Genome and Epigenome Editing for Stress-Tolerant Crops E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Fachliteratur
- Sprache: Englisch
Provides a timely overview of the use of CRISPR and non-coding RNA technologies to develop climate-resilient crops
With mounting challenges from climate change, expanding populations, and resource limitations, the need for resilient and sustainable agricultural systems has never been greater. Genome and Epigenome Editing for Stress-Tolerant Crops summarizes advanced techniques for creating crops that can withstand both biotic and abiotic stressors. Edited by renowned biologist Jen-Tsung Chen, this authoritative volume discusses the coordination of CRISPR/Cas technology with ncRNA-based epigenetics to enhance stress tolerance and improve crop quality.
In addition to offering insights into genetic and molecular advances, contributions by experts in the field present key methodologies and applications that bridge multiple omics technologies with genome editing for impactful agricultural outcomes. Addressing emerging tools and strategies that could be instrumental in achieving the United Nations Sustainable Development Goals (SDGs) and advancing sustainable agriculture, Genome and Epigenome Editing for Stress-Tolerant Crops:
- Provides an in-depth overview of CRISPR/Cas and non-coding RNA strategies to develop stress-tolerant crops.
- Integrates multiple omics approaches, including genomics, transcriptomics, and metabolomics for comprehensive crop improvement.
- Discusses strategies for resilience against both abiotic and biotic stressors, such as drought, salinity, pests, and pathogens.
- Offers practical applications of CRISPR and RNA technologies for high-yield, high-quality crop development.
- Presents recent research advancements in epigenetic regulation to fine-tune plant stress responses.
- Discusses future directions in plant science to inspire new research and experimental designs.
Genome and Epigenome Editing for Stress-Tolerant Crops is essential reading for advanced undergraduate and graduate courses in plant biology, molecular genetics, and agricultural biotechnology. It is also a valuable reference for researchers, plant breeders, and scientists working on crop improvement and climate-resilient agriculture initiatives.
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Table of Contents
Cover
Table of Contents
Title Page
Copyright Page
List of Contributors
Preface
1 Mitigating Heat Stress Response in CRISPR/Cas‐Mediated Edited Crops by Altering the Expression Pattern of Noncoding DNA
