Root Nodules

145 - 66 mya Cretaceous

Paleosols Flowering Plants Leaves Mycorrhiza Animals

Roots

As these flowering plants take over from the woody plants, there was a plethora of different shapes and sizes, from shrubs and bushes to annual, biennials and perennial plants. Each has different rooting structures, but they would be generally bushier then than the trees.

There are two main sorts of roots. Taproots, dominant in Dicots have a main central root upon with, small, lateral roots called root hairsched. Dicots include roses, geraniums, soybeans, carrots, and oak trees beetroot, parsley All Monocots, like marigold, and banana, have a fibrous root system, which were different from previous root systems, as they would have provided a lot more spread for aggregates and pores to increase aerobic metabolism a bigger rhizosphere. The soil must have changed when these smaller flowering plants took over from the trees.

What changes would come as a result of these more fibrous roots?

In addition to roots, monocots develop runners and rhizomes, which are creeping shoots. Runners serve vegetative propagation, have elongated internodes, run on or just below the surface of the soil and in most case bear scale leaves. Rhizomes frequently have an additional storage function and rhizome producing plants are considered geophytes Geophytes are plants typically with underground storage organs, where the plants hold energy and water, which we often call ‘a bulb’ This is a short axial body bearing leaves whose bases store food.

Fungal Association: We see the arrival of two more sorts of mycorrhiza associating with roots in this period, and more specific to certain plants.

Root Nodules

Could new Nitrogen-fixing nodules on plants help produce a wide variety of flowers to help explain Darwin’s abominable mystery? Or are they just a footnote?

In the late 19th century, it was discovered that legumes could establish a root nodule symbiosis with various soil bacteria. "Since ancient times, legumes (Fabaceae) have been known as “nitrogen accumulators” important for soil fertility (Hirsch, 2009). The anatomy of legume plants, including nodules, was already well known in the 17th century. However, it took until the late 19th century to discover that nodules provide a unique trait fixing nitrogen". (Huisman & Geurts 2020) The nodules on their roots had been dissected in the 17th century, but it was another 200 years before the two were put together.

“Nitrogen bacteria teach us that Nature, with her sophisticated forms of the chemistry of living matter, still understands and utilizes methods which we do not as yet know how to imitate.”
Fritz Haber, 1918, who invented the Haber-Bosch pressure cooking process to 'fix' airborne nitrogen as ammonia.

Many of us are familiar with plants we call legumes, like alfalfa beans, soybean, clover peas, and pulses. That is unless you live in the Antipodes, Australia and New Zealand, who do not have legumes. This indicates the late arrival of these plants after these countries and Antarctica had split off from Pangea around 100 -90 mya.

Nitrogen-fixing bacteria (NFB) became associated with certain plant roots, living inside them and usually creating nodules, and providing the plant with available nitrogen - more than from lightening. Some flavonoids , released by legume roots, attract rhizobia bacteria. These bacteria need high levels of oxygen to ‘fix’ nitrogen. It takes a lot of energy to break the bond between nitrogen atoms in the nitrogen molecules predominant in air.

Deep Homology

Originating around 110-100 MYA, this innovation suggests ‘deep homology’ in symbiotic N2-fixation driven by a single and necessary evolutionary innovation (Werner et al 2014). ‘Deep homology’ is where there are similar parts or processes in widely different organisms. Their anatomy may be very different but they use similar recipes or "algorithms" to fix the nitrogen. This term was first coined by a French zoologist called Geoffrey in the early 1820s but not accepted until the end of the 20th century. Yet, while coming from the same recipe, in some clades this innovation was lost. The complexity of nodulation in the nitrogen-fixing clade explains a single evolutionary origin (Griesman et al 2014) but that this single gain was “followed by massively parallel loss of nitrogen-fixing root nodules triggered by events at geological scale." (Velzen et al 2018)

Evolution

Was there one or many sources for N-fixing?

The symbiotic NFB invade the root hairs of host plants, where they multiply and stimulate formation of root nodules - enlargements of plant cells and bacteria in intimate association. Within the nodules the bacteria convert free nitrogen to ammonia, which the host plant utilizes for its development.

