SEQUENCING AZOLLA

https://experiment.com/projects/azolla-a-little-fern-with-massive-green-potential
 http://www.azollagenome.net/


Wetlands biologist Asan Baghevzadeh, grabs a handful of the aquatic azolla plant in the Anzali wetlands on Iran’s Caspian Sea coast near Bandar-e-Anzali. (AP Photo/Hasan Sarbakhshian)

the AZOLLA GENOME PROJECT
http://www.reddit.com/r/science/comments/27oxbt/science_ama_series_im_dr_kathleen_pryer_a/
http://www.economist.com/news/science-and-technology/21604532-finding-genome-extraordinary-plant-aquatic-alfalfa
http://www.cleveland.com/opinion/index.ssf/2014/06/an_ancient_solution_to_a_moder.html
An ancient solution to a modern problem
by Kathleen Pryer  /  June 07, 2014

Fifty million years ago, the Earth was so warm that turtles and alligators thrived in lush forests at the poles. Much of the North Pole was covered in a rather less charismatic life form: the floating, duckweed-like fern, Azolla. Recent geological evidence from Arctic Ocean seabeds reveals 50-million-year-old sediments that are composed almost entirely of Azolla fossils for an 800,000-year span. This interval, known as the “Arctic Azolla event,” was a period when Azolla repeatedly blanketed the ocean surface, forming dense mats of vegetation. Then something really interesting happened. As these Azolla plants died and became part of the sediment, they took atmospheric carbon down with them. Global atmospheric levels of CO2 fell significantly, precipitating Earth’s initial shift from a greenhouse world toward the current icehouse climate that we’re now worried will melt.

Left: ACEX Lomonsov Ridge drilling location. Right: The 50 million year old core filled with brown layers of Azolla remains,
Left: ACEX Lomonosov Ridge drilling location. Right: The 50 million year old core filled with brown layers of Azolla remains.

Azolla is still with us, floating on the surface of ponds, lakes and rice paddies. Though tiny — one Azolla plant could comfortably sit on top of your smallest fingernail — it can double its entire body mass in less than two days. Some researchers think this makes it a promising alternative for biofuel production and carbon-capture efforts. But Azolla does yet another interesting trick — it captures all the nitrogen fertilizer it needs from the atmosphere around it. Since the dawn of agriculture, Asia’s farmers have known about, and deliberately exploited, the benefits of growing Azolla as a companion plant with rice. The floating fern thrives in rice paddies, fixing nitrogen and other nutrients, constantly improving the soil composition and providing a natural, green fertilizer that significantly bolsters rice productivity.

The secret here is that Azolla isn’t just a plant; it’s a “superorganism,” a symbiotic collaboration of a plant and a microbe. In a special protective cavity inside each leaf, Azolla hosts a microbe called Nostoc that spends its entire life converting atmospheric nitrogen into food for its host. Azolla and Nostoc have clearly demonstrated a prodigious ability to combat global warming, and to produce precious nitrogen that could help feed the world in a more sustainable way. Even though they have been co-evolving for nearly 100 million years, we­­ know very little about them and how they communicate. Wouldn’t it be great to understand this symbiotic relationship better, and to be able to understand the biological “conversation” between the host and the microbe? Because it is classified as a “lowly fern,” Azolla has been sidelined in plant genome studies. Repeated appeals to granting agencies for funding to unlock the know-how embodied by this superorganism have been met with responses like “too unconventional” or “too risky.”


