Biotechnology in Plant and Environmental Health

The Role of Biotechnology in Plant and Environmental Health

Objectives

Provide definitions of biotechnology.
Highlight opportunities for biotechnological solutions to agricultural and environmental problems.
Use nitrogen fertilizer use as an example.

What is Biotechnology?

Biotechnology is technology that utilizes biological systems, living organisms, or parts thereof to develop or create different products.
It involves the exploitation of biological processes for industrial and other purposes, especially through genetic manipulation.
It's about finding biological solutions to human problems.

The Nitrogen ‘Problem’

Synthetic nitrogen fertilizers are environmentally very costly to produce.
Much of the applied nitrogen is lost to the air, water, and land, causing massive environmental problems.
N2O is 265 times more potent than CO2.
Nitrogen fertilizer use has increased more than 5-fold in 40 years.
Higher nitrogen inputs do not necessarily mean higher yield.
Excessive N-inputs have a range of negative environmental impacts.
Human activities create more reactive nitrogen than all natural processes combined.
Much of the reactive N is lost to the atmosphere or washed into waterways rather than taken up by plants.
Biogeochemical cycling of N and P have been radically altered by human activities.
Only a small proportion of applied P fertilizers is taken up by plants – most is immobilised in the soil or washed into waterways.
Nutrient Input Levels are Massively Unsustainable.
A concerted global effort to reduce N and P use is required just like that done for ozone depletion

The Nitrogen ‘Solution’

Take inspiration from nature (evolution) to better supply nitrogen to crops.
Biological nitrogen fixation: The conversion of atmospheric N2 to NH3, a form that can be used by plants.
Explore biotechnological solutions.

Challenges in the Nitrogen ‘Solution’

Biological N-fixation is restricted to bacteria and archaea and does not occur in eukaryotes.
Symbiotic N-fixation is restricted mainly to legumes.
Can biological or symbiotic N-fixation be leveraged to improve NUE in non-legume crops such as cereals?

Crash Course in N-Fixation and Nodulation

N-fixation is genetically very complex: It requires the concerted action of 22-57 different genes.
N-fixation is energetically costly: consumes 8 electrons and at least 16 ATP per N2 fixed.
Nitrogenase is very sensitive to O2 levels: Energy is often expended to maintain low O2.
Specific compounds (flavonoids) released by the plant are detected by the rhizobia.
Expression of nodulation (nod) genes is induced and nod factors are released by rhizobia.
Nod factors induce nodule formation in the plant.
The plant maintains an environment in the nodule suitable for N-fixation.
Highly species specific: One plant species, one ‘group’ of rhizobia.
The initiation, formation, and function of legume-rhizobia root nodules is intricate and complex.
Legume-rhizobia nodulation is a highly evolved, ancient relationship.

Five Ways Biotechnology can Solve the Nitrogen ‘Problem’

Inoculation of leguminous plants with the right symbiotic N-fixing microbial partner.
Introduction of nitrogen fixation genes directly into plants.
Engineering of non-legume plants to nodulate and establish symbiotic N-fixation.
Development of new tailored associations between N-fixing microbes and non-legume plants.
Leveraging soil ecology to promote free-living N-fixation and improving biological nitrogen cycling.

Inoculation of Legumes With Rhizobia

Inoculation is widely used on legumes in Australia and other countries where legumes have been introduced.
Concept of inoculants in Australia dates back to the 1890’s.
Legume inoculants were first commercialised in the 1950’s.
Early use of legumes in Australia was plagued by poor productivity.
Technical challenges were overcome to generate effective inoculants:
- Bacterial strain acquisition and maintenance.
- Formulation and carriers – e.g. peat.
- Carrier sterilisation.

Inoculation of Legumes With Rhizobia: Benefits and Limitations

Benefits: Easy to do. Robust - exploits ancient evolutionary relationships.
Limitations: Restricted to leguminous plants.
Ongoing challenges:
- Improving shelf life and formulation of inoculants.
- Finding ‘elite’ inoculant strains.
- ‘Old’ biotechnology

Engineering Nitrogen Fixing Cereals

Introduce nitrogenase genes directly into plants  Most direct approach.
Can N-fixation genes be transferred to a non-N-fixing bacterium? Yes!
Example:
- Klebsiella pneumoniae --> Escherichia coli (nif)
This discovery initiated 50 years of research into N-fixation in non-legumes.

Engineering Nitrogen Fixing Cereals: Challenges

Expression of prokaryotic genes in a eukaryote host. Solved
Sensitivity of nitrogenase to O2. Solved.
- Mitochondria are specialised bacteria.
- Maintain a low O2 concentration.
- Contain abundant ATP.
Complexity of nitrogenase synthesis.
- Nif genes have been successfully cloned into mitochondria.
- A few components of the nitrogenase gene have been expressed in plant mitochondria.
- Mitochondrial genes may be able to substitute for nif genes.
- A lot of work remains and we are probably 20 or more years away from N-fixing cereal plants.

Engineering Nitrogen Fixing Cereals: Future Challenges & Benefits

Will plants containing nif genes be capable of fixing sufficient nitrogen?
Will N-fixing plants be high yielding?
Will N-fixing plants be a biosecurity risk?

Benefits: Potentially robust – once optimised all plants containing nif genes should be able to fix nitrogen.
Limitations: Extremely technically challenging. System must be individually engineered and optimised for every crop.

