Our Problem: Waterlogging
When people think of global warming and climate change, drought is one of the most common effects that comes to mind—evidenced by the amount of research and programs based on drought. Scientists, and even many iGEM teams, have focussed a lot on the effects of drought.
However, climate change models are also suggesting that the frequency and severity of heavy precipitation may increase in the near future all over the world—waterlogging is an unfortunate effect that climate change has on our planet.[1] Global warming causes waterlogging to be even more unpredictable in its nature, making it troublesome to solve. Around 10% of the arable land in the world is affected by waterlogging. This results in a loss in yield in barley, lentils, chickpeas—it can even result in upto 50% loss in wheat.[2][3]
Waterlogging refers to an excessive accumulation of water in the soil near plant roots that negatively affects plant and soil health. In plants, waterlogged soil causes exaggerated stress responses, releasing harmful amounts of ethylene. This results in the destruction of macromolecules and inhibition of photochemical processes—causing low crop productivity, loss of chlorophyll, and cell death.[4][5]
Moreover, waterlogged soil is often depleted in nutrients due to leaching and is enriched in deposited salts. Together, this makes land affected by waterlogging unusable in the long term.
Having personally witnessed the effects of waterlogging in a teammate’s farm—a ruined harvest and sludge-filled land—the team was even more motivated to tackle this issue. Looking into the issue further, we realised that the human population is expected to grow dramatically and farmers must increase their production by a staggering 60% by 2050 in increasingly limited arable land. Preventing land degradation due to waterlogging becomes an immediate necessity. The loss of 16,000 to 26,000 million kg of food can be prevented in India alone, by just fending off waterlogging.[6][7]
How did we go about choosing our project?
We began in January by dividing our team into six groups. Each group brainstormed to come up with ideas. These ideas would be worked upon in terms of a rubric we made, focussing primarily on feasibility, relevance, and innovation. Through a series of discussions amongst ourselves and professors in our institute, we noted the potential difficulties and tried to incorporate inputs that we received. This process revealed several fatal flaws in certain ideas, and the team voted to drop them in favour of working on other ideas with more potential. Rinse and repeat; this led to us having three ideas at hand. We then created a detailed rubric, styled in the form of the iGEM Medal Criteria, to help us analyse the merits and limitations of each idea. Ultimately, we conducted a final presentation of ideas, and the team voted Hydrazome as the idea we’d work on for the iGEM competition.
A Concise Description of our Project
Our project Hydrazome aims to help plants deal with the stress of waterlogging. When water collects and fills up the gaps in between the soil, it creates a hypoxic environment. This environment then triggers the enhanced synthesis of an amino acid called ACC (1-aminocyclopropyl-1-carboxylic acid) in the roots of the plant. While part of the ACC gets secreted through the roots, the remaining is transported to the shoot region, where it gets oxidised to form ethylene. [1][5]
Initially, this ethylene is synthesized in small amounts, which is beneficial to combat these stressful conditions - the first peak. Prolonged exposure to stress leads to a sharp increase in ethylene production - the second peak.[5] This second peak of ethylene is what is detrimental to the survival of the plant.
Certain plant growth-promoting rhizobacteria (PGPR) can produce an enzyme known as ACC deaminase (acdS) - which breaks ACC down into ammonia and α-ketobutyrate.[8] This reduces the levels of ACC, which can get transported to the shoot and converted to ethylene. We plan on introducing this structural gene into our chassis organism - Azospirillum brasilense sp7. This gene would be primarily expressed under conditions of waterlogging by placing it under the control of a hypoxia-inducible promoter.
We’re investigating the role of exopolysaccharides produced by rhizobacteria in helping them adhere to plant roots. We will also be looking into its role in nutrient and water retention for the purpose of extreme condition resistance.[9]
For the purpose of biosafety, we will be working on a toxin-antitoxin system to prevent horizontal gene transfer.
Why is Hydrazome a biofertiliser?
This year our team decided to work on making our project into a product that could be conveniently used by farmers. Azospirillum brasilense is already prevalent in the biofertiliser industry.[10][11] However, we wanted to enhance its utility and also allow it to be used to overcome stressful conditions faced by the plants.
Our chassis organism Azospirillum brasilense has been shown to associate with the roots of multiple species of plants.[12] This, in addition to the fact that genetic manipulation of plants could be more time-consuming, led us to the conclusion that using a microbial chassis would be more effective than a plant chassis. This would allow us to have a ‘one size fits all’ solution - avoiding the need to modify multiple species of plants.
By virtue of it being an organism, Azospirillum has the ability to provide the plant with several useful chemical compounds. These compounds serve the roles that are otherwise fulfilled by chemical fertilizers.
