylation is a co-translational event. The two unglycosylated polypeptides formed by polysome run-off are slightly smaller than the two polypeptides formed by in vitro translation of isolated RNA, indicating that a signal peptide may be present on these polypeptides. Run-off synthesis with rough ER produces a pattern of four polypeptides similar to the one obtained by in vivo labeling. The two abundant glycopolypeptides formed by polysome run-off. This result indicates the existence of a second glycosylation event for the abundant polypeptides. Inhibition of glycosylation by Triton X-100 during chain-completion with rough ER was used to show that these two glycosylation steps normally occur sequentially. Both glycosylation steps are inhibited by tunicamycin. Analysis of carhohydrate to protein ratios of the different polypeptides and of trypsin digests of polypeptides labeled with [(3)H]glucosamine confirmed the conclusion that some glycosylated polypeptides contain two oligosaccharide chains, while others contain only one. An analysis of tryptic peptide maps shows that each of the unglycosylated polypeptides is the precursor for one glycosylated polypeptide with one oligosaccharide chain and one with two oligosaccharide chains.

Localization of vicilin peptidohydrolase in the cotyledons of mung bean seedlings by immunofluorescence microscopy.

Vicilin peptidohydrolase, the protease that hydrolyzes the reserve proteins in the cotyledons of mung bean (Vigna radiata) seedlings, has been localized intracellularly by immunofluorescence microscopy using monospecific antibodies against the enzyme and rhodamine-coupled goat-anti-rabbit immunoglobulin G's. The enzyme can first be visualized after 3 days of seedling growth and is associated with small foci within the cytoplasm of the storage parenchyma cells farthest from the vascular bundles. On the 4th day of growth, the protease is also present in the numerous large protein bodies within these cells. Vicilin peptidohydrolase is known to be synthesized de novo starting on the 3rd day of growth. Our observations are therefore consistent with the interpretation that the enzyme is synthesized in the cytoplasm and subsequently transported to the protein bodies.

Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides in the protein bodies of developing pea cotyledons.

Cotyledons of developing pea seeds (pisum sativum L.) were labeled with radioactive amino acids and glucosamine, and extracts were prepared and separated into fractions rich in endoplasmic reticulum (ER) or protein bodies, The time-course of synthesis of the polypeptides of legumin and vicilin and the site of their assembly into protein oligomers were studied using immunoaffinity gels and sucrose density gradients. When cotyledons were pulse-labeled (1-2 h), newly synthesized vicilin was present as a series of polypeptides with M(r) 60,000-65,000, and newly synthesized vicilin was present as series of polypeptides with M(r) 75,000, 70,000, 50,000, and 49,000. These radioactive polypeptides were found primarily in the ER (Chrispeels et al., 1982, J Cell Biol., 93:5- 14). During a subsequent chase period, newly synthesized reserve proteins were initially present in the protein bodies in the above-named polypeptides. Between 1 and 20 h later, radioactive legumin subunits (M(r) 40,000 and 19,000) and smaller vicilin polypeptides (M(r) 34,000, 30,000, 25,000, 18,000, 14,000, 13,000, and 12,000) appeared in the protein bodies. The appearance of these labeled polypeptides in the protein bodies was not the result of a slow transport from the ER (or cytoplasm). Newly synthesized legumin and vicilin polypeptides were assembled into oligomers of 8S and 7S, respectively, in the ER. They appeared in the protein bodies in these oligomeric forms before the appearance of the smaller polypeptides (M(r) less than 49,000). These results indicate that the smaller vicilin polypeptides (M(r) less than 49,000) arise delayed posttranslational processing of some or all of the larger vicilin polypeptides. The precursors of legumin are completely processed in the protein bodies 2-3 h after their synthesis. The processing of the vicilin precursors is much slower (6-20 h) and only a fraction of the precursor molecules are processed. As a result both large (M(r) more than 49,000) and small polypeptides of vicilin accumulate in the protein bodies, whereas legumin accumulates only as polypeptides of M(r) 40,000 and 19,000.

