N-Fix - too good to be true?


November 2013
 
Berillo wheat variety
CC photo by Wheat initiative on Flickr
The holy grail of gene technology has long been the creation of plants which can fix nitrogen from the air to provide their own nitrogen fertiliser.  Scientific and financial resources have been poured into attempts to develop such GM crops in Britain. 

There's no doubt that world agriculture desperately needs to find an alternative to our current dependence  on artificial nitrogen fertilizers.  They’re expensive, energy- and fossil fuel-hungry, climate-, environment- and health-damaging. 

Natural conversion of nitrogen gas in the air to a form usable by plants is carried out by soil bacteria.  Scientists who are aware of the complexity of nitrogen-fixation in such bacteria have reservations about whether the process can be translated by GM into higher plants.  Besides the 20 genes involved (each structured to express in bacteria not in plant cells) and the enzymes needed (some of which are assembled from separately generated components and some of which incorporate iron and molybdenum ions), the reaction itself can only take place if oxygen is excluded.  The energy costs and metabolic contortions needed to achieve such novel reactions and conditions in a plant are so extensive that achieving a robust crop at the end of the day may be a “pipe-dream” (Institute of Science in Society). 

However, researchers at Newcastle University have been following another line of inquiry.  They've come up with 'N-fix' technology. 

According to the press release, N-fix is “a natural, nitrogen seed coating” which is “environmentally friendly and can be applied to all crops”.  It involves “neither genetic modification nor bio-engineering” but uses “naturally occurring nitrogen-fixing bacteria”.  N-fix is based on a bacterium found originally in sugar-cane, which can be induced to insert into plant cells: it potentially provides every cell in the plant with the ability to fix nitrogen from the air. 

This latest fertiliser-generating technology has passed through preliminary tests in the laboratory and glass-house.  The next stages will be field trials “to produce robust efficacy data”, and then regulatory approval.  It is anticipated that N-fix crop plants will be commercially available within the next 2-3 years. 

N-fix certainly seems to tick all the boxes: a natural bacterium doing its natural thing inside conventional plants, involving nothing more than a natural coating on the seed. 

Or, is there a box missing? 

The Institute of Science in Society tried to find out more about what N-fix actually involves.  It found:
  1. A paper published in 2006 describing a technique to induce nitrogen-fixing bacteria to colonise the inside of root cells in experimental model plants.
  2. A patent dated 2008 for the technique described in the 2006 paper
  3. A very recent power-point presented by 'Azotic Technologies' (the N-fix license-holding company), to promote the technology, although the information given is unreferenced and unpublished.
Beyond that, the only description available is what’s contained in the Newcastle University press release.  The media coverage of the announcement was wide, but the release specified that no additional resources were available, and requests for more information went unanswered. 

Thus, the story so far seems to be:

A technique was invented for enabling the invasion of plant cells by a microbe.  This was quickly patented, after which no information entered the public domain and no further quality control by peer review was applied to the science.  Two hundred species of novel microbe-harboring crops are currently being prepared for commercialisation.  N-fix is going to solve all the problems arising from the use of artificial nitrogen fertiliser, feed people in poor areas, and will be on our dinner plates in a couple of years. 

The missing box is, of course, safety testing. 

N-fix is being pushed as so natural that the question of safety just doesn't arise.  (COMMENT  If your memories of the GM issue go back to the 1990s, you'll be getting a feeling of déjà vu.) 

What sort of safety questions does N-fix raise? 

Natural infection of sugar-cane by natural N-fix bacteria follows physical damage to the root.  The bugs lodge in the walls of root cells and are transported into other parts of the plant in the xylem (water transport channels).  They are, therefore, naturally limited to sites outside the living cells.

Note.  In natural nitrogen-fixing legumes such as clover and peas, the bacteria dwell in protective nodules formed by the root: the microbes are in very close proximity to, but not inside, the root cells to which they supply nitrogen.

Azotic's 'natural' N-fix bacteria contained in its 'natural' seed coating, seem to manage to get into the plant without physical damage.  Once inside, Azotic's N-fix are able to breach the cell wall, enter the cell itself, and proceed to multiply and spread throughout the plant inside the plant's cells.  This seems to have no natural parallel. 

