Genetics and Plant Breeding Project Proposal

Project Description – Project Proposal

• Starting Point
• Objectives and work programme
• Project- and subject-related list of publications
• Supplementary information on the research context
• Requested modules/funds

Answer:

Project Proposal

Investigating the Role of Circular RNAs (circRNAs) in Drought Stress Tolerance of Soybean

1         Starting Point

State of the art and preliminary work

Introduction: Role of Circular RNAs (circRNAs) in Enhancing Drought Tolerance in Soybeans

In the field of agriculture, drought stress is an imminent threat that has a significant impact on important crops like soybeans and tomatoes. These crops, which are essential for human consumption and livestock feed, suffer the brunt of this problem, demanding a vital focus on growing drought-resistant cultivars (Taheri et al., 2022). Understanding the molecular complexities behind drought tolerance becomes critical in the search for drought-resistant crop cultivars. Soybean (Glycine max) is a widely farmed legume that accounts for nearly half of total world grain legume production. Chickpea, another important legume, accounts for around 7% of total output.

Both are excellent nutritional sources for human diet. Nevertheless, the challenge of drought stress mainly hinders production of soybean, leading to reductions in total biomass, pod count, seed quantity, seed weight, and total seed yield of a plant (Toker et al., 2007; Jha et al., 2020).

In response to the critical demand for drought-resistant crops, our research aims to uncover the potential of Circular RNAs (circRNAs) in improving soybean drought resistance. We aim to uncover the subtle regulatory functions performed by circRNAs in strengthening soybeans against the negative effects of drought by using an innovative greenhouse-based plant breeding strategy.

We aim to explore the present status of circRNA research, comprehending their molecular complexities and their uses, in order to commence this investigation. Simultaneously, we address the numerous issues provided by drought stress in soybeans, taking into account the negative effects on a variety of critical factors. We hope not only to contribute to current agricultural difficulties, but also to the larger arena of plant breeding, by examining the landscape of plant breeding methodologies used to address these challenges.

In summary, our work aims not just to mitigate the immediate effects of drought stress on soybean output, but also to expand the common knowledge base, encouraging a more resilient and sustainable agricultural future.

Soybean

Soybean (Glycine max L.) originated in eastern Asia, probably in north and central China. It is an important oil seed crop and are the largest source of edible oil accounting for roughly around 50% of total oil seed production in the world and economically important leguminous crop as it provides more than 25% of global protein requirement. According to the FAO stats by the year 2032, soybean production is predicted to peak 415 Mt, which is twice the total of remaining oil seeds which are currently at 189 Mt (Michele et al., 2023). Soybean was a neglected crop until WWII. Germany developed soy oil lard substitute and meat substitute (Soybean Plant Biology, n.d.). Soybean has multiple uses, it is used for oil production, food for humans, feed for animals, serves as a forage and cover crop and fixes atmospheric nitrogen. It is proven to have an oil, protein and carbohydrate content of 21, 40%, and 34% respectively (Nazareth, 2009). Food products such as soymilk, soy sauce, soy chunks, soy flour, soy meat, tofu etc. have gained popularity in the market for their nutrient content. It has medicinal properties, due to the presence of β-carotene, vitamin-c, high calories, essential amino acid, it can be used in anti-hypersensitive, antimicrobial, antioxidant, anti-diabetic and anticancer activities which are highly beneficial for human health. Soybean production has been increasing over the years, but it is not considered as significant as the major crops like wheat, maize, rice etc. due to its environmental constrains.

Circular RNAs (circRNAs): Key Regulators in Plant Biology and Breeding

Next-generation high-throughput sequencing methods, along with fast bioinformatics tools, have transformed genomics by revealing non-coding areas of the genome (Satamet al., 2023). This discovery has led to the recognition of numerous non-coding RNAs or ncRNAs that act pivotal roles in orchestrating growth and development of plant, as well as its responses to environmental stressors at both stage of mRNA and protein synthesis. Circular RNAs or circRNAs, long noncoding RNAs or lncRNAs, and short RNAs or sRNAs have become key influencers in regulating gene expression, developing novel possibilities for advancing crop development (Summanwar et al., 2020).