Role of the endoplasmic reticulum in the synthesis of reserve proteins and the kinetics of their transport to protein bodies in developing pea cotyledons.

Developing pea (Pisum sativum L.) cotyledons were labeled with radioactive amino acids, glucosamine, and mannose in pulse an pulse-chase experiments to study the synthesis, glycosylation, and transport of the reserve proteins vicilin and legumin to the protein bodies. Tissue extracts were fractionated on sucrose gradients to isolate either the endoplasmic reticulum (ER) or the protein bodies. Immunoaffinity gels were used to determine radioactivity in the reserve proteins (legumin and vicilin). After pulse-labeling for 45 min with amino acids, about half the total incorporated radioactivity coincided closely with the position of the ER marker enzyme NADH-cytochrome c reductase at a density of 1.13 g . cm-3 on the sucrose gradient. Both radioactivity and enzyme activity shifted to a density of 1.18 g . cm-3 in the presence of 3 mM MgCl2 indicating that the radioactive proteins were associated with the rough ER. Approximately half of the incorporated radioactivity associated with the rough ER was in newly synthesized reserve protein and this accounted for 80% of the reserve protein synthesized in 45 min. Trypsin digestion experiments indicated that these proteins were sequestered within the ER. In pulse-chase experiments, the reserve proteins in the ER became radioactive without appreciable lag and radioactivity chased out of the ER with a half-life of 90 min. Radioactive reserve proteins became associated with a protein body-rich fraction 20-30 min after their synthesis and sequestration by the ER. Pulse-chase experiments with radioactive glucosamine and mannose in the presence and absence of tunicamycin indicated that glycosylation of vicilin occurs in the ER. However, glycosylation is not a prerequisite for transport of vicilin from ER to protein bodies. Examination of the reserve protein polypeptides by SDS PAGE followed by fluorography showed that isolated ER contained legumin precursors (Mr 60,000-65,000) but not the polypeptides present in mature legumin (Mr 40,000 and 19,000) as well as the higher molecular weight polypeptides of vicilin (Mr 75,000, 70,000, 50,000, and 49,000). The smaller polypeptides of vicilin present in vicilin extracted from protein bodies (Mr 12,000-34,000) were absent from the ER. The results show that newly synthesized reserve proteins are preferentially and transiently sequestered within the ER before they move to the protein bodies, and that the ER is the site of storage protein glycosylation.

Transport of proteins to the plant vacuole is not by bulk flow through the secretory system, and requires positive sorting information.

Plant cells, like other eukaryotic cells, use the secretory pathway to target proteins to the vacuolar/lysosomal compartment and to the extracellular space. We wished to determine whether the presence of a hydrophobic signal peptide would result in the transport of a reporter protein to vacuoles by bulk flow; to investigate this question, we expressed a chimeric gene in transgenic tobacco. The chimeric gene, Phalb, used for this study consists of the 1,188-bp 5' upstream sequence and the hydrophobic signal sequence of a vacuolar seed protein phytohemagglutinin, and the coding sequence of a cytosolic seed albumin (PA2). The chimeric protein PHALB cross-reacted with antibodies to PA2 and was found in the seeds of the transgenic plants (approximately 0.7% of total protein), but not in the leaves, roots, or flowers. Immunoblot analyses of seed extracts revealed four glycosylated polypeptides ranging in molecular weight from 29,000 to 32,000. The four polypeptides are glycoforms of a single polypeptide of Mr 27,000, and the heterogeneity is due to the presence of high mannose and endoglycosidase H-resistant glycans. The PHALB products reacted with an antiserum specific for complex plant glycans indicating that the glycans had been modified in the Golgi apparatus. Subcellular fractionation of glycerol extracts of mature seeds showed that only small amounts of PHALB accumulated in the protein storage vacuoles of the tobacco seeds. In homogenates made in an isotonic medium, very little PHALB was associated with the organelle fraction containing the endoplasmic reticulum and Golgi apparatus; most of it was in the soluble fraction. We conclude that PHALB passed through the Golgi apparatus, but did not arrive in the vacuoles. Transport to vacuoles is not by a bulk-flow mechanism, once proteins have entered the secretory system, and requires information beyond that provided by a hydrophobic signal peptide.

