User:Pfluorescens/Gunnera

Cyanobacterial symbiosis
Several species of Gunnera are known to host nitrogen-fixing cyanobacteria from the genus Nostoc, namely strains of Nostoc punctiforme. Nostoc punctiforme is the only species of Nostoc that readily engages in this intimate symbiosis. First thought to be a cyanobacteria from genus Scytonema or Anabaena, Nostoc provide fixed nitrogen to the plant, while the plant provides fixed carbon to the microbe. The bacteria enter the plant via glands found at the base of each leaf stalk and initiate an intracellular symbiosis. This intracellular interaction is not only unique among angiosperms; Gunnera are also the only land plants known to harbor cyanobacteria intracellularly. In addition to other symbiotic relationships between nitrogen-fixing bacteria and land plants that have been better studied, such as in the case of legumes and nitrogen-fixing rhizobia, examining the interaction between Gunnera and Nostoc may provide further insights to allow the creation of novel symbioses between crop plants and cyanobacteria, improving crop growth in locations lacking fixed nitrogen in the soil.

Evolutionary origins of the symbiosis
In thanks to fossil pollen records as well as phylogenetic analyses, Gunnera are believed to have evolved 90 to 100 million years ago during the Cretaceous period. This period was marked by an increased sea level, which caused the formation of seaways that would extend far into continents. Fossil pollen records show that Gunnera resided close to these shallow seaways, and high temperatures paired with heavy sunlight contributed to these waters being largely anoxic. The anoxic environment contributed to further denitrification of these already nutrient-deficient wetlands, and these conditions may provide evidence for the prevalence and success of nitrogen-fixing bacteria in these environments. It would be beneficial for Gunnera to form an association with Nostoc due to the bacteria's sheer abundance and its ability to fix some of the nitrogen gas that was lost from soil denitrification. It is currently unknown if this symbiosis was initiated and took the same form as it does today, due to a lack of reliable Gunnera macrofossil remains. The global loss of seaways and subsequent aridity accounted for the extinction of many animals that were exclusive to these habitats, and this large climatic shift may explain Gunnera moving to higher altitudes, while retaining that global distribution. Nostoc was certainly common in the brackish seaway environments of the Cretaceous period, and the cyanobacteria occupies a multitude of niches today, so it is unlikely that Nostoc evolved any specialized traits as a direct consequence of this symbiosis. It is more likely the case that the plant host evolved to develop specialized glands to recruit the bacteria symbiont, since Nostoc is able to photosynthesize in its free-living form and is not dependent on Gunnera for carbon. This proposed evolution of Gunnera is merely speculative, and the underlying mechanisms for bacterial recruitment are still unknown.

Host specificity
As a genus, Nostoc seemingly shows low host specificity since it is able to engage in symbioses with a plethora of different plants and animals. It was first believed that all species of Nostoc are able to successfully infect Gunnera, but in reality, the Nostoc-Gunnera symbiosis has stringent specificity. Nostoc punctiforme strains taken from Gunnera have little success infecting other plants, likely due to Gunnera being anatomically and phylogenetically distinct from other plant genera. Nostoc from the environment infect a plant individual; Nostoc is not vertically transmitted between plant generations. Nostoc does not spread through the plant from one gland to the next, each gland is infected independently and the Nostoc remains sequestered in the surrounding plant tissue. The initial attachment of the cyanobacteria to host seems to be non-discriminatory, but successful infection is dependent on the bacteria's ability to form hormogonia. The presence of fimbriae have often been the difference between successful infection and no infection in animal cells, but in the case of Nostoc and Gunnera, there are no links to physical bacterial structures determining infection success. There is little information on how Gunnera separates a potential symbiont from an invader; in addition, how compounds in the host's mucilage are used for communication with Nostoc are relatively unknown. Different strains of Nostoc have varying success in colonizing a Gunnera host; however, all known species of Gunnera have been found to form an endosymbiotic relationship with Nostoc. Nostoc punctiforme is a novel strain for studying the Nostoc-Gunnera symbiosis since it is able to infect all Gunnera species, and its genome has been completely sequenced.

Distinct secretory glands
In the case of most plant-cyanobacterial associations, the cyanobiont is situated extracellularly in the plant host, but with the help of specialized stem glands, this is not the case with Gunnera. The stem glands of Gunnera were initially believed to always form as part of the regular development of the plant; in actuality, these specialized glands are formed when the plant is faced with a nitrogen-poor environment. This initial misconception was due to the prevalence of secretory glands in naturally growing Gunnera plants. Cell division within the plant stems forms the secretory glands. Progenitor cells in the glands make papillae. These papillae are arranged together to form channels that connect the gland to the environment by bursting through the epidermal layer. About 5 to 8 papillae comprise the channel lining, with one larger papillae centrally located in the channel. These channels act as highways for Nostoc to travel and initially attach to the glands. In nitrogen-limited conditions, the plant redistributes resources from the leaves to the petioles to help form these stem glands, and in nitrogen-rich environments, sucrose stores contribute to the formation of "callus-like tissue," where the glands would otherwise form. Stem glands are continuously being formed at the base of new leaves, since older glands lose their ability to take on more bacteria. Phloem is charged with the transport of resources in and out of the glands. Anthocyanin buildup causes the glands to take on a reddish color. Gunnera stem glands are unique in plant-cyanobacteria symbioses since the glands do not form due to Nostoc colonization, but rather due to a nitrogen-limited environment. In other symbioses, there is an exchange of signals between the plant and the cyanobiont before the formation of infection sites, such as in the case of nodule formation in Rhizobium–legume symbioses. In naturally growing Gunnera plants, every single stem gland is occupied by cyanobacteria.