1.1 Introduction
1.2 Impact of Climate Change and Heat Stress on Crop Productivity
1.3 Strategies for Mitigating Heat Stress Response Through Gene Editing and Derivative Technologies
1.4 Conclusions and Future Perspectives
References
2 Design Future Crops with Stress Resilience by CRISPR/Cas Reprogramming Noncoding RNAs
2.1 Introduction
2.2 Noncoding RNAs Associated with Drought in Plants
2.3 Noncoding RNA Associated with Salt Stress Tolerance in Plant
2.4 Important to Edit Noncoding RNA by CRISPR/Cas to Reprogram Them
2.5 Gene‐Editing Mechanism in Plants with CRISPR/Cas Technology
2.6 Functions of Different Cas Proteins
2.7 Different Cas9 Variants for Genome Editing in Plants
2.8 Success in CRISPR/Cas‐Editing Noncoding RNA Genes in Plants
2.9 Challenges Associated with Editing Noncoding RNA in Plants
2.10 Conclusion and Future Directions
References
3 Improving Plant Abiotic Stress Tolerance by Modulating LncRNAs Using CRISPR/Cas9 Technology
3.1 Introduction
3.2 The CRISPR‐Cas Technology
3.3 Mechanistic Overview of CRISPR/Cas9‐Based Editing
3.4 Impact of lncRNAs on Plant Abiotic Stress
3.5 CRISPR/Cas lncRNA Editing Improves Plant Abiotic Stress Tolerance
3.6 Future Directions and Perspective
3.7 Conclusion
References
4 CRISPR/Cas‐Modified Long Noncoding RNAs for Regulating Plant Abiotic Responses
4.1 Introduction
4.2 Long Noncoding RNA (lncRNA) from Plants May Also be Related to Stress
4.3 Conclusion
References
5 Functional Analysis of Plant Noncoding Genomes Using CRISPR‐Cas9‐Mediated Approaches
5.1 Introduction
5.2 Noncoding Genome in Plants
5.3 CRISPR‐Cas9 Technology
5.4 Applications of CRISPR‐Cas9 in Plant Genomics
5.5 Designing CRISPR‐Cas9 Experiments for Noncoding Regions
5.6 Techniques for Characterizing CRISPR‐Cas9‐Mediated Mutations
5.7 Future Prospects
5.8 Conclusion
References
6 Small RNA‐Mediated Plant Protection Constructed byCRISPR/Cas Genome Editing
6.1 Introduction
6.2 Principles of Small RNA‐Mediated Plant Defense Mechanisms
6.3 CRISPR/Cas‐Mediated Engineering of Small RNA Pathways
6.4 Case Studies: Small RNA‐Mediated Plant Protection Enhanced by CRISPR/Cas
6.5 Challenges and Limitations
6.6 Future Prospects
6.7 Conclusion
References
7 Plant Immunity Released by CRISPR/Cas Technology‐Mediated Modifying Long Noncoding RNAs
7.1 Introduction
7.2 Plant Immunity
7.3 Long Noncoding RNAs and CRISPR/Cas Technology
7.4 Enhancing Plant Immunity Through lncRNA Modification
7.5 Key Roles of lncRNAs in Plant Immunity
7.6 Application of CRISPR/Cas Technology‐Mediated Modifying Long Noncoding RNAs
7.7 Ethical Regulations, Bioethical Uses, and Future Prospects
7.8 Conclusion
References
8 Plant Stress Memory Rewrite by CRISPR/Cas‐Mediated Approaches
8.1 Introduction
8.2 Understanding Plant Stress Response
8.3 Plant Stress Memory and Its Affecting Factors
8.4 Molecular and Epigenetic Mechanism of Plant Stress Memory
8.5 Exploring CRISPR/Cas Approaches to Write Plant Stress Memory
8.6 Genome‐Editing Technique Based on CRISPR/Cas9
8.7 The CRISPR‐Cas Genome Modification Mechanism
8.8 Ethical Issues
8.9 Future Prospects
8.10 Conclusion
References
9 Salinity‐Tolerant Crop Breeding Through Reprogramming Noncoding RNAs Mediated by CRISPR/Cas9 Technology
9.1 Introduction
9.2 Negative Impact of Salinity Stress on Plants
9.3 Why Developing Salinity‐Tolerant Crops Is a Necessity?
9.4 Breeding for Salt‐Tolerant Crops Against Salinity Stress
9.5 An Overview of ncRNAs in Plants
9.6 Mechanistic Insights of CRISPR/Cas9 Tool
9.7 Targeting/Reprogramming ncRNAs Using CRISPR/Cas9 Tools
9.8 Concluding Remarks
References
10 Genetic Engineering of
Cis
‐Regulatory Elements by CRISPR/Cas Technology for Crop Improvement
10.1 Introduction
10.2
CRISPR
/Cas System Advancements
10.3 CRISPR/Cas9 Application for Improving Crop Quality
10.4
Cis
‐regulatory Components in Introns, Promoters, and Intergenic Areas
10.5 Natural Variation Affecting
Cis
‐Regulatory Elements in Crops by InDels, SNPs, and Transposable Elements
10.6 Ways to Use the
CRISPR
/Cas‐Editing System for Transgene‐Free Genetic Engineering
10.7 The Genome‐Wide Association Study’s (GWAS) Identification of CREs that are Vital to Agriculture
10.8 Customized CRE Alterations
10.9 CRE Editing with
CRISPR
/Cas
10.10 Methods for Improving Horticultural Crops Through
CRISPR
/Cas‐Mediated Cis‐Engineering Applications
10.11 Opportunities and Challenges
10.12 Conclusion
References
11 Harnessing CRISPR Technology to Edit Noncoding RNAs for Enhanced Disease Resistance in Crops
11.1 Introduction
11.2 A Brief Overview of CRISPR/Cas Technology
11.3 Overview of Noncoding RNAs
11.4 CRISPR/Cas‐Edited Crops for Disease Resistance and Limitations
11.5 Modulation of Noncoding RNAs in CRISPR/Cas‐Edited Crops
11.6 Example of CRISPR‐Cas‐Edited Crops with Modified Noncoding RNAs
11.7 Future Perspectives and Challenges
11.8 Conclusion
References
12 Temperature‐Smart Crops Through Edited Noncoding RNAs Using CRISPR/Cas Technology
12.1 Introduction
12.2 The Function of ncRNAs in Response to Temperature Stress in Plants
12.3 CRISPR/Cas Technology for Editing Noncoding RNAs
12.4 CRISPR/Cas9 Editing of Noncoding RNAs (ncRNAs) for Cold and Heat Tolerance
12.5 Applications in Cold and Heat Tolerance
12.6 Case Studies: Temperature‐Smart Crops Developed Through ncRNA Editing
12.7 Challenges and Future Directions
12.8 Future Prospects for Temperature‐Smart Crops
12.9 Conclusions
References
13 Technical Advancements in Functional Mutagenesis of Plant Long Noncoding RNAs Using CRISPR/Cas Technology
13.1 Introduction
13.2 The Complex Architecture of Plant lncRNAs
13.3 The Diverse and Dynamic Functions of Plant lncRNAs
13.4 Utility and Challenges of CRISPR/Cas System for Deciphering lncRNA Functions
13.5 Functional Analysis of Different Plant lncRNAs Using CRISPR/Cas System
13.6 Conclusion and Future Directions
References
14 Plant Gene Silencing Modified by CRISPR/Cas‐Reprogrammed Small Interfering RNAs for Regulating Abiotic Stress Responses
14.1 Introduction
14.2 CRISPR/Cas System: Principles and Applications
14.3 Role of Small Interfering RNAs in Gene Silencing
14.4 CRISPR/Cas‐Mediated Reprogramming of siRNAs for Gene Silencing
14.5 Regulation of Abiotic Stress Responses Through Gene Silencing
14.6 Conclusion
References
15 Functional Analysis and Modification of Plant MicroRNA by CRISR/Cas System for Stress Tolerance
15.1 Introduction
15.2 miRNA Role in Plants
15.3 In Silico Approaches to Identifying miRNA
15.4 CRISPR‐Based Editing of miRNA in Plants for Stress Control
15.5 Conclusion and Future Prospects
References
16 Employment of RNA Interference and CRISPR/Cas Machinery for Disease Resistance in Crops
16.1 Introduction
16.2 The Editors of Nature
16.3 The Mechanisms of Silence: Understanding RNA Interference
16.4 Advancing Agriculture: Engineered RNA Interference for Enhanced Crop Performance
16.5 RNAi Approaches: Targeted Silence to Combat Crop Diseases
16.6 Sculpting Genetic Frontiers: CRISPR and Its Triumphs in Crop Disease Management
16.7 Conclusion
References
17 Antiabiotic Stress Capacities of Crops Gained by CRISPR/Cas‐Edited Small RNAs
17.1 Introduction
17.2 CRISPR/Cas Technology: Mechanism and Applications
17.3 CRISPR/Cas‐Mediated Editing of sRNA for Stress Tolerance
17.4 Application of CRISPR/Cas‐Edited sRNA in Stress Tolerance
17.5 Challenges and Future Prospects of CRISPR/Cas‐Edited Small RNAs for Abiotic Stress Resistance in Crops
17.6 Conclusion
References
Index
End User License Agreement
List of Tables
Chapter 1
Table 1.1 Summary of the potential CRE targets in different crops for the d...
