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Segregation of Nitrogen Fixation and Oxygenic Photosynthesis in the Marine Cyanobacterium Trichodesmium

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Science  16 Nov 2001:
Vol. 294, Issue 5546, pp. 1534-1537
DOI: 10.1126/science.1064082

Abstract

In the modern ocean, a significant amount of nitrogen fixation is attributed to filamentous, nonheterocystous cyanobacteria of the genusTrichodesmium. In these organisms, nitrogen fixation is confined to the photoperiod and occurs simultaneously with oxygenic photosynthesis. Nitrogenase, the enzyme responsible for biological N2 fixation, is irreversibly inhibited by oxygen in vitro. How nitrogenase is protected from damage by photosynthetically produced O2 was once an enigma. Using fast repetition rate fluorometry and fluorescence kinetic microscopy, we show that there is both temporal and spatial segregation of N2 fixation and photosynthesis within the photoperiod. Linear photosynthetic electron transport protects nitrogenase by reducing photosynthetically evolved O2 in photosystem I (PSI). We postulate that in the early evolutionary phase of oxygenic photosynthesis, nitrogenase served as an electron acceptor for anaerobic heterotrophic metabolism and that PSI was favored by selection because it provided a micro-anaerobic environment for N2 fixation in cyanobacteria.

Nitrogenase, the enzyme that catalyzes the reduction of atmospheric N2 to ammonia, is irreversibly inhibited upon exposure to molecular oxygen (1,2). Cyanobacteria produce molecular oxygen via photosynthesis and have evolutionary adaptations that protect nitrogenase from oxygen; these adaptations include either a temporal separation, in which N2 fixation occurs in the dark, or a spatial segregation, in which N2 fixation is confined to a specialized cell, the heterocyst, in which only PSI remains active. The major bloom-forming N2-fixing organisms (diazotrophs) in modern oceans belong to the genus Trichodesmium. This genus is characterized by nonheterocystous filaments (trichomes), which form colonies.Trichodesmium are unusual among cyanobacteria because they fix nitrogen only during the photoperiod, while simultaneously producing O2 (3, 4). How nitrogenase is protected from damage by photosynthetically produced O2 and how this process is regulated has been an enigma since Dugdale et al. first identified these organisms as light-dependent diazotrophs 40 years ago (3–8). In Trichodesmium, nitrogenase is localized in subsets of consecutively arranged cells in each trichome, which also contain photosynthetic components (8, 9,10) and comprise 15 to 20% of all cells (9–14). Here, we demonstrate that a combined temporal and spatial segregation of N2 fixation and oxygen evolution provides a window of opportunity that permits the cells to fix nitrogen for only a few hours during the photoperiod.

Using fast repetition rate fluorometry (FRRF) (15), oxygen production, and carbon and N2 fixation, we found that changes in the activity of photosystem II (PSII) reveal a temporal separation between N2 fixation and photosynthesis during the photoperiod. In the field, photosynthetic carbon fixation increased in the morning but declined at midday, when nitrogenase activity peaked (Fig. 1C) (16). High N2-fixation rates were measured for ∼6 hours surrounding the middle of the photoperiod. When N2 fixation declined, photosynthetic 14C uptake increased again (Fig. 1A). This inverse relationship was even more pronounced in laboratory cultures, where an almost complete temporal separation of N2 fixation and 14C fixation was also observed (Fig. 2, A and C). Moreover, the period of high N2 fixation was characterized by a decline in gross photosynthetic production, which resulted in a negative net production of oxygen (Fig. 2D). The photochemical quantum yield [variable fluorescence/maximal fluoresence (F v/F m)] of PSII varied inversely with N2 fixation in both field and cultured populations (Figs. 1 and 2). During the photoperiod,F v/F m was 50 to 60% lower at the peak of N2 fixation, increasing to maximum values at the end of the photoperiod, when N2 fixation declined (Figs. 1B and 2, A and C). This characteristic diel pattern in the quantum yields was observed under both subsaturating and saturating irradiances (Figs. 1B and 2, A and C) but disappeared when N2 fixation was inhibited in cells grown with nitrate (Fig. 2B).

Figure 1

(A to D) Diel changes in N2 fixation, carbon uptake, and fluorescence patterns measured by a FRRF fluorometer on surface populations ofTrichodesmium spp. (both colonies and free filaments) collected from the Arafura and Timor Seas from 29 October to 15 November 1999. (A) Representative pattern of N2fixation (squares) (as measured by acetylene reduction) and acid-stable14C uptake (triangles) for T. thiebautiicolonies obtained on 7 and 8 November 1999. (B toD) Data represent measurements made on sea water from 3 m (filtered through a 200-μm net) during 7 and 8 November (solid symbols) and 11 and 12 November (open symbols), using a continuous flowthrough FRR fluorometer. Microscopic observations showed mostly free filaments and small colonies of Trichodesmiumspp. in these samples. (B) Photochemical quantum yields (F v/F m). Data shown are a composite of continuous FRRF measurements on 7 and 8 November (solid diamonds) and the average and standard deviations from the total samples of handpicked colonies between 29 October and 15 November 1999 (solid squares). (C) Oxidation rates of QA (τ]. (D) Redox state of the PQ as estimated from δF mF m =F m (ST – MT)].

