An active maize Adhl-F gene, a Ds-induced mutant of this gene, and two independent Ac-induced revertant alleles have been isolated. The Ds mutant differs from the progenitor allele in having a 405-base pair insertion flanked by a direct repeat of 8 bp. The repeat is a duplication of the 8 bp existing at the point of insertion in the 5' untranslated region of the gene. The insertion sequence is AT-rich (A, adenine; T, thymine) and has 11-bp inverted repeat sequences at its termini. In the revertants the insertion with its inverted repeats is deleted, but the 8-bp direct repeats remain in modified form. These results establish that the 405-bp sequence is a Ds element. The Adh1 messenger RNA level is low in the Ds mutant, and it appears that new sites for transcription initiation or RNA processing or both are used. There are at least 30 sequences in the maize genome related to the Ds element.
In vivo C02 fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin (Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N2 fixation. PEP carboxylase activity was predominantly located in the bacteroid-containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO2 fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO2 fixation in mature nodules was 7.9 nmol hour-1 mg fresh weight-', similar to rates of N2 fixtion and reported values for amino acid translocation.The results suggest that the oxaloacetete used as the primary "carbon skeleton" acceptor for ammonia assimilation and amino acid synthesis in lupin nodules is provided via the PEP carboxylase reaction rather than through the tricarboxylic acid cyde. The source of PEP is presumably glycolysis, while the major source of CO2 is inferred to be respirtion.The symbiotic association which develops when legume roots are infected with an appropriate strain of Rhizobium is able to convert atmospheric N2 into ammonia. The ammonia is then assimilated into amino acids which are used for plant growth (1).The pathway for ammonia assimilation has been elucidated for lupin and other legumes (9,11,12).In lupin, asparagine is the main compound exported from the nitrogen-fixing nodule tissue, together with smaller amounts of aspartate, glutamine, glutamate, and threonine (11,13 (4), as are also the root and nodule enzymes for ammonia assimilation (9, 11-13). In addition, it has been shown that PEP carboxylase is present in the root nodules of broad bean, and that bean nodules take up CO2 (8).In this paper we present results showing that both in vitro PEP carboxylase activity and in vivo C02 fixation are found in detached lupin root nodules at rates suggesting that PEP carboxylase may be the main source of oxaloacetate for ammonia assimilation.MATERIALS AND METHODS Lupinus angustifolius L. cv. Bitter Blue, inoculated with Rhizobium strain NZP 2257, was grown and harvested as previously described (14).For each experiment, detached nodules from at least three plants were mixed and divided into a sufficient number of samples of 50 to 200 mg fresh wt for separate duplicate measurements of acetylene reduction and PEP carboxylase activity and quadruplicate measurements of CO2 fixation.Acetylene reduction activity was assayed as before (14). In vivo C02 fixation was measured by incubating detached nodules at 25 C in a 25-ml Erlenmeyer flask containing a small piece of moist filter paper. The assay was initiated by the injection of 100 ,ll of 14CO2 (Radiochemical Centre, Amersham; specific radioactivity 5-10 Ci/mol) ...
Labeling studies using detached lupin (Lupinus angustifofius) nodules showed that over times of less than 3 minutes, label from 13,4-'4Clglucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with 11-14C1-and 16-"4Cjglucose, while with IU-_4C1-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the "carbon skeletons" for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction.A comparson of 14cO2 release from nodules supplied with 11-14C1-and 16-14Cjglucose indicated that the oxidative pentose phosphate pathway accounted for less than 6% of glucose metabolism. Several enzymes of the oxidative pentose phosphate and glycolytic pathways were assayed in vitro using the 12,000g supernatant fraction from nodule homogenates. In all cases, the specific activities were adequate to account for the calculated in vio fluxes.Three out of four diverse treatments that inhibited nodule nitrogen fixation also inhibited nodule CO2 fiXation, and in the ease of the fourth treatment, replacement of N2 with He, it was shown that the normal entry of label from exogenous 14CO2 into the nodule amino acid pool was strongly inhibited.In lupin nodules, N2 fixation and asparagine synthesis proceed in accordance with the over-all stoichiometric relationship: ATP,NADPH N2 + oxaloacetate -' asparagine (7, 10) In a previous paper (3) we suggested that the oxaloacetate needed for asparagine synthesis is provided by the PEP' carboxylase reaction, and we showed that the in vivo CO2 fixation activity and in vitro PEP carboxylase activity of lupin nodules were sufficient to provide oxaloacetate at the rates required (3).In this paper we report further evidence supporting the PEP carboxylase scheme, based on the short term labeling patterns of detached nodules supplied with "C-labeled glucose. In addition, we show several treatments that inhibit N2 fixation by detached nodules also inhibit CO2 fixation and the incorporation of exogenous "CO2 into nodule amino acids.
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