Several?

The “single gain–parallel loss” hypothesis is used for the following review. From the origination, species of eight lineages establish N-fixing nodules.

“Various species of the plants Fagales (Beeches and Birches) and the Cucurbitales (Cucumbers) and their sister in the monophyletic group or clade Rosales (Nettles) can establish a nitrogen-fixing nodule symbiosis with Actinobacteria of the genus Frankia and are named accordingly actinorhizal plants. The remaining two lineages, legumes (Fabaceae, Fabales) and Parasponia (Cannabaceae, Rosales), establish a nodule symbiosis with bacteria that are collectively known as rhizobia. The occurrence of two distinct classes of diazotrophic microsymbionts suggests that at least two major switches in microsymbiont partner have occurred. Frankia species have intrinsic characteristics to protect nitrogenase from oxidation and can fix nitrogen in a free-living form, whereas rhizobia are dependent on the mechanisms provided by the plant. In line with this, it is thought that Frankia was the ancestral microsymbiont in the nitrogen-fixing clade.(Heisman & Geurts 2020)

Common Ancestor?

Most researchers now believe nodulation did not evolve multi times as we once thought. “Nodules harbouring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages either with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies using Parasponia species (Cannabaceae), the only non-legumes that can establish nitrogen-fixing nodules with rhizobium…. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules…. Parallel loss of these symbiosis genes indicates that these non-nodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ~100 million years ago in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen fixing nodules in crop plants." (Velzen et al 2018) This means agricultural scientists have to concentrate on how this trait evolved only once when they try to find a way to replicate this trait in grain crops. To help them, it may be worth looking at why & how the evolution of N-Nodules happened when it did - and why some died out.

N-Fixing

N-fixing by plants would have been a major evolutionary advantage. We have seen that nitrogen-fixing bacteria (NFB) living in soil have been around for nearly 500 million years. and that that arbuscular mycorrhiza have been around nearly as long. Yet the two did not come together for another 250-300 million years. Nodule formed bacteria were a significant development.

The free-living bacteria in the soil, for 500 are called Azotobacter. However, this second nodule forming N-fixers include symbiotic bacteria, like Rhizobium associated with leguminous plants, Frankia, associated with certain dicots and certain Azospirillum species, associated with cereal grasses, which we’ll see in another 50 million years.

Mycorrhiza

It seems that successful nodule formation has something to do with arbuscular mycorrhiza. Some parts of mycorrhiza mechanism are found in NFBs. Studies indicate “the Arbuscular Mycorrhiza AMF + NFB combinations yield a considerable gain in P. gonoacantha (Mimosa) shoot weight compared with the treatments that only included inoculating with bacteria or AMF. The results also confirm that the treatment effects among the AMF + NFB combinations produced different shoot dry weight/root dry weight ratios. We conclude that AMF is not necessary for nodulation and that this dependence improves species development because plant growth increases upon co-inoculation" (Junior et al 2017)

Viruses

In particular, the effect of viruses on the legume-rhizobia symbiosis may impact soil nutrient cycles. Viruses in soil ecosystems are likely play important roles in the cycling rates of other nutrients, such as nitrogen (e.g., proteins and nucleic acids), sulfur (e.g., proteins), and phosphorus (e.g., polyphosphate, cellular membranes, and nucleic acids).

Viruses, also called ‘phages’, can infect a range of rhizobia and are indigenous to most soils and had been around since the origin of soils. Applying them to soils can reduce nodulation by rhizobia and also influence nodulation competition, leading to increased bacterial plasticity. Remember the viruses are lumps of D/RNA, moving close to bacterial genes. This movement of genetic material , called ‘transduction’, between distantly related species contributes to genetic plasticity in bacteria. It may well be this that provides novel advantages in terms of natural selection in a changing environment. Thus – in a strange way – the viruses provide a critical mechanism for diversification and speciation of bacteria. This has been said to account for the rapid spread of antibiotic resistance in bacteria. It is likely an important mechanism for transferring gene sets in various soil environments, although there are only a few studies on transduction rates in soils. .

