The dnaJ (hsp40) locus in Rhizobium leguminosarum bv. phaseoli is required for the establishment of an effective symbiosis with Phaseolus vulgaris.

P121R25 is a Tn5-induced mutant of the effective Rhizobium leguminosarum bv. phaseoli strain P121R that is unable to use glutamate as the sole carbon and nitrogen source and is defective in symbiotic nitrogen fixation. Enzymatic analysis showed that three enzymes implicated in glutamate metabolism (glutamate dehydrogenase, 2-oxoglutarate dehydrogenase, and glutamate synthase) were affected by this mutation. Sequencing of the chromosomal locus bordering the Tn5 in P121R25 indicated the presence of the dnaK and dnaJ genes in an arrangement similar to that described in R. leguminosarum bv. viciae (GenBank accession number Y14649). The mutation was located in the dnaJ (hsp40) gene.

Production of a diagnostic monoclonal antibody in perennial alfalfa plants.

The increasing use of monoclonal antibodies (mAbs) in diagnostic reagents necessitates efficient and cost-effective mAb production methods. In blood banks, one of the most routinely used reagents is the anti-human IgG reagent used for the detection of non-agglutinating antibodies. Here we report the production of a functional, purified anti-human IgG, through the expression of its encoding genes in perennial transgenic alfalfa. Transgenic plants expressing the light- and heavy-chain encoding mRNAs were obtained, and plants from crosses were found to express fully assembled C5-1. The purification procedure yielded mainly the H2L2 form with specificity and affinity identical to those of hybridoma-derived C5-1. The ability to accumulate the antibody was maintained both in parental F1 lines during repeated harvesting and in clonal material; the antibody was stable in the drying hay as in extracts made in pure water. Also, plant and hybridoma-derived C5-1 had similar in vivo half-lives in mice. These results indicate that plant C5-1 could be used in a diagnostic reagent as effectively as hybridoma-derived C5-1, and demonstrates the usefulness of perennial systems for the cost-effective, stable, and reliable production of large amounts of mAbs.

An alfalfa rubisco small subunit homologue shares cis-acting elements with the regulatory sequences of the RbcS-3A gene from pea.

A genomic clone of RbcS was isolated from an alfalfa (Medicago sativa L. cv. Apica) genomic library and characterized. Although this clone has structural features similar to a functional gene, the second exon is interrupted by a stop codon and thus is not fully translatable in the plant. Sequence analysis of the 5' and 3' noncoding regions of RbcSK-1A showed a high sequence homology to the flanking sequences of the RbcS-3A gene from pea. The regions of homology contain many important cis-regulatory elements shown to be essential for regulation of the RbcS-3A gene in pea. The promoter of this alfalfa rubisco clone was used in a translational fusion to test its ability to control the expression of the GUS reporter gene in an homologous nuclear background. High levels of GUS enzyme activity were recorded. These strong levels are comparable to some exceptionally high levels produced in other studies following the use of photosynthesis gene promoters in fusions with the GUS reporter gene.

Differential accumulation of two glycine-rich proteins during cold-acclimation alfalfa.

Two mRNAs, MsaCiA and MsaCiB, encoding for proteins harboring glycine-rich motifs, accumulate in alfalfa during cold acclimation. Fusion polypeptides containing the amino acid sequences deduced from these mRNAs were produced in Escherichia coli and used to raise antibodies. Each antibody cross-reacted specifically with soluble polypeptides, MSACIA-32 and MSACIB, respectively. These polypeptides were detectable only in crowns of cold-acclimated plants, even though MsaCiA mRNA accumulated in both crows and leaves during cold acclimation. The analysis of parietal proteins showed that several MSACIA-related proteins, with a molecular mass of 32, 41 and 68 kDa, did accumulate in leaf cell walls and one of 59 kDa crown cell walls. This diversity is most probably due to a tissue-specific maturation of MSACIA. A discrepancy was found between the time-course of accumulation of MSACIB and the one of the corresponding transcript. These results indicate that timing and localization of MSACIA and MSACIB expression are different, and suggest that this differential expression involves both transcriptional and post-transcriptional events. Comparisons made among six cultivars of contrasting freezing tolerance suggest that low tolerance could be explained by failure to accumulate proteins like MSACIA and MSACIB at a sufficient level.