Azospirillum - Our Smart Chassis
Azospirillum brasilense is a well characterized plant growth promoting rhizobacterium that is found associated with plant roots.[12] It is a microaerobic diazotroph - this means that in conditions of limited oxygen it is able to fix atmospheric nitrogen into ammonia that plants can utilize.[13] Using its flagella Azospirillum is able to swim to such microenvironments in the soil - it is microaerophilic. Phytohormones secreted by Azospirillum help plant roots in the enhanced absorption of nutrients and water.[14] E. coli has only five chemoreceptors, while our chassis, on the other hand, has forty-eight receptors that guide its movement through the soil, making it more sensitive to environmental changes.[15]
Azospirillum can naturally induce the production of antioxidants in plants - thus helping them overcome the stress generated by ROS.[16] It facilitates the absorption of certain nutrients by plants, which is otherwise inhibited during conditions of salinity.
Why Genome Integration?
During our preliminary research we found that a sister species, Azospirillum lipoferum 4B contains the acdS gene in a chromid. However, it was seen that A. lipoferum could lose this chromid when it was grown for many generations.[17] This is why we have decided to introduce acdS into the genome, from where it is less likely to be lost. This also overcomes the challenge of requiring an antibiotic marker in order to maintain the plasmid.
A. lipoferum is also comparatively much more difficult to work with and maintain in a laboratory as it requires continuous subculturing. This is why we decided to work with A. brasilense as our chassis organism.[18]
Project Background
Climate Change and Heavy Precipitation Projections
Observations of heavy precipitations across very diverse regions in the world have been occurring at a much higher rate recently, as predicted by early theory and model predictions from decades ago.[19] Climate models from the recent past indicate that increasing temperatures will intensify the water cycle - which will increase evaporation. [20] This then will result in more recurring and severe storms in certain areas, but will also contribute to drying over some other areas.[21] As a result, storm-prone areas will be likely to experience increases in precipitation and an increased risk of flooding, whereas areas far away from storm tracks will experience less precipitation and an increased risk of drought.[20]
Identifying drought and flooding as being the two sides of the disastrous impact of climate change, our project was developed to keep both sides in control. Goal 15 of the UN’s Sustainable Development Goals - Take urgent action to combat climate change and its impacts - inspired us to direct our solution’s design in such a way that our genetically modified biofertiliser is useful in all circumstances - for healthy plants, as well as for plants stressed because of drought or waterlogging.
Azospirillum’s natural ability to enhance drought-induced stress in plants is well-established and experimented. [23] In addition, our investigation into the role of exopolysaccharides in the water-holding capacity of Azospirillum-inoculated soils would help us enhance this ability. The primary engineering objective of our project - the introduction of hypoxia-regulated ACC deaminase - will help plants be more resilient towards waterlogging and flooding induced stress.
Enzymatic Responses to Biotic and Abiotic Stresses
The role of ethylene in biotic and abiotic stress responses is well characterised. [24] Stress responses in plants eventually affect the plant through ethylene as the effector. Stress induces the activity of S-adenosyl-L-methionine synthase [25], which then upregulates the production of S-adenosyl-L-methionine (SAM). SAM is then converted to ACC with the help of the enzyme ACC synthase. ACC synthase hence increases the amount of substrate ACC available to produce higher amounts of ethylene in plant tissues. [26] In the case of waterlogging, the hypoxic environment created in the rhizosphere induces stress responses in plants.
Ethylene Peaks During Waterlogging
The phytohormone ethylene is an important regulator of normal plant growth and development. A multitude of stressful conditions like heavy metals, extreme temperatures, excessive or insufficient water lead to an excessive amount of ethylene being produced as a stress response. One of the models that describe “stress ethylene” production includes ethylene being produced in two peaks. The first ethylene peak is of a much smaller magnitude compared to the second peak and is primarily responsible for initiating plant defense and protection mechanisms. This small peak is thought to consume all the pre-existing pool of ACC in stressed plant tissue. The second peak of ethylene is of a comparatively much larger magnitude and is due to the synthesis of additional ACC by the plant in response to stress. This peak is generally detrimental to plant growth and is often involved in initiating processes such as senescence, chlorosis and leaf abscission, ultimately leading to the death of the plant. [5]
Loss in Crop Yield Due to Waterlogging
As mentioned in brief above, waterlogging of soil creates multiple hypoxic microenvironments within the rhizosphere. Waterlogging has also been found to promote soil nitrogen loss through denitrification [27], reduced soil nitrogen mineralisation [28][29]. In general, since nitrogen is the limiting nutrient for the growth of crops, these aftereffects have a disastrous impact on crop yield. [30]
Agriculture is the main occupation in India, with 70% of the rural households still depending primarily on agriculture for their livelihood and 82% of farmers being small and marginal. It is one of the most critical sectors in India, contributing to 18% of India’s GDP.