Circular RNAs, or circRNAs, represent a subtype of non-coding RNA characterized by the absence of 5′ caps or 3′ tails. Their unique circular structure arises from a ligation process of 3′-5′ head-to-tail portion during back-splicing. Ranging in length from 100 nucleotides to several kbs, circRNAs can originate from both coding genes and non-coding genes. Characterization studies reveal their presence in various forms, including exonic circRNAs derived from exons, intronic circRNAs from introns, and intergenic circRNAs from intergenic regions. The expression patterns of circRNAs exhibit cells, tissues, and development stage dependence. Notably, circRNAs display enhanced stability compared to linear type RNAs, suggesting their involvement in biological processes through distinct mechanisms.

Role of circRNA include splicing/transcription, interaction with RNA binding proteins, miRNA inhibition by acting as miRNA sponges (Venkatesh et al., 2021). Their role as miRNA sponges is emphasized, as circRNAs contain miRNA binding sites, influencing gene regulation in both plants and mammals. The intricate interplay between circular-RNAs, micro-RNAs, and messenger RNAs is termed circularRNA-microRNA-mRNA interaction (Chu et al., 2018; Zhang & Dai, 2022).

CircRNA Biogenesis

Circular RNAs (circRNAs) are formed through back-splicing at canonical splicing sites, mediated by canonical splicing machinery or base pairing between inverted repeat elements. Lariat precursors during exon-skipping or debranched intron lariats contribute to circRNA generation. Linear RNase R-based library preparation enhances circRNA enrichment in sequencing studies. Bioinformatics tools, categorized as pseudo-reference-based or fragment-based, aid circRNA identification. Locus-specific profiling using genomic back-splice sites validates and quantitates circRNAs. While reverse transcription and PCR validate circRNAs, droplet digital PCR and RT-qPCR quantify them. A comprehensive understanding requires a combination of genome-wide and locus-specific approaches (Zhang & Chen, 2020).

Current Insights into Plant circRNAs

The current understanding of circular RNAs (circRNAs) in plants is distinguished by substantial advances in genomics and molecular biology. These non-coding RNA molecules have received attention because to their distinctive circular shape and crucial activities in governing vegetative and reproductive growth and development, as well as responses to both abiotic and biotic stresses in plants. While the majority emphasis in current plant circular-RNA research lies in their identification through large-scale sequencing, only a limited subset of circRNAs has clearly defined molecular functions. Unique features in circRNA biosynthesis and functions are beginning to emerge, with just a fraction in plants harbouring miRNA binding sites (Lu et al., 2015). This lack of understanding highlights the necessity for more research into plant circRNAs to uncover their unique characteristics and functional relevance (Zhang & Dai, 2022).

Circular RNAs are gaining prominence as a rising star in the field of plant biology, with enormous potential for use in plant breeding programmes (Lai, 2018). Because of their stability and specificity in expression patterns, they are appealing candidates for genetic modification to improve desired characteristics in crops. CircRNAs’ distinct properties, including as their potential role as miRNA sponges and participation in stress responses, make them ideal candidates for crop enhancement methods (Zhang & Chen, 2020). Unlocking the activities of circRNAs in plants holds the prospect of establishing novel ways for breeding crops with enhanced resilience, productivity, and tolerance to a variety of environmental problems.

Role of Circular RNAs (circRNAs) in Plant Stress Responses

Circular RNAs (circRNAs) have made considerable advances in plant systems (Liu et al., 2017) beginning with Arabidopsis (A. thaliana) with convincing indicators of RNA circles (Pan et al., 2018). Subsequent research discovered a large number of circRNAs, approximately 12,000 in rice (Oryza sativa) and approximately 6000 in Arabidopsis, (Ye et al., 2015) confirming their presence in both monocotyledonous and dicotyledonous plants. The increasing panorama of circRNA research now includes tomato, wheat, kiwifruit, tea, barley, maize, and soybean (Zhao et al., 2019).

