Assignment: Advancing Crop Resilience: Genetic Engineering Against Climate Change Induced Heat Stress The following assignment is divided into two parts: I. THE WRITTEN PART -...

Assignment:


Advancing Crop Resilience: Genetic Engineering Against Climate Change Induced Heat Stress

The following assignment is divided into two parts:

I.


THE WRITTEN PART - Firstly, make sure you make the correct changes to the written section of the

Background information PART 2

II.


PRESENTATION PART - Secondly,

Create a PowerPoint slide of 5 to 6 slides using


ONLY


The

Background information PART 2(WRITTEN PART)

Follow Instructions below:

1. 
   
   For the written part Make sure that you make all the necessary changes including
   using the comments
   given in the
   Background information PART 2 SECTION.
   
   

2. 
   
   When creating the presentation slides make sure you follow the steps of
   Background Information PART 2.
   
   
   

3. 
   
   Make sure that the slides flow and connect with
   
   BOTH
   
   the
   Background information PART XXXXXXXXXXSECTION AND The RESEARCH SECTION.
   Ensure that what you are writing makes sense.
   
   

4. 
   
   Make sure that the
   STRATEGIES
   OF Background information PART 2 CONNECTS WITH THE RESEARCH SECTION
   
   

5. 
   
   Make sure that you make all the necessary changes including
   using the comments
   given in the
   Background information PART 2 SECTION.
   
   

6. 
   
   Keep the slides simple – less is more.
   
   


7. 
   
   Text – the audience should be listening, not reading. Stick to bullet points and elaborate verbally(THE WRITTEN PART)
   
   

8. 
   
   Also, for images of the presentation, use the methods of the Presentation Image Instructions attachment document that explains the citation and reference of images.
   
   

Background Information

Part 1

Define Climate and its factors, focusing on Temperature.

Climate change is the shift in weather conditions and patterns over long periods of time. Climate change

can be caused by many factors ranging from increase greenhouse gas release to natural changes like volcanic eruptions

that cool the atmosphere with ash that blocks the sunlight.

These changes can have a negative impact on the growing seasons of essential crops. Crops that rely on critical precipitation or ideal temperatures cannot grow properly and have reduced yield.

The loss

in plant production could devastate many regions worldwide

that already struggling with food security
. One of the most important factors of climate change

is how to negate temperature increase via increasing the resilience of plants.

Statistical data for how temperature has changed over past years.

The statistical data shows that from the 1978, the temperature worldwide was steadily exponentially increasing, reaching 1 C above average in 2020.

1 C does not seem to have much impact, but regional areas worldwide can have temperature changes ranging from 1 C to 4 C, which can significantly affect the environment.

How has the change in temperature caused heat stress in rice and

potatoes, and what has its effect been on them?

Potatoes are known to be the third most produced crop at 388 million tons along with rice, the second most produced at 782 million tons

worldwide. Both crops are susceptible to rising temperatures. The impact on potatoes has been shown to reduce yield growth by 18–32% during high temperatures at 30 Celsius over the growing season (Lal et al., XXXXXXXXXXbillion people worldwide rely on potatoes as a main food source, a loss of 10% would leave one hundred million people without their staple.

Temperature changes have been shown to affect the plant by reducing the size of the leaves, the number of branches of the potatoes, and even the height of the plant. The other effects of heat stress also start hindering the level of water that the plant can retain, resulting in drought-related symptoms that prevent the growth of sprouts and roots. Therefore, it is significant for the development of potato plants that are genetically altered to combat this ever-growing problem before a large amount of the crop becomes unable to grow in subtropical regions.

Rice, the main staple for 3.5 billion people, is also affected by the temperature due to its requirement for plantation in water covered fields during its growth period. The rising temperature can lead to reduced water from excess evaporation leading to unsuitable ground for rice to grow.

Even with enough water, the temperature can make it difficult for the rice to germinate, resulting in complete crop failure. The impact of heat stress on plant performance is mainly related to strength, period, and timing, which interferes with plant growth in high-temperature areas (Shrestha et al., 2022).

Part 2

➢


Describe genetic engineering and how it is performed in plants.
[1]

To tweak the genetic makeup of plants, scientists first pinpoint and [2]snag
a gene that carries the trait they want to see in the crop. This gene[3]
gets tucked into a vector, [4]a DNA taxi, which ferries the gene into the plant cell. Techniques like using a bacterium or shooting genes directly into cells help do this. Once inside, if the gene takes root, those cells grow into plants, which are
[5]checked to ensure the new trait is showing up and sticking around for the long haul. Genetic engineering opens possibilities for crops that 4 can better guard off bugs, give bigger harvests, or pack a healthier punch (Kumar, 2020).