Figure 2

Diel patterns in N2 fixation, in quantum efficiencies of PSII and in respiratory oxygen consumption and photosynthetic oxygen evolution in cultures of Trichodesmiumstrain IMS101. (A and B) Axenic cultures grown under a 14:10 hour light/dark cycle (L/D) under 40 μmol of quanta m−2 s−1. (C and D) Culture grown at 12:12 L/D under 80 μmol of quanta m−2s−1. (A and C) Quantum yields (triangles) and acetylene reduction rates (diamonds) of culture grown under diazotrophic conditions with no inorganic nitrogen source. (B) Quantum yields (triangles) of culture grown on 200 μM NO3 exhibiting no N2 fixation as measured by the acetylene reduction method. (D) Oxygen consumption and evolution as measured on a Clark-type O2 electrode. Dark respiration (triangles), gross photosynthesis (circles), and net oxygen evolution (open circles).

We used FRRF to determine temporal changes in the redox state of photosynthetic electron transport (PET) components. The rate of oxidation of the primary electron acceptor in PSII, quinone A (QA ), declined from sunrise to sunset, which suggested that the electron transfer components downstream of QA [e.g., at the plastoquinone (PQ) pool] are chemically reduced (Fig. 1, C and D) (17). The retardation of electron flow led to lower quantum yields and lower rates of photosynthetic oxygen production (Fig. 1B).

Blocking linear electron transport on the acceptor side of PSII with the inhibitors 3-3,4-dichlorophenyl-1′,1′-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which poise the PQ pool in either an oxidized or reduced state, respectively (18), caused an immediate decline in nitrogenase activity when applied to cultures under aerobic conditions [Web fig. 1 (19)]. Under anaerobic conditions, however, nitrogenase activity was inhibited by DBMIB, which affects both photosynthetic and respiratory pathways (20), but was not inhibited by DCMU, which inhibits only QA oxidation. These results reveal that PET is not an immediate source of electrons for nitrogenase; dark respiration, although required for N2fixation, is inadequate as an oxygen-scavenging mechanism (21); and linear PET is required for N2 fixation under aerobic conditions [Web fig. 2 (19)]. The differential effect of DCMU under aerobic and anaerobic conditions reveals that nitrogenase is protected from oxygen by electrons supplied by PSII. This phenomenon strongly implies that oxygen is scavenged by PSI via the Mehler reaction (22) [Web fig. 2 (19)].

We used fluorescently tagged primary antibodies to nitrogenase and to D1, a core protein of the oxygen-evolving PSII reaction center (23), to examine the pattern of segregation of N2 fixation and oxygenic photosynthesis on a cellular level. D1 was present in most cells in a trichome, including those containing nitrogenase (Fig. 3C). Because the turnover of D1 is extremely rapid (24), the presence of this protein strongly implies that oxygen production and N2 fixation are not simply spatially segregated. Moreover, when N2 fixation is maximal, H2O2(produced primarily by the reduction of O2by the Mehler reaction) is present in most cells in the trichomes, including central zones where nitrogenase is clustered (25) (Fig. 3D).

Figure 3

(A) A surface bloom ofTrichodesmium spp. from the Arafura Sea. Inset: Puff and tuft formations of T. thiebautii colonies. (B) Nitrogenase localization in a single IMS101 trichome visualized with an epifluorescent microscope (Olympus U-MWU; BP 330 to 385, DM 400, BA 420) using a fluorescent secondary antibody Alexa-350 (Molecular Probes). Insert: Natural population of T. erythraeum probed as above. (C) Trichodesmium IMS101 probed simultaneously with fluorescently tagged primary antibodies to both D1 and nitrogenase and viewed on a confocal laser microscope (Zeiss LSM410) at 488/528 nm and 568/600 to 620 bandpass excitation/emission for D1 (green) and nitrogenase (red), respectively. Thelarge image is the composite overlay of both channels. Insert: Nitrogenase label only. (D) Colonies of T. erythraeum, collected from surface waters of the Arafura Sea and stained with DAB, showing the intracellular distribution of H2O2 as a brown stain throughout the cells during peak hours of N2 fixation (13:30 to 14:30). Insert: Midsection of a single trichome stained with brown deposits. (E to H) Trichomes of culturedTrichodesmium IMS101 viewed with a microscope for two-dimensional measurements of in vivo chlorophyll fluorescence kinetics (45). (E and G) Trichomes from the early hours (00:00 to 04:00) of the photoperiod when N2 fixation is low. (F and H) Trichomes from hours of high N2 fixation (5 to 7 hours into the photoperiod). (E and F) Total chlorophyll fluorescence. Inserts: Trichomes photographed with a nonamplified high-resolution camera showing the pattern of normal and bright filaments (i.e., high total fluorescence to very lowF v/F m) for the corresponding sampling times. Scale bar indicates relative fluorescence yield. (G and H) False color images of the two-dimensional distribution of PSII efficiency, F v/F malong the trichomes.