To look at an example of the crop loss caused due to waterlogging in India[31]:
“The extent of the yield reduction is influenced by the physiological stage of growth at which waterlogging occurs, the time and duration of waterlogging, temperature, the fertility of soil and the kind of crop (Gupta et al., 2006). The cotton is very sensitive to waterlogging and 77 percent yield reduction due to this, the corresponding figures for paddy, wheat and sugarcane are 42 per cent, 38 per cent and 61 per cent (Joshi, 1994).”
Waterlogging is a double blow to our farmers: it decreases agricultural productivity by 30% and leaves a saline layer that makes the land untenable for a year. In addition to this, waterlogging is highly unpredictable, which makes any preventive measure impractical; this can result in farmers losing their entire livelihood in one blow. Such a financial shock cannot be absorbed by the majority of our farmers and any effort alleviating this problem is very important to our farmer community.
12% of the world’s arable land is estimated to be waterlogged frequently, leading to approximately 20% crop yield reduction.[32] Waterlogging in the near future is predicted to increase due to global climate change, especially in irrigated regions such as the Yangtze Watershed and other areas in China as well as irrigated areas of the United States, Pakistan, Argentina, and Europe. Therefore, tackling the issue of waterlogging is also a global challenge and needs to be addressed.
In spite of being an agricultural powerhouse, India does not score well on the Global Food Security Index (2021). Goal 2 of the UN’s Sustainable Goals - end hunger, achieve food security and improved nutrition and promote sustainable agriculture - motivates us to ensure that our project is planned with the needs of our country’s farmers in mind. They are our primary stakeholders, and our primary consumers.
ACC: From Plant Roots to PGPB
Under hypoxic conditions caused by waterlogging, plants respond to this stress with an increased amount of ACC being produced in the roots. A significant amount of this is exuded from plant roots, following which the ACC can be hydrolysed to ammonia and α-ketobutyrate by ACC deaminase-producing bacteria. Eventually, the amount of ACC gets decreased inside plant tissue and an equilibrium is maintained through continuous exudation of additional ACC into the rhizosphere.
ACC oxidase (which converts ACC to ethylene) and ACC deaminase (which converts ACC to α-ketobutyrate) compete for access to ACC. The amount of ethylene in plants under waterlogged conditions was shown to be reduced if ACC deaminase reacts with ACC before ACC oxidase [5]. Depending on the concentration gradient of ACC across the soil, ACC which gets exuded, is deaminated to form α-ketobutyrate and ammonia. Through this method, symbiotic bacteria which live in the rhizosphere, lower the levels of ethylene stress (by 60%–90%) [1] by producing ACC deaminase and contribute to the growth and development of plants under waterlogged conditions.
ACC as a Source of Nutrition
As per the foundational paper authored by Bernard Glick et. al 1998, - A Model For the Lowering of Plant Ethylene Concentrations by Plant Growth-promoting Bacteria [33] - plant-growth-promoting rhizobacteria help reduce stress ethylene levels by degrading ACC into α-ketobutyrate and ammonia. An important fact to consider here is that the breakdown of ACC provides the rhizobacterium with a new nitrogen source to utilise. We performed an analysis of the enzymatic breakdown of ACC in Azospirillum brasilense Sp7, and came up with the following pathway:
Project Design
Hypoxia Inducible Promoters
Our project deals with the expression of genes under conditions of waterlogging, and hypoxia inducible promoters (HIP) play a pivotal role in achieving such regulated expression.
During our literature review we were unable to find an existing well characterized HIP for Azospirillum brasilense sp7 and therefore we decided to work three probable promoters - exaA, fdhF and FNR promoter.
The exaA promoter is present upstream to the quinoprotein alcohol dehydrogenase gene in A. brasilense sp7. The activity of this promoter is found to be induced significantly in the presence of glycerol, fructose, primary and secondary long chain alcohols(C4-C6). Being a motile microaerophilic bacteria, A. brasilense is expected to switch to fermentative pathways under hypoxic conditions and produce alcohols which are expected to induce the activity of the exaA promoter. During waterlogging, the plant roots might also undergo anaerobic metabolism which might lead to the exudation of such inducers into the rhizospheric environment.[34]
The fdhF promoter is a well characterized E. coli hypoxia promoter [35] that is present on the iGEM registry. We decided to test the inducibility of this promoter in Azospirillum brasilense by using a lacZ reporter construct and cloning it in a broad host range mobilizable vector.
The FNR promoter is a hypoxia-inducible promoter found in the FNR (Fumarate and Nitrate Reductase) regulon native to E. coli. The FNR protein is a transcriptional activator for a number of genes that are involved in anaerobic respiration. Azospirillum brasilense has nitrogenase genes [36] that are regulated by the FNR/CRP complex [37] and so it was hypothesized that this promoter would show regulated activity in Azospirillum.