But to sustainably produce food for a world population of more than 7 billion people — all while reducing pollution and greenhouse gases — we need to do some risky research. Novel ideas and innovative approaches that could reveal just how nature “does what it does” naturally might help to revolutionize current farming practices. The cost of continuing to do the same old, same old makes little economic sense. Specifically, we need a more sustainable source of nitrogen. By 2015, roughly 200 million tons of industrially produced, nitrogen-rich fertilizer will be needed to grow the world’s food, a process that will consume vast amounts of fossil fuel and exacerbate our CO2 problems. Azolla and Nostoc have great potential to reduce the world’s reliance on fossil fuels, while scrubbing a bunch of CO2 out of the atmosphere in the bargain. We’re not talking about a lot of money to do this. Genomic sequencing of this unique Azolla-Nostoc system would cost well under $1 million. That’s far less than the $8 billion each year that U.S. farmers pay for nitrogen fertilizer — much of which finds its way into rivers and streams, damaging delicate water systems. This small step toward potentially helping crops to use less synthetic nitrogen could benefit farmers’ bottom lines, the environment and the prices we pay for food. I’d like to see the genome of the Azolla superorganism sequenced so that we can understand the language that codes for the molecular machinery underlying this symbiotic partnership, and possibly tailor it to suit our needs. Knowing how this works might even enable us to engineer crops to fix their own nitrogen — an achievement that could truly revolutionize modern agriculture. Not often does such a small price promise a big gain.

{Kathleen Pryer is a professor of biology at Duke University. She is also president of the American Fern Society and past president of the American Society for Plant Taxonomists. For more information, check out the website azollagenome.net}

Azolla fern
close-up of Azolla

ORIGIN STORIES
http://www.azollagenome.net/blog/awesome-breakthrough-news-for-azolla
http://www.scientificamerican.com/article/how-scientists-uncovered-arctic-clues-to-a-past-where-a-tiny-fern-changed-the-planet/
How Scientists Uncovered Arctic Clues to a Past Where a Tiny Fern Changed the Planet
Researchers attempt to puzzle out how Earth got its ice caps
by Jennifer Huizen and ClimateWire  /  Jul 14, 2014

This may come as a shock, but only 55 million years ago, our planet had no polar ice caps; in fact, it nearly became a steamy, runaway greenhouse world, with CO2 levels exceeding 2,500 ppm. Then, all of a sudden, something intervened, causing a shift. Atmospheric carbon dioxide began to drop, steadily generating today’s world, with ice caps at both poles. But why did this happen? And better yet, could whatever triggered this drastic switch be used to temper today’s climate? Good questions. But the answers lay buried deep in the Arctic, hidden to researchers. “Even up to 2004, the Arctic was still a big unknown,” recalled Jonathan Bujak, a renowned palynologist, a person who studies dust and fine particles, such as fossil spores and pollen grains. “But as the ice sheets began to recede, we finally had our chance.”

A research project called the Arctic Coring Expedition linked to the Integrated Ocean Drilling Project headed north, hoping to find traces of this phenomenon to explain how it had happened. What the researchers found to be the likely culprit was a complete surprise. Encompassing the period of time in question was a 26-foot-thick column of fossilized ferns, a species so small it can fit on your fingernail but is capable of doubling its mass in two days. It is called Azolla. “Quite frankly, we were all shocked,” Bujak said. “What was this freshwater fern doing in Arctic waters, thousands of kilometers from shore, and how had it possibly had the power to begin to change our climate?” Bujak wasn’t the only person asking questions about Azolla. At the same time, a botanist from Duke University, Kathleen Pryer, was appealing to the government for funding to sequence the fern’s genome.

Sediment cores (Acex)
The cores contain layers of fossils and minerals

Crowdfunding a genome
Pryer worried that because no fern genome had ever been sequenced, what might be learned about these ancient plants—some of the oldest known vegetative of life forms on Earth—was highly limited. But because ferns don’t carry the same economic clout as major agricultural crops do, her requests were denied. This didn’t stop her. Pryer knew there was a global need to decipher Azolla‘s genetics. It had been used for thousands of years as a fertilizer in rice paddies, fixing nitrogen at unheard-of rates. Over time, people from all spectrums of science had already begun expanding its uses to include wastewater treatment, bioremediation, a source of food for both humans and livestock and even a biofuel. On top of all this, Azolla has the capacity to trap an astounding amount of carbon, at maximum rates sequestering 60 tons a year of CO2 per hectare, equivalent to the emissions of almost two hours of flight by a Boeing 747. “This tiny fern houses within it secrets on how to sequester carbon, fix nitrogen and ultimately increase food production without sending the Earth to hell in a handbasket,” Pryer said. “Someone had to open the gates for all other work, and we decided the only way to do this would be to look to the masses for help.”

