Immunological studies of glutamine synthetase in Frankia-Alnus incana symbioses

Abstract

We have investigated the presence and form of glutamine synthetase (GS) in Frankia vesicle cluster preparations of two actively nitrogen‐fixing Frankia‐Alnus incana root‐nodule symbioses and in cultured Frankia sp. strain CpI1 (HFP070101). The symbioses contained Frankia CpI1 or the local source of Frankia. We used Western‐blot analysis with antisera raised against three types of GS. In symbiotic Frankia GS protein was not detected at a significant level when either antisera against Rhodospirillum rubrum GS or antisera against Rhizobium meliloti GSII were used. In cultured Frankia CpI1 GSI was detected both when grown with NH4+ or N2 as nitrogen source, and GSII was detected when grown on N2. Antiserum raised against the nodule‐specific GSn1 of Phaseolus vulgaris crossreacted with a 43‐kDa polypeptide corresponding to plant GS in root‐nodule extracts from Alnus, and with a 41‐kDa polypeptide corresponding to GSII in cultured Frankia CpI1 grown on N2. We conclude that both GSI and GSII are repressed in symbiotic Frankia and that NH4+ produced through nitrogen fixation is assimilated by the plant in Frankia‐Alnus incana symbioses. It thus appears that vesicle formation, synthesis of nitrogenase and synthesis of GS are separately regulated in symbiotic Frankia and that the plant has to supply symbiotic Frankia with organic nitrogen in some form in addition to the carbon.

Primary metabolism in N2-fixing Alnus incana?Frankia symbiotic root nodules studied with 15N and 31P nuclear magnetic resonance spectroscopy

“…These primary metabolic steps are most likely localized to the plant part of the nodule. An initial labeling of Gln and a-amines within Frankia seems unlikely because neither of the two different forms of GS (Schultz and Benson 1990) appears to be expressed in Frankia during symbiosis (Lundquist and Huss-Danell 1992) and no GS activity was detected in symbiotic vesicles (Blom et al 1981). Expression of GS was localized to the plant cells harboring Frankia in A. glutinosa root nodules (Guan et al 1996).…”

“…The decrease in nitrogenase activity was avoided by reducing the concentration of O 2 in the atmosphere, which suggests that nitrogen assimilation could be involved in maintaining a stable O 2 environment for the highly O 2 -labile nitrogenase. The primary assimilation of NH 4 + into amino acids appears to be confined to the plant cell surrounding Frankia since neither of Frankia's two forms of GS (Schultz and Benson 1990) are expressed in the symbiotic stage in Alnus root nodules (Lundquist and Huss-Danell 1992). To what extent nitrogenase activity in Frankia is dependent on a continuous supply of carbon from the host, and whether this is intimately linked to the production of NH 4 + is not clear.…”

Novel Expression Pattern of Cytosolic Gln Synthetase in Nitrogen-Fixing Root Nodules of the Actinorhizal Host, Datisca glomerata

“…Stem exudates from well-watered plants were collected early in the morning by microcentrifugation of small-diameter cut stems at 3,000 rpm for 5 min followed by 4,000 rpm for 5 min, using a swinging-bucket rotor. To obtain an extract enriched in Frankia cell amino acids, a differential filtration method was modified from Lundquist and Huss-Danell (1992). Nodules harvested 6 weeks PI were homogenized on ice in 50 mM Tris buffer, pH 8 (1 g nodule/30 mL buffer) or in 50 mM phosphate buffered saline, pH 7.4.…”

Close linkage of genes encoding glutamine synthetases I and II in Frankia alni CpI1

“…In CpI1 cells growing in combined nitrogen-free medium or in media containing amino acids degraded to glutamate, total GS activity increases severalfold, with GSII accounting for over 90% of the total activity (44,47). In cells growing on ammonia or on amino acids degraded to ammonia, neither GSII activity nor the polypeptide itself is detectable (26,38,44,47). GSII is thus an inducible enzyme that is made in response to perceived nitrogen limitation.…”

“…In culture, GSI is made under all conditions tested, and GSII is made under conditions that signal N limitation (13,44,47), but apparently neither is expressed in symbiosis (26). The transcriptional control of Frankia ginII may thus have similarities to the nitrogen-regulated transcriptional control of GSII in gram-negative bacteria, with additional levels of control in symbiosis.…”