Thus, N-fix bacteria appear to have all the infectious qualities of the perfect pathogen and, indeed, they are known to cause plant disease when present at high levels, because they produce plant growth hormones. At low levels of infection the extra hormone can boost growth, but at high levels the plants are over-stimulated and become sickly. 

Pictures of Azotic's N-fix in the 2006 paper show plant roots cells chock-a-block with bacterial colonies.  Why aren't the plants succumbing to high plant hormone levels?  Perhaps the 'natural' N-fix bacteria in the 'natural' artificial seed coating aren't so natural after all? 

According to the 2006 paper, the mechanism for N-fix bacteria penetration through the cell membrane is by sucrose-induced 'endocytosis' (i.e. the cell 'swallows' the bugs by enfolding them in a membrane).  Sucrose-induced endocytosis is a natural mechanism by which plant cells rapidly take in sucrose from their surrounding.  It's clearly not natural for bacteria to sneak in by this route. 

Endocytosis of the N-fix bugs was found to happen only when the plants were inoculated with isolated bacteria.  This suggests that, once inside the plant cells, the rate of reproduction must be ferocious to fill up the root tissues as they were seen to do.  The possibility of rapid evolution to another, possibly pathogenic, variety under these circumstances is very real, and the transmission of  N-fix bacteria by sap-sucking insects is already known to occur. 

There are several other safety alarm bells ringing for N-fix.  Breaching of the cell wall seems to involve hijacking the plant's own cell-softening agents, 'expansins'.  Expansins are implicated in the allergenic potential of pollen grains.  Neither N-fix nor the protective mucoid coating it produces to block out oxygen have ever been a meaningful part of our diet.  The unprecedented quantities of N-fix bacteria inside the plant cells using up plant nutrients and generating their own by-products will affect the nutritional quality of the plant and will reduce the root exudates which are a vital part of soil health.  Moreover, the plants' metabolism and immune responses will alter significantly in response to the presence of the invading bacteria. 

The Institute of Science in Society points out that plants growing in healthy, chemical-free, organic soils are not limited by nitrogen availability: numerous free-living and symbiotic natural nitrogen-fixing microbes exist in organically-managed soils. 

Interestingly, there is a lesson from the far past on the dangers of artificial introduction of nitrogen-fixing bacteria into the cells of a higher organism.  In the 1970s, scientists on much the same track as N-fix but with less sophisticate resources at their disposal succeeded in introducing nitrogen-fixing bacteria into soil fungi which live in close association with pine tree roots.  The idea was to boost tree growth by supplying extra nitrogen direct to the roots.  When applied to pine trees, the concept worked to a degree.  However, in the case of one strain of fungus, the nitrogen-fixing modification made it pathogenic and it penetrated to all parts of the plant, and killed it.  Fortunately, this aberrant fungus never got our of the laboratory, or it could have destroyed whole forests. 

OUR COMMENT 


Is the novel insertion of whole bacteria inside plant cells just an extreme form of GM?  Plant-bacteria hybrids clearly pose a risk to your health, your food supply and the environment.  All safety-testing is being sidestepped.   

Get ready to demand full environmental stress trials which test both crop and microbe outcomes, and full life-long animal feeding trials followed by clinical trials before any N-fix technology comes anywhere near your dinner plate. 

Also there is just too much secrecy surrounding N-fix: demand transparency.
 

SOURCES
  • Nitrogen-fixing for All Crops Not the Answer, Institute of Science in Society Report, 23.09.13
  • World changing technology enables crops to take nitrogen from the air,  Nottingham University Press Release, 25.07.13
  • David Straton, The Genetic Engineering Debate, 1977
  • Edward Cocking, et al., 2005, intracellular colonization of roots of Arabidopsis and crop plants by Gluconacetobacter diazotrophicus, In Vitro Cell Developmental Biology 0 Plant 42
  • Raúl Pedraza, 2008, Recent advances in nitrogen=fixing acetic acid bacteria, International Journal of Food Microbiology
  • Ed Etxeberia, et al., 2005, Sucrose-inducible Endocytosis as a Mechnaism for Nutrinet Uptake in Heterotrophic Plant Cells, Plant Cell Physiology 47(3)
  • JA. D. Arencibia, et al., 2006, Induced-Resistance in sugarcane Against Pathogenic Bacteria Xanthomonas albilineans Mediated by an Endophytic Interaction, Sugar Technology 8(4)

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