The Nostoc Bryophyte Symbiotic Experimental System

This chapter will review the results of studies primarily utilizing an association between the nitrogen-fixing cyanobacterium Nostoc punctiforme (strain PCC 73102, synonym ATCC 29133) and the bryophyte hornwort Anthoceros punctatus that is routinely reconstituted in the laboratory with the separately pure-cultured partners. The focus will be more on mechanisms of the interaction than on diversity of symbionts; the latter is an expanding topic which has recently been examined using molecular genetic techniques (West and Adams 1997; Costa et al. 2001; Rasmussen and Nilsson 2002). The complete genome sequence of N. punctiforme strain ATCC 29133 is now known (http://genome.jgi-psf.org/microbial/). Therefore, results and concepts will be discussed and hypotheses offered in the context of the N. punctiforme genome sequence, as well as genetic, physiological and biochemical traits of the two partners. When appropriate, comparisons will also be made to other cyanobacterial-plant systems.

Nostoc punctiforme has an extraordinarily wide range of physiological properties, vegetative cell developmental alternatives and ecological niches (Meeks et al. 2002; Meeks 2003). It is amenable to genetic manipulation, including random transposon mutagenesis (Cohen et al. 1994) and targeted gene replacement (Hagen and Meeks 1999). These collective characteristics enhance the scientific value of its genome sequence (Meeks et al. 2001). N. punctiforme represents a subgroup of cyanobacteria that can grow in complete darkness as a respiratory heterotroph, a metabolic mode that may be required in symbiotic association. Two of the developmental alternatives are essential to its symbiotic interactions; the differentiation of motile filaments called hormogonia, which serve as the infective units of plant associations, and of heterocysts, the sole sites of nitrogenase expression in nearly all Nostoc strains (Fig. 1). The third vegetative cell developmental alternative is the formation of resting cells termed akinetes. Akinetes can be detected in the senescing regions of plant associations, but their role, if any, in symbiotic interactions has not been addressed. Inclusive of the vegetative cell cycle, the four developmental directions of a N. punctiforme vegetative cell are numerically unparalleled in the bacterial world. They provide multiple stages of a cell cycle for developmental regulation, including by environmental signals from symbiotic partners. The N. punctiforme genome contains a remarkably high number of sensory transduction systems with approximately 156 sensor histidine kinases, 103 response regulator proteins, and 51 serine/threonine protein kinases, plus 7 putative adenylate/guanylate cyclases (Meeks et al. 2001; Meeks 2005). The environmental signals that modulate the activity of these sensory systems are largely unknown; nevertheless, there are more than ample regulatory proteins to serve as potential targets for signals from symbiotic partners.

Fig. 1. Phase contrast photomicrographs of three free-living developmental states of N. punctiforme. A Ammonium grown filaments lacking any differentiated cells. B Di-nitrogen grown filaments containing nitrogen-fixing heterocysts (het) present in a non-random spacing pattern. C Motile hormogonium filaments showing the smaller cell size relative to N2 or NH4+ grown cultures. Panels A and C reproduced from Meeks et al. (2002)

Fig. 1. Phase contrast photomicrographs of three free-living developmental states of N. punctiforme. A Ammonium grown filaments lacking any differentiated cells. B Di-nitrogen grown filaments containing nitrogen-fixing heterocysts (het) present in a non-random spacing pattern. C Motile hormogonium filaments showing the smaller cell size relative to N2 or NH4+ grown cultures. Panels A and C reproduced from Meeks et al. (2002)

Nostoc punctiforme shows broad symbiotic competence within the phylogenetic spectrum of plants, including the bryophyte hornworts (Enderlin and Meeks 1983) and liverworts (Joseph and Adams 2000), gymnosperm cycads, from whence it was isolated (Rippka et al. 1979), and the angiosperm Gunnera spp. (Johansson and Bergman 1994), as well as the non-lichen fungus, G. pyriforme (Kluge et al. 2002). Photographs of the plant associations, illustrating the different compartments in which the cyanobacterium is localized, are presented in Fig. 2. It is difficult to imagine that N. punctiforme successively developed specific adaptive processes for each plant group as members of the group emerged over evolutionary time. Rather, it is logical to hypothesize that the plants must have independently evolved strategies to control key regulatory and metabolic pathways of the cyanobacterium that are normally expressed in free-living growth. For example, a shift to a heterotrophic mode of carbon and energy metabolism, the differentiation of hormogonia and heterocysts, and N2 fixation are basic processes that Nostoc expresses apart from a plant partner, but the responses are enhanced in the presence of the plant partner (Meeks 1998). Thus, the transition from free-living to symbiotic growth of Nostoc can best be characterized as changes in the degree of a response. In contrast, when rhizobia associate with leguminous plants, the vegetative cells change into a bacteroid state and induce nitrogenase synthesis and activity, neither of which do they express apart from the plant partner (van Rhijn and Vanderleyden