Chapter 2
Table 2.1 Summary of reports published in recent years where noncoding RNA ...
Chapter 4
Table 4.1 lncRNAs involved in abiotic stress response in monocotyledons and...
Chapter 6
Table 6.1 Summary of small RNA classes and their functions in plant defense...
Table 6.2 Case studies of CRISPR/Cas‐mediated small RNA pathway modificatio...
Chapter 7
Table 7.1 Application of CRISPR/Cas technology‐mediated modifying long nonc...
Chapter 8
Table 8.1 Various stressors, their first encounter and recurrence stages, t...
Chapter 9
Table 9.1 Representative examples of ncRNAs involved in mediating salinity ...
Chapter 10
Table 10.1 List of targeted genes for improving crops through CRISPR/Cas‐me...
Chapter 11
Table 11.1 Exploitation of CRISPR‐Cas9 to counteract crop bacterial disease...
Table 11.2 List of viral resistance in plants introduced via CRISPR‐Cas9 ag...
Chapter 12
Table 12.1 Examples of noncoding RNAs (ncRNAs) involved in temperature stre...
Table 12.2 CRISPR/Cas systems used in ncRNA editing for temperature stress ...
Table 12.3 Potential applications of edited ncRNAs in temperature‐smart cro...
Chapter 13
Table 13.1 Knockout of different plant lncRNAs using the CRISPR/Cas system....
Chapter 14
Table 14.1 Types of Cas Proteins and their functions.
Table 14.2 Successful modification of siRNAs for targeted gene silencing.
Table 14.4 Challenges and perspectives of CRISPRi.
Chapter 15
Table 15.1 Key experiments exploring the variation in miRNA and their targe...
Table 15.2 Transcriptome sequence retrieval and assembly tools for miRNA id...
Table 15.3 Trimming tools for preprocessing of raw sequence data.
Table 15.4 Databases for identifying noncoding and repetitive sRNAs.
Table 15.5 Sequence alignment tools that might help in novel miRNA identifi...
Table 15.6 Authorized miRNA databases having already authenticated and vali...
Table 15.7 Plant miRNA databases that help in miRNA prediction.
Table 15.8 Databases/tools that can provide secondary structure of miRNAs f...
Table 15.9 miRNA and mRNA interaction analysis tools.
Table 15.10 Prediction tools for miRNA targets.
Table 15.11 Functional annotation and pathway analysis of identified miRNA ...
Table 15.12 miRNAs targeted using CRISPR/Cas systems.
Chapter 16
Table 16.1 Different developments in crop plants using various types of nc‐...
Table 16.2 Deployment of disease resistance in various crops through RNAi a...
Table 16.3 Target genes and corresponding phenotypes for disease resistance...
Chapter 17
Table 17.1 Abiotic stress and crop response gained by edited small RNAs.
List of Illustrations
Chapter 2
Figure 2.1 Pipeline for CRISPR/Cas gene editing of noncoding RNA genes in pl...
Figure 2.2 CRISPR/Cas9 gene‐editing mechanism.
Chapter 3
Figure 3.1 lncRNA‐mediated abiotic stress adaptations in plants. Cold‐respon...
Figure 3.2 Comparison of crop‐breeding approaches. Genome editing: precisely...
Figure 3.3 Flow chart to generate mutagenized plant by CRISPRs‐Cas (clustere...
Figure 3.4 lncRNA‐mediated stress responses in the plant. Under drought stre...
Chapter 4
Figure 4.1 Classification of the regulatory ncRNAs consists of microRNAs (mi...
Chapter 5
Figure 5.1 CRISPR‐Cas9 experiment for noncoding regions and functional analy...
Chapter 7
Figure 7.1 Major technical limitations; (a) ethical concerns with CRISPR/Cas...
Chapter 8
Figure 8.1 Concept flowchart of plant stress memory rewrite by CRISPR‐Cas.
Figure 8.2 Ethical issues in plant stress memory rewrite by CRISPR‐Cas.
Chapter 9
Figure 9.1 Different effects of salinity stress in plants.
Figure 9.2 Outline of CRISPR/Cas9 technology to develop salt‐tolerant crops....
Chapter 10
Figure 10.1 The process of altering plant genes using CRISPR/Cas9.
Figure 10.2 Publication data of CRISPR/Cas from 2016 to 2020.
Figure 10.3 The structure of plant genes, distinguishing between coding (exo...
Chapter 11
Figure 11.1 Three classes of programmable nucleases. (a) ZFN, (b) TALEN, and...
Figure 11.2 The CRISPR/Cas9 system.
Figure 11.3 Classification of known CRISPR‐Cas systems.
Figure 11.4 A historical chronology of the CRISPR‐Cas9 system’s component di...
Figure 11.5 Mechanism of action of CRISPR‐Cas9.
Figure 11.6 Applications of CRISPR‐Cas9 in breeding technologies.
Chapter 12
Figure 12.1 Types of noncoding RNAs and their numerous key functions.
Figure 12.2 CRISPR/Cas mechanism on ncRNAs for temperature‐smart crops.