Cell wall protein in plants: autoradiographic evidence.

Autoradiography of phloem-parenchyma tissue from carrots, which was allowed to incorporate radioactive proline and then plasmolyzed, indicates that a stable protein moiety is associated with the cell wall.

Aquaporins: water channel proteins of plant and animal cells.

Certain biological membranes, such as the erythrocyte plasma membrane, have a high osmotic water permeability, and such membranes have long been suspected of harboring water channels. The molecular identity of these channels has now been established with the purification of water-channel proteins and the cloning of the genes encoding them. Homologous water-channel proteins, called 'aquaporins', are present in plants and animals. These channels are water selective and do not allow ions or metabolites to pass through them. Their discovery is providing new insights into how plant and animal cells facilitate and regulate the passage of water through their membranes.

Tonoplast and Soluble Vacuolar Proteins Are Targeted by Different Mechanisms.

The delivery of proteins to the vacuole and its limiting membrane (the tonoplast) by the secretory system is thought to be a dissociative process in which vesicles bud from one compartment and fuse with another. We studied the transport kinetics of phytohemagglutinin (PHA) and tonoplast intrinsic protein (TIP) in mesophyll protoplasts obtained from transgenic tobacco plants transformed with genes encoding these two proteins. In pulse-chase experiments, arrival of PHA in the vacuole was found to be slower (completed 24 hr after synthesis) than the arrival of TIP in the tonoplast (completed 6 hr after synthesis). Brefeldin A and monensin block protein transport by interfering in specific vesicle transport steps. Brefeldin A prevents anterograde vesicle transport between the endoplasmic reticulum and the Golgi, whereas monensin inhibits correct sorting in the trans-Golgi network by disrupting the proton gradient across the membrane. Both inhibitors blocked the transport of PHA to the vacuole and altered the rate at which its complex glycan is processed by Golgi enzymes. Neither drug stopped the arrival of TIP in the tonoplast, suggesting that the flow of vesicles continues in the presence of these inhibitors. We suggest that soluble proteins like PHA and membrane proteins like TIP reach their vacuolar destinations by different paths.

A new lectin in red kidney beans called PvFRIL stimulates proliferation of NIH 3T3 cells expressing the Flt3 receptor.

A new legume lectin has been identified by its ability to specifically stimulate proliferation of NIH 3T3 fibroblasts expressing the Flt3 tyrosine kinase receptor. The lectin was isolated from conditioned medium harvested from human peripheral blood mononuclear cells activated to secrete cytokines by a crude red kidney bean extract containing phytohemagglutinin (PHA). Untransfected 3T3 cells and 3T3 cells transfected with the related Fms tyrosine kinase receptor do not respond to this lectin, which we called PvFRIL (Phaseolus vulgaris Flt3 receptor-interacting lectin). When tested on cord blood mononuclear cells enriched for Flt3-expressing progenitors, purified PvFRIL fractions maintained a small population of cells that continued to express CD34 after 2 weeks in suspension cultures containing IL3. These cultures did not show the effects of IL3's strong induction of proliferation and differentiation (high cell number and exhausted medium); instead, low cell number at the end of the culture period resulted in persistence of cells in the context of cell death. These observations led to the hypothesis that PvFRIL acts in a dominant manner to preserve progenitor viability and prevent proliferation and differentiation.

Molecular characterization of a new arcelin-5 gene.

