Expression and Localization of Plant Protein Disulfide Isomerase.

A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules.

Molecular characterization and expression of an alfalfa protein with sequence similarity to mammalian ERp72, a glucose-regulated endoplasmic reticulum protein containing active site sequences of protein disulphide isomerase.

A complementary DNA clone (G1) containing sequence similarity to the mammalian lumenal endoplasmic reticulum protein ERp72 was isolated from an alfalfa (Medicago sativa L.) cDNA library by screening with a cDNA encoding human protein disulphide isomerase (PDI), which contains two thioredoxin-like active site regions which are highly conserved in ERp72. The polypeptide encoded by G1 consists of 364 amino acids, possesses a putative N-terminal secretory signal sequence and two regions, 113 amino acids apart, identical to the active sites of PDI and ERp72. G1 appears to be encoded by a small gene family in alfalfa, whose transcripts are constitutively expressed in all major organs of the plant. In alfalfa cell suspension cultures, G1 transcripts were markedly induced by treatment with tunicamycin, but not in response to calcium ionophore, heat shock or fungal elicitor. A similar expression pattern was observed for transcripts encoded by B2, a recently cloned alfalfa cDNA with strong sequence similarity to PDI. We discuss potential roles of plant proteins resembling vertebrate PDI and ERp72.

Molecular characterization and expression of an isocitrate dehydrogenase from alfalfa (Medicago sativa L).

A putative isocitrate dehydrogenase (IDH) cDNA from alfalfa has been cloned and sequenced. The derived amino acid sequence of 433 residues contains the isocitrate and isopropylmalate dehydrogenase signatures, is 63% identical to yeast mitochondrial NADP-IDH and shares high sequence identity with peptides of pig heart NADP-IDH. The sequence contains a potential N-terminal leader with similarities to a thylakoid transit peptide. IDH transcripts and NADP-IDH activity were detected in all alfalfa tissues examined, their levels depending upon the tissue type and its developmental stage. Transcripts and enzymatic activity were not induced on exposure of cell suspension cultures to a fungal elicitor. IDH is encoded by a small gene family in alfalfa.

Molecular cloning of a putative plant endomembrane protein resembling vertebrate protein disulfide-isomerase and a phosphatidylinositol-specific phospholipase C.

cDNA clones containing sequence similarity to the multifunctional vertebrate protein disulfide-isomerase (PDI, EC 5.3.4.1) were isolated from an alfalfa (Medicago sativa L.) cDNA library by screening with a cDNA sequence encoding human PDI. The polypeptide encoded by a clone designated B2 consisted of 512 amino acids and was characterized by a 24-amino acid hydrophobic leader sequence, two regions with absolute identity to the vertebrate PDI active site (Ala-Pro-Trp-Cys-Gly-His-Cys-Lys), and a C-terminal endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu). The overall identity of the B2 sequence to that of human PDI was 35% at the amino acid level (79% when conservative substitutions were included) and 39% at the nucleotide level; this included homology between B2 and the region of human PDI believed to be involved in binding estrogens. The deduced amino acid sequence of B2 was also 35% identical to that of a rat form I phosphatidylinositol-specific phospholipase C. Lysates from Escherichia coli cells harboring an expression plasmid bearing the B2 sequence contained significantly elevated levels of PDI activity. Southern analysis indicated the presence of a small PDI-related gene family in alfalfa, of which B2 appeared to correspond to a single gene. An approximately 2-kilobase B2 transcript was expressed in all alfalfa organs tested. In alfalfa cell suspension cultures, B2 transcripts were strongly induced by tunicamycin but not by exposure to fungal elicitor.

Isolation and characterization of an Arabidopsis biotin carboxylase gene and its promoter.

In the plastids of most plants, acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a multisubunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protien (BCCP), and carboxytransferase (alpha-CT, beta-CT) subunits. To better understand the regulation of this enzyme, we have isolated and sequenced a BC genomic clone from Arabidopsis and partially characterized its promoter. Fifteen introns were identified. The deduced amino acid sequence of the mature BC protein is highly conserved between Arabidopsis and tobacco (92.6% identity). BC expression was evaluated using northern blots and BC/GUS fusion constructs in transgenic Arabidopsis. GUS activity in the BC/GUS transgenics as well as transcript level of the native gene were both found to be higher in silique and flower than in root and leaf. Analysis of tobacco suspension cells transformed with truncated BC promoter/GUS gene fusions indicated the region from -140 to +147 contained necessary promoter elements which supported basal gene expression. A positive regulatory region was found to be located between -2100 and -140, whereas a negative element was possibly located in the first intron. In addition, several conserved regulatory elements were identified in the BC promoter. Surprisingly, although BC is a low-abundance protein, the expression of BC/GUS fusion constructs was similar to 35S/GUS constructs.

Structure and expression of an Arabidopsis acetyl-coenzyme A carboxylase gene.

Acetyl-coenzyme A carboxylase (ACCase) catalyzes the formation of malonyl-coenzyme A, which is used in the plastid for fatty acid synthesis and in the cytosol for several pathways including fatty acid elongation and flavonoid synthesis. Two overlapping Arabidopsis genomic clones were isolated and sequenced to determine the entire ACCase-coding region. Thirty introns with an average size of 94 bp were identified by comparison with an alfalfa ACCase cDNA sequence. The 10-kb Arabidopsis ACCase gene encodes a 251-kD polypeptide, which has 80% amino acid sequence identity with alfalfa ACCase and about 40% identity with ACCase of rat, chicken, yeast, and the diatom Cyclotella. No chloroplast transit peptide sequence was observed, suggesting that this Arabidopsis gene encodes a cytosolic ACCase isozyme. ACCase gene transcripts were detected by RNase protection assays in Arabidopsis root, leaf, silique, and seed. Genomic DNA blot analysis revealed the presence of a second related Arabidopsis ACCase gene.