Tomato circular-RNAs have been demonstrated to play a role in chilling responsive activities, while grape circRNAs are implicated in the regulation of cold-stress (Zuo et al., 2016). This heightened interest extends to soybeans, where tissue-specific expression patterns have been observed. Nevertheless, the precise functions of circular-RNAs in soybeans under abiotic stress conditions remain largely unexplored (Zhao et al., 2019).

Recent research has revealed a link between circRNAs and stress reactions. According to Wang et al (2017), in wheat, 62 circRNAs had distinct expression patterns in response to water-loss stress, but ~160 tomato circRNAs reacted to cold damage, indicating a regulatory role in low-temperature stress (Zuo et al., 2016).

Cold acclimation, an important stage in plant adaptation to cold temperatures, involves complex changes in sucrose production, photosynthetic efficiency, and carbon and nitrogen metabolism. Several studies have found circRNAs to be involved in these processes, functioning as potential miRNA sponges. In soybeans, 2134 circRNAs included predicted binding sites for 92 miRNAs, indicating a part in the complex molecular dance of cold acclimation (Zhao et al., 2019).

Exploring circRNAs in Leguminous Crops under Drought Stress Efficient algorithms have made it possible to investigate drought stress tolerance mechanisms in leguminous crops (Khan et al., 2019). While the significance of coding genes in conferring drought stress tolerance is partially understood, the function of nc-RNAs, particularly circRNAs, in case of leguminous crops like chickpea and soybean remains largely unexplored. The existence of circRNAs in agricultural transcriptomes has been suggested by in silico analysis, with variable

expression under drought stress and control settings. Notably, circRNAs have been found as miRNA sponges in soybean that target critical drought-responsive genes (Dasmandal et al., 2020).

Functional Understanding of circRNAs:

While circRNA-miRNA-mRNA networks have been built, the sponge functions of circRNAs in plants, unlike in mammals, remain unproven. Functional investigations of circRNAs in plants are limited; however, there is some evidence suggesting that they exert influence on gene expression at both the transcriptional and posttranscriptional stages by interacting with miRNAs (Bhar & Roy, 2023). Transgenic experiments in rice indicated that overexpression of a circRNA construct might adversely affect its parental gene, indicating that circRNAs play a multifaceted function in gene expression control (Song et al., 2021). CircRNAs are increasingly being hypothesised to play critical roles in plant responses to environmental stressors, as seen in rice, wheat, kiwifruit, and potato (Yu et al., 2019).

In summary, the present level of knowledge in plant biology on circRNAs, particularly in the context of soybeans and drought stress, serves as a framework for our research. The early findings highlight the possible regulatory functions of circRNAs in plant stress responses and pave the way for further investigation into their particular contributions to soybean resilience under drought circumstances.

1         Objective and work program

1.1         Total duration of the research

3 years

1.2         Objectives of the project

The primary objective of the present study is to enhance our understanding of the regulatory role of circRNAs in soybeans under drought-stress conditions. We intend to offer insights for circRNA-based techniques in soybean breeding for increased drought stress resistance by defining drought-responsive circRNAs, connecting their expression with physiological parameters, and modifying circRNA to assess its influence on drought tolerance.

Objectives of the study includes:

• Characterize circRNA expression in drought-stressed greenhouse soybeans.
• Identify drought-responsive circRNAs.
• Assess circRNA regulatory roles in key soybean drought pathways.
• Correlate circRNA levels with drought-related physiological parameters.

• Use molecular techniques to manipulate circRNA, evaluate impact on drought tolerance.
• Evaluate heritability and stability of circRNA-induced drought tolerance.
• Integrate findings into a comprehensive circRNA regulatory network model.
• Offer insights for circRNA-based strategies in breeding for improved soybean drought tolerance.

The following research questions will be investigated in detail:

What specific profiles of circRNA expression are observed in soybean plants under drought stress?

Which circRNAs demonstrate varying expression patterns in reaction to drought stress conditions in soybeans?