Arcelins are insecticidal proteins found in some wild accessions of the common bean, Phaseolus vulgaris. They are grouped in six allelic variants and arcelin-5 is the variant with the highest inhibitory effect on the development of Zabrotes subfasciatus larvae. Characterization of the protein and its genes resulted in the identification of three polypeptides and the isolation of two genes that encode the Arc5a and Arc5b polypeptides. Here we describe a new gene, Arc5-III. The protein it encodes has 81% amino acid identity with the derived amino acid sequences of Arc5-I and Arc5-II. The Arc5-III gene is highly expressed in developing seeds and at a much lower level in roots. Data obtained by a combination of two-dimensional gel electrophoresis, protein sequencing and MALDI-TOF mass spectrometry analysis support the conclusion that Arc5-III encodes a polypeptide present in Arc5c band. Using ion-exchange chromatography, three fractions containing arcelin-5 polypeptides were eluted by increasing the salt concentration. The three fractions contain various amounts of the three arc-5 polypeptides and inhibit the growth of Zabrotes subfasciatus larvae differentially, suggesting differences in insecticidal activity among the arcelin-5 isoforms.

Projection structure of a plant vacuole membrane aquaporin by electron cryo-crystallography.

The water channel protein alpha-TIP is a member of the major intrinsic protein (MIP) membrane channel family. This aquaporin is found abundantly in vacuolar membranes of cotyledons (seed storage organs) and is synthesized during seed maturation. The water channel activity of alpha-TIP can be regulated by phosphorylation, and the protein may function in seed desiccation, cytoplasmic osmoregulation, and/or seed rehydration. Alpha-TIP was purified from seed meal of the common bean (Phaseolus vulgaris) by membrane fractionation, solubilization in diheptanoylphosphocholine and anion-exchange chromatography. Upon detergent removal and reconstitution into lipid bilayers, alpha-TIP crystallized as helical tubes. Electron cryo-crystallography of flattened tubes demonstrated that the crystals exhibit plane group p2 symmetry and c222 pseudosymmetry. Since the 2D crystals with p2 symmetry are derived from helical tubes, we infer that the unit of crystallization on the helical lattice is a dimer of tetramers. A projection density map at a resolution of 7.7 A revealed that alpha-TIP assembles as a 60 A x 60 A square tetramer. Each subunit is formed by a heart-shaped ring comprised of density peaks which we interpret as alpha-helices. The similarity of this structure to mammalian plasma membrane MIP-family proteins suggests that the molecular design of functionally analogous and genetically homologous aquaporins is maintained between the plant and animal kingdoms.

The role of weak protein-protein interactions in multivalent lectin-carbohydrate binding: crystal structure of cross-linked FRIL.

Binding of multivalent glycoconjugates by lectins often leads to the formation of cross-linked complexes. Type I cross-links, which are one-dimensional, are formed by a divalent lectin and a divalent glycoconjugate. Type II cross-links, which are two or three-dimensional, occur when a lectin or glycoconjugate has a valence greater than two. Type II complexes are a source of additional specificity, since homogeneous type II complexes are formed in the presence of mixtures of lectins and glycoconjugates. This additional specificity is thought to become important when a lectin interacts with clusters of glycoconjugates, e.g. as is present on the cell surface. The cryst1al structure of the Glc/Man binding legume lectin FRIL in complex with a trisaccharide provides a molecular snapshot of how weak protein-protein interactions, which are not observed in solution, can become important when a cross-linked complex is formed. In solution, FRIL is a divalent dimer, but in the crystal FRIL forms a tetramer, which allows for the formation of an intricate type II cross-linked complex with the divalent trisaccharide. The dependence on weak protein-protein interactions can ensure that a specific type II cross-linked complex and its associated specificity can occur only under stringent conditions, which explains why lectins are often found forming higher-order oligomers.

Bean [alpha]-Amylase Inhibitor Confers Resistance to the Pea Weevil (Bruchus pisorum) in Transgenic Peas (Pisum sativum L.).

