Design

Introduction

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The problem we are looking to solve is how to engineer wheat plants that have an increased tolerance to heat stress. We propose to do this by genetically engineering the plants to carry genes that code for proteins that confer greater heat and drought tolerance to the plants. This section describes plant responses to heat stress as well as our proposed genetic engineering solution.

Heat Shock Response

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Proteins folded into their functional conformation are said to be in their native state. Proper protein folding depends on many factors including temperature, pH, and presence of cofactors. At higher temperatures, molecules have greater kinetic energy which can disrupt the intermolecular interactions that keep proteins folded in their native state. When proteins are no longer properly folded, they can become non-functional, which can have serious effects on the cell and even cause cell death.

Chaperones are a class of proteins that help other proteins fold properly and achieve their native state. The type of chaperones that help proteins fold when temperatures rise are heat shock proteins (HSPs). HSPs are activated by the binding of cis-acting regulatory elements (heat shock elements or HSEs), near the promoters of HSPs, to trans-acting regulatory factors called heat shock factors (HSFs), which increases the transcription of HSP genes.

Heat Stress in Wheat

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Heat stress occurs when plants experience higher than ideal temperatures. At higher temperatures, cell membranes have increased membrane fluidity, proteins have reduced stability, and structural changes can occur in chromatin^[1]. These changes at the cellular level can lead to many negative effects on overall plant health^[2].

Some of the negative effects of heat stress in wheat include:

• poor seed germination
• decrease in grain filling duration
• reduction in grain number per head
• deactivation of the RuBisCO enzyme
• decrease in photosynthetic capacity
• premature leaf senescence
• decrease in chlorophyll content
• decreased crop yield

Heat-Inducible Promoters

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Genes are expressed through transcription by RNA polymerase. RNA polymerase first binds to promoters before transcribing the coding and non-coding sequences of the gene. Some promoters are constitutive, meaning RNA polymerase can bind under any condition, and thus the promoters and corresponding genes are continuously active. Other promoters require specific conditions for RNA polymerase to bind. The promoters we will be working with are heat-inducible promoters. Heat shock factors must first bind to the heat shock element in order to recruit RNA polymerase to the promoter. After RNA polymerase binds to the promoter, transcription of our target genes can proceed.

ACC Deaminase

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Protein Function: 1-aminocyclopropane-1-carboxylate deaminase (ACCD) breaks down 1-aminocyclopropane-1-carboxylate (ACC), an ethylene precursor, into α-ketobutyrate and ammonia^[3]. Ethylene is a plant hormone involved in the stress response pathway of plants, including for heat stress^[4]. At low levels (10 g/L), ethylene can promote growth, but at high levels (>25 g/L), ethylene can slow and even stop root and shoot growth^[3].

In response to stresses, like high temperature, ethylene is synthesized and initiates a signal cascade that leads to the activation of several genes through transcription factors known as ethylene response factors or ERFs^[5]. Genetically engineered plants with reduced ethylene receptors (decreased ethylene sensitivity) have been shown to have longer lasting flowers and slower fruit ripening^[4]. Inhibiting the ethylene pathway in heat-shocked wheat plants has been shown to reduce the number of kernel abortions and inhibit the reduction in kernel weight^[6].

Regulation: ACCD expression and enzyme activity is directly induced by its substrate ACC, but also other amino acids (L-alanine, DL-alanine, D-serine). ACCD is competitively inhibited by L-isomers of amino acids (L-alanine, L-serine, L-homoserine, L-α aminobutyric acid). The ACCD gene is negatively regulated by leucine, which is synthesized from α-ketobutyrate. Pyridoxal phosphate (PLP) is a cofactor of ACCD^[3].

Production: Plant-associated bacteria synthesize ACCD which helps lower the ethylene content in plants^[3]. High ethylene concentration reduces plant growth, so these microbes help the plants grow under stressful conditions.

Protein Structure: ACCD is a protein of weight 105-112 kDa depending on the species^[3]. In its native state, the ACCD protein can be found in a homodimer or homotrimer structure.