Nod factor

The term ‘nodulating’ gives its name to the mechanism of how this symbiosis between bacteria and roots works. It is called the ‘Nod’ factor. Each nodulating species today is the result of over 100 million years of evolution that has accumulated further complexity. Those which survived, and dispersed widely at a later date, provided the wherewithal for the hugely successful class of nutrient symbioses that transformed the earth’s capability to grow crops.

Nodulation’ begins with the exchange of chemical signals . The plants secrete flavanoids– smelly sorts of chemicals aromatic compounds. These go into the rhizosphere and activate ‘nod genes’ in the Rhizobia bacteria. These nod genes help create a bacterial chemical signal, Nod factor. It is a heavy duty sugar that binds to a specific plant enzyme that works on the root hairs making them curl and trap Rhizobia. The bacteria move through the infection thread and usually end up in a cell where they fix the N. (Coleman et al 2018 p34) This soil signalling helps develop the ‘symbiotic’ relationship – one where both parties benefit. There are a lot of these relations in soil, where many of the creatures and other organisms are helping each other eg mycorrhiza.....

Fungal and bacterial symbioses

Legumes have developed fungal and bacterial symbioses. The fungal symbioses provide phosphate nutrients while the bacteria provide the other main nutrient, nitrogen. They have several similarities. The signals that initiate nodulation and mycorrhisation in legumes partially overlap. Legume genes have been identified that are required for the establishment of both AM and root nodule symbiosis. They are referred to as the common SYM genes, and provide a first glimpse at signalling cascades essential for nodulation and mycorrhiza development. “Based on these signalling cascades, it is tempting to speculate that the root nodule symbiosis, where fossil records date back to the late Cretaceaous, adopted and modified more ancient signal transduction pathways leading to AM formation, already been in place 400 million years ago. What are the common aspects of recognition of mycorrhizal fungi and Rhizobium by the host, and signalling that leads to an symbiosis between the two – and plant?" (Manchanda & Garg 2007)

SYM genes

SYM genes refer to:

Genes that contribute to a developmental pathway
SYM-3 and SYM-4 are genes that contribute to a developmental pathway that is redundant with a MEC-8-dependent pathway. Animals that are homozygous for mutations in mec-8 and either sym-3 or sym-4 have a defect that causes them to stop developing just before or after hatching. SYM-3 encodes a protein with an unknown function, and SYM-4 encodes a WD-repeat protein.
Genes involved in nodule formation
The formation of nodules on legume roots is a complex process that involves genes from both the host and the bacterial genome. A plasmid called Sym-plasmid, which is found in symbiotic bacteria that live in the root nodules of legumes, contains symbiotic determinants, including nodulation and nitrogen fixation genes.
Genes that may be related to terrestrial habitats
SYM homologues have not been found in green algae, which may indicate that SYM genes are related to terrestrial habitats

Why Symbiosis now?

Back to the same question, only now in molecular form: “What promoted the plant SYM (symbiotic) signal now?” Why did it not happen before? What was in the environment to make SYM signalling successful? Both mycorrhiza and free-living N-fixing bacteria had been around for ages, yet only now link up via SYM? Why did this period around 100mya encourage this nodule formation in legumes?

How do such complex mechanism like this evolve? There are three evolutionary stages of innovation: potentiation, actualization and refinement. During potentiation, mutations arise, which enable future development but do not the actual physical changes, analogous to the emergence of the N2-fixation precursor. The next stage actualizes the emergence of the rudimentary phenotype, the multiple origins of N2-fixation states of various types. Lastly, refinement stages add mutations to fine-tune the nodules (Quandt et al 2013)

The nitrogen reduction process that fixes atmospheric nitrogen into ammonia which these bacteria employ is basically the same chemical reaction that free-living bacteria employ - which evolved about 300 million years earlier.
N2+16H20 + 16ATP+8e-- = 2NH3 +H2 +16ADP +16Pi +8H+

Holy Grail

The world of mycorrhiza fungi and nitrogen free living nitrogen fixing bacteria coming together has been studied extensively because these symbioses have ‘great potential for agricultural applications'. I consider that it has been the 'holy grail' for agricultural scientists over the last 50 years to try and work out how to get these N-nodules into the big grain crops, like legumes do. 80% of all living land plants form AM, yet the nitrogen-fixing root nodule symbiosis with Rhizobia is almost exclusively restricted to legumes. Many agricultural scientists have looked to see how to transfer these N-nodules to a range of crop plants, and failed.