Bruchid larvae cause major losses of grain legume crops through-out the world. Some bruchid species, such as the cowpea weevil and the azuki bean weevil, are pests that damage stored seeds. Others, such as the pea weevil (Bruchus pisorum), attack the crop growing in the field. We transferred the cDNA encoding the [alpha]-amylase inhibitor ([alpha]-AI) found in the seeds of the common bean (Phaseolus vulgaris) into pea (Pisum sativum) using Agrobacterium-mediated transformation. Expression was driven by the promoter of phytohemagglutinin, another bean seed protein. The [alpha]-amylase inhibitor gene was stably expressed in the transgenic pea seeds at least to the T5 seed generation, and [alpha]-AI accumulated in the seeds up to 3% of soluble protein. This level is somewhat higher than that normally found in beans, which contain 1 to 2% [alpha]-AI. In the T5 seed generation the development of pea weevil larvae was blocked at an early stage. Seed damage was minimal and seed yield was not significantly reduced in the transgenic plants. These results confirm the feasibility of protecting other grain legumes such as lentils, mungbean, groundnuts, and chickpeas against a variety of bruchids using the same approach. Although [alpha]-AI also inhibits human [alpha]-amylase, cooked peas should not have a negative impact on human energy metabolism.

Uptake and apparent digestion of cytoplasmic organelles by protein bodies (protein storage vacuoles) in mung bean cotyledons.

The large protein bodies of the storage parenchyma cells of mung bean (Vigna radiata) cotyledons contain vesicles measuring 0.2 to 2.0 mum in diameter. The vesicles contain ribosomes, ribosomes, membranous elements which may be derived from the endoplasmic reticulum and occasionally Golgi bodies and mitochondria. The vesicles can be seen by transmission electron microscopy in thin sections of plastic embedded specimens and in replicas of freeze-fractured preparations. Serial sections show that the vesicles are completely separated from the protein body membrane and are not invaginations of that membrane. Vesicles with cytoplasmic structures are seen most frequently in 2 to 4 day old seedlings. The vesicles may be formed when undulations of the protein body membrane are so deep as to permit the pinching-off of a portion of the cytoplasm, resulting in its subsequent isolation from the cytoplasm within the protein body. The digestion of the storage protein in the protein body is accompanied by the disappearance of the ribosomes and the membranous elements in the vesicles. We interpret this disappearance of the cytoplasmic structures in the vesicles as being due to their digestion by the protein body hydrolases (ribonuclease, proteinase and lipolytic enzymes). The uptake of cytoplasmic structures by the protein bodies continues after the reverse proteins have been digested. Cytochemical staining shows that the protein bodies and especially the vesicles are rich in acid phosphatase, a known marker of lytic activity in cells. The evidence presented here indicates that the protein bodies are the intracellular sites at which the digestion of cytoplasmic structure occurs. Protein bodies should therefore be considered not only as compartments for the hydrolysis of the stored protein, but also as autophagic organelles involved in the degradation of cytoplasmic macromolecules. The term protein bodies is well established, but the term protein storage vacuoles may describe these organelles more precisely.

Secretion of phytohemagglutinin by monkey COS cells.

The entire coding region of a gene, which encodes a polypeptide of phytohemagglutinin (PHA-L), obtained from a library of genomic DNA of the common bean Phaseolus vulgaris cv. Greensleeves, was introduced into the SV40 expression vector pJC119. Monkey COS1 cells were transfected with the recombinant clone and the synthesis, glycosylation, and transport of PHA-L studied and compared with the normal processes in bean cotyledons. In the bean, phytohemagglutinin is synthesized on the rough endoplasmic reticulum and transported via the Golgi complex to protein bodies, vacuole-like organelles. Phytohemagglutinin was synthesized and glycosylated at the ER and processed in the Golgi apparatus of the transfected COS1 cells. After passing the Golgi apparatus, PHA-L was slowly secreted into the culture medium (half-time of 3-6 h), a result indicating that the signals for targeting proteins beyond the Golgi apparatus in plant cells are different from those in animal cells. PHA, which is stored in protein bodies in the plant cells, is secreted by animal cells. Tunicamycin inhibited both glycosylation and secretion of PHA by the COS1 cells, a finding indicating an essential role of the oligosaccharides for transport of PHA in these cells in contrast to the situation found in bean cotyledons. PHA, secreted into the culture medium, was partially sensitive to endo H, a result indicating the presence of one high-mannose and one complex oligosaccharide chain, a situation identical to that in beans.