A cold-induced gene from Medicago sativa encodes a bimodular protein similar to developmentally regulated proteins.

A new cold-regulated (COR) gene, msa CIC, was isolated by differential screening of a cDNA library from cold-acclimated crowns of alfalfa (Medicago sativa L. cv. Apica). Transcripts of msa CIC were not detectable in unacclimated alfalfa and accumulated to higher levels in cold-acclimated plants of the cold-tolerant cv. Apica than in those of the cold-sensitive cv. CUF-101. The DNA sequence analysis of a full-length cDNA clone revealed that msa CIC encodes for a putative protein (MSACIC) of 166 amino acids with distinct proline-rich and hydrophobic domains. Protein sequence comparisons indicated that MSACIC is similar to a group of bimodular proteins that are developmentally regulated in other plant species.

A new cold-induced alfalfa gene is associated with enhanced hardening at subzero temperature.

When alfalfa (Medicago sativa L. cv Apica) plants grown at room temperature are transferred to 2 degrees C, the temperature at which 50% of the plants fail to survive (LT50) decreases from -6 to -14 degrees C during the first 2 weeks but then increases to -9 degrees C during the subsequent 2 weeks. However, when plants are kept for 2 weeks at 2 degrees C and then transferred to -2 degrees C for another two weeks, the LT50 declines to -16 degrees C. These changes in freezing tolerance are paralleled by changes in transcript levels of cas15 (cold acclimation-specific gene encoding a 14.5-kD protein), a cold-induced gene. Cold-activation of cas15 occurs even when protein synthesis is inhibited by more than 90%, suggesting that cold-initiated events up to and including the accumulation of cas15 transcripts depend on preexisting gene products. cas15 shows little homology to any known gene at the nucleotide or amino acid level. The deduced polypeptide (CAS15) of 14.5 kD contains four repeats of a decapeptide motif and possesses a bipartite sequence domain at the carboxy terminus with homology to the reported nuclear-targeting signal sequences. Although the relative amount of cas15 DNA as a fraction of the total genomic DNA is similar in cultivars with different degrees of freezing tolerance, its organization in the genome is different. The possible role of cas15 in the development of cold-induced freezing tolerance is discussed.

Modification of Phospholipid Catabolism in Microsomal Membranes of [gamma]-Irradiated Cauliflower (Brassica oleracea L.).

Acceleration of membrane deterioration has been observed recently during storage of [gamma]-irradiated cauliflower (Brassica oleracea L., Botrytis group). In the present study, the activity of microsome-associated lipolytic enzymes was investigated in cauliflower florets exposed to 0 or 4 kilograys of [gamma] radiation and stored for 8 d at 13[deg]C. Radiolabeled breakdown products obtained from the metabolism of (16:0/18:2*)-phosphatidylcholine and (16:0/16:0)-phosphatidyl-[N-methyl-3H]choline by microsomal membranes indicated that phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase were associated with the membranes. The rate of phosphatidylcholine catabolism by the membranes increased slowly in control cauliflower during storage. [gamma] irradiation caused an immediate rise in phosphatidylcholine catabolism that remained higher than that of the controls during subsequent storage. Collectively, the data suggest that enhancement of membrane lipolytic activity results from free-radical-induced stress. Rapid increase of the membrane-associated phospholipase D activity may be a key event leading to accelerated membrane deterioration following [gamma] irradiation.

Absorption and assimilation of nitrate and ammonium ions by jack pine seedlings.

