Basil S. Shorrosh

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.

Antisense expression and overexpression of biotin carboxylase in tobacco leaves

The plastid acetyl-coenzyme A carboxylase (ACCase) catalyzes the first committed step of fatty acid synthesis and in most plants is present as a heteromeric complex of at least four different protein subunits: the biotin carboxylase (BC), the biotin carboxyl carrier protein, and the [alpha] and [beta] subunits of the carboxyltransferase. To gain insight into the subunit organization of this heteromeric enzyme complex and to further evaluate the role of ACCase in regulating fatty acid synthesis, BC expression was altered in transgenic plants. Tobacco (Nicotiana tabacum) was transformed with antisense-expression and overexpression tobacco BC constructs, which resulted in the generation of plants with BC levels ranging from 20 to 500% of wild-type levels. Tobacco plants containing elevated or moderate decreases in leaf BC were phenotypically indistinguishable from wild-type plants. However, plants with less than 25% of wild-type BC levels showed severely retarded growth when grown under low-light conditions and a 26% lower leaf fatty acid content than wild-type plants. A comparison of leaf BC and biotin carboxyl carrier protein levels in plants with elevated and decreased BC expression revealed that these two subunits of the plastid ACCase are not maintained in a strict stoichiometric ratio.

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.

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

A λgt11 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 α-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 inEco RI,Hind III, andBam 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.

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.

Sequence analysis and developmental expression of an alfalfa protein disulfide isomerase

Protein disulfide isomerase (PDI) aids in the folding of newly synthesized proteins in the lumen of the endoplasmic reticulum by catalyzing correct disulfide bond formation [ 1 ]. Vertebrate PDI is also the/%subunit of prolyl 4-hydroxylase [2] and a component of the microsomal triglyceride transfer protein complex [3]. To initiate a study of the structure and function(s) of PDI in plants, we screened a cDNA library constructed from poly (A) RNA isolated from alfalfa (Medicago sativa L.) cell suspension cultures, with a 546 bp fragment from a human PDI cDNA clone. Three (B2, L1 and G1) out of 30 positive clones were further characterized by full sequence analysis. All three contained the thioredoxin-like putative catalytic sites characteristic of vertebrate PDI. The protein encoded by the G1 cDNA was found to be similar to the vertebrate endomembrane protein ERp72, a glucose-regulated protein [4]. The protein encoded by the B2 cDNA shared sequence similarity with the G1 protein, but was much more similar to vertebrate PDI, and exhibited PDI activity on expression in Escherichia coli [5]. We here report the nucleotide and deduced amino acid sequences of the L1 cDNA (Fig. 2). The molecular weight of the encoded L1 protein Fig. 2. Developmental expression of B2/L1 mRNA(s) in alfalfa plant organs. Total RNA (10 #g) was resolved by electrophoresis on formaldehyde gels, transferred to nitrocellulose and probed with the full-length 1.8 kb cDNA insert of the B2 clone. Numbers 1 through 6 are approximately equal to the age of the plant in weeks. For number 7, the plants were approximately 9 weeks old. The blot was also probed with an alfalfa 25S rRNA cDNA [6], which detects an approximately 3.4 kb transcript, as a control for loading and transfer efficiency.