Genome Integration - How?
We have planned on performing genome integration by using suicide vectors compatible to A. brasilense sp7 and introducing our desired constructs into the regions of redundant/duplicated genes in the genome. The beta lactamase gene was found to be highly amplified in the Azospirillum genome, scattered in the chromosomal as well as plasmidic DNA [38]. Therefore the sites of this gene might be suitable for the integration. pSUP202 is a mobilizable suicide vector that has previously been used for gene knockouts in the genome of Azospirillum brasilense sp7 and is therefore the preferred choice of vector [39].
The idea behind genome integration is to amplify the redundant gene in two fragments and insert our construct in between these two fragments and then ligate it into the pSUP202 backbone. Once pSUP202 is conjugated into Azospirillum it is expected that homologous recombination will take place and our intended construct will be exchanged into the genome.
Exopolysaccharides
Exopolysaccharides (EPS) are heteropolysaccharides secreted by many bacterial species such as Azospirillum brasilense, Bacillus subtilis, and even cyanobacterial species such as Nostoc. They are typically produced in response to extracellular environmental stressors [40]. The biofilm produced by EPS allows for microorganisms to remain in close proximity to each other, and with respect to A. brasilense this allows for better plant-bacterial interactions. EPS keeps the bacteria within the rhizosphere, allowing it to fix nitrogen easily and alleviate abiotic stresses of the plants. EPS has also been shown to perform heavy metal sequestration [41].
With respect to A. brasilense, one of the properties of EPS is its ability to absorb nutrients, water, and consolidate the soil. An issue such as waterlogging, where soil can be laden with water following which drought-like conditions are experienced, can be benefited by having such a polymer in its composition.
We plan on overexpressing native A. brasilense genes involved in EPS production (exoB, exoB2, and exoC) and comparing the nature of the EPS produced by them. Our aim is to produce an exopolysaccharide of high viscosity; this will help with root associations, soil consolidations, and water absorption.
Our Biocontainment Strategy
Movement of genetic material between two organisms can happen via vertical transfer (which is the transmission of genetic material from parent to offspring) and horizontal transfer. While introducing GMOs in the environment, HGT is an important thing to consider from the biosafety point of view because spread of the antibiotic resistance genes to human pathogens can cause a surge in diseases that are difficult to tackle.
Administering our chassis as a biofertiliser in the soil without any biocontainment strategy can be risky as it can potentially lead to the transfer of unwanted genes in the soil bacteria. During the later part of our project cycle we realised that we would realistically not perform our intended genome integrations in the limited time we had. Therefore the plasmid biocontainment strategy we have decided to use is a type II Toxin-Antitoxin (TA) system where both the toxin and antitoxin are proteins. A stable toxin and its labile antitoxin are usually the two components of this module. Plasmid coded toxin-antitoxin systems are essential for post-segregational killing, also called as addiction.[42]
The toxin protein is inhibited post-translationally by the binding of an antitoxin protein. One such toxin-antitoxin system is the RnlA and RnlB system found in E. coli K12 chromosome as an anti-phage mechanism. Under normal growth conditions, the RnlA toxin is inhibited by its cognate antitoxin, RnlB. However, RnlB disappears during T4 phage infections which renders the RnlA toxin, an endoribonuclease, active. RnlA cuts off phage RNA and prevents infection.[43] LsoA-LsoB is a toxin-antitoxin system found in a cryptic plasmid pOSAK1 in E. coli O157:H7 which shares a weak homology with the RnlA-RnlB system [44]. Interestingly, the Dmd protein found in T4 bacteriophage directly binds to both RnlA and LsoA and inhibits their toxicity.[44]
The idea is to encode the lsoA/rnlA toxin in the artificially introduced plasmid along with the gene of interest and the Dmd anti-toxin in the genome of Azospirillum brasilense Sp7. This would mean that the plasmid of interest is fatal to the recipient soil bacteria while it does not affect our chassis. A mechanism also has to be set in place to ensure that the amount of antitoxin produced in the genome is enough to neutralise the toxin element produced in the plasmid. This is where the promoter efficiency of both toxin and antitoxin elements comes into the picture. The efficiency of promoters can be controlled indirectly using upstream activation sequences (UAS). An UAS is located upstream of the core promoter and serves as a binding site for specific transcriptional activators. While the core promoter is responsible for pre-initiation complex (PIC) recruitment and assembly, the UAS provides additional stability and regulation of PIC formation. [45] UAS’s with varying efficiencies could be used to control efficiencies of the promoter for both toxin and antitoxins.
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