What potential regulatory functions do the identified circRNAs play in crucial molecular pathways linked to drought stress tolerance in soybeans?

Is there a correlation between the levels of circRNA expression and physiological parameters associated with the adaptation of soybean plants to drought stress?

How do molecular techniques for circRNA manipulation impact soybean drought stress tolerance, and what are the underlying mechanisms?

What is the heritability and stability of drought-tolerant traits conferred by circRNA-mediated mechanisms across subsequent generations of soybean plants?

How can the findings be integrated into a comprehensive circRNA regulatory network model, elucidating their involvement in soybean drought stress response?

What valuable insights can be derived for circRNA-based strategies in soybean breeding programs aimed at improving drought stress tolerance?

1.3        Work programme and proposed research methods

Plant Materials and Stress Treatments:

Seed Sterilization and Germination: Surface-sterilize the soybean cultivars SRR2545896 (control, CT) and SRR2545900 (drought stress, DS) seeds for 10 minutes with 1% NaClO. After sterilization, rinse the seeds three times for two minutes each in distilled water. Immerse the seeds in tap water for a duration of 12 hours, followed by a germination period of 4 days within a dark incubator set at a temperature of 25°C.

Transplantation and Growth Conditions: Fill each plastic container with humus soil and five germinating seeds. Adjust the pots to a temperature of 26 °C during the day and 18 °C at night, with a light schedule of 14 hours during the day and 10 hours at night. Maintain a light intensity of 300 mol•m2•s1 photosynthetic photon flux density and a relative humidity ranging from 50% to 60%. The photoperiod should consist of 16 hours of light followed by 8 hours of darkness.

Experimental design:

Group Division and Dehydration Stress: Divide the seedlings that have attained the second stage characterized by fully expanding trifoliate leaves, stage into two groups at random: one group for the treatment of artificial dehydration stress with PEG 6000 to simulate drought stress (DS) and the other group served as the control without stress treatment (CT) (Wang et al., 2020).

Harvesting and Sample Preparation: Harvest the leaf tissues from both the stressed and control groups 12 hours after treatment. Gather representative leaves from three distinct seedling pots for each treatment. Consolidate the leaves obtained from all three pots to create one sample set for each respective treatment.

Sample Preservation: Immediately after collection, transfer both stressed and control samples into snap-frozen condition in liquid nitrogen. Maintain the frozen samples at -80°C until their required use.

2. Characterize circRNA Expression Profiles in Soybeans under Drought Stress

RNA Extraction and construction of circRNA libraries for sequencing

For total RNA extraction, both control and PEG-stressed leaves are processed using Trizol, followed by acid-phenol-chloroform extraction. To construct the circular-RNA sequencing library, the entire RNA content from each sample is utilized. Pre-treat five micrograms of total RNA with the CircRNA Enrichment Kit for circRNA enrichment. Subsequently, following the manufacturer’s instructions, employ the a Total RNA Library Prep Kit to generate RNA libraries from the pre-treated RNAs. Using the BioAnalyzer, ensure library quality and measure it. Denature the libraries into single-stranded DNA molecules, amplify them in situ as clusters, and then sequence them using the Illumina HiSeq Sequencer for 150 cycles (Wang et al., 2016; Wang et al., 2020).

Identification of CircRNAs

Utilize the Illumina HiSeq Sequencer for paired-end sequencing, employing in-house Perl scripts for quality filtering. Eliminate low-quality reads containing adapters and poly-N sequences from the raw data to acquire clean reads. Employ the BWA-MEM program to align the clean reads to the reference genome/transcriptome.

To detect and identify circRNAs, employ the CIRI method and leverage Ensembl genomic annotations. The CIRI algorithm executes a two-step data filtration process. In the initial stage, it recognizes paired chiastic clipping signals in the Sequence Alignment Map generated by BWA-MEM, utilizing GT-AC signals as the paired chiastic clipping signals. Subsequently, in the second phase, CIRI conducts another search of the SAM alignment to identify additional junction readings. Simultaneously, it implements further filtering to diminish false positive possibilities by removing improperly mapped reads from homologous genes or repetitive regions.