Chapter 13
Figure 13.1 Different types of plant lncRNAs according to their genomic loca...
Figure 13.2 lncRNAs regulate plant gene expression via diverse mechanisms. T...
Figure 13.3 lncRNA functional knockout strategies using the CRISPR/Cas syste...
Chapter 14
Figure 14.1 Detailed view of the CRISPR structure and its components.
Figure 14.2 Detailed view of CRISPR‐mediated gene editing in plants.
Figure 14.3 A detailed process of siRNA synthesis.
Chapter 15
Figure 15.1 miRNA biogenesis and function in plants. The production of plant...
Figure 15.2 miRNA targeting using the CRISPR/Cas system. The CRISPR/Cas syst...
Figure 15.3 CRISPR/Cas‐driven precision editing of miRNA regions and its bio...
Chapter 16
Figure 16.1 Techniques in plant breeding and genome editing for the enhancem...
Figure 16.2 RNAi and CRISPR‐Cas9 genome‐editing mechanisms. (a) RNAi pathway...
Figure 16.3 Comparative approaches of gene silencing for crop improvement. (...
Figure 16.4 Techniques for gene silencing and gene editing in crop improveme...
Chapter 17
Figure 17.1 Overview of CRISPR technology.
Guide
Cover Page
Table of Contents
Title Page
Copyright Page
List of Contributors
Preface
Begin Reading
Index
WILEY END USER LICENSE AGREEMENT
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Genome and Epigenome Editing for Stress‐Tolerant Crops
Edited by
Jen‐Tsung Chen
National University of Kaohsiung
Kaohsiung, Taiwan
This edition first published 2025© 2025 John Wiley & Sons Ltd
All rights reserved, including rights for text and data mining and training of artificial intelligence technologies or similar technologies. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions.
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Library of Congress Cataloging‐in‐Publication DataNames: Chen, Jen‐Tsung, editor.Title: Genome and epigenome editing for stress‐tolerant crops / edited by Jen‐Tsung Chen.Description: First edition. | Hoboken, NJ : Wiley, 2025. | Includes bibliographical references and index.Identifiers: LCCN 2024058673 (print) | LCCN 2024058674 (ebook) | ISBN 9781394280018 (hardback) | ISBN 9781394280032 (adobe pdf) | ISBN 9781394280025 (epub)Subjects: LCSH: Plant genetics. | Plants–Effect of stress on. | Epigenetics.Classification: LCC QK981 .G484 2025 (print) | LCC QK981 (ebook) | DDC 581.3/5–dc23/eng/20250205LC record available at https://lccn.loc.gov/2024058673LC ebook record available at https://lccn.loc.gov/2024058674
Cover Design: WileyCover Image: © Til Vogt/Shutterstock
List of Contributors
Mariam AkhtarAgricultural Biotechnology DivisionNational Institute for Biotechnology andGenetic Engineering (NIBGE)Constituent College of Pakistan Institute ofEngineering and Applied Sciences (PIEAS)Faisalabad, Pakistan
Imran AminAgricultural Biotechnology DivisionNational Institute for Biotechnology andGenetic Engineering (NIBGE)Constitute College of Pakistan Institute ofEngineering and Applied Sciences (PIEAS)Faisalabad, Pakistan
Afreen AnisFaculty of Agriculture and Allied SciencesC.V. Raman Global UniversityBhubaneswar, India
Muhammad Jawad Akbar AwanAgricultural Biotechnology DivisionNational Institute for Biotechnology andGenetic Engineering (NIBGE)Constituent College of Pakistan Institute ofEngineering and Applied Sciences (PIEAS)Faisalabad, Pakistan
Asad AzeemDepartment of Plant Breeding and GeneticsUniversity of LayyahLayyah, Pakistan
Sana BasharatCollege of Tropical Agriculture and ForestryHainan UniversityHaikou, Hainan, ChinaKey Laboratory of Tropical HorticulturalCrop Quality RegulationCollege of HorticultureHainan UniversityHaikou, Hainan, ChinaHainan Yazhou Bay Seed LaboratorySanya Nanfan Research Institute of HainanUniversitySanya, ChinaFang Zhiyuan Academician Team InnovationCenter of Hainan ProvinceSanya, China
Andriy BilichakAgriculture and Agri‐Food CanadaMorden Research and Development CentreMorden, Manitoba, Canada
Muhammad Ismail BuzdarAgricultural Biotechnology DivisionNational Institute for Biotechnology andGenetic Engineering (NIBGE)Constituent College of Pakistan Instituteof Engineering and AppliedSciences (PIEAS)Faisalabad, Pakistan
Himanshushekhar ChaurasiaMechanical Processing DivisionICAR‐Central Institute for Research on CottonTechnologyMumbai, Maharashtra, India
Privilege ChikoveFaculty of Agriculture and Allied SciencesC.V. Raman Global UniversityBhubaneswar, India
Ananya ChoudhuryFaculty of Agriculture and Allied ScienceC. V. Raman Global UniversityBhubaneswar, India
Parinita DasDivision of Agricultural BioinformaticsICAR‐Indian Agricultural Statistics ResearchInstituteNew Delhi, Delhi, IndiaDiscipline of BioinformaticsThe Graduate SchoolICAR‐Indian Agricultural Research InstituteNew Delhi, Delhi, India
Soumyashree DashFaculty of Agriculture and Allied SciencesC.V. Raman Global UniversityBhubaneswar, India
Swatismita DeoFaculty of Agriculture and Allied ScienceC. V. Raman Global UniversityBhubaneswar, India
Dhaarani V.Faculty of Agriculture and Allied ScienceC. V. Raman Global UniversityBhubaneswar, India
Tushar K. DuttaDivision of NematologyICAR‐Indian Agricultural Research InstituteNew Delhi, Delhi, India
