Since the discovery of the GS/GOGAT-catalyzed pathway for glutamate biosynthesis,
extensive studies have unequivocally shown that this pathway is the main
route of soil nitrogen assimilation as well as photorespiratory ammonium ion
reassimilation in plants (see for reviews Hirel and Lea, 2001; Ireland and Lea,
1999; Lam et al.
, 1995; Lea and Ireland, 1999; Miflin and Habash, 2002; Stitt et al.
2002). Plants possess two types of ferredoxin- and NADPH-dependent GOGAT
isozymes (Fd-GOGAT and NADPH-GOGAT). Genes encoding Fd- and NADHGOGAT
isozymes and their regulation of expression have been extensively discussed
in other reviews (Hirel and Lea, 2001; Ireland and Lea, 1999; Lam et al.
1995; Lea and Ireland, 1999; Miflin and Habash, 2002; Stitt et al.
, 2002). The
Fd-GOGAT isozymes (two isoforms encoded by two different genes in Arabidopsis
) constitute the majority of the GOGAT activity in plants, accounting
for over 90% and ~70% of total GOGAT activity in Arabidopsis
leaves and roots,
respectively (Ireland and Lea, 1999; Somerville and Ogren, 1980; Suzuki et al.
2001). The significant role of Fd-GOGAT in ammonium ion assimilation, particularly
of photorespiratory ammonium ion, was demonstrated by a number of
genetic and molecular approaches. Many plant mutants, defective in growth
under photorespiratory conditions, were based on mutations in genes encoding
Fd-GOGAT (Ireland and Lea, 1999; Somerville and Ogren, 1980). Notably, although Arabidopsis
possesses two Fd-GOGAT isozymes, mutations in one are sufficient to
cause sensitivity to enhanced photorespiration (Somerville and Ogren, 1980). This
nonredundant function was explained by two contrasting patterns of expression
of the genes encoding these isozymes (Coschigano et al.
, 1998). The significant role of Fd-GOGAT in reassimilating photorespiratory ammonium ion was also
demonstrated in transgenic tobacco plants with reduced Fd-GOGAT due to
antisense expression (Ferrario-Mery et al.
, 2000). When transferred from CO2
conditions to ambient air to enhance photorespiration, the plants accumulated
significantly higher levels of ammonium ion as well as the two GOGAT substrates,
glutamine and α-ketoglutarate, than control plants (Ferrario-Mery et al.
2000). This suggests that glutamine and α-ketoglutarate were less efficiently converted
into glutamate in the transgenic plants, causing a less-efficient incorporation
of photorespiratory ammonium ion into glutamine. In addition, the reduced Fd-GOGAT expression was also associated with altered levels of leaf amino acids,
implying that a number of amino acid biosynthesis pathways are affected and
may be regulated in response to changes in ammonium ion and/or glutamine
levels (Ferrario-Mery et al.
Constituting a minor proportion of the total plant GOGAT activity, NADPHGOGAT
received less attention than the Fd-GOGAT. However, several lines of
evidence indicate that, despite being a minor isozyme, the NADPH-GOGAT
activity in plants is not redundant. NADPH-GOGAT is unable to compensate
for Fd-GOGAT shortage, implying a distinct metabolic function (Ireland and
Lea, 1999; Somerville and Ogren, 1980). Moreover, plant genes encoding
NADPH-GOGAT generally exhibit contrasting expression patterns compared
to Fd-GOGAT genes. While Fd-GOGAT is abundantly produced in photosynthetic
leaves, NADPH-GOGAT is produced in nonphotosynthetic organs, such
as roots, senescing leaves, and nodules formed in legume roots (see Lancien et al.
, 2002 and references therein). This suggests that in contrast to the major
function of Fd-GOGAT in reassimilation of photorespiratory ammonium ion,
NADPH-GOGAT functions mainly in primary nitrogen assimilation and in
nitrogen transport from source to sink.
To study the function of NADH-GOGAT, its activity was reduced by up to
87% in transgenic alfalfa plants, using antisense constructs controlled either by an
AAT-2 promoter with enhanced expression in nodules, or by a nodule-specific
leghemoglobin promoter (Cordoba et al.
, 2003; Schoenbeck et al.
, 2000). The transgenic
plants were chlorotic and exhibited altered symbiotic phenotypes compared
to controls. In addition, nodule amino acids and amides levels were lower, while
sucrose levels were higher in the transgenic plants than in control plants, implying
thatNADPH-GOGAT represents a major rate-limiting enzyme for the incorporation
of ammonium ion and sugars into amino acids in nodules.
The functional role of NADPH-GOGAT was also studied in an Arabidopsis
T-DNA insertion within the single Arabidopsis
gene encoding this enzyme that
abolished expression of the gene (Lancien et al.
, 2002). In contrast to
reduced levels of Fd-GOGAT, which exhibited metabolic and growth defects
under conditions of enhanced photorespiration (see above), the Arabidopsis
T-DNA mutant lacking NADPH-GOGAT exhibited metabolic and growth defects
when photorespiration was repressed. Based on these results, NADPH-GOGAT
and Fd-GOGAT appear to play nonredundant roles in the assimilation of nonphotorespiratory
ammonium (derived from soil nitrogen or nitrogen fixation)
and photorespiratory ammonium into glutamate, respectively.
The metabolic function of NADPH-GOGAT was also studied by constitutive
expression of the alfalfa enzyme in transgenic tobacco plants (Chichkova et al.
, 2001). Shoots of the transgenic plants contained higher total carbon and
nitrogen than wild-type plants when administered either nitrate or ammonium
ion as sole nitrogen sources. In addition, the transgenic plants contained higher
dry weight than control plants upon entering flowering. These results are consistent
with the rate-limiting role of NADPH-GOGAT in nitrogen assimilation and
also with the importance of nitrogen assimilation for plant growth (Chichkova et al.