The Holy Grail of agricultural science is to find a way to biologically scion on to the stocks of grain plants the nodule N-fixing, mainly found in legumes. If only we could inoculate/inject/introduce nitrogen fixing bacteria into major food crops like grain, rice and corn, then – the story goes – our food problems will be solved. It was the Holy Grail 50 years ago, when I worked in agricultural research, and still is. The hunt for this intensifies as N fertilisers are becoming more expensive. Despite lots of research and many more recent genetic technologies, the grail remains elusive.

"Although exciting progress has been made, many big challenges must be overcome before the engineering of Bacterial N-Fixation in non-legume plants becomes a reality. " (Guo et al 2023)

Copy Recipe

Many agricultural scientists across the world are looking for ways to copy the recipe. Nitrogen is everywhere, and makes up 4/5 of our atmosphere. These bacteria turn nitrogen it into ammonia and other nitrogen compounds that help the plants grow- particularly to help make chlorophyll the essential pigment for absorbing light and gives plants their characteristic green colour. This is essential part of photosynthesis for plants to grow.

Nitrogen compounds are also important elements of how plants hold energy (ATP) and transfer their messages across the plant and into next generation – as DNA. These bacteria require a lot of energy (16 moles of adenosine triphosphate - ATP) to ‘fix’ each mole of nitrogen These organisms obtain this energy by oxidizing organic molecules.

This ‘fixing’ of nitrogen is a valuable asset for plant growers as it means they do not have to buy nitrogen fertilisers. The fertilisers are one the main contributors to global warming from farm sources – contributing about a third of the total ‘farm footprint’.

Fertilisers

The world uses vast amounts of nitrogen fertiliser. As part of the ‘Green Revolution’ massive amounts of nitrogen fertiliser are applied and soon show their benefit as really. If a field looks bright green, that means it has had loads of nitrogen fertiliser. Some say that it has enabled another two billion people to live on this planet. This may well be true. For some reason the same number - of about 2 billion people- are still badly nourished.

We are also increasingly concerned about these nitrogen fertilisers as they require a lot of energy to produce. The nitrogen is fixed using a process called ‘Haber – Bosch’, by making ammonia - a molecule with one nitrogen atom and 3 atoms of hydrogen. This needs vast amounts of energy, especially to break the di-nitrogen bond of airborne nitrogen. It is reckoned that around 1-2% of all the world’s energy use is to break this bond. Basically, ammonia is made in big pressure cookers, needing both pressure and temperature to push the atoms together. As we grow more concerned about global warming, these fertilisers are being seen as a major contributor to that.

Global Nitrogen Challenge

Nearly 40% of all GHG from farming worlwide come from fertilisers worldwide according to the Stern Report (Annex 7.g). Even growing biofuels like maize makes no sense when using so much nitrogen fertiliser to grow the crop. There are major concerns about the amount of nitrous oxides emitted from soils after fertiliser application, and much pollution to water courses bringing about over nourishment (eutrophication) causing algal blooms which kill off fish.. The United Nations talk of the Global Nitrogen Challenge which needs much more attention, both scientifically and politically. Would it not be wonderful if we could reduce that reliance on nitrogen fertilisers. Like legumes have done..

Search

Some legumes are better at fixing nitrogen than others. Common beans are poor fixers (less than 50kg /ha) and fix less than their nitrogen needs. Maximum economic yield for beans in New Mexico requires an additional 30–50kg/ha of N fertiliser. If beans are not nodulated, yields often remain low, regardless of the amount of nitrogen applied. Nodules apparently help the plant use fertilizer nitrogen efficiently.