➢


Discuss and briefly describe CRISPR Cas9 technology and how it benefits genome editing and modification.

CRISPR/Cas9, a game-changer in genetic engineering, lets
[6]
us nip genes precisely. It is like using molecular scissors for pinpoint edits—cutting, adding, or altering DNA sequences. This tech has revolutionized research, offering a straightforward way to modify organisms, study gene functions, and potentially correct genetic disorders. It is faster, cheaper, and more accurate than previous methods. It empowers scientists to explore and manipulate the genome with unprecedented ease and precision, opening new avenues in medical and agricultural research (Sun, 2019).[7]

➢


How CRISPR-Cas 9 can be helpful to combat Heat stress and make heat resistant plant

CRISPR-Cas9 can combat heat stress in plants by editing genes that regulate temperature tolerance, enabling the development of heat-resistant varieties.
[8]This precision tool targets and modifies specific parts of the plant's DNA associated with heat response mechanisms, such as heat shock proteins,
[9]
improving the plant's ability to withstand higher temperatures. This approach is promising for creating crops that can maintain productivity and quality in the face of climate change-induced heat stress.

➢


Temperature tolerance in plants uses joint research in genetic engineering. Here are the three possible strategies we will address in our research:

The second strategy is gene silencing, which will be discussed in detail in the rice section. CRISPR-Cas9 system functions by accurately targeting and cutting specific DNA sequences in the genome, allowing for gene silencing. By designing a guide RNA that matches the gene's DNA sequence of interest, CRISPR-Cas9 can precisely disrupt the gene's function, effectively "silencing" it.

Additionally, this part will be delved into detail in the rice section. The third strategy is Gene expression, which uses the CRISPR-Cas9 system for regulation by targeting gene promoters or regulatory elements to boost or repress the expression of specific genes. This gene tinkering allows scientists to study the function of genes and create 6 plants that can better adapt to environmental stresses, like heat, by altering how genes are turned on or off.

Research

●



Rice varieties with enhanced OsERA 1(Oryza sativa
ENHANCED RESPONSE TO ABA1) mutant lines exhibit rice growth in drought-stressed environments


(Ogata et al., 2020).

Numerous physiological and molecular studies have demonstrated the essential functions that phytohormone signalling pathways, including auxin, brassinosteroids, and abscisic acid (ABA), play in modulating the drought stress response in plants. ABA primarily handles the plants' responses to reduced water availability. Endogenous ABA levels rise in dehydrated cells, resulting in various developmental and physiological reactions, including stomatal closure and the activation of dehydration-responsive gene expression. Research has revealed that the β-subunit of the protein farnesyltransferase, encoded by Arabidopsis thaliana ENHANCED RESPONSE TO ABA1 (ERA1), controls both the dehydration response and ABA signalling. The study examines how the OsERA1 gene affects rice's response to drought stress. It shows that rice osera1 mutant lines with frameshift mutations produced by CRISPR/Cas9 have higher ABA sensitivity and more capacity to respond to drought stress by controlling stomatal activity. It finds that OsERA1 is a negative regulator of rice responses to drought stress and ABA and of primary root development in non-stressed circumstances.

Methods:


Using the vector pCAMBIA, the researcher used CRISPR-cas9 with target gRNAs PAM sites and Restriction sites. The vector was introduced in
Agrobacterium tumefaciens
strain LBA4404, which helped to trance the immature rice (Oryza sativa
L. ssp
japonica
cv. Nipponbare) embryos. The transformation was confirmed using PCR with primer to amplify the Cas9 fragment to detect mutation around the target site. After finding the positive transformation, Cas9-free with a mutant line for further reproduction of the OsERA 1 mutant lines of the rice plant. The mutant lines were then subjected to different physiological tests, initially grown in controlled conditions like CO2, heat, and regulated ABA. After seed germination, two mutant lines of rice and one wild type(control) were grown in mild drought stress conditions.

Results and Conclusion:


At 35 to 44 days following seeding, the relative growth rates of the two mutant plants were much lower than those of the WT plants. According to these results, two mutant plants respond to drought stress more strongly than WT plants. Through more rapidly stomatal closure, the osera1 mutant plants react to drought stress more quickly than WT plants. This data support the idea that OsERA1 is a negative regulator of the plant's reaction to drought stress. Like
Arabidopsis thaliana
ERA1, OsERA1 plays a crucial role in rice's drought stress response as it functions as a negative regulator of responses to ABA and drought stress.