SBPase

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Protein Function: SBPase is a phosphatase enzyme in the Calvin Cycle that removes a phosphate group from sedoheptulose 1,7-bisphosphate (S1,7BP) to produce sedoheptulose 7-phosphate (S7P). The Calvin Cycle is the light-independent portion of photosynthesis that fixes carbon dioxide to produce three carbon sugars (DHAP) that will eventually form glucose.

Regulation: Increased production of SBPase in plants has shown to lead to increased photosynthesis and starch accumulation, in regular conditions and when plants are under cold stress^{[7, 8]}. Increasing SBPase copy number has been shown to improve photosynthesis rate and increase seed size in wheat^[9]. SBPase is regulated by pH (inhibited at lower pH), divalent cation (Mg²⁺) concentrations, and light (through a ferredoxin chain). At 400 ppm CO₂ (the [CO₂ in the atmosphere]), there is no excess of SBPase, unlike other enzymes in the Calvin Cycle, so altering its concentration can greatly affect yields^[10].

Protein Structure: SBPase is encoded in the nuclear genome of plants and is synthesized in the cytosol. The protein is translated with an N-terminal extension, known as the transit peptide, which directs the enzyme to the chloroplast. In its native state the SBPase protein is a homodimer. The SPBase protein in wheat has 393 amino acids.

Choline Monooxygenase

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Protein Function: Choline monooxygenase (CMO) is one enzyme in the plant pathway that synthesizes glycine betaine (GB). CMO converts choline into betaine aldehyde which is then converted to GB by betaine aldehyde dehydrogenase. GB maintains high ionic strength and helps drive protein folding. CMO is an enzyme already expressed in wheat (Triticum aestivum) plants^[11].

Tobacco plants with engineered high levels of GB from increased copies of betaine aldehyde dehydrogenase were shown to have greater heat tolerance^[12]. Arabidopsis thaliana transformed with the bacterial version of CMO was also shown to have higher GB levels and increased tolerance to heat stress^[13].

Regulation: Ferredoxin is a cofactor of CMO, thus the CMO reaction is oxygen and quinone dependent.

Protein Structure: CMO is 65 kDa and 556 amino acids long. CMO has homologues in bacteria and animal species.

Gene Circuits & Construct Development

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We designed four different constructs:

1. The first was a control construct that included a constitutively active promoter (CaMV35S) and the gene for a green fluorescent protein (GFP).
2. The second construct contained a heat-inducible promoter (TaHsp70d), the SBPase gene, and the gene for a red fluorescent protein (IRFP) in frame with the SBPase gene. This means that the SBPase and IRFP genes will be transcribed and translated into one polypeptide.
3. The third construct also contained the TaHsp70d promoter, the ACCD gene, and the gene for a blue fluorescent protein (BFP) in frame with ACCD.
4. The final construct contained the TaHsp70d promoter, the CMO gene, and the gene for a yellow fluorescent protein (YFP) in frame with CMO.

Protoplasts & Proof of Concept

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Our genetic constructs were first constructed in E. coli, isolated and purified, then transfected into protoplasts. Protoplasts are plant cells that have had their cell walls digested. The cell walls of plants are made of cellulose which are cross-linked sugar molecules. Protoplasts can be made by treating plant cells to a cellulase enzyme that degrades the cellulose cell wall. Protoplasts are easier to transform and transfect than normal plant cells. The cell wall can reduce the efficiency of a transformation and transfection.

Protoplasts are conducive to both transient and stable transformation, and regeneration of whole plants from protoplasts is well researched ^{[16, 17]}. Protoplasts are also useful for specific gene editing experiments with techniques like CRISPR-Cas9^[16].

By transfecting wheat protoplasts with our constructs, we tested our genetic circuits in wheat cells under heat stress. We can demonstrate that our constructs were successfully synthesized, and that the expression of ACCD and SBPase can be induced in wheat cells under heat stress by measuring the fluorescence from the protoplasts after heat shock.

We were unable to transform entire wheat plants and create a transgenic line of wheat due to time and resource restrictions. However, by demonstrating that our genetic system works in wheat protoplasts this shows a proof of concept that we would be able to confer increased heat tolerance to entire wheat plants.