Robert G. Gregerson

Primary assimilation of nitrogen in alfalfa nodules: molecular features of the enzymes involved

Abstract The primary assimilation of symbiotically fixed nitrogen (N) in alfalfa root nodules involves complex intermingling with carbon (C) metabolism. Integrated functioning of both cytosolic and organelle-associated enzymes is required to link N assimilation with C metabolism. Understanding how N and C metabolism are controlled in root nodules requires fundamental knowledge of how the plant genes involved are regulated. While significant progress has been made in understanding the regulation of glutamine synthetase (GS), much less is known about the genes controlling other enzymatic steps in this process. To that end we have isolated, purified and characterized the root nodules enzymes aspartate aminotransferase (AAT), phosphoenolpyruvate carboxylase (PEPC) and glutamate synthase (NADH-GOGAT). Moreover the cDNAs encoding these crucial enzymes were isolated and characterized. While the most prominent forms of GS associated with N assimilation in nodules are located in the cytosol, AAT and NADH-GOGAT appears to be organelle-associated. The deduced amino acid sequence suggested and immunogold labeling showed that nodule-enhanced AAT-2 is located in amyloplasts. Comparison of the deduced amino acid sequence of nodule-enhanced NADH-GOGAT to the N-terminal sequence of the processed protein indicated that NADH-GOGAT has a 101 amino acid presequence. However, it is unclear as to which organelle ADH-GOGAT is targeted. Cytosolic phosphoenolpyruvate carboxylase (PEPC), which can be expressed in legume root nodules at levels comparable to those detected in leaves of C4 plants, provides a substantial amount of carbon for malate, aspartate and asparagine biosyntheses. RNA blots showed that GS, AAT, PEPC, and NADH-GOGAT mRNAs were enhanced about 15-fold during the development of effective alfalfa nodules. By comparison, the expression of GS, AAT and PEPC mRNAs was reduced by 65% in ineffective nodules. NADH-GOGAT was different from GS, AAT, and PEPC in that expression had an absolute requirement for a factor(s) related to effective nodules. The data suggest that NADH-GOGAT plays a key role in regulating N assimilation. Moreover, plastids in nodules play a major role not only in C metabolism but also in N metabolism.

Aspartate Aminotransferase in Alfalfa Nodules: Localization of mRNA During Effective and Ineffective Nodule Development and Promoter Analysis

Aspartate aminotransferase (AAT) plays a critical role in the assimilation of symbiotically fixed nitrogen into aspartate and asparagine in legume root nodules. The enzyme occurs as a cytosolic form (AAT1) and a plastid form (AAT2) in alfalfa nodules. To elucidate the functional role of each isozyme in root nodule metabolism further, in situ hybridization was used to determine the pattern of transcript accumulation from the two genes. AAT2 transcripts were localized to infected cells throughout the symbiotic zone of effective alfalfa nodules; however, expression was reduced in ineffective nodules. The AAT1 gene was expressed in the uninfected cells of the invasion zone and symbiotic zone, the nodule parenchyma, and nodule vascular bundles of both effective and ineffective nodules. The AAT1 and AAT2 promoters were evaluated in transgenic alfalfa plants containing promoter β-glucuronidase (GUS) gene fusions. Histochemical staining patterns agreed with results from in situ localization. The distribution patt...

Cloning and Developmental Expression of a Nodule-Enhanced Sucrose Synthase cDNA from Alfalfa

The carbon (C) cost for symbiotic nitrogen (N) fixation is quite high. The ultimate source of C for N2 fixation is sucrose derived from leaves. For use in root nodules, sucrose must be cleaved to glucose and other smaller carbon compounds. Here we report the isolation and characterization of a full length cDNA encoding the enzyme sucrose synthase (SS; EC 2.4.1.13). This SS cDNA shows greatest expression in effective nitrogen-fixing nodules. It is also expressed in stems and roots with little expression in leaves and cotyledons. While maximum expression of SS in nodules appears to require active nitrogenase, the gene is also expressed in ineffective nodules, albeit at reduced levels. Measurement of starch concentrations in nodules shows that SS expression is not coupled to starch biosynthesis. In situ hybridization studies show that expression of SS occurs in both infected and uninfected cells and in the nodule meristem.

A Unique Textbook for Teaching Courses in Bioinformatics [review of Discovering Genomics, Proteomics, and Bioinformatics, 2nd ed. (Cummings, B.; 2006)]

Discovering Genomics, Proteomics, and Bioinformatics, 2nd ed., by A.M. Campbell and L.J. Heyer. Published by Benjamin cummings, 2006, ISBN: 978-0805382198, 464 pages.