NITROGEN FIXATION UNDER PHYSIOLOGICAL AND SALT-STRESSING CONDITIONS
2. ROLE OF BORON IN FREE-LIVING NITROGEN-FIXING MICROORGANISMS
2.1. Boron in cyanobacteria
Among dinitrogen fixing microorganisms, Cyanobacteria, or blue-green algae, form a remarkable group because they have oxygenic photosynthesis that probably made cyanobacteria responsible for the major evolutionary transformation of the biosphere, leading to the development of aerobic metabolism on Earth three billion years ago. In evolutionary terms, they represent a link between bacteria and green plants.
Their cellular organisation, known as prokaryotic is characterised by a lack of membrane bound organelles, however, their principal mode of nutrition, oxygen evolving photosynthesis is similar to that which operates in all other nucleate algae and higher plants. Therefore, the cyanobacteria provide a biologically simple model for studying problems in mineral nutrition, especially of boron and calcium not only in relation to nitrogen fixation but also to photosynthesis and typical plant processes.
The requirement of boron is not a general feature in cyanobacteria. For example, evidence is presented that boron is not required for the growth of Anacystis nidulans (Synechococcus PCC7942) (Martínez et al., 1986). Furthermore, Anabaena PCC7119 a dinitrogen-fixing cyanobacterium, growing in the presence of combined nitrogen was not affected by boron deficiency.
However, when this microorganism was grown under dinitrogen-fixing conditions lacking any boron, inhibition of growth and deficiency of photosynthetic pigment proteins were observed (Mateo et al., 1986).
Inhibition of growth resulting from boron deficiency was reversible by B addition or by supply of combined nitrogen. These findings are consistent with the pre-ceding data and suggest that boron is only required by Anabaena when cells are growing under dinitrogen-fixing conditions.
The study of dinitrogen fixation by the acetylene reduction method indicated that nitrogenase activity of boron deficient cells was reduced to about 40% of those activities observed in the boron supplied cells within the first two hours of
culture. There was no detectable nitrogenase activity in cultures after 24 h of boron deficiency. At this time other metabolic processes (such as photosynthesis) were not affected by boron deficiency. Therefore, boron may have a role in dinitrogen-fixation in cyanobacteria. However, nitrogenase synthesis was not affected by boron deficiency, indicating other role of B not directly related with the enzyme activity (García-González et al., 1988).
Nitrogen fixation is a anaerobic process and all nitrogenase components are rapidly destroyed by oxygen. Heterocysts are specialised cells present in some filamentous cyanobacteria when grow in the absence of a combined nitrogen source.
These cells are capable of aerobically fix N2 because they maintain the reducing environment required for cyanobacterial nitrogenase activity. There may be several complementary mechanisms that enhance the effectiveness of heterocysts as sites for dinitrogen fixation: the general reducing environment, the high activities of some enzymes of oxidative penthoses pathway (OPP), a lack of photosynthetic oxygen evolution, enhanced superoxide dismutase, etc. However, the most conspicuous of these mechanisms is the presence of a thick envelope in the heterocyst. This envelope is comprised of a inner laminated layer, a central homogeneous layer consists of specific glycolipids that are absent in vegetative cells (Lambein and Wolk, 1973). It has been suggest that these glycolipids provide a barrier to the diffusion of oxygen.
Examination of boron starved cultures clearly shows important changes in the morphology and ultra structure of the heterocysts (Figure 1) not only in Anabaena but also in other cyanobacteria (Bonilla et al., 1990). It was proposed that boron might be involved in stabilising the heterocyst envelope. Quantification by HPLC of glycolipids in heterocyst envelopes of B-deficient cultures showed that the amount of these components was less than 15% of that in the control after 24 hours of B deprivation (García-González et al., 1991). These results clearly show that boron
Table 1. Effects of boron deficiency on dry weight (mg mL–1), protein (µgg–1dw) and pigment contents (µgg–1dw) of Anabaena PCC7119 after 96 h of growth in media containing NO3
–or under nitrogen fixing conditions (N2).
Dry weight Protein Chlorophyll Phycobiliproteins
NO3
–+B 1.20 ± 0.20 496 ± 31 11.9 ± 3.0 097.3± 16.5
NO3
––B 1.12 ± 0.20 482 ± 28 11.4 ± 2.0 093.8± 18.0
N2+B 0.40 ± 0.10 437 ± 32 11.3 ± 2.0 115.4 ± 22.0
N2–B 0.16 ± 0.05 208 ± 15 06.5± 0.8 042.9± 7.0
Table 2. Effects of boron deficiency on nitrogenase activity (µmol C2H4mg–1chl h–1) of Anabaena PCC 7119.
1 h % 2 h % 3 h % 24 h %
inhib. inhib. inhib. inhib.