Post-translational processing of two alpha-amylase inhibitors and an arcelin from the common bean, Phaseolus vulgaris.

Mass spectrometric methods were used to investigate the proteolytic processing and glycopeptide structures of three seed defensive proteins from Phaseolus vulgaris. The proteins were the alpha-amylase inhibitors alphaAI-1 and alphaAI-2 and arcelin-5, all of which are related to the seed lectins, PHA-E and PHA-L. The mass data showed that the proteolytic cleavage required for activation of the amylase inhibitors is followed by loss of the terminal Asn residue in alphaAI-1, and in all three proteins, seven or more residues were clipped from the C-termini, in the manner of the seed lectins. In most instances, individual glycoforms could be assigned at each Asn site, due to the unique masses of the plant glycopeptides. It was found that alphaAI-1 and alphaAI-2 differed significantly in their glycosylation patterns, despite their high sequence homology. These data complement the previous X-ray studies of the alpha1-amylase inhibitor and arcelin, where many of the C-terminal residues and glycopeptide residues could not be observed.

Characterization of beta-fructosidase, an extracellular glycoprotein of carrot cells.

Seedlings and suspension-cultured cells of carrot (Daucus carota) contain a cell wall associated as well as a soluble form of beta-fructosidase (beta F). These two forms have different pH optima: 4.6 for cell wall beta F and 5.6 for soluble beta F. Soluble beta F is relatively more abundant in the seedlings and cell wall beta F is relatively much more abundant in the cultured cells. Protoplasts of cultured cells have only the soluble form (pH optimum 5.6) indicating that the cell wall associated form is indeed extracellular in situ. Cell wall beta F was purified to homogeneity and has an Mr = 63,000. Antibodies raised against the deglycosylated enzyme cross-reacted with two soluble enzyme forms: in cultured cells, the soluble enzyme has an Mr = 58,000 and, in seedlings, there are two forms of Mr = 58,000 and 52,000. Treatment of purified cell wall beta F with endoglycosidase H and trifluoromethanesulfonic acid (complete deglycosylation) indicated that the enzyme probably has one high mannose and two complex glycans. This was confirmed by HPLC analysis of [3H]GlcNAc- and [3H]fucose-labeled glycopeptides obtained after trypsin digestion of radioactively-labeled beta F. The amino acid composition shows that cell wall beta F has 18.6% glycine.

cDNA cloning, biochemical characterization and inhibition by plant inhibitors of the alpha-amylases of the Western corn rootworm, Diabrotica virgifera virgifera.

We report the characterization and cDNA cloning of two alpha-amylase isozymes from larvae of the Western corn rootworm (Diabrotica virgifera virgifera LeConte). Larvae raised on artificial media have very low levels of amylase activity, and much higher levels are found in larvae raised on maize seedlings. At pH 5.7, the optimum pH for enzyme activity, the alpha-amylases are substantially but not completely inhibited by amylase inhibitors from the common bean (Phaseolus vulgaris) and from wheat (Triticum aestivum). Using the reverse transcriptase polymerase chain reaction (RT-PCR), we cloned two cDNAs with 83% amino acid identity that encode alpha-amylase-like polypeptides. Expression of one of the two cDNAs in insect cells with a baculovirus vector shows that this cDNA encodes an active amylase with a mobility that corresponds to that of one of the two isozymes present in larval extracts. The expressed enzyme is substantially inhibited by the same two inhibitors. We also show that expression in Arabidopsis of the cDNA that encodes the amylase inhibitor AI-1 of the common bean results in the accumulation of active inhibitor in the roots, and the results are discussed with reference to the possibility of using amylase inhibitors as a strategy to genetically engineer maize plants that are resistant to Western corn rootworm larvae.