We used a microscope equipped for two-dimensional measurement of in vivo chlorophyll fluorescence kinetics (26) to further examine the spatial heterogeneity in photosynthetic activity of PSII within individual cells and between trichomes. A combination of actinic radiation, saturating flashes, and a pulsed measuring light was applied to the microscopic field, enabling high spatial resolution of measured and derived fluorescence parameters for individual cells within the trichomes. In cultures measured during the early and late stages of the photoperiod, and in nitrate-grown or stationary-phase cultures, the total fluorescence yield was homogeneous in 85% of the trichomes (Fig. 3E), although zonations were observed inF v/F m (Fig. 3G). In nitrogen fixing cultures, total fluorescence was high (Fig. 3F) and the quantum yield of photochemistry in PSII was low (Fig. 3H). The lower quantum yields were a consequence of a proportionately larger increase in the initial dark-adapted fluorescence (F o) than in F m, implying that PSII reaction centers are reduced on the acceptor side (15). The bright inactive zones were nonuniformly distributed and were seen in whole filaments, on the tips of filaments, and in central areas of trichomes (Fig. 3F). Cells could turn photosynthetic activity (i.e., variable fluorescence) on and off within 10 to 15 min, illustrating that inTrichodesmium, in contrast to fully evolved heterocystous cyanobacteria, all cells are photosynthetically competent, but individual cells modulate oxygen production and consumption during the photoperiod. Moreover, the increased occurrence of inactive photosynthetic zones during the hours of high N2 fixation is evidence of both temporal and spatial segregation of the two processes.

Our results (Figs. 1 to 3) demonstrate a combined spatial and temporal segregation of N2 fixation from photosynthesis and suggest a sequential progression of photosynthesis, respiration, and N2 fixation in Trichodesmium over a diel cycle. These pathways are entrained in a circadian pattern (27) that is ultimately controlled by the requirement for an anaerobic environment around nitrogenase (28). Light initiates photosynthesis, providing energy and reductant for carbohydrate synthesis and storage, stimulating cyclic and pseudocyclic (Mehler) electron cycling through PSI, and poising the PQ pool at reduced levels (Figs. 1A and 2, A and C) [Web fig. 2 (19)]. High respiration rates (29) early in the photoperiod (Fig. 2D) supply carbon skeletons for amino acid synthesis (the primary sink for fixed nitrogen) but simultaneously reduce the PQ pool further, sending negative feedback to linear PET [Web fig. 2 (19)]. The reduced PQ pool leads to a down-regulation of PSII (Figs. 3, F and H, and 1, C and D). However, linear electron flow to PSI is never abolished [Web fig. 2 (19)]. The down-regulation of PSII opens a window of opportunity for N2 fixation during the photoperiod, when oxygen consumption exceeds oxygen production. As the carbohydrate pool is consumed, respiratory electron flow through the PQ pool diminishes, intracellular oxygen concentrations rise, the PQ pool becomes increasingly oxidized, and net oxygenic production exceeds consumption (Figs. 1A and 2, C and D). Nitrogenase activity is lost until the following day.

The combination of spatial and temporal segregation of N2 fixation and oxygenic photosynthesis during the photoperiod appears to reflect the evolutionary history of N2 fixation in cyanobacteria. Nitrogenase is an ancient enzyme that almost certainly arose in the Archean Ocean before the oxidation of the atmosphere by oxygenic photoautotrophs (30,31). We propose that under the prevailing anaerobic conditions of that period in Earth's history, N2 served as a readily accessible electron sink for anaerobic heterotrophs. In contemporary diazotrophic microbes, including cyanobacteria, the reductants for nitrogenase are provided by respiratory electron flow. With the evolution of cyanobacteria and the subsequent generation of molecular oxygen, oxygen-protective mechanisms in diazotrophs would be essential. Indeed, phylogenetic trees of diazotrophic cyanobacteria, based on nifH gene sequences, suggest that Trichodesmium branched out very early (32). A full temporal separation, in which nitrogen is only fixed at night, then developed in unicellular cyanobacterial diazotrophs and in some nonheterocystous filamentous diazotrophs (7). Finally, in yet other filamentous organisms, complete segregation of N2 fixation and photosynthesis was achieved with the cellular differentiation and evolution of heterocystous cyanobacteria (33). It is remarkable that the pathway adopted by Trichodesmium has persisted in the oceans through the present time. This persistence suggests that the tempo of evolution in marine diazotrophic cyanobacteria is extremely slow.

  • * To whom correspondence should be addressed. E-mail: irfrank{at}imcs.rutgers.edu

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