1. Assessing Drought Stress Tolerance:

Various physiological parameters will be examined to determine physiological responses in soybean plants under drought stress circumstances. Water content, chlorophyll levels, and stomata conductance are among the factors to be assessed.

Water Content Measurement: Determine the water content in drought-stressed soybean plants by weighing the plant tissues before and after drying.

Chlorophyll Level Analysis: Determine the influence of drought stress on photosynthetic activity by measuring chlorophyll levels in plant tissues using spectrophotometry.

Stomata Conductance Analysis: Evaluate stomata conductance as an indicator of the plant’s ability to regulate water loss. To measure stomata conductance in response to drought stress, employ either a porometer or a leaf gas exchange device.

Correlation Analysis: Use correlation analysis to explore potential links between circRNA expression levels and physiological responses. Establish statistical correlations between circRNA expression data and water content, chlorophyll levels, and stomatal conductance using acceptable statistical approaches.

Manipulate circRNA Expression to Enhance Drought Tolerance in Soybeans

Use genetic manipulation techniques to alter circRNA expression.

Genetic Manipulation: To alter circRNA expression, use RNA interference (RNAi) constructs or circRNA overexpression vectors. Transgenic soybean plants with altered circRNA expression should be created.

Validation of Expression: Using molecular method qRT-PCR to validate circRNA expression levels in modified plants. Confirm the effective manipulation of circRNA in transgenic plants.

Drought Stress Evaluation: Drought stress is applied to both modified and control plants. Drought stress tolerance in modified and control soybean plants is assessed and compared.

2. Assess Heritability and Stability of Drought-Tolerant Traits

To evaluate the heritability and stability of drought-tolerant traits conferred by circRNA-mediated mechanisms across subsequent generations of soybean plants, a comprehensive multigenerational research approach will be employed. The objective is to understand the persistence and transmission of the desirable traits induced by circRNA manipulation in soybeans under drought stress conditions.

Generational Propagation:

Initial Generation (G0):

1. Development of Modified and Control Groups: Develop transgenic soybean plants with altered circRNA expression through genetic manipulation techniques such as RNA interference (RNAi) constructs or circRNA overexpression vectors. Simultaneously, maintain a control group with unaltered circRNA expression.
2. Characterization and Evaluation: Assess the circRNA expression levels and drought tolerance characteristics in the initial generation (G0) plants under controlled conditions. Conduct thorough physiological assessments and record relevant data.

Subsequent Generations (G1, G2, G3, etc.):

1. Generational Propagation: Propagate soybean plants from both the modified (RNAi and overexpression) and control groups through subsequent generations (G1, G2, G3, etc.).
2. Monitoring CircRNA Expression: Continuously monitor circRNA expression levels in each generation using techniques such as RNA extraction and RT-qPCR. Assess whether the modified circRNA expression remains stable across generations.
3. Drought Stress Evaluation: Subject soybean plants from each generation to drought stress conditions to evaluate drought tolerance characteristics. Employ physiological parameters, including water content, chlorophyll levels, and stomatal conductance, to assess the plants’ responses.

Statistical evaluation: Employ ANOVA (Analysis of Variance) to determine the significance of differences in circRNA expression and drought tolerance traits between the modified and control groups across generations. Calculate the heritability of circRNA-induced drought-tolerant traits by comparing the variability of traits within and between generations. This analysis provides insights into the genetic contribution to the observed traits.

• Provide Insights and Practical Applications:

Discuss the findings in relation to soybean drought stress tolerance. Highlight major findings from multi-generational research and circRNA modification.

Provide insight on prospective circRNA-mediated techniques in soybean breeding projects. Investigate the implications for crop enhancement and sustainable farming techniques.

Based on the study’s findings, suggest future research directions. Discuss potential breakthroughs and topics for further research.

1.4       Handling of research data

Data processing is critical for sustaining accuracy and reproducibility throughout the research process. The following protocols will be implemented:

Collection of data and storage: Plant growth characteristics, circRNA expression levels, physiological examinations, and sequencing findings should all be recorded and stored in a safe, backed-up database.