In the search we have made significant advances in our understanding of the various tools of examination - morphology, molecular genetics, biochemistry, and physiology - of nodulation and the variability and conservation between different nodulating species. We are looking into nitrogen fixation genes (commonly referred to as nif genes) that encode the components of nitrogenase – that breaks the strong dinitrogen bond - and other proteins controlling the process. About 20 nif genes have been found. So a bit of GM is not doing the trick.

I would suggest to future researchers: Look at the circumstances which bought about the evolution of NFBs 100 mya. Find out about all those NFBs which have fallen by the wayside since, and see whether there is anything there to rejuvenate. There is no one ‘gene’ or process which will work but it is a stacked combination of processes with nearly 300 (nif) genes that have evolved over 100 my.

Different Ways of looking

There is also different ways of looking at the issue. There are three main sorts of NFBs – symbiotic ones like Rhizobia in the roots, those ‘associated with roots like Azospirilium, and free-living sorts like Azotobacter and Clostridium feeding off root exudates. Recent studies have shown bacterial ‘associations’ with various cereal crops like rice, wheat can be beneficial. But their contribution is harder to measure than symbiotic nodules. Researching bacteria that are ‘associated’ or ‘free-living’ gets away from patent issues as to who owns the plant end product. There is another way, and that is by incorporating Rhizobium into biofertilizers, which can be inoculated on the cereal plant host at planting. Another reason Rhizobia should be added to biofertilisers is their ability to ‘solubise phosphates’- releasing phosphates adsorbed to soil particles, making them available for plant growth (Hajjam et al 2016)

Legumes regulate the N-fixing symbiosis in response to exogenous nitrogen sources better than actinorhizal plants, like Frankia. Legumes evolved a mechanism to respond to changes in the amount of nearby soil N nutrients: the more there are, the less nodule formation. This would save the ‘carbon cost’ for full reliance on nodules - estimated to be more than 30% of the total photosynthates (Huisman & Guerts 2020)

Legumes clearly had massive advantages in that they could live without available nitrate nutrient and probably on low phosphate availability too. This means they could live in poor soils but, in the process of growing, turn them into much better ones. This would have enabled them to extend soils in dry conditions. This could well have been in association with the ‘soil crust’, described earlier in this same period.

Role of Soil Animals

What is the role of soil animals in this? After all, we are learning how all the soil components react with each other. Bacterial and fungal grazing by soil invertebrates such as Collembola and nematodes is known to increase the amount of soluble nitrate in the soil by breaking down the organic molecules - a process called N-mineralisation. “The microbial biomass and ratio of bacterial to fungal biomass tended to increase with increasing soil moisture. Collembola feeding activity and growth increased with increasing soil moisture conditions. Collembola significantly enhanced N mineralisation in soil at water potentials of −11.8 and −0.5 kPa. The greatest relative increase in N mineralisation attributed to Collembola occurred in the −11.8 kPa treatment. The change in contribution of the Collembola to N mineralization with soil moisture was most likely induced by changes in Collembola feeding activity and microbial community structure. The growth in body length of the Collembola was significantly greater at higher moisture conditions than at the lowest moisture condition, indicating that increases in both metabolic activity and biomass of the Collembola population contributed to the enhanced N mineralization." (Kaneda & Kaneko 2010)

This seemingly insignificant bit of research is quite important. That relationship between springtail and bacteria and fungi has been going on for hundreds of million of years. Further research could help show how that relation of springtails with free-living N-fixing bacteria may have changed with the advent of Nodule forming Bacteria. The hypothesis that collembola affect rhizobia and mycorrhizas of soybean (Glycine max) was studied in pot and field experiments. ”When a high density of the collembolan species Folsomia candida, was added to pots, the number of nodules per plant increased 52%". (Lussenhop 1996) However, they could not replicate the results in field trials, but put that down to the lower concentration of Collembola. Nor could they explain the result.