Subham GhoshDivision of Agricultural BioinformaticsICAR‐Indian Agricultural Statistics ResearchInstituteNew Delhi, Delhi, IndiaDiscipline of BioinformaticsThe Graduate SchoolICAR‐Indian Agricultural ResearchInstituteNew Delhi, Delhi, India
Ifrah ImranAgricultural Biotechnology DivisionNational Institute for Biotechnology andGenetic Engineering (NIBGE)Constitute College of Pakistan Institute ofEngineering and Applied Sciences (PIEAS)Faisalabad, Pakistan
Anam IshtiaqAgricultural Biotechnology DivisionNational Institute for Biotechnology andGenetic Engineering (NIBGE)Constitute College of Pakistan Institute ofEngineering and Applied Sciences (PIEAS)Faisalabad, Pakistan
Abul Kalam Mohammad Aminul IslamDepartment of Genetics and Plant BreedingFaculty of AgricultureBangabandhu Sheikh Mujibur RahmanAgricultural UniversityGazipur, Bangladesh
Faiz Ahmad JoyiaCentre of Agricultural Biochemistry andBiotechnology (CABB)University of AgricultureFaisalabad, Pakistan
Angeline JurryFaculty of Agriculture and Allied SciencesC.V. Raman Global UniversityBhubaneswar, India
Soumita KarmakarFaculty of Agriculture and Allied SciencesC.V. Raman Global UniversityBhubaneswar, India
Emanpreet KaurAgriculture and Agri‐Food CanadaMorden Research and Development CentreMorden, Manitoba, Canada
Kadiyala KavyaDepartment of Genetics and Plant BreedingCollege of Post Graduate Studies inAgricultural SciencesCentral Agricultural University (Imphal)Umiam, Meghalaya, India
Ajay Kumar KeotBiological Sciences and Technology DivisionCSIR‐North East Institute of Science andTechnology (NEIST)Jorhat, Assam, IndiaAcademy of Scientific and InnovativeResearch (AcSIR)Ghaziabad, India
Jahan KhanAgricultural Biotechnology DivisionNational Institute for Biotechnology andGenetic Engineering (NIBGE)Constituent College of Pakistan Institute ofEngineering and Applied Sciences (PIEAS)Faisalabad, Pakistan
Muhammad Sarwar KhanCentre of Agricultural Biochemistry andBiotechnology (CABB)University of AgricultureFaisalabad, Pakistan
Marium KhatunDepartment of Genetics and Plant BreedingFaculty of AgricultureBangabandhu Sheikh Mujibur RahmanAgricultural UniversityGazipur, Bangladesh
Abhishek KumarFaculty of AgricultureMaharishi Markandeshwar UniversityMullana, Ambala, Haryana, India
Niraj KumarBiological Sciences and TechnologyDivision, CSIR‐North East Institute of Scienceand Technology (NEIST)Jorhat, Assam, IndiaAcademy of Scientific and InnovativeResearch (AcSIR)Ghaziabad, India
Sharia Yesmin LabonnoDepartment of Genetics and Plant BreedingFaculty of AgricultureBangabandhu Sheikh Mujibur RahmanAgricultural UniversityGazipur, Bangladesh
Louie Cris LoposAgriculture and Agri‐Food CanadaMorden Research and Development CentreMorden, Manitoba, Canada
Muhammad Arslan MahmoodAgricultural Biotechnology DivisionNational Institute for Biotechnology andGenetic Engineering (NIBGE)Constitute College of Pakistan Institute ofEngineering and Applied Sciences (PIEAS)Faisalabad, Pakistan
Alagu ManickaveluDepartment of Genomic ScienceCentral University of KeralaKasaragod, Kerala, India
Riwandahun MarweinBiological Sciences and Technology DivisionCSIR‐North East Institute of Science andTechnology (NEIST)Jorhat, Assam, IndiaAcademy of Scientific and Innovative Research(AcSIR)Ghaziabad, India
Srutirekha MishraFaculty of Agriculture and Allied ScienceC. V. Raman Global UniversityBhubaneswar, India
Sneha MurmuDivision of Agricultural BioinformaticsICAR‐Indian Agricultural Statistics ResearchInstituteNew Delhi, Delhi, India
Ghulam MustafaCentre of Agricultural Biochemistry andBiotechnology (CABB)University of AgricultureFaisalabad, Pakistan
Rubab Zahra NaqviAgricultural Biotechnology DivisionNational Institute for Biotechnology andGenetic Engineering (NIBGE)Constitute College of Pakistan Institute ofEngineering and Applied Sciences (PIEAS)Faisalabad, Pakistan
Bhupati NayakFaculty of Agriculture and Allied ScienceC. V. Raman Global UniversityBhubaneswar, India
Fernanda Freitas de OliveiraGenetics DepartmentFederal University of ParanaCuritiba‐PR, Brazil
Jardel de OliveiraDepartment of Agronomy ‐ PostgraduateProgram in Agronomy ‐ Plant ProductionSão Paulo Western UniversityPresidente Prudente‐SP, Brazil
Pritilagna PanigrahiFaculty of Agriculture and Allied ScienceC. V. Raman Global UniversityBhubaneswar, India
Aqsa ParvaizDepartment of Biochemistry andBiotechnologyThe Women University MultanMultan, Pakistan
Laxmipriya PatiFaculty of Agriculture and Allied ScienceC. V. Raman Global UniversityBhubaneswar, India
Liu PingwuCollege of Tropical Agriculture and ForestryHainan UniversityHaikou, Hainan, ChinaKey Laboratory of Tropical Horticultural CropQuality RegulationCollege of HorticultureHainan UniversityHaikou, Hainan, ChinaHainan Yazhou Bay Seed LaboratorySanya Nanfan Research Institute of HainanUniversitySanya, ChinaFang Zhiyuan Academician Team InnovationCenter of Hainan ProvinceSanya, China
PoojaDepartment of Plant PathologyChaudhary Charan Singh (CCS) HaryanaAgricultural UniversityHisar, Haryana, India
Janapareddy RajeshDepartment of Seed Science and TechnologyHemvati Nandan Bahuguna GarhwalUniversitySrinagar, Uttarakhand, India
Noru Rajasekhar ReddyDepartment of Genetics and Plant BreedingCollege of AgricultureKerala Agricultural UniversityThiruvananthapuram, Kerala, India