1995). These morphological and physiological transitions to the rhizobial symbiotic growth state are best characterized as a new kind of a response. Moreover, the Nostoc colonize structures or areas (called symbiotic cavities) of the plants that are present at all times and change very little in the symbiotic state. Conversely, rhizobia induce the formation of a root nodule, which can be considered as a new plant organ, structured to protect nitrogenase from oxygen, while supplying sufficient oxygen for rhizobial respiration (van Rhijn and Vanderleyden 1995). The fact that Nostoc, and related heterocyst-forming species, carry their own oxygen protective mechanism may account for the relative simplicity of their symbiotic associations. These differences in degree, versus kind of response, and simple structures, versus a new organ, contribute to our conclusion that the interactions in Nostoc-based symbioses are primarily (but not exclusively) unidirectional from the plant to the cyanobacterium (Meeks 1998), in contrast to the extensive signal exchange between partners that characterizes the rhizobia-legume associations (Perret et al. 2000).

The gymnosperm cycad Cycas

The gymnosperm cycad Cycas

The bryophyte homwort Anthoceros The angiosperm Gunnera

Fig. 2. Photographs of representatives of the three terrestrial groups of plant partners with which N. punctiforme will form a symbiotic association. The gymnosperm Cycas sp. showing the vegetative fronds (A) and (B) an excised coralloid root with the tip of one lobe sectioned to show the ring-shaped cyanobacterial cavity (nc). The bars in panels A and B are 0.5 m and 0.5 cm, respectively. The bryophyte hornwort Antho-ceros punctatus showing the gametophyte (G) and attached sporophyte (S) generations in panel C and D pure cultured gametophyte tissue reconstituted in the laboratory with N. punctiforme localized in slime cavities (nc). The bars in panels C and D are both 1.0 cm. The Angiosperm Gunnera spp. depicting a seedling with distinctive stem glands (E) the sites of Nostoc entry into the stem and (F) a tangential section of a stem of a giant Gunnera chilensis plant showing numerous symbiotic cavities occupied by Nostoc (nc). The bars in panels E and F are both 1.0 cm. Reproduced from Meeks and Elhai (2002)

Fig. 2. Photographs of representatives of the three terrestrial groups of plant partners with which N. punctiforme will form a symbiotic association. The gymnosperm Cycas sp. showing the vegetative fronds (A) and (B) an excised coralloid root with the tip of one lobe sectioned to show the ring-shaped cyanobacterial cavity (nc). The bars in panels A and B are 0.5 m and 0.5 cm, respectively. The bryophyte hornwort Antho-ceros punctatus showing the gametophyte (G) and attached sporophyte (S) generations in panel C and D pure cultured gametophyte tissue reconstituted in the laboratory with N. punctiforme localized in slime cavities (nc). The bars in panels C and D are both 1.0 cm. The Angiosperm Gunnera spp. depicting a seedling with distinctive stem glands (E) the sites of Nostoc entry into the stem and (F) a tangential section of a stem of a giant Gunnera chilensis plant showing numerous symbiotic cavities occupied by Nostoc (nc). The bars in panels E and F are both 1.0 cm. Reproduced from Meeks and Elhai (2002)

As originally defined by deBary (1879), the term symbiosis simply described the living together of differently named organisms. It is now most often applied to mutualistic interactions in which all of the organisms involved clearly benefit from the intimate physical association, as opposed to parasitic interactions in which one partner benefits and the other is slowly harmed. The mutualistic terminology may appropriately reflect the rhizobia-legume associations where the heterotrophic rhizobia benefit from a physical association with a photoautotrophic plant, while the plant benefits from the nitrogen fixation activity of the bacterium. The benefit to a photoautotrophic Nostoc in association with a photoautotrophic plant is not obvious, except, perhaps, as a shelter to avoid grazing predation. As an alternative to mutualistic and parasitic, we have described nitrogen-fixing Nostoc associations as a form of symbiosis more precisely conceptualized as plant domestication of a cyanobacterial ammonium-producing factory (commensal) (Meeks and Elhai 2002). The cyanobacterium neither benefits nor is harmed by the association. Consequently, the working model we are testing does not draw regulatory or structural gene analogies to rhizobial nodulation (nod) genes that are active only during association of the bacteria with legumes (van Rhijn and Vanderleyden 1995; Perret et al. 2000), although general strategies and chemical signals may be similar.

