Plant Science 150 2000 181 – 189
Differential regulation of nitrogenase activity by ionic and osmotic stresses and permeable sugars in the Cyanobacterium Anabaena sp.
strain L-31
Tonina A. Fernandes, Shree Kumar Apte
Cell Biology Di6ision, Bhabha Atomic Research Centre, Trombay, Bombay
400 085
, India Received 28 April 1999; received in revised form 14 September 1999; accepted 14 September 1999
Abstract
Nitrogenase acetylene reduction activity in Anabaena sp. strain L-31 is significantly enhanced by the addition of sucrose, but is inhibited upon addition of sodium chloride. The positive effect of sucrose is not a general osmotic stress effect since
non-permeable osmolytes mannitol or polyethylene glycol PEG do not influence nitrogenase activity. Unlike enteric bacteria, Anabaena cells take up and metabolise sucrose and incorporate products of its catabolism into proteins. Cultures inhibited in
photosynthesis retain the ability to take up sucrose but do not show acetylene reduction activity, even when supplemented with sucrose. Addition of an inhibitor of transcription rifampicin or of a repressor of nitrogenase biosynthesis ammonium chloride
abolishes the positive effect of sucrose on acetylene reduction activity. Cultures grown with permeable sugars glucose, fructose and sucrose show significantly higher levels of dinitrogenase reductase Fe-protein of nitrogenase complex while those grown
with NaCl lack the protein. Fe-protein content is not affected by non-permeable solutes. Thus, exogenous sucrose elevates dinitrogenase reductase synthesis but does not appear to support the requirement of reductant for nitrogenase activity. The data
substantiate our previous finding that the ionic and osmotic stresses differentially regulate cyanobacterial nitrogenase activity and explain the relatively superior osmotolerance of diazotrophic cyanobacterial strains, as compared with their sensitivity to salinity
stress. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Anabaena; Ionicosmotic stress; Differential effects; Nitrogen fixation www.elsevier.comlocateplantsci
1. Introduction
Among the environmental stresses, salinity and drought osmotic stress rank as the most detri-
mental for crop productivity [1]. Apart from the crop plants, these stresses also adversely affect the
growth and metabolism of agriculturally impor- tant bacteria especially nitrogen fixers, phosphate
solubilisers, etc. thereby further limiting crop pro- ductivity. Both salinity and drought impose a wa-
ter stress on all living cells and cause turgor loss. Salinity stress, in addition, has an ionic compo-
nent that is especially deleterious to plant cells [2]. Generally, heterotrophic microbes like enteric
bacteria and yeast show an identical response to ionic and osmotic stresses [3]. In contrast, pho-
toautotrophic microbes like cyanobacteria exhibit differential sensitivity to ionic and osmotic stresses
[4]. In view of the close similarity of their physiol- ogy and metabolism to plants and their possible
phylogenetic
relationship with
chloroplasts, cyanobacteria are considered good model systems
for analysis of plant responses to environmental stresses [5,6]. In addition, information on the sen-
sitivity of cyanobacterial nitrogen fixation to envi- ronmental stresses has an important bearing on
the potential of these microbes as nitrogen biofer-
Corresponding author. Tel.: + 91-22-550-50002348; fax: + 91- 11-5505-151.
E-mail address
:
bsaraspara.barc.ernet.in S. Kumar Apte 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 8 3 - 1
tilisers for crop plants growing in stressful environments.
Our laboratory has been studying the re- sponses
of heterocystous
nitrogen-fixing An-
abaena strains to salinity stress and sucrose — hitherto
considered an
osmotic stress
in cyanobacteria. In Anabaena strains tolerance to
NaCl and sucrose are not obligatorily linked i.e. salt-sensitive strains exhibit significant tolerance
to sucrose and vice-versa [4]. Cyanobacterial halotolerance is primarily determined by the
ability of cyanobacteria to exclude Na
+
[7,8] while osmotolerance is facilitated by the specific
need-based expression of certain osmoresponsive genes and synthesis of osmotic stress proteins, or
OSPs [5,9,10] leading to the accumulation of compatible solutes like glucosylglycerol, sucrose,
trehalose and betaines [11 – 13]. Both halotoler- ance as well as osmotolerance of Anabaena
strains can be experimentally upgraded, respec- tively, by treatments which curtail Na
+
influx or induce expression of OSPs [5]. The most striking
difference in the response of Anabaena strains to ionicosmotic stresses relates to nitrogenase activ-
ity. We have shown earlier that exogenously added NaCl severely inhibits while sucrose re-
markably enhances acetylene reduction activity by 2.5- to 3-fold [4].
These findings have raised certain questions, such as: 1 do osmotic stresses affect cyanobac-
terial N
2
fixation the same way as sucrose? 2 If not, does sucrose act as osmotic stress or are
Anabaena cells permeable to sucrose? If sucrose is taken up. 3 Is it accumulated as compatible
solute? or 4 Is it utilised to support growth and nitrogenase activity in light and in dark? 5
Is the positive effect of sucrose on nitrogenase activity related to nitrogenase biosynthesis or its
activity? etc.
The present study has addressed the aforemen- tioned questions to reveal the molecular basis of
the differential effects of sucrose and NaCl on cyanobacterial nitrogenase activity. Our data
show that while NaCl represses the synthesis of dinitrogenase reductase Fe-protein, sucrose and
other permeable sugars enhance dinitrogenase re- ductase synthesis. Non-permeable osmolytes such
as polyethylene glycol PEG and mannitol nei- ther enhance dinitrogenase reductase synthesis
nor
affect nitrogenase
activity in
Anabaena strains.
2. Materials and methods