Engineering Nodulation in Cereals

Challenge: complex genetics required to initiate and maintain symbiosis.
Many rhizobia contain all the genes necessary to form nodule symbioses with plants.
Do any non-legumes contain genes for establishing symbioses with microbes?

Engineering Nodulation in Cereals: Mycorrhizal Associations

The same genetic pathway is involved in rhizobia and mycorrhizal symbioses.
Mycorrhizal associations (450 million years) evolved long before nodule symbioses (~110 million years).
Recruitment of the mycorrhizal symbiosis signaling pathway was a key step in the evolution of nitrogen-fixing nodulation.
The majority of land plants contain this pathway.

Engineering Nodulation in Cereals cont.

Can we engineer the common SYM pathway to work with N-fixing rhizobia?
Can we engineer N-fixing rhizobia to produce mycorrhizal-like symbiosis signals?
Rhizobial and mycorrhizal fungal symbioses induce the same genetic pathway in plants.
Rhizobia and mycorrhizal fungi use very similar or even identical signal molecules to initiate symbiosis (lipochitooligosaccharides).
Benefits: Building on ancient evolutionary relationships.
Limitations: Need to manipulate complex plant genetics that are not fully understood.

Engineering N-fixing Bacteria to associate with Cereals

Many free-living, epiphytic, and endophytic bacteria can fix nitrogen.
Inoculation of plants with these bacteria is not straight forward.
Products like Ultrisha® N, Azotobacter salinestris, and Methylobacterium symbioticum are being explored.
It is claimed that the use of these microbial products allows reduction in N fertilisation of ~30 Units per hectare.

Engineering N-fixing Bacteria to associate with Cereals - Process

Identify: Screen thousands of soil bacteria to identify potential N-fixing plant symbionts.
Reprogram: Genetically engineer strains to produce more nitrogen and better associate with plants.
Produce Nitrogen: 'Optimize' the interaction of the bacteria with target plants to fix nitrogen under diverse environmental conditions.
Distribute at Scale

Engineering N-fixing Bacteria to associate with Cereals - Benefits and Limitations

Benefits: Bacteria are highly diverse and easy to genetically manipulate. N-fixation is present in a very diverse range of bacteria.
Limitations: Gains in terms of nitrogen supply are currently very modest. Requires the use of genetically modified bacteria.
53 trials across 10 US states were conducted using a range of associative N-fixing products – including Ultrisha and ProveN; 51 of these trials showed no yield benefit

Over-promising and Under-delivering

Ultrisha® N example shows the bacterium in this product cannot fix nitrogen – it is marketed globally as being able to.
Proven® 40 example shows the published rate of N-fixation at a level inconsequential for plant growth.
Products backed by poor science risk damaging the credibility of the biologicals industry more broadly.

Improving Nitrogen Use Efficiency by Harnessing Soil Ecology

Natural biological processes control the availability of many soil nutrients but are frequently overlooked.
High levels of mineral fertilizer overshadow or inhibit biological activities.
Nitrogen supply to plants can be enhanced through soil ecology by two primary mechanisms:
1. Activity of N-fixing bacteria (free-living and plant-associated).
2. Enhanced nitrogen cycling through soil food web dynamics.

Microbes and Soil Nutrient Cycling

Nitrogen-fixation: N2 NH4^+
Nitrification: NH4^+ NO3^-
Denitrification: NO3^- N2O + N2
N-cycling: NH4^+ Org-N
Mineralisation; Assimilation
Nitrogen cycling is driven exclusively by complex interactions between microorganisms.
Abundant and diverse microbiomes are very efficient at retaining and cycling nitrogen.
The right soil physical and chemical conditions are required to improve biological N-supply.

Improving Nitrogen Use Efficiency by Harnessing Soil Ecology cont.

In symbiotic N-fixation, the plant maintains the environment for optimal N-fixation.
In free-living N-fixation, the environment determines the conditions for N-fixation.
Management practices play a critical role in determining this environment.
Mechanism 1: leveraging the activity of free-living N-fixing bacteria.

Improving Nitrogen Use Efficiency by Harnessing Soil Ecology cont. 2

Mechanism 2: Enhancing nitrogen cycling through soil food web dynamics.
A complex and diverse soil food web means that most mineralized nitrogen is recycled and not lost to leaching or volatilization.

Improving Nitrogen Use Efficiency by Harnessing Soil Ecology - Fast vs. Slow Cycle

It is possible to influence the balance of the soil food web through modifying inputs of ‘soil food’.
Balance in the soil food web can influence rates of N-mineralisation.
Rates of N-mineralisation can to some extent be modulated through manipulating the soil food web

Improving Nitrogen Use Efficiency by Harnessing Soil Ecology -Benefits and Limitations

Benefits: Leverages natural processes working with nature. Genetic modification is not required. Can work on any crop/soil.
Limitations: Ecological systems are very complex. Our understanding of how to manipulate soil ecosystems is limited.

Summing Up

There are many different potential biotechnological strategies to improve nitrogen supply to plants.
Each strategy has its own unique benefits, limitations, and challenges.
Huge potential exists to harness biotechnological solutions to the nitrogen ‘problem’.
Ecological or agroecosystem-based approaches have the greatest potential to improve N-use efficiency.