Jack pine (Pinus banksiana Lamb.) seedlings were grown in a shaded or unshaded light regime with either NO(3) (-)- or NH(4) (+)-N as the sole N source. After three months, seedlings grown with NH(4) (+)-N were larger than seedlings grown with NO(3) (-)-N. Irradiance had a greater effect on growth of ammonium-fed seedlings than on growth of nitrate-fed seedlings.At all times from 6 to 24 h following incorporation of (15)N, soluble, insoluble, and total (15)N contents of shoots and roots were higher in ammonium-fed seedlings than in nitrate-fed seedlings. The pattern of (15)N accumulation in shoots was similar to that in roots. After 6 and 24 h of (15)N incorporation, unshaded, ammonium-fed seedlings had 8.8 and 2.8 times greater total (15)N contents, respectively, than unshaded, nitrate-fed seedlings. In response to shading, ammonium-fed seedlings increased their total uptake of (15)N per unit root weight, whereas nitrate-fed seedlings did not. No nitrate or (15)NO(3) (-) was detected in any plant tissue. Nitrate-fed plants had higher NH(4) (+), Asp, and Gln concentrations in needles and higher gamma-aminobutyric acid and Arg concentrations in stems. Accumulation of (15)N in roots was not affected by the pH of the (15)N solution or by the N source fed to the seedlings before the period of (15)N incorporation. Thus NO(3) (-) transport into roots, rather than its reduction or transport within the plant, seems to be the factor limiting the growth of jack pine supplied with NO(3) (-)-N as the sole N source.

Induction of Senescence-Like Deterioration of Microsomal Membranes from Cauliflower by Free Radicals Generated during Gamma Irradiation.

Membrane deterioration differs in aging and senescent tissues. Involvement of free radicals in the process is generally recognized. Little is known about the physiological effects of gamma irradiation on plant tissues. Degradation of microsomal membranes by the action of free radicals, generated in vivo by gamma rays, was investigated. Cauliflower florets (Brassica oleracea L., Botrytis group) were exposed to 2 or 4 kiloGray of gamma radiation. Membrane deterioration was assessed during 8-day storage at 13 degrees C. Some senescence was indicated in nonirradiated controls by a parallel depletion of lipid phosphate and protein. Irradiation caused an immediate increase in tissue electrolyte leakage and a small increase in the free fatty acid content of membranes. In irradiated samples, leakage of electrolytes and the ratios of sterol to phospholipid and of free fatty acid to phospholipid increased with storage. During this period, membrane protein was progressively lost and the lipid phosphate-to-protein ratio increased markedly. Polyunsaturated fatty acids were selectively depleted from the free fatty acid fraction for all treatments, suggesting lipoxygenase activity. No change in lipid saturation was observed in the polar lipid fraction. The results suggest an enzyme-catalyzed senescence-like membrane deterioration, probably induced by chemical deesterification of phospholipids by free radicals generated during irradiation.

Purification and Properties of Glutamine Synthetase in Leaves and Roots of Pinus banksiana Lamb.

A method is described for the purification of glutamine synthetase (GS; EC. 6.3.1.2) from the leaves and roots of Pinus banksiana Lamb., a conifer which utilizes ammonium as its primary nitrogen source. The enzyme was purified to apparent homogeneity by a procedure involving salt fractionation as well as ion-exchange, size exclusion, and affinity chromatography. Since the final preparation produced two bands on SDS polyacryamide gels but only one band on a nondenaturating gel, it is concluded that the two subunits (44 and 40 kilodaltons, respectively) are part of a single enzymatic protein which shows GS activity. The pH optimum for leaf GS ranged between 6.2 and 6.5, one pH unit lower than the values reported for higher plants which utilize primarily nitrate nitrogen. Magnesium requirements for GS in P. banksiana were different for leaves and roots, showing V(max)/2 values of 2.5 and 8 millimolar, respectively at 5 millimolar ATP. Furthermore, K(m) values for ammonium were higher for the enzyme in leaves (33.1 micromolar) than in roots (19.2 micromolar). K(m) values for ATP and for glutamate, on the other hand, were similar for the two tissues. A polyclonal antibody was produced against the purified leaf GS. Western blots of leaf homogenates produced two bands, the lighter one being more abundant. The same pattern was found when immunodetection was performed using an anti GS IgG produced against purified GS from Phaseolus nodules thus indicating common antigenic determinants. At least 30% of total GS was recovered in a plastid-fraction of dark-grown calli produced from the basal part of P. banksiana hypocotyls.

Tissue and Cellular Distribution of Glutamine Synthetase in Roots of Pea (Pisum sativum) Seedlings.