The above conceptualization of unidirectional flow of information in a commensal interaction leads to the hypothesis that the regulatory circuits and structural gene targets in these nitrogen-fixing associations uniquely evolved in the cyanobacterial lineage. We have suggested a similar evolutionary scenario in the context of vegetative developmental alternatives (Meeks et al. 2002; Meeks 2005). Therefore, the genes encoding the processes cannot be identified in the genome sequence by purely comparative bioinformatics approaches. Rather, functional assays will be necessary to fish the respective genes from the genome.

As guides in devising functional assays, we have developed working models. The interactions between N. punctiforme and A. punctatus, while most likely operating as a continuum, can be experimentally separated into two sequential stages, each with substages (Fig. 3). The initial stage is establishment of the association and involves the differentiation and behavior of hormogonium filaments, the infective units. The second stage is development of a functional association involving growth and metabolic alteration of vegetative cells, and the differentiation and behavior of heterocysts. This model is supported by mutants we have isolated of N. punctiforme that are separately defective in establishment or functional development of an association (Table 1). The physiological roles of the products of the mutated genes will be discussed in subsequent sections.

Hormogonium

Fig. 3. Schematic of the continuum of interactions between A. punctatus and N. puncti-forme leading to a N2-fixing symbiotic association. The interactions are depicted as unidirectional from plant to cyanobacterium. The numbers refer to experimentally distinct substages that are described in the text. Reproduced with modifications from Meeks (2003)

Fig. 3. Schematic of the continuum of interactions between A. punctatus and N. puncti-forme leading to a N2-fixing symbiotic association. The interactions are depicted as unidirectional from plant to cyanobacterium. The numbers refer to experimentally distinct substages that are described in the text. Reproduced with modifications from Meeks (2003)

Table 1. Phenotypes of selected N. punctiforme mutants altered in symbiotic infection or function. Details of the mutated genes and their functional roles are in the text

Strain

Symbiotic

Acetylene

Gene

Stage

coloniesa

reductionb

induced

affected

byc

per g FW

colony

ATCC 29133d

0.21±0.04

6.3±1.2

12.4±3.3

--

--

UCD 398 (sigH)d

1.2±0.2

8.0±3.9

10.1±4.1

HIF

infection

UCD 328 (hrmAf

1.6±0.1

6.1±1.1

8.6±1.3

HRF

infection

UCD 444 (ntcA) f

0

0

0

--

infection

UCD 416 (hetF)

0.26±0.06

0

--

function

UCD 464 (hetR)f

0.36±0.04

0

0

--

function

"Number of symbiotic colonies visible in a dissecting microscope 2 weeks after co-culture, normalized to mg dry wt of A. punctatus tissue counted and |g Chl a of the Nostoc inoculant bIn situ acetylene reduction activity given as nmol ethylene formed per g fresh weight of gametophyte tissue or as pmol ethylene formed per symbiotic Nostoc colony cHIF is hormogonium inducing factor and refers to exudate of A. punctatus containing a factor(s) that induces hormogonium differentiation; HRF is hormogonium repressing factor and refers to an aqueous extract of A. punctatus that contains a factor(s) that represses hormogonium differentiation "Campbell et al. (1998) eCohen and Meeks (1996) fWong and Meeks (2002)

"Number of symbiotic colonies visible in a dissecting microscope 2 weeks after co-culture, normalized to mg dry wt of A. punctatus tissue counted and |g Chl a of the Nostoc inoculant bIn situ acetylene reduction activity given as nmol ethylene formed per g fresh weight of gametophyte tissue or as pmol ethylene formed per symbiotic Nostoc colony cHIF is hormogonium inducing factor and refers to exudate of A. punctatus containing a factor(s) that induces hormogonium differentiation; HRF is hormogonium repressing factor and refers to an aqueous extract of A. punctatus that contains a factor(s) that represses hormogonium differentiation "Campbell et al. (1998) eCohen and Meeks (1996) fWong and Meeks (2002)

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