The results offer a genetic improvement plan for rice and advance the understanding of the function of ABA signalling in the drought stress response in rice. The production of crops is substantially affected by the increased frequency of extreme weather events brought on by climate change, and drought is a critical abiotic stressor affecting rice yield in lowland rainfed rice agroecosystems. The results highlight the use of OsERA1 mutations to engineer enhanced drought tolerance in rice and show the potential of CRISPR/Cas9-targeted mutagenesis in functionally evaluating the role of OsERA1 in plants. Moreover, it sheds light on the potential role of alternative transcripts and specific regions of the OsERA1 gene in ERA1 function. Overall, the study enhances our understanding of the genetic mechanisms involved in the plant response to drought stress and provides critical information for efforts to engineer drought tolerance in rice genetically.

Drawback/future research opportunity:


The two osera1 mutant lines may have low crop yields and hence not be appropriate drought-tolerant lines for agricultural uses since they exhibit increased stomatal closure under drought stress. Thus, the researcher proposes that it could be worthwhile to investigate the effects of the mutation of the OsERA1 promoter area using CRISPR/Cas9. In addition, the two osera1 mutations can be combined with additional alleles that compensate for lower yield to develop enhanced drought tolerance.

(

Add images and visual data from the research paper on plant growth in different conditions, images of gene modification)

●



OsNAC092 (Transcription factors, class of regulatory gene) has vital functions in rice drought stress, and Knockout of OsNAC092 using CRISPR-Car9 has increased rice drought tolerance


(Wang et al., 2022)

Transcription factors, particularly those belonging to the NAC family, are a significant class of regulatory proteins involved in that response. Examples of these NAC transcription factors in action include wheat and soybean, which control genes linked to stress tolerance. Furthermore, salt and drought sensitivity are impacted by specific mutations in rice transcription factors, such as OsNAC041 and OsNAC006, respectively. Reactive oxygen species (ROS) build up in plants under unfavourable development circumstances, which causes oxidative damage. With the use of many enzymes, glutathione aids in cellular homeostasis and ROS reduction. The study focused on OsNAC092's function under drought stress. Using CRISPR-Cas9 technology to develop mutant plants and analyze their expression, researchers discovered that OsNAC092 regulates genes linked to drought tolerance, improving rice's resistance to drought at the phenotypic, physiological, and molecular levels.

Methods:


After surface sterilizing, seeds of osnac092 mutants and wild-type rice (Nipponbare, Oryza sativa L. japonica) were cultivated on hormone-free 1/2 MS agar medium containing sucrose and agar. The Agrobacterium-mediated seed callus approach was used to alter mutant plants, which were kept under specific culture conditions. To assess the osnac092 mutant's reaction, drought stress treatments were administered throughout the germination and seedling phases. To comprehend the regulatory function of OsNAC092, promoter prediction and expression profile analysis were carried out. The pZHY988 vector and the CRISPR-Cas9 technique achieved targeted mutagenesis of OsNAC092.


Physiological measures, such as glutathione content and enzyme activity, were used to evaluate stress responses. To compare the gene expression patterns of wild-type and mutant plants under normal and drought circumstances, transcriptome analysis was carried out using RNA-seq. Understanding the molecular pathways behind OsNAC092's drought resistance required quality control, alignment, gene expression calculations, differential gene screening, and various mining studies.

Results:


The qRT-PCR findings showed that OsNAC092 was sensitive to unfavourable stress; dryness and salt induced the most noticeable response (increased expression). Overall, OsNAC092 expression was reduced by temperature changes; however, at 42 C, some expression recovered following an initial decline. The expression of OsNAC092 was stimulated by gibberellin A3 (GA3), ABA, plant hormones (indole acetic acid—IAA), and H2O2 treatments. After mannitol was used to germinate mutant line seeds to mimic drought stress. The results showed that the osnac092 plants were more tolerant to drought stress at the germination stage than the WT. The osnac092 mutants exhibited better drought resistance than the WT plants. Eight-week-old WT and osnac092 plants with consistent growth were selected for drought stress testing. After 14 days of drought treatment, the WT plants began to wilt severely, while the osnac092 mutant line was less affected.

Future Opportunity:


Experimental results indicated the metabolic pathways involving OsNAC092, and further analysis of their regulation under drought stress will promote the molecular breeding of drought-tolerant rice
.