Pryer’s team began a crowdfunding project with the help of the online site Experiment.com, hoping to gain $15,000 to sequence the fern’s genes. Two weeks ago, one of the largest sequencing laboratories in the world, the Beijing Genomics Institute, signed on to help Pryer’s dream come true. That means Azolla‘s playbook on how to save the Earth’s climate could become open access in as little as one year. Pryer said they have added an additional bonus round to their fundraising, to keep those already involved in the loop and also to prepare the perfect sample to send to China to be sequenced, providing the most beneficial results. The campaign ends this week. “We’ll have the Rolls-Royce of Azolla samples to send, thanks to this extra round,” Pryer said.

Ancient plant and a mysterious woman
It took a real adventurer to find Azolla. Jeanne Baret, the first woman to circumnavigate the globe, was likely the first Westerner to identify the plant. Beginning as a contracted house worker for Philibert Commerson, the naturalist for Louis XV, Baret continued her work for him when Commerson was allotted a stipend for an assistant on a circumnavigation voyage.

Disguised as a man, she helped Commerson collect a good deal of his plant samples. Her efforts needed to be particularity fierce, as she constantly was in the position of having to defend her masculinity against the sailors on board, who became increasingly suspicious about her gender. A 2001 paper by Vassar College’s Lizabeth Paravisini-Gebert recounts that “voyagers witnessed him [Baret] accompanying his master on all his expeditions amidst the snows and icy hills of the Straits of Magellan, carrying with courage and strength provisions, weapons and portfolios of plants.”

Eventually, Baret’s identity was discovered, but little fuss was made over the mishap by the superiors on board, and Baret continued her work. On the return voyage, Commerson died in 1773 in what is today Mauritius. Baret chose to stay on the island, marrying a former petty officer. When their samples returned to France, another famous French scientist of the time, Jean-Baptiste Lamarck, come across them and attempted to classify the tiny plant, mistaking it for a member of a family of flowering plants. He had, of course, not seen Azolla in all its glory, floating in open water. When Baret finally returned to France in the 1780s, she faced an unexpected homecoming. Due to the intervention of the ship’s master and some of Commerson’s friends, she was not only pardoned by the courts, but given an annual pension from France’s navy, which referred to her as a “femme extraordinaire.”

In 1878, German naturalist Heinrich Aton de Bary used Azolla to first illustrate his definition of the term symbiosis, or two unlike biological identities living together in unison. He used the example of Azolla paired with lichen to exemplify his new term but also noted a bacteria that seemed to be inherent to the fern, serving as an even more extreme example of symbiosis. With their spongy, lobe-like leaves only a fraction of an inch long, Azolla float on the surface of bodies of fresh water, dangling long tendrils below. In these leaves, Azolla have created a microenvironment, co-evolving with tiny bacteria called cyanobacteria for an estimated 100 million years.

Over time, the bacteria lost the ability to live independently of the fern, but their photosynthetic machinery increased its nitrogen-fixing capability by a factor of between 12 and 20. The bacteria became the powerhouse of the fern leaf, super-concentrating its photosynthetic power, while gaining shelter and a continuous food source from the fern. “To these bacteria, they know no other home than the inside of an Azolla leaf. To each species of Azollabelongs a different species of accompanying bacteria, and the bacteria is passed on from one fern to the next in their spores,” said Francisco Carrapico, a cell biologist and Azolla expert at the University of Lisbon. “It’s the ideal relationship.”

Being able to fix nitrogen so well also makes the fern a fantastic carbon sequesterer. But this still didn’t explain what Azolla was doing in the Arctic. A research team based at Utrecht University was set up to study the question, called the Darwin Azolla Project. This brought together many different types of scientists from around the world, and finally, a proposed explanation to Azolla‘s Arctic presence arose. “We remained dumbfounded,” Bujak said, “that was, until Carrapico piped up that we also needed to consider the fern’s carbon-capturing power in the context of this time period.”