The effect of nitrate application on glutamine synthetase activity in roots of pea (Pisum sativum L.) seedlings (2 weeks old) was studied. Separation of organelles from root fragments by sucrose density-gradient centrifugation revealed that both nitrite reductase and glutamine synthetase activities increased in root plastids as a response to nitrate application and that no such response was induced by ammonium application. Glutamine synthetase activity was also found to increase in plastids with distance from apex in nitrate-treated plants, the highest specific activity being located in the fourth 1-centimeter segment. Separation by SDS-PAGE and characterization by Western blotting showed that cytosolic glutamine synthetase contains one subunit polypeptide (28 kilodaltons) and that plastid glutamine synthetase contains both the 38-kilodalton subunit and a heavier subunit. When nitrate was present in the nutrient solution, the heavier subunit increased in abundance in protein fractions obtained from purified root plastids.

Nitrate content, amino acid composition and growth of yellow birch seedlings in response to light and nitrogen source.

Yellow birch (Betula alleghaniensis Britt.) seedlings were grown for three months in a greenhouse at two radiant flux densities-full light (FL) and 50% shade (LL)-and with three nitrogen sources- ammonium only (NH(4) (+)), nitrate only (NO(3) (-)) and a 1:1 mixture of ammonium and nitrate (NH(4) (+)/NO(3) (-))-in a completely randomized factorial design. The total biomass of seedlings grown under low light (LL) did not vary significantly with nitrogen source; although NO(3) (-)-treated seedlings were smaller and had a significantly lower (P

The activity, characterization and distribution of the nitrogen assimilation enzyme, glutamine synthetase, in jack pine seedlings.

The extraction of glutamine synthetase (GS) from jack pine (Pinus banksiana Lamb.) tissue was facilitated by solubilization of the tissue with 1% or more Nonidet P-40 detergent. In contrast with procedures commonly used to extract GS from other plant tissues, highest recovery of GS was obtained when jack pine tissues were subjected to ultrasonic homogenization in the absence of PVP. Chromatography on DEAE-Sephacel showed that jack pine needles possess two isoforms of GS. Isoform GS(1), which is generally associated with the cytoplasm, eluted at 90 mM KCl and accounted for 80% of total GS activity. Isoform GS(2), which eluted at 280 mM KCl, is generally associated with the chloroplast and is thought to be active in the primary assimilation of ammonium in leaves. Thus GS(2) activity may be important if conifers are to avoid ammonium toxicity under circumstances, such as exposure to nitrous oxides, where nitrate reductase activity is induced in needles. In June, 72% of total GS activity was located in needles. Near the end of the growing season in August, however, only 1% of total GS activity was found in needles whereas 79% was found in roots.

Isoenzymes of Glutamine Synthetase in Roots of Pea (Pisum sativum L. cv Little Marvel) and Alfalfa (Medicago media Pers. cv Saranac).

Cell organelles have been isolated from protoplast lysates and total homogenates obtained from root tips of Pisum sativum L. (cv Little Marvel) and Medicago media Pers. (cv Saranac) grown in hydroponics with nitrate nutrient solutions. Density-gradient and differential centrifugation procedures have been used to prepare mitochondria-and plastid-enriched fractions in which glutamine synthetase (GS) activity was estimated. Even when purified protoplasts were gently ruptured, significant breakage of plastids occurred during preparation as shown by the high proportion of nitrite reductase recovered in the soluble fraction. Of the total GS activity recovered, up to 20% was associated with the plastid fraction, depending on the source of plant material and the GS assay utilized; when corrected for recovery of the plastid marker nitrite reductase, it was calculated that 15 to 57% of alfalfa and 14 to 64% of pea root GS was located in the plastids. A true biosynthetic assay in which glutamine production was monitored by high performance liquid chromatography was devised to estimate the physiological significance of the transferase and the semibiosynthetic assays currently used for activity measurements. When compared with the true and semibiosynthetic assays, the transferase assay for GS appeared to underestimate the root plastid enzyme. Root plastid GS was partially purified by ion-exchange chromatography, and results show that the isoenzyme found in root plastids is different from chloroplastic or cytosolic GS.