Maria RehmanCentre of Agricultural Biochemistry andBiotechnology (CABB)University of AgricultureFaisalabad, PakistanDepartment of BiotechnologyUniversity of SargodhaSargodha, Pakistan
Rabia RehmanAgricultural Biotechnology DivisionNational Institute for Biotechnology andGenetic Engineering (NIBGE)Constitute College of Pakistan Institute ofEngineering and Applied Sciences (PIEAS)Faisalabad, Pakistan
Anna ReraFaculty of Agriculture and Allied SciencesC.V. Raman Global UniversityBhubaneswar, India
Lellapalli RitheshDepartment of Plant PathologyKerala Agricultural UniversityThiruvananthapuram, Kerala, India
Aryadeep RoychoudhuryDiscipline of Life SciencesSchool of SciencesIndira Gandhi National Open UniversityNew Delhi, Delhi, India
Jyoti Prakash SahooFaculty of Agriculture and Allied SciencesC.V. Raman Global UniversityBhubaneswar, India
Subhashree Priyadarshinee SahooFaculty of Agriculture and Allied ScienceC. V. Raman Global UniversityBhubaneswar, India
Arfa SaifullahCentre of Agricultural Biochemistry andBiotechnology (CABB)University of AgricultureFaisalabad, Pakistan
Muhammad Waseem SajjadAgricultural Biotechnology DivisionNational Institute for Biotechnology andGenetic Engineering (NIBGE)Constitute College of Pakistan Institute ofEngineering and Applied Sciences (PIEAS)Faisalabad, Pakistan
Santanu SamantaDepartment of BiotechnologySt. Xavier's College (Autonomous)Kolkata, West Bengal, India
Abhik SarkarDivision of Agricultural BioinformaticsICAR‐Indian Agricultural Statistics ResearchInstituteNew Delhi, Delhi, IndiaDiscipline of BioinformaticsThe Graduate SchoolICAR‐Indian Agricultural ResearchInstituteNew Delhi, Delhi, India
Sumi SarkarDepartment of Genetics and Plant BreedingFaculty of AgricultureBangabandhu Sheikh Mujibur RahmanAgricultural UniversityGazipur, Bangladesh
Manash Pratim SarmahDepartment of ZoologyUniversity of Science & TechnologyBaridua, Meghalaya, India
Tiago Benedito dos SantosDepartment of Agronomy ‐ Program inAgronomy ‐ Plant ProductionSão Paulo Western UniversityPresidente Prudente‐SP, Brazil
IffatShaheenDepartment of AgricultureBahauddin Zakariya UniversityMultan, Pakistan
Nimra ShahidDepartment of BotanyUniversity of NarowalNarowal, Punjab, Pakistan
Muhammad ShareefDepartment of BotanyUniversity of NarowalNarowal, Punjab, Pakistan
Sehrish ShehzadiCentre of Agricultural Biochemistry andBiotechnology (CABB)University of AgricultureFaisalabad, Pakistan
Nyashadzashe ShengeziFaculty of Agriculture and Allied SciencesC.V. Raman Global UniversityBhubaneswar, India
Saqib SiddiqueAgricultural Biotechnology DivisionNational Institute for Biotechnology andGenetic Engineering (NIBGE)Constitute College of Pakistan Institute ofEngineering and Applied Sciences (PIEAS)Faisalabad, Pakistan
Hidam Bishworjit SinghDepartment of BiotechnologyGauhati UniversityGuwahati, Assam, India
Dhanawantari L. SinghaRabindranath Tagore UniversityHojai, Assam, India
Dipro SinhaDivision of Agricultural BioinformaticsICAR‐Indian Agricultural Statistics ResearchInstituteNew Delhi, Delhi, India
Subhalaxmi SwainFaculty of Agriculture and Allied ScienceC. V. Raman Global UniversityBhubaneswar, India
SwarnalataTripathyFaculty of Agriculture and Allied ScienceC. V. Raman Global UniversityBhubaneswar, India
Swapan Kumar TripathyCollege of AgricultureOdisha University of Agriculture andTechnologyBhawanipatna, India
Muhammad WaseemCollege of Tropical Agriculture and ForestryHainan UniversityHaikou, Hainan, ChinaKey Laboratory of Tropical Horticultural CropQuality RegulationCollege of HorticultureHainan UniversityHaikou, Hainan, ChinaHainan Yazhou Bay Seed LaboratorySanya Nanfan Research Institute of HainanUniversitySanya, ChinaFang Zhiyuan Academician Team InnovationCenter of Hainan ProvinceSanya, China
DilianeHarumi YaguinumaDepartment of Agronomy ‐ PostgraduateProgram in Agronomy ‐ Plant ProductionSão Paulo Western UniversityPresidente Prudente‐SP, Brazil
RidaZahidAgricultural Biotechnology DivisionNational Institute for Biotechnology andGenetic Engineering (NIBGE)Constitute College of Pakistan Institute ofEngineering and Applied Sciences (PIEAS)Faisalabad, Pakistan
Preface
In recent decades, the world has faced serious crises mainly caused by the continuously expanding human population, the resulting environmental pollution, and the shortage of food and natural resources. Undoubtedly, these conditions can be worse under the scenario of climate change globally, such as a rising temperature that can lead to shortages of water resources and, consequently, increase the risk of agricultural and ecological droughts. To ensure sufficient agricultural nutrition and food production, scientists are expected to solve the problems by developing new crop breeding methods, particularly focusing on identifying the stress‐tolerant traits and uncovering underlying in‐depth machinery. Importantly, these approaches can advance agricultural biotechnology and crop breeding focusing on acquiring future crops with climate‐resilient capacities, which might greatly ensure nutrition and food security under the challenge of upcoming global climate change. Therefore, nowadays, the demand to organize crop breeding programs for developing climate‐resilient crops is inevitably increasing.