Researchers hadn’t considered this property a likely factor in the fern’s Arctic success, and for good reason. Even with abundant carbon and nitrogen to consume, the size of the plant and its limited access to fresh water make it almost inconceivable that it could even survive in the Arctic, let alone muster up enough power and mass to change the Earth’s entire climate, saving our planet, perhaps, from a Venus-like, overheated oblivion. As with most good science stories, as soon as one question had been answered, another had to be asked. If Azolla had grown to such proportions that it could have affected the climate to such a degree, what had stopped the so-far invincible fern in its tracks and led to the initial climate plunge? The more the team looked, the more they found evidence that made the Azolla saga even more unbelievable.

The Arctic Ocean 49 million years ago (left) and 50 million years ago (right).
The Arctic Ocean immediately prior to the Arctic Azolla Event (left) and during the event (right). Illustrations from Barke et al. (2012).

ARCTIC AZOLLA EVENT
http://www.rcom.marum.de/English/Arctic_Coring_Expedition_2004.html
http://news.bbc.co.uk/2/hi/science/nature/5034026.stm
Arctic’s tropical past uncovered
by Rebecca Morelle

Fifty-five million years ago the North Pole was an ice-free zone with tropical temperatures, according to research. A sediment core excavated from 400m (1,300ft) below the seabed of the Arctic Ocean has enabled scientists to delve far back into the region’s past. An international team has been able to pin-point the changes that occurred as the Arctic transformed from this hot environment to its present cold status. The findings are revealed in a trio of papers published in the journal Nature.

Unlocked secrets
Until now, our understanding of the Arctic’s environmental history has been limited because of the difficulties in retrieving material from the harsh, ice-covered region. But in 2004, the Arctic Coring Expedition (Acex) used ice-breaking ships and a floating drilling rig to remove 400m-long cylinders of sediment from the bottom of the ocean floor. The cores were taken from the 1,500km-long (930 miles) Lomonosov Ridge, which stretches between Siberia and Greenland. The core holds layer upon layer of compressed fossils and minerals, which when studied can tell the story of millions of years of Arctic history. The bottom end of the cylinder helped scientists to uncover what had happened to the Arctic during a dramatic global event known as the Palaeocene-Eocene Thermal Maximum, which occurred about 55 million years ago. “This time period is associated with a very enhanced greenhouse effect,” explained Appy Sluijs, a palaeoecologist from Utrecht University in the Netherlands, and the lead author on one of the papers. “Basically, it looks like the Earth released a gigantic fart of greenhouse gases into the atmosphere – and globally the Earth warmed by about 5C (9F). “This event is already widely studied over the whole planet – but the one big exception was the Arctic Ocean.” The core revealed that before 55 million years ago, the surface waters of the Arctic Ocean were ice-free and as warm as 18C (64F). But the sudden increase in greenhouse gases boosted them to a balmy 24C (75F) and the waters suddenly filled with a tropical algae, Apectodinium. When current climate models were applied to this period of the Earth’s history, said Dr Sluijs, they predicted North Pole temperatures to be about 15C (27F) lower than the core shows.

Azolla covered large areas of the Arctic Ocean surface 50 million years ago
Azolla mats covered large areas of the Arctic Ocean between 49 and 50 million years ago

Blanket layers
The second of the three papers, led by paleaoecologist Henk Brinkhuis, also from Utrecht University, reports that the Arctic Ocean underwent another transformation about 50 million years ago. The water changed from salty to fresh, and the ocean became covered with a thick layer of freshwater fern, called Azolla. “We assume from climate models from the early Eocene Period that there was lots of fresh water coming into the basin via precipitation and giant Canadian and Siberian river run-offs,” said Professor Brinkhuis. “And, at a certain point, this gave rise to this whopping great growth of Azolla.” He believes the prolific growth of this fern, may be linked to the later drops in temperature in the area. “When you have so much of this plant in this giant sea, you have a mechanism to pump out carbon dioxide from the atmosphere. It is sort of an anti-greenhouse effect,” he said. “We argue that this sits right on the break from the really warm hot house period into the time when the ice house begins.”