(

Add images and visual data from the research paper on plant growth in different conditions, images of gene modification)

Potato Heat Resistant. (4 slides,4 minutes)






(Pranav)

●



Transgenic expression in potato plant.

The study conducted involved the introduction of the CBF3 gene from Arabidopsis thaliana into potato plants (Solanum tuberosum) under the control of different promoters. The findings indicated that high temperatures, precisely 40°C or higher, significantly induced the expression of AtCBF3 in the transgenic potato lines. After heat stress, several physiological and molecular parameters were assessed, revealing notable differences between the transgenic lines and wild-type plants.

●



Identifying the gene responsible for heat stress.

The gene responsible for potato heat stress is investigated through molecular genetics techniques such as quantitative PCR (Q-PCR) and semi-quantitative RT-PCR. These methods help analyze gene expression levels under different conditions, such as high-temperature stress. Protein extraction and Western blot analysis also detect specific proteins associated with heat stress response, such as heat-shock protein 70 (HSP70) or D1.

Identifying the gene responsible for heat stress in potatoes is crucial for understanding this crop's molecular mechanisms underlying heat tolerance. This knowledge can help develop strategies to enhance potato cultivars' resilience to high-temperature stress, thereby improving yield and productivity.

Identifying the gene responsible for heat stress involves various molecular biology techniques such as genetic transformation, gene expression analysis (Q-PCR, RT-PCR), protein extraction, and Western blot analysis. These methods enable researchers to examine gene expression and protein levels changes in response to high-temperature stress.

This study uses leaf samples from potato plants (Solanum et al.) for gene expression analysis and protein extraction. Specifically, the fourth leaf from the top of the seedlings is utilized for the experiments.

●



Methods used to study the mechanism.

Genetic transformation:


The study introduces constructs containing the AtCBF3 gene from Arabidopsis thaliana into potato plants. This is achieved through genetic transformation, where foreign DNA is inserted into the plant genome using Agrobacterium tumefaciens as a vector.

Gene expression analysis:


To assess the expression levels of the AtCBF3 gene and its downstream target genes in response to high-temperature stress, molecular biology techniques such as quantitative PCR (Q-PCR) and semi-quantitative RT-PCR are employed. Q-PCR allows for precise quantification of gene expression levels, while semi-quantitative RT-PCR provides a qualitative assessment.

Protein analysis:


The study also involves analyzing specific proteins associated with heat stress response levels, such as heat-shock protein 70 (HSP70) or D1. This is done through protein extraction from plant tissues, followed by Western blot analysis. Western blotting allows for the detection and quantification of target proteins using specific antibodies.

Physiological and biochemical measurements:


Various physiological and biochemical parameters related to heat stress response are measured, including changes in calcium ion (Ca2+) concentration and the levels of proline and soluble sugars. These measurements provide insights into the mechanisms by which plants respond to high-temperature stress.

By employing these methods, researchers can understand the molecular mechanisms underlying heat stress response in potato plants and elucidate the role of the AtCBF3 gene in enhancing heat tolerance.

Results

●



Induction of AtCBF3 Expression

: High temperatures (40°C or higher) significantly induce the expression of AtCBF3 in the transgenic potato lines.

●



Enhanced Photosynthesis and Antioxidant Defense.

●


Increased net photosynthetic rate (Pn) and maximal photochemical efficiency of photosystem II (Fv/Fm) in transgenic lines compared to wild type.

●


Higher accumulation of the D1 protein, which is involved in photosynthesis, in transgenic lines.

●


Reduction in the accumulation of reactive oxygen species in transgenic lines, indicating improved antioxidant defense.

●



Gene Expression Analysis:


Q-PCR assay confirms the upregulation of genes involved in photosynthesis and antioxidant defense in transgenic lines under heat stress.

●



Effect on Heat-Shock Protein 70 (HSP70):


Contrary to expectations, the accumulation of HSP70 increased in the wild-type line but decreased in transgenic lines under heat stress.

Conclusion

●


Potato plants expressing AtCBF3 exhibit enhanced tolerance to high temperatures due to improved photosynthesis and antioxidant defense mechanisms, as evidenced by gene expression analysis and physiological measurements.

●


The mechanism of enhanced tolerance may not solely depend on the regulatory pathways involving HSP70. The study concludes that AtCBF3 can enhance the heat resistance of potato plants and suggests that the CBF pathway may play a role in mediating potato heat stress response. These findings contribute to understanding crops' genetic mechanisms underlying heat stress tolerance. They may facilitate the development of heat-tolerant potato varieties through breeding or genetic engineering approaches.