Infection process
After gland development concludes, these secretory glands produce a low pH mucilage that attracts Nostoc from the surrounding soil. This excretion contains lots of polysaccharides that do not appear to solely attract Nostoc, as other bacteria and organisms are drawn to it. The cyanobacteria first infect the gland surface and assemble. Next, the bacteria differentiate into long vegetative cell chains called hormogonia and enter the gland through intercellular channels. Nostoc hormogonia typically show positive phototaxis, but instead of moving towards the light, hormogonia extend into the dark interior of the gland. While the polysaccharide rich mucilage is a non-specific attractant for many bacteria, it is hypothesized that some phenolic compound created deep within the gland tissue is the actual initial attractant for Nostoc, and higher concentrations of this compound in the intercellular channels explains the negative phototaxis exhibited by the hormogonia. The ability for Nostoc to form the filamentous hormogonia is paramount for successful symbiosis, since cyanobacteria strains that are unable to form hormogonia cannot infect the host. Other types of bacteria become more scarce as Nostoc enters the gland, suggesting that Gunnera has an antimicrobial substance that Nostoc is specifically resistant to, implying some recognition mechanism the host uses to differentiate the symbiont from a threat. Host cells lining the surface of the channels secrete a mucilage containing carbohydrates that helps the cyanobacteria continue to grow inside the plant, while the bacteria is still extracellularly located. Nostoc causes the host channel cells to further divide. From inside the channels, Nostoc fills in the spaces between host cells and the Gunnera cell wall is dissolved, allowing the cyanobacteria to enter and reside intracellularly. It has been proposed that Nostoc makes an enzyme called cellulase to do this, because cell wall degradation only occurs in plants infected by Nostoc. Once the bacteria has colonized the host cells, the plant cell wall is regenerated, sealing the bacteria. Cyanobacteria are not known to produce enzymes that degrade plant cell walls, and there may be other microorganisms involved in helping Nostoc colonize the host cells. After intracellular infection is established, the bacteria differentiates into heterocysts, which are specialized cells that are the sites for nitrogen fixation. Inside the host cell, the bacteria is no longer in the motile, filamentous form, and completely differentiated into heterocysts, filling much of the host cell. The bacteria differentiates from vegetative cells into heterocysts at a higher rate than any other known cyanobacteria.

Resource exchange
The Nostoc-Gunnera symbiosis is facultative in that both the plant host and cyanobacteria can live without one-another, suggesting that the nutrient exchange must be largely beneficial to both host and symbiont. While initially a photoautotroph, fixing atmospheric nitrogen for its own needs, the Nostoc-Gunnera demonstrates how Nostoc transforms into a "heterotrophic supplier" of nitrogen to its Gunnera host. This change is marked by a decrease in nitrogen-storing cyanophycin in the heterocysts. In return, Gunnera supplies the bacteria with fixed carbon acquired through photosynthesis, in the form of soluble sugars. While Nostoc is able to undergo photosynthesis when it is free-living, it is reliant upon the photosynthate provided by Gunnera when it is in symbiosis, due to the dark environment of the plant interior. In the Nostoc-Gunnera symbiosis, carbohydrates composed of glucose and fructose specifically are supplied by the host plant to assist the cyanobacteria in its ability to fix atmospheric nitrogen by nitrogenase. This differs from other symbioses in which sucrose also contributes to cyanobacterial nitrogenase activity. Nitrogenase synthesis occurs exclusively in the heterocysts; the heterocysts encased in the host cell protect the nitrogenase from inactivation by oxygen. The anaerobic conditions within the heterocysts are perfect for nitrogen assimilation. These cells serve as sites for nitrogen fixation where atmospheric nitrogen gas is reduced to ammonia, before being supplied to the plant. Despite occupying only a small proportion of a plant's tissue volume, Nostoc is able to supply all of the nitrogen requirements for small Gunnera species, such as G. albocarpa, and most of the nitrogen needs for larger Gunnera varieties, such as G. tinctoria. Gunnera tinctoria plants that were infected with Nostoc punctiforme have shown higher growth and nitrogen levels than uninoculated plants. G. tinctoria mass and leaf size has been shown to increase tenfold after Nostoc colonization, which may indicate that the bacteria offers further benefits than nitrogen to the plant host. Gunnera 's unique ability to form an intimate relationship with Nostoc provides it an ecological advantage in nutrient poor soils.