Future predictions
Further up the core, the first evidence of ice formation emerges. “Five hundred thousand years above where the Azolla was found, we found the first drop stones,” explained Professor Brinkhuis, who is also a co-author on the third paper which details Arctic ice-formation. “These are little stones that come from icebergs, icesheets or sea ice. So it must have been cold enough to have ice. “Before we did this, it was thought that the ice field in the Northern Hemisphere only began about three million years ago; but now we have pushed that back to 45 million years ago. Although the data tells us how the world changed from one with greenhouse conditions to one with ice house conditions millions of years ago, it may also help scientists to predict what will result from the present changes in climate. Appy Sluijs points out that the data reveals that some of the climate models used to detail the Arctic’s history got things wrong; and, as they are the same models that predict our future climate, they may need adjusting. Kate Moran, lead author of one the papers and professor of oceanography and ocean engineering at University of Rhode Island, agrees: “We anticipate that our data will be used by climate modellers to give us better information about how climate change occurs and possibly where global climate might be leading. “Today’s warming of the Arctic can, in all likelihood, be attributed to mankind’s impact on the planet; but, as our data suggest, natural processes operating in the past have also resulted in a significant warming and cooling of the Arctic.”


Specimen of fossil Azolla (left) which is identical to those of the same age from the ACEX cores. Both have leaves (circled above in red) and tendrils (circled in blue) that are identical to those of modern Azolla (right). The illustrated fossil is from the Green River Formation of Garfield County, Colorado, dated between 50.5 and 55.5 Ma (million years) and was kindly provided by Dr Ian Miller of the Denver Museum of Science.

WHY FERNS?
http://www.ijehse.com/content/12/1/66
http://www.biotechnologyforbiofuels.com/content/7/1/30
http://theazollafoundation.org/azolla/the-arctic-azolla-event-2/
http://blogs.biomedcentral.com/gigablog/2014/06/16/guest-posting-lets-crowdfund-a-fern-genome-that-will-blow-your-mind/
by  / June 16, 2014

We need a fern genome. Why? Because considering the 470 million year history of plants on land, most species belongs to bryophytes, lycophytes, ferns and gymnosperms, which eventually yielded to the infamous flowering plants 90 million years (Myr) ago. Ferns, the third largest of these five radiations-and the sister group to all seed plants-is the only lineage for which a reference genome has not yet been sequenced. That doesn’t sound right, does it? Without a fern genome, we cannot fully comprehend the processes that govern the evolution of plant genes and genomes, and the patterns underlying major evolutionary transitions in land plants will still remain elusive.

Important guidelines in selecting candidates for genome sequencing have included economic relevance to humans, ability to answer important biological questions, and a small genome size.Azolla, an inconspicuous aquatic fern, passes all three criteria with flying colors. In this blog post, we will show you how Azolla genomic studies can not only drastically improve our understanding of plant evolution, host-symbiont interactions and nitrogen metabolism, but also have broad implications for sustainable agriculture and alternative biofuel production. We also present our citizen science crowdfunding attempt to initiate the Azolla Genome Project.

Azolla is a most unusual fern. It is a minute, aquatic plant that floats on the surface of ponds and lakes, bearing little resemblance to a typical fern. In addition, Azolla’s 740Mb genome is tiny compared to other fern genomes (740Mb vs. >10Gb), making Azolla an ideal first genome to be sequenced in ferns. Azolla is also a superorganism: within specialized cavities in each of its tiny leaves, Azolla harbors the nitrogen-fixing cyanobacterium Nostoc azollae. The Azolla-Nostocinteraction is unique among known plant-bacteria endosymbiotic associations in that the symbiosis is maintained even during sexual reproduction, whereby the Nostoc cyanobiont is vertically transmitted to subsequent generations. Because of such vertical transmission, the cyanobiont phylogeny mirrors the Azolla species phylogeny.

Azolla and Nostoc have co-evolved for nearly 100 Myr. Nostoc has entirely lost its autonomy, as it cannot be cultured when isolated from the host plant. Recent sequencing has revealed that Nostoc azollae is undergoing genomic downsizing, suggesting it might be in the early stages of evolving into a plant organelle devoted to nitrogen fixation. Loss-of-function in the cyanobiont is most prominent in genes related to nutrient uptake and metabolism, response to environmental stimuli and DNA repair. However, function has been retained in key symbiotic processes, such as nitrogen-fixation and cell differentiation.

