Regulation of cellular differentiation in filamentous cyanobacteria in free-living and plant-associated symbiotic growth states

Abstract

Certain filamentous nitrogen-fixing cyanobacteria generate signals that direct their own multicellular development. They also respond to signals from plants that initiate or modulate differentiation, leading to the establishment of a symbiotic association. An objective of this review is to describe the mechanisms by which free-living cyanobacteria regulate their development and then to consider how plants may exploit cyanobacterial physiology to achieve stable symbioses. Cyanobacteria that are capable of forming plant symbioses can differentiate into motile filaments called hormogonia and into specialized nitrogen-fixing cells called heterocysts. Plant signals exert both positive and negative regulatory control on hormogonium differentiation. Heterocyst differentiation is a highly regulated process, resulting in a regularly spaced pattern of heterocysts in the filament. The evidence is most consistent with the pattern arising in two stages. First, nitrogen limitation triggers a nonrandomly spaced cluster of cells (perhaps at a critical stage of their cell cycle) to initiate differentiation. Interactions between an inhibitory peptide exported by the differentiating cells and an activator protein within them causes one cell within each cluster to fully differentiate, yielding a single mature heterocyst. In symbiosis with plants, heterocyst frequencies are increased 3- to 10-fold because, we propose, either differentiation is initiated at an increased number of sites or resolution of differentiating clusters is incomplete. The physiology of symbiotically associated cyanobacteria raises the prospect that heterocyst differentiation proceeds independently of the nitrogen status of a cell and depends instead on signals produced by the plant partner.

FIG. 1.

Photomicrographs of vegetative and heterocyst-containing filaments of the cyanobacterium N. punctiforme. (A) Phase-contrast image of vegetative filaments grown in the presence of ammonium. No heterocysts are visible. (B) Phase-contrast image of filaments grown in the absence of any combined nitrogen source in the culture medium. Heterocysts, identified by arrowheads, are present at well-spaced intervals. (C) Epifluorescence image of the same filaments as in panel B. Excitation was at 510 to 560 nm (green), exciting phycoerythrin, and emission was greater than 600 nm. Heterocysts have negligible fluorescence, while vegetative cells have intense combined fluorescence from phycobiliproteins and chlorophyll a. Bar, 10 μm.

FIG. 2.

Photographs of plant partners showing the location of symbiotic cavities housing associated cyanobacteria. (A and B) Sporophyte (S) and gametophyte (G) generations of the bryophyte Anthoceros punctatus (A) and Nostoc colonies (nc) (B) within the gametophyte tissue. Bar, 1.0 cm. (C and D) Azolla caroliniana floating sporophyte leaves and trailing submerged roots (C). The cavity of a dorsal leaf housing symbiotic Nostoc/Anabaena is shown in a vertical position. A, Nostoc/Anabaena filaments; hc, Azolla hair cells (D). Photographs copyright Gerald Peters. Panel D reprinted from reference 142 with permission of the author. Bar, 0.25 cm and 0.25 mm in panels C and D, respectively. (E and F) Stem and leaves of the cycad Cycas taiwaniana (E) and ventral view and cross section of a coralloid root cluster from C. taiwaniana (F). Nostoc is present in a green annular ring (nc) within the root. Bar, 0.5 m and 0.5 cm in panels E and F, respectively. (G and H) Seedling of stoloniferous Gunnera manicata showing the location of the stem gland (sg) (G) and tangential stem section of a giant Gunnera chilensis showing location of the Nostoc colonies (nc) (H). Photographs copyright Warwick Sylvester. Panel G reprinted from reference 22 with permission of the publisher. Bar, 1.0 cm.

FIG. 3.

Metabolic interactions between heterocysts and vegetative cells. A lighter vegetative cell exchanges metabolites (thin lines) with a darker heterocyst bound by its characteristic thick envelope. The heterocyst has polar plugs at either end. Thick lines indicate metabolic pathways. The dotted line indicates a pathway whose existence is uncertain. Carbon dioxide is fixed in vegetative cells through the dark reactions of photosynthesis (PS), and the resulting triose is metabolized to pyruvate through the partial tricarboxylic acid cycle to isocitrate and then via IDH to α-ketoglutarate (αKG). The α-ketoglutarate combines with glutamine (Gln) via glutamate synthase (GOGAT) to form glutamate (Glu). In heterocysts, carbohydrate from vegetative cells enters the oxidative pentose phosphate (OPP) pathway to produce reductant (H:) used to support the activity of nitrogenase (Nase) to produce ammonium and concurrently yield α-ketoglutarate. Ammonium combines with glutamate, derived from the vegetative cell, through a reaction catalyzed by GS to form Gln. These reactions (if confirmed) should serve to reduce the level of α-ketoglutarate in the vegetative cell (small type) and increase it in heterocysts (large type). See the text for a discussion of the proposed cellular levels of α-ketoglutarate.

FIG. 4.

Distribution of randomly spaced heterocysts. (A) Calculated distribution of heterocysts if each cell differentiates with a probability of 0.1 (inspired by references 207 and 209). The graph shows the fraction of intervals with a given number of vegetative cells intervening between two heterocysts, according to the formula P_n = a(1 − a)ⁿ, where n represents the number of intervening vegetative cells and a is the probability that a specific cell differentiates. The average heterocyst spacing is (1 − a)/a, which in this case is one heterocyst every 9 vegetative cells. (B) Graphical derivation of the formula that predicts the distribution of heterocysts given random differentiation.

FIG. 5.

