SYMBIOSIS - Cyanobacteria - page 380

analysis of the symbiont N . azollae 0708 which is clustered with Cylindrospermopsis raciborskii CS-505
and Raphidiopsis brookii D9 rather than with other Anabaena species.
ii) Exchange of nutrients during symbiosis : Initial characterization of Azolla - A . azollae symbiosis
revealed that A . caroliniana plants free of the symbiont could be obtained by treatment with
combination of antibiotics (aureomycin, penicillinG, streptomycin, bacitracin, polymyxin-B-sulphate).
Such Azolla plants required combined nitrogen for their growth (Peters and Mayne, 1974a). This
substantiates that the symbiont fi xes nitrogen and meets the nitrogen requirements of the host as
well as its own. The nitrogenase activity as determined by ARAs has indicated that A . azollae is
responsible for the fi xation of nitrogen and there exists a considerable exchange of metabolites
between the fern and symbiont (Peters and Mayne, 1974b). Hill (1975) for the fi rst time documented
that the symbiont differentiates heterocysts at high frequency from near zero at the growing point
to nearly 20-30% of all cells in mature leaves. The vegetative cells also undergo an increase in their
cell size. The developmental pattern of Anabaena could be correlated with that of the fern as the cells
of the symbiont at the apex remain small and devoid of heterocysts and cannot fi x nitrogen. Soon
after the colonization of leaf cavities, the symbiont differentiates heterocysts at high frequency and
fi xes nitrogen (Hill, 1977). N 15 -labelling experiments assisted by ARAs confi rmed that A . azollae in
symbiosis with Azolla fi xes nitrogen and supports growth of the host plant in a nitrogen defi cient
medium (Peters et al. , 1977). Further, [ 13 N 2 ] N 2 incorporation studies of Meeks et al . (1987) have
confi rmed that the symbiont fi xes nitrogen.
Newton and Cavins (1976) detected high levels of free ammonia in the intracellular nitrogenous
pool of Azolla plants growing in N 2 -free media with the symbiont. Thus this free ammonia seems
to serve as the major source of nitrogen for the symbiotic relationship. Symbiont-free Azolla plants
or Azolla plants with symbionts grown in nitrate media did not contain free ammonia to that level
indicating that the free ammonia has been derived out of nitrogenase of the algal cells.
The distribution of ammonia assimilating enzymes such as glutamate dehydrogenase and
glutamine synthetase in between the host plant and symbiont revealed that the former contributed
to nearly 75% of the enzyme pool suggesting that both the partners in this association have the
capacity to synthesize glutamate either through GDH or GOGAT pathways (Ray et al ., 1978).
A defi nite developmental role for ensuring Azolla - Anabaena symbiosis has been assigned to
the hair cells residing in the cavity of the host plant. These hairs have been shown to establish
elaborate cell wall in growths that are characteristic of transfer cells (Duckett et al ., 1975). Peters et
al . (1978) suggested that the hairs in mature and immature cavities might be involved in nitrogen
assimilation and the release of fi xed nitrogen respectively. Scanning and transmission electron
microscopy of A . azollae symbiotic in A. pinnata revealed that the fi laments are loosely entangled
and are seen adhering to the protruding hairs and folded cell walls of the cavities. The presence of
frequent invaginations or unbranched two-celled hairs and the cytoplasm of the mature hair cells
contain a transparent network with blebs and vesicles. These structures appear to help in exchange
of metabolites between the symbiotic partners (Neumüller and Bergman, 1981). These hair cells are
postulated to play an important role in transferring fi xed nitrogen to the host due to the presence
of high activity of ammonia-assimilating enzymes (Uheda, 1986).
Two major isoforms of glutamine synthestase (GS) are known in higher plants, i.e. GS1 and
GS2. The former is known to occur in the cytoplasm while the latter is in the plastids/chloroplasts.
Differential roles for these two isoforms have been indicated. GS1 in roots helps in the assimilation of
ammonia derived from nitrate reduction while GS2 helps in the assimilation of ammonia produced
during photorespiration (Cren and Hirel, 1999; Ireland and Lea, 1999; Lancien et al ., 2000; Tobin and
Yamaya, 2001). Uheda et al . (2004) demonstrated the location of GS2 isoform of the enzyme in mature