Analysing of Data and Integrate Findings into a Regulatory Network Model:

Data Integration:

Combine sequencing data that includes profiles of circRNA expression during drought stress.

Integrate qRT-PCR results to validate the expression levels of specific circRNAs.

In order to offer an in-depth understanding of plant responses to drought stress, integrate physiological measures like as water content, chlorophyll levels, and stomatal conductance measurements.

Data Interpretation:

Analyze the combined data to find relationships between circRNA expression levels and physiological responses to drought stress.

Recognize circRNAs that have consistent expression patterns across sequencing, qRT-PCR, and physiological evaluations, indicating possible involvement in drought stress tolerance.

Investigate the potential involvement of circRNA expression patterns in stress-responsive pathways or molecular mechanisms related to drought tolerance.

To validate or extend known processes related to circRNA participation in drought stress responses, compare integrated findings with existing understanding.

Develop a comprehensive regulatory network model illustrating circRNA-mediated interactions. Utilize visualization tools to depict relationships among circRNAs, miRNAs, and target mRNAs.

Publication and Documentation:

The outcomes of the research will be documented in a detailed report, which will include raw data, analysis details, and interpretations.

Upon publication, all protocols, code, and analysis processes will be made accessible for review.

The findings of this study will have a significant impact on soybean breeding and will be published in renowned international journals. Furthermore, we intend to submit our findings at both national and international conferences, therefore advancing soybean breeding and consumption.

1. Project- and subject-related list of publications

Bhar, A., & Roy, A. (2023). Emphasizing the Role of Long Non-Coding RNAs (lncRNA), Circular RNA (circRNA), and Micropeptides (miPs) in Plant Biotic Stress Tolerance. Plants, 12(23), 3951.

Chu, Q., Bai, P., Zhu, X., Zhang, X., Mao, L., Zhu, Q. H., & Ye, C. Y. (2020). Characteristics of plant circular RNAs. Briefings in bioinformatics, 21(1), 135-143.

Dasmandal, T., Rao, A. R., & Sahu, S. (2020). Identification and characterization of circular RNAs regulating genes responsible for drought stress tolerance in chickpea and soybean. Indian Journal of Genetics and Plant Breeding, 80(01), 1-8.

Jha, U. C., Bohra, A., & Nayyar, H. (2020). Advances in “omics” approaches to tackle drought stress in grain legumes. Plant Breeding, 139(1), 1-27.

Khan, N., Bano, A., Rahman, M. A., Guo, J., Kang, Z., & Babar, M. A. (2019). Comparative physiological and metabolic analysis reveals a complex mechanism involved in drought tolerance in chickpea (Cicer arietinum L.) induced by PGPR and PGRs. Scientific reports, 9(1), 2097.

Liu, T., Zhang, L., Chen, G., & Shi, T. (2017). Identifying and characterizing the circular RNAs during the lifespan of Arabidopsis leaves. Frontiers in plant science, 8, 1278.

Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods, 25(4), 402-408.

Lu, T., Cui, L., Zhou, Y., Zhu, C., Fan, D., Gong, H., … & Han, B. (2015). Transcriptome-wide investigation of circular RNAs in rice. Rna, 21(12), 2076-2087.

Milanesi, P. M., Steffen, P. R., Huzar-Novakowiski, J., Mezomo, M. P., Stefanski, F. S., Tonin, R. J., … & Fochesatto, M. (2023). Temporal dynamics of Asian soybean rust according to sowing date and fungicide application, and its effects on soybean yield in the Northwest Mesoregion, Rio Grande do Sul, Brazil. Indian Phytopathology, 1-13.

Nazareth, Z. M. (2009). Compositional, functional and sensory properties of protein ingredients prepared from gas-supported screw-pressed soybean meal. Iowa State University.

Pan, T., Sun, X., Liu, Y., Li, H., Deng, G., Lin, H., & Wang, S. (2018). Heat stress alters genome-wide profiles of circular RNAs in Arabidopsis. Plant molecular biology, 96, 217-229.