To support such demand, modern plant molecular biotechnology has been upgraded fast to achieve high‐throughput, high‐resolution, smart, and precision manners such as multiple omics‐based functional genomics, CRISPR/Cas9‐mediated genome editing, RNA technology, and so on. In the post‐genomics era, the approaches of multiple omics continue to mine huge amounts of genetic resources and accumulate interesting key genes, and definitely need efficient ways of genetic engineering to benefit crop breeding and agricultural production. In the past decade, an emerging genetic engineering technology, that is, genome editing technology, has the power to release the potential of plant genomes through the precise delivery of tolerant genes for crop improvement. The system of genome editing has upgraded fast, and now, CRISPR/Cas9 has become the dominant technology. Thus, the integration and coordination of multiple omics and CRISPR/Cas9 for organizing omics‐CRISPR breeding strategies inevitably become the next wave of important tasks in plant science. In addition, in recent years, RNA technology/epigenetic regulation has been introduced into diverse fields of plant science and has been proven to its great potential in mitigating plant stress responses as well as being a fine‐tuning regulator in some ways to advance crop improvement and breeding. The role of noncoding RNAs (ncRNAs) in the management of CRISPR‐edited crop production has become an important topic. In the future, achieving climate‐resilient agriculture needs the coordination of some crucial technologies involving multiple omics, CRISPR/Cas9, and functional RNA technology/epigenetics.
In this book, the ways for vertical integration and horizontal coordination of the crucial technologies were comprehensively discussed to advance crop breeding programs toward stress‐resilient agriculture.
This book presents the coordinated CRISPR‐noncoding RNAs (ncRNAs) strategies for combating diverse stressors and complicated or multiple stress conditions. It covers both abiotic and biotic stress, including stressors of salinity, temperature, drought, heavy metals, pests, pathogens, and so on, and proposes strategies to develop stress‐tolerant crops with high‐yield and high‐quality traits through the integration or coordination of the mainstream technologies, that is, multiple omics, CRISPR/Cas, and ncRNA‐based epigenetics.
This book is an ideal reference to integrate the emerging field of multiple omics, CRISPR/Cas, and ncRNA‐based epigenetics by sharing crucial aspects of methods, applications, and future directions. It opens doors for students and researchers to efficiently overview these critical subtopics of plant science and technology and thus realize the concept and, hopefully, inspire the ideas of future experiments and the exploration of the knowledge and, eventually, lead to better development of future crops by scientists, plant biologists, and crop breeders.
The book editor, Dr. Jen‐Tsung Chen, appreciates all contributors for their valuable chapters and the staff of Wiley for their instruction and assistance.
1Mitigating Heat Stress Response in CRISPR/Cas‐Mediated Edited Crops by Altering the Expression Pattern of Noncoding DNA
Emanpreet Kaur, Louie Cris Lopos, and Andriy Bilichak
Agriculture and Agri‐Food Canada, Morden Research and Development Centre, Morden, Manitoba, Canada
1.1 Introduction
Heat stress, a significant constraint in crop production, is becoming increasingly threatening with the onset of climate change and the increased frequency of extreme heat waves. Plants are highly vulnerable to temperature fluctuations beyond their optimum range for growth, development, and reproduction (Govindaraj et al., 2018). Previously, elevated temperatures, especially heat waves, have already substantially reduced the regional yields of rice, wheat, and corn in Asia, North America, India, and Europe (Hassan et al., 2021). The Intergovernmental Panel on Climate Change’s Sixth Assessment Report (IPCC’s AR6) indicated that the global surface temperature rose by 1.09 °C from 1850–1900 to 2011–2020, with an anticipated rise of at least 0.2 °C per decade without substantial mitigation (IPCC, 2021). This could potentially compromise our capacity to attain food security in the future; the current predictive models suggest that yield losses among staple crops could be as high as 8% per 1 °C increase in global temperature (Zhao et al., 2017; Lee et al., 2024). While adaptive agricultural practices such as altering planting dates, improving irrigation availability and efficiency, utilizing shade structures, growing in controlled environments, and applying osmoprotectants could alleviate the effects of heat stress on crop yield, these approaches are not always cheap and flexible, especially at higher production levels. The deployment of thermotolerant cultivars that can withstand elevated temperatures without yield or agronomic penalties is still seen as the most sustainable solution to feeding the ever‐growing human population amidst rising global temperatures.