Molecular cloning, characterization, and elicitation of acetyl-CoA carboxylase from alfalfa.

Acetyl-CoA carboxylase [ACCase; acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] catalyzes the ATP-dependent carboxylation of acetyl CoA to produce malonyl CoA. In plants, malonyl CoA is needed for plastid localized fatty acid biosynthesis and for a variety of pathways in the cytoplasm including flavonoid biosynthesis. We have determined the full nucleotide sequence of an ACCase from alfalfa, which appears to represent a cytoplasmic isozyme. Partial cDNAs were isolated from a cDNA library of suspension culture cells that had been elicited for isoflavonoid phytoalexin synthesis. The full-length sequence was obtained by primer extension and amplification of the cDNA with synthetic primers. The sequence codes for a protein of 2257 amino acids with a calculated M(r) of 252,039. The biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase domains, respectively, show approximately 72%, 50%, and 65% sequence similarity to those of animal, diatom, and yeast ACCase sequences. ACCase enzyme activity and transcripts are induced severalfold upon addition of yeast or fungal elicitors to alfalfa cell cultures.

Stress responses in alfalfa (Medicago sativa L.) XIX. Transcriptional activation of oxidative pentose phosphate pathway genes at the onset of the isoflavonoid phytoalexin response.

We have isolated cDNA clones encoding the pentose phosphate pathway enzymes 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from alfalfa (Medicago sativa L.). These exhibit extensive nucleotide and amino acid sequence similarity to the corresponding genes from bacteria, Drosophila and mammals. Transcripts encoding both enzymes are expressed at high levels in roots and nodules. Exposure of alfalfa suspension cells to an elicitor from yeast cell walls results in co-ordinated increases in transcription rates for both genes, followed by increased steady state transcript levels but only slightly increased extractable enzyme activities, at the onset of accumulation of isoflavonoid phytoalexins. Levels of NADPH and NADP remain relatively constant in alfalfa cells following elicitation. The rapid transcriptional activation of 6PGDH and G6PDH does not therefore appear to be a response to altered pyridine nucleotide redox state. These genes appear to respond to early events in elicitor-mediated signalling rather than to subsequent elicitor-induced changes in secondary metabolism. Hydrogen peroxide, a potential signal for elicitation of anti-oxidative genes in biologically stressed plant cells, did not induce 6PGDH or G6PDH transcripts or enzymatic activity.

The pea chloroplast membrane-associated protein, IEP96, is a subunit of acetyl-CoA carboxylase.

Two forms of acetyl-CoA carboxylase (ACCase) have been characterized in pea (Pisum sativum L.) leaves; a heteromeric chloroplast enzyme and a homomeric, presumably cytosolic enzyme. The biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and beta-carboxyltransferase (CT) subunits of the plastidial-ACCase have recently been characterized and cloned. To further characterize the carboxyltransferase, an improved assay for CT was developed and used to follow its partial purification. CT activity co-purifies with ACCase activity during gel permeation chromatography. However, upon anion-exchange chromatography or native PAGE, CT separates from the BC and BCCP subunits of plastidial-ACCase and ACCase activity is lost. In addition, it is demonstrated that a previously sequenced pea chloroplast cDNA of unknown function (IEP96) with a predicted molecular weight of 91 kDa encodes the alpha-CT subunit of the MS-ACCase. Antibodies raised against the first 404 amino acids of IEP96 protein detected a polypeptide with molecular weight of 91 kDa that co-eluted during gel permeation chromatography with plastidial CT and ACCase activities. These antibodies also immunoprecipitated the activities of both ACCase and CT with the concomitant precipitation of the beta-CT subunit. Furthermore, antibodies against beta-CT immunoprecipitated the IEP96 protein. Two-dimensional PAGE and DEAE purification of ACCase protein demonstrated that the beta-CT forms a tight association with the IEP96 protein. Pea leaf was fractionated into soluble and membrane fractions and the alpha-CT subunit was primarily associated with the membrane fraction. Together, these data demonstrate that IEP96 is the alpha-CT subunit of pea chloroplast ACCase.

Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves.

A novel cereal storage protein: molecular genetics of the 19 kDa globulin of rice.

A lambda gt11 cDNA library, constructed from poly(A)+ RNA isolated from immature rice seed endosperm, was screened with affinity-purified antibodies against the rice storage protein called alpha-globulin (previously), or the 19 kDa globulin (our term). A positive clone was isolated and sequenced and shown to encode a 21 kDa precursor for the 19 kDa globulin, based on the identity of portions of the inferred amino acid sequence and the sequence of three cyanogen bromide peptides of the 19 kDa globulin. Analysis of genomic DNA by Southern blotting using the cDNA clone probe revealed one hybridizing band in Eco RI, Hind III, and Bam HI digests. This strongly suggests that the 19 kDa globulin is encoded by a single-copy gene. Because of its single-copy nature and its abundance of Arg and lack of Lys, the 19 kDa rice globulin appears to be a particularly attractive target for genetically engineering increased Lys content in rice seeds.