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’

1. Inoculation of leguminous plants with the right symbiotic N-fixing microbial partner.
2. Introduction of nitrogen fixation genes directly into plants.
3. Engineering of non-legume plants to nodulate and establish symbiotic N-fixation.
4. Development of new tailored associations between N-fixing microbes and non-legume plants.
5. 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

1. Expression of prokaryotic genes in a eukaryote host. Solved
2. Sensitivity of nitrogenase to O2. Solved.
   • Mitochondria are specialised bacteria.
   • Maintain a low O2 concentration.
   • Contain abundant ATP.
3. 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

1. Will plants containing nif genes be capable of fixing sufficient nitrogen?
2. Will N-fixing plants be high yielding?
3. 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

1. Identify: Screen thousands of soil bacteria to identify potential N-fixing plant symbionts.
2. Reprogram: Genetically engineer strains to produce more nitrogen and better associate with plants.
3. Produce Nitrogen: 'Optimize' the interaction of the bacteria with target plants to fix nitrogen under diverse environmental conditions.
4. 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.