Breeding for thermotolerance is associated with complexities originating from this trait’s polygenic nature and the complex genetic regulatory network that controls its manifestation (Yeh et al., 2012). Currently, we are progressing in explaining the mechanisms and regulatory control of thermotolerance in model species such as Arabidopsis (Arabidopsis thaliana), but given the diversity of thermotolerance phenotypes among different plant species, this may not be sufficient (Yeh et al., 2012). Elucidating species‐specific thermotolerant mechanisms will greatly benefit the development of thermotolerant cultivars.
Gene‐editing platforms, particularly clustered regularly interspaced palindromic repeats (CRISPRs)/CRISPR‐associated protein 9 (Cas9) and their derivatives, have been beneficial in generating novel thermotolerant phenotypes and functional elucidation. In plant breeding, the most mainstream gene‐editing approach is to mutate the coding region of a candidate protein‐coding gene in a targeted manner to generate phenotypic variability to select upon. To a lesser extent, the mutation usually results in abolished gene function (knockout), gain‐of‐function, or neofunctionalization. Unfortunately, negative pleiotropic effects may also result when targeting genes involved in complex gene networks.
An emerging approach in genome editing for crop improvement is targeting noncoding DNA, particularly cis‐regulatory elements (CREs). These noncoding DNA segments regulate gene expression when interacted with trans‐regulatory elements. Contrary to the mainstream approach of modifying gene products, this approach aims to quantitatively modify the levels and/or the temporal–spatial gene expression to achieve phenotypic diversity while at the same time minimizing or avoiding pleiotropic effects (Swinnen et al., 2016). The transcriptional rewiring associated with the selection of CRE variants during the domestication of our staple crop species (Swinnen et al., 2016) and the diverse phenotypic changes induced when editing CREs (reviewed in Saeed et al. (2022)) greatly emphasize the phenotypic contributions of CREs in particular and noncoding DNA in general, making a strong case for the utility of this approach in accelerating crop improvement.
This chapter will review approaches for improving thermotolerance by modifying CREs and other functional, noncoding DNA elements using genome‐editing tools, particularly those derived from CRISPR/Cas9. Since thermotolerance is a quantitative polygenic trait tightly regulated by a complex regulatory network, we believe this approach could greatly benefit its development.
1.2 Impact of Climate Change and Heat Stress on Crop Productivity
1.2.1 A Physiological Impact of Heat Stress on Plant Growth and Development
Heat stress is a significant environmental factor affecting crop yield and quality. The definition of heat stress depends on the natural habitat and is species‐specific (Yeh et al., 2012). Mild temperature increase generally induces plant development and early flowering and alters immunity (Hua, 2013; Verhage et al., 2014; Capovilla et al., 2015; Gangappa et al., 2017). Plants can adapt to suboptimal temperatures through thermomorphogenesis – a range of morphological adaptations, including hypocotyl elongation, upward leaf movement (thermonasty), petiole elongation, reduced stomatal density, and formation of smaller and thinner leaves (Quint et al., 2016; Casal & Balasubramanian, 2019). Eventually, plants can cool themselves through open rosette structures and transpiration (Crawford et al., 2012; Park et al., 2019).
Extreme temperatures can cause irreversible plant damage, significantly affecting crop development and profitability and seriously threatening national and global food security (Lesk et al., 2022). Temperature stress disrupts photosynthesis, water metabolism, nutrient cycling, protein synthesis, reproduction, and the functionality of various enzymes, phytohormones, pollen development, and signaling molecules, resulting in sizeable reductions in yields (Crafts‐Brandner & Salvucci, 2002; Zinn et al., 2010; Mishra et al., 2023). Molecular mechanisms of thermosensing and signaling have been primarily explored in Arabidopsis with the basic Helix‐Loop‐Helix (bHLH) transcription factor (TF) PHYTOCHROME INTERACTING FACTOR 4 (PIF4) considered as the core of temperature signaling pathways (Gangappa et al., 2017; Casal & Balasubramanian, 2019). One of the key downstream targets of the PIF4 pathway involved in promoting growth is YUCCA8 (YUC8), which encodes a rate‐limiting enzyme in auxin biosynthesis and is critical for thermomorphogenesis (Franklin et al., 2011; Lee et al., 2014).
At the same time, the PIF4‐independent signaling pathways (PIF4 and PIF7 alike) were discovered to directly stimulate auxin biosynthesis and trigger thermomorphogenesis in a brassinosteroid‐dependent manner (Chung et al., 2020; Fiorucci et al., 2020; Vu et al., 2021). The active form of the photoreceptor phytochrome B (phyB) senses elevated temperature and is converted from the active Pfr form to the inactive Pr form. The nuclear export of the phyB‐Pr upon warmth releases PIF4 inhibition, initiating thermomorphogenesis (Legris et al., 2016; Qiu et al., 2019). Additionally, other mechanisms of temperature sensing were reported in Arabidopsis. The translation of PIF7 mRNA is enhanced by elevated temperature through the relaxation of the PIF7 mRNA hairpin structure, leading to the accumulation of PIF7 protein (Chung et al., 2020). Also, the transcriptional repressor EARLY FLOWERING 3 (ELF3) aggregates into inactive condensates during warming, contributing to sensing elevated temperatures (Jung et al., 2020).
Heat can damage photosynthetic machinery by inhibiting enzymes like RUBISCO and denaturing chloroplastic proteins. This leads to stomatal closure, limiting CO2 uptake and resulting in the buildup of reactive oxygen species (ROS), which cause cellular damage, affecting lipids, proteins, and nucleic acids (