+B 22.5 ± 2.5 57.1 ± 5.4 104.1 ± 10.8 836.0 ± 57.5
–B 20.2 ± 2.4 10.3 32.8 ± 2.9 42.5 056.1± 4.8 46.1 007.2± 1.2 99.1
is an essential element for stabilizing the inner glycolipid layer of the heterocyst envelope. Therefore, the result of B deficiency is the inhibition of nitrogenase activity by the massive oxygen entry inside the heterocyst.
In response to boron deficiency, there is also a short-term increase in the activ-ities of the mechanisms that protect nitrogenase in heterocysts against oxygen inhibition: increases in SOD, catalase and peroxidase (García-González et al., 1988), as well as respiration, and the OPP (García-González et al., 1990).
2.2. Boron in Frankia
A nitrogen-fixing bacterium with structural and functional similarities to hetero-cystous cyanobacteria is the actinomycete Frankia. Bacteria of the genus Frankia form so-called actinorhizal symbioses with several non-leguminous shrubs and trees termed actinorhizal plants, wherein the endophytic form of the microsymbiont develops the N2-fixing activity (Wall, 2000).
Similar to cyanobacteria, but different to rhizobia, Frankia strains isolated from
Figure 1. Heterocysts of Anabaena PCC 7119 grown in the presence (+B) or in the absence (–B) of boron. Scanning electron microscopy (upper side) shows collapsed heterocysts in B-deficient fila-ments. Degeneration of the envelope and the absence of the glycolipid layer show by transmission electron microscopy (bottom side) in –B indicates a role of the micronutrient in the stabilisation of these components.
nodules can fix N2when cultured without a nitrogen-combined source. Nitrogenase in free-living cultures or in symbiotic state is localised inside the specialised vesicles that differentiate from some filament tips (Huss-Danell, 1997). The N2-fixing vesicle is in many ways structurally and functionally analogous to the heterocyst (Zehr, 1998). Therefore, based on the similarity to heterocysts, B has been demonstrated to be essential not only for the development of the actinorhizal symbioses but also for the differentiation of N2-fixing vesicles of Frankia as in heterocystous cyanobac-teria (Bolaños et al., 2002a).
Frankia BCU110501, a strain isolated from Discaria trinervis nodules (Chaia, 1998) was unable to grow (Figure 2) in B deficient conditions. Filaments of B-deficient cultures are sorter than normal, and development of functional N2-fixing vesicles is inhibited in the absence of the micronutrient (Figure 3).
The protection of nitrogenase activity against oxygen diffusion is attributed to the resistance properties of the lipidic multilaminate vesicle wall (Parsons et al., 1987), which can change its thickness by modifying the number or lipidic monolayers in response to different pO2(Harris and Silvester, 1992). The analysis of lipids showed that vesicles have a higher content of glycolipids and neutral lipids than vegeta-tive cells, being the major proportion long-chain polyhydroxy fatty acids or alcohols
Figure 2. Liquid cultures of Frankia BCU110501 in media with (+B) or without (–B) boron.
Figure 3. Filaments of Frankia BCU110501 developed in the presence (+B) or in the absence (–B) of boron. Boron deficiency leads to short filaments that do not develop N2-fixing vesicles (highlighted by arrowheads in +B).
(Tunlid et al., 1989). A very high concentration of the hopanoid bacteriohopanetetrol is also present (Berry et al., 1991). All of these constituents of the vesicle envelope are compounds rich in diol groups which can interact with borate ions. The appear-ance of B-deficient vesicles is similar to that reported for B-starved heterocysts (García-González et al., 1991), which is due to the loss of the inner laminated layer of the heterocyst envelope. That layer is composed of glycolipids with long-chain polyhydroxyl alcohols (Lambein and Wolk, 1973) stabilized by boric acid.
The place were the lipidic envelope is supposed to be (Torrey and Callaham, 1982) is very narrow inside B-deficient vesicles, suggesting also a thinner laminated envelope. Therefore, B can play a role in the stabilisation of vesicle envelope (Figure 4), as the micronutrient does in the heterocysts.
Contrary to cyanobacteria, the micronutrient is also needed for Frankia vegeta-tive growth when the bacteria are cultivated in media containing combined nitrogen.
Boron deficient filaments in either culture are thinner and with a twisted appear-ance that indicate an altered surface (Figure 5). The inner laminated layer of the heterocyst envelope stabilized by B is composed of lipids not found in vegetative cells (Nichols and Wood, 1968), while vesicle envelope is enriched in lipids that are also constitutive of filaments. This particular difference could explain why B is also needed for the structure and growth of vegetative cells of Frankia BCU110501.
Figure 4. Bis(diol) borate complex (A). Hypothetical model for bacteriohopanetetrol and bacterio-hopanetetrol phenylacetate linkage by boron in the envelope of Frankia vesicles and filaments (B).