Satam, H., Joshi, K., Mangrolia, U., Waghoo, S., Zaidi, G., Rawool, S., Thakare, R.P., Banday, S., Mishra, A.K., Das, G. and Malonia, S.K. (2023). Next-generation sequencing technology: Current trends and advancements. Biology, 12(7), 997.

Song, Y., Bu, C., Chen, P., Liu, P., & Zhang, D. (2021). Miniature inverted repeat transposable elements cis-regulate circular RNA expression and promote ethylene biosynthesis, reducing heat tolerance in Populus tomentosa. Journal of Experimental Botany, 72(5), 1978-1994.

Summanwar, A., Basu, U., Rahman, H., & Kav, N. N. (2020). Non-coding RNAs as emerging targets for crop improvement. Plant Science, 297, 110521.

Taheri, S., Gantait, S., Azizi, P., & Mazumdar, P. (2022). Drought tolerance improvement in Solanum lycopersicum: An insight into “OMICS” approaches and genome editing. 3 Biotech, 12(3), 63.

Toker, C., Lluch, C., Tejera, N. A., Serraj, R., & Siddique, K. H. M. (2007). 23 Abiotic Stresses. Chickpea breeding and management, 474.

Venkatesh, T., Thankachan, S., Kabekkodu, S. P., Chakraborti, S., & Suresh, P. S. (2021). Emerging patterns and implications of breast cancer epigenetics: an update of the current knowledge. In Epigenetics and Reproductive Health (pp. 295-324). Academic Press.

Wang, X., Chang, X., Jing, Y., Zhao, J., Fang, Q., Sun, M., & Li, Y. (2020). Identification and functional prediction of soybean CircRNAs involved in low-temperature responses. Journal of plant physiology, 250, 153188.

Wang, Y., Yang, M., Wei, S., Qin, F., Zhao, H., & Suo, B. (2017). Identification of circular RNAs and their targets in leaves of Triticum aestivum L. under dehydration stress. Frontiers in Plant Science, 7, 2024.

Ye, C. Y., Chen, L., Liu, C., Zhu, Q. H., & Fan, L. (2015). Widespread noncoding circular RNA s in plants. New Phytologist, 208(1), 88-95.

Yu, Y., Zhang, Y., Chen, X., & Chen, Y. (2019). Plant noncoding RNAs: hidden players in development and stress responses. Annual review of cell and developmental biology, 35, 407-431.

Zhang, P., & Dai, M. (2022). CircRNA: a rising star in plant biology. Journal of Genetics and Genomics.

Zhang, P., Li, S., & Chen, M. (2020). Characterization and function of circular RNAs in plants. Frontiers in Molecular Biosciences, 7, 91.

Zhao, W., Chu, S., & Jiao, Y. (2019). Present scenario of circular RNAs (circRNAs) in plants. Frontiers in plant science, 10, 379.

Zuo, J., Wang, Q., Zhu, B., Luo, Y., & Gao, L. (2016). Deciphering the roles of circRNAs on chilling injury in tomato. Biochemical and biophysical research communications, 479(2), 132-138.

Summary

This study proposal focuses on the effect of Circular RNAs (circRNAs) in increasing drought stress tolerance in soybeans. Recognizing drought as a severe hazard to critical crops, notably soybeans, the study seeks to identify the regulatory activities of circRNAs in drought-stressed soybean plants. The project outlines a three-year work program that includes characterizing circRNA expression profiles, identifying drought-responsive circRNAs, assessing their regulatory roles, and manipulating circRNA expression to evaluate its impact on drought tolerance. Multigenerational studies will also be conducted to determine the heritability and durability of circRNA-induced characteristics. By incorporating data into a circRNA regulation network model, the study hopes to not only contribute to minimizing immediate drought impacts on soybean productivity, but also to enhance the larger area of plant breeding by providing insights for more robust and sustainable agriculture methods. The proposed study will further our understanding of circRNA activities in soybeans, paving the path for novel crop development options.