I'll have a go. We know collembola have been eating bacteria since 500mya and mycorrhiza since 400mya Perhaps there is something in the signalling (SYM above) which also attracts collembola to the nodules and they help distribute both fungal and bacterial spores - like thay always have. The potential would seem to be enormous and well worth more research.

I have been asked if I could measure mycorrhizal improvements, and there are ways to measure both ecto and endomycorrhiza. But I've come to the conclusion it is better to improve ‘Soil Health, and then the creatures and spores will follow. But I would like to test it properlys I was asked recently 'Do you thinkk Regenerative Farming is working?' I had to answer that I'd need a few farms, with controls of various sorts, on different soils over a period of 5-10 years to test the effects. When I was a research soil scientist there were probably a couple of dozen researchers in 4 or 5 research institutes to do this sort of thing. Now there are barely any in the UK, although more in the EU, America, India and China.

Let’ consider this further, as the rewards are immense. Instead of expensive, polluting N-fertilisers, is there a better way be help farmers improve the nitrogen uptake? The way to encourage these little soil creatures is keep the soil stable and moist - as in No-dog systems.

Imagine that, instead of all this stuff about gene editing and N-fixing cereals, we could just make sure the soil is looked after better and these insignificant creatures may just supply the answer.

UK Soil Animals Research

In the UK, we lost much of land-based research following the first sell-off – the Plant Breeding Institute - in the late 1980s. The then Prime Minister Margaret Thatcher believed that only ‘pure’ research should attract government funds. Any ‘applied’ research should be funded by the industry. Agricultural science is by definition an applied science so it was expected that ‘industry’ should fund most as it. This ‘near market’ research was first identified in the Barnes Report, a Minister’s Report which has never been published. I asked the minister responsible at the time Baroness Trumpington, but even she could not find a copy. With the exception of some sectors like potatoes, the UK agriculture industry did not pick up this sort of research.
We have lost three quarters of our land-based research stations (from 32 to 8) in the last 35 years. Could we reduce significant amounts of GHGs by the simple expediency of looking after our soil and encouraging those barely visible springtails to do a lot of the work - just as they have for 400 million years?

We could be doing looking at how N-fixing, phosphate solubising nodules impact in the wider soil as we develop biofertilisers. However when I have made the case for Azotobacter biofertilisers, I have been told by the 'main Nitrogen man' that it is 'being oversold'. I reply that the bacteria need to be 'stacked', ie. that several 'diazotrophs' are used together. Rhizobial inoculants, applied frequently as biofertilizers, play an important role in sustainable agriculture. However, inoculants often fail to compete for nodule occupancy against native rhizobia with inferior nitrogen-fixing abilities, resulting in low yields. (Suarez et al 2021) Much more research needed on biofertilisers which seems to have concentrated in the South rather than Northern soils. This is what .’public money for public goods’ could be doing. We will pick up on this later (under 'Now')

Rotation Rotation Rotation

We could learn from what people throughout history have done. When I was taught to be an agricultural zoologist we were often reminded that the best control of pests and diseases was 'rotation, rotation, rotation'.

The use of legumes in rotations has long been recognised. The three-field system is a regime of crop rotation in which a field is planted with one set of crops one year, a different set in the second year, and left fallow in the third year. A set of crops is rotated from one field to another. The technique was first used in China in the Eastern Zhou period hundreds of years round 500BC and was adopted in Europe in the medieval period. Rotations today

Imagine the effect of those legumes 100 million years ago? It would have led to much wider fertilisation of the soil.
Rhizobia bacteria can not only fix nitrogen but also solubise phosphates, and that spreads out from the nodules to make the soil a lot more fertile.
No wonder this is the holy grail for agricultural researchers. If somebody does come up with a scientific 'fix' they will be rich and famous. However, I wont be because I would suggest we have to look more holistically, at why and how N-nodules evolved in this period and the role of of the other soil inhabitants.

Mycorrhiza (AMF) (late Devonian)

Mycorrhiza (EcM) (Jurassic)

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