Spacing of heterocysts. Distances between heterocysts, measured as the number of intervening vegetative cells, are given, and their relative frequencies by actual count (solid line) and by calculation presuming no influence of one cell on another (dashed line) (see Fig. 4 for the equation defining P, the probable heterocyst frequency). The value of a, the probability that a cell differentiates, was taken to be the heterocyst density (heterocysts per total cells) from the given data. Graphs inspired by reference 215, using data taken from reference 221. The frequencies of interheterocyst intervals of length zero are not shown in the first two panels. (A) Wild-type Anabaena strain PCC 7120 grown on nitrate and shifted to no fixed nitrogen for 24 h (P = 9.5%). (B) patS Anabaena strain PCC 7120 mutant grown on nitrate and shifted to no fixed nitrogen for 24 h (P = 16.1%). (C) patS Anabaena strain PCC 7120 mutant grown on ammonium and shifted to nitrate for 96 h (P = 5.6%). Owing to the dispersed nature of the data under the conditions in panel C, the frequencies have been displayed as a rolling average, averaging three intervals at a time centered about the given number.

FIG. 6.

Physical interpretation of the one-stage model of heterocyst spacing: Each line represents a filament consisting of many cyanobacterial cells. The color within each cell represents its nitrogen status: the darker the color, the greater the amount of available nitrogen. (A to D) A filament is suddenly starved for nitrogen. Each cell draws on nitrogen reserves, postulated to be available in different cells to different degrees. When a cell has depleted its reserves to the extent that a critical level of starvation is reached (∗), it becomes committed to heterocyst differentiation. (E to G) Commitment is postulated to have two effects. The committed cell releases a signal (N) that diffuses to adjacent cells and prevents them from differentiating. In this interpretation, the signal is postulated to be a nitrogenous substance that feeds adjacent cells (symbolized by a darkening of their color). In addition, commitment prevents the committed cell from responding to its own inhibitor. Cells distant from the committed cells continue to starve, until one reaches the critical level. The position of the first cells that initiate differentiation is not critical to spacing. Spacing is determined by lateral inhibition.

FIG. 7.

Two-stage model of heterocyst spacing. (A) In response to nitrogen deprivation, four contiguous cells at similar stages in their cell cycles (shown here beginning cell division) initiate differentiation. The string of differentiating cells (dark) is resolved by competitive interaction to a single heterocyst (thick envelope). (B) Either nitrogen deprivation or the presence of a plant signal activates NtcA protein in all cells. The presence of activated NtcA protein and passage of the cell through a critical stage in the cell cycle induce hetR to a middle level of expression, mediated through HetF. Some initiate cells attain a PatA-dependent state (?; perhaps activation of HetR), characterized by a further induction of hetR expression. In these cells, PatS is highly expressed and diffuses to adjacent cells, where it is taken up and inhibits HetR expression. Cells where the positive intracellular HetR feedback loop (thick line) dominates become heterocysts. Cells where the negative intercellular PatS effect dominates revert to vegetative status. As they do, their own positive-feedback loop weakens as PatS inhibits HetR expression and those cells produce less PatS (dotted lines). Cells that are not passing through the critical stage of the cell cycle when nitrogen deprivation takes hold do not initiate differentiation and remain vegetative cells.

FIG. 8.

Photomicrograph of filaments of N. punctiforme in the N₂-dependent vegetative growth state and at different stages of the hormogonium cycle. Phase-contrast images of vegetative filaments (A) and hormogonia (B to D) are shown. Note the process of heterocyst differentiation by the end cells of the hormogonium filaments starting at T₄₈(C), with the appearance of mature heterocysts and an increase in size of vegetative cells by T₈₄(D); this is followed by vegetative cell growth and division and eventually by differentiation of intercalary heterocysts. Bar, 10 μm.

FIG. 9.

Photomicrographs of symbiotically associated cyanobacteria. (A and B) Nostoc associated with Anthoceros; (C) Nostoc/Anabaena associated with Azolla. Panel A is a phase-contrast image and panels B and C are epifluorescence images with excitation light near 510 to 560 nm and emission greater than 600 nm. Arrowheads point to heterocysts in panel C. Bar, 10μm. Compare the cell sizes to those in Fig. 1 and 8. Panels A and B reprinted from reference with permission of the publisher. Panel C reprodinted from reference with permission of the publisher (kindly provided by Gerald Peters).

FIG. 10.

Photomicrograph of Nostoc/Anabaena in association with Azolla. The bright-field image shows intact filaments, heterocyst frequency, and vegetative cell intervals. Bar, 10 μm. Reprinted from reference with permission of the publisher (kindly provided by Gerald Peters).

FIG. 11.

Electron micrograph of a portion of a Nostoc colony in association with Anthoceros. There are six heterocysts (H) distinguishable from nine vegetative cells in this field of view. Cells contain carboxysomes (Cb), where Rubisco is localized, and cyanophycin granules (Cy). Bar, 10 μm. Reprinted from reference with permission of the publisher (kindly provided by Yvonne Weeden-Garcia).

FIG. 12.

Model illustrating heterocyst strings (C), differentiation sites (F), and vegetative cell intervals (I). The filament is shown, where green cells represent vegetative cells and yellow cells represent heterocysts. A sample calculation for each of four parameters is explained further in the text. H_T is the fraction of total cells that are heterocysts (7/22 in this example). C is the contiguity, i.e., the average number of heterocysts per string (7/4 in this example). F is the fraction of cells that are heterocyst strings or foci (4/19 in this example). I is the ratio of vegetative cells to foci of differentiating cells (15/4 in this example).