Nevertheless, this somewhat well characterized Azolla-Nostoc symbiosis is just the tip of Azolla’s microbiome iceberg. In addition to Nostoc, an entire community of other bactobionts exists withinAzolla’s specialized leaf cavities, including other Nostoc species, Arthrobacter, Agrobacterium andCorynebacterium. Interestingly, some of these non-Nostoc symbionts appear to also have nitrogen-fixing capabilities, suggesting that they may contribute to Azolla’s capacity for nitrogen fixation. However, this microbial community is very poorly characterized: the diversity and identity of these other bacteria, their degree of interaction with their host, their mode of inheritance, and most critically, their role in enabling Azolla’s ‘superpower’ (as you will see next), have not yet been established.

This superorganism’s ‘superpower’
Why should we care about Azolla (other than for its fascinating coevolution story with its microbiome)? We believe that Azolla has enormous, yet long-overlooked, green potential. As we all know, we have screwed up our planet. The climate is getting hotter, water is getting less abundant and dirtier, energy is in crisis, and food production is unable to keep up with growing population demands. To help us get out of this situation, we need to learn how nature “does what it does” naturally. One peek into the buried Azolla literature and you would be amazed by its potential to help bring down atmospheric CO2 level, clean up contaminated water, provide alternative biofuels, and boost sustainable agriculture—a perfect storm!

Mountain building and changing oceanography further reduced CO2 which caused temperatures to fall even more after the Azolla Event.
Mountain building and changing oceanography further reduced CO2 which caused temperatures to fall even more after the Azolla Event.

Global warming and biofuels — Fossil data from the Arctic Ocean show that about 50 Myr ago, there was an enormous Azolla bloom spanning nearly a one million-year-interval, known as the “Arctic Azolla event”. It is estimated that Azolla covered 4,000,000 km2 of freshwater in the Arctic, which translates into over 1012 tons of carbon sequestration. The massive atmospheric CO2drawdown by Azolla during this period cooled the earth, and shifted the Early Eocene greenhouse world towards our present icehouse climate.
Can Azolla do it again? Azolla is fast-growing (capable of doubling its biomass every other day), easy to harvest, contains low-levels of lignin, and does not compete for arable lands with other crops, conditions that are perfect for biofuel-based, alternative energy production. Perhaps we can summonAzolla again to help us combat global warming?

Phytoremediation and wastewater treatment — Water shortage is undoubtedly one of the most serious crises we are facing today. The availability of potable water has been greatly impacted by industrial and agricultural pollution. In the past two decades, Azolla has been shown to be capable of hyperaccumulating a great variety of heavy metal pollutants—arsenic, gold, cadmium, chromium, copper, lead, mercury, nickel—to as much as 2% of the dry weight. In addition, Azolla can degrade 60-90% of bisphenolA (BPA) from aqueous solution, and when tested in swine wastewater, it is able to effectively decontaminate superfluous ammonium and phosphorus, thereby suppressing algal blooming and eutrophication. Azolla’s astonishing ability to remove various hazardous pollutants, grow rapidly and be easily harvested; makes this fern a very promising phytoremediation agent for wastewater treatment.

Nitrogen fixation and sustainable agriculture — to feed the world’s growing population, we will need roughly 200 million tons of industrially produced, nitrogen-rich fertilizer by 2015, a process that will consume vast amounts of fossil fuel and greatly exacerbate our CO2 problems. To make things even worse, over 20% of these applied nitrogen fertilizers will runoff into delicate water systems, fueling phytoplankton blooms and causing extensive ‘dead zones’. Azolla—with its symbiotic, nitrogen-fixing microbiome—offers an alternative and much more sustainable solution. Compared to the more familiar legume-Rhizobia system, the AzollaNostoc symbiosis fixes almost three times more nitrogen per hectare. Asia’s farmers have known this for thousands of years, growing Azolla with rice to bolster its productivity. Indeed, a recent field experiment has shown that incorporating Azolla, duck and fish into rice farming can more than double the net revenue. Rice will continue to be one of the most important crops in the world. With the recent publication of 3,000 rice genomes, we can definitely foresee a significant leap in rice genetic breeding. However, here we want to also champion Azolla—rice’s best companion—that, in addition to enhancing rice’s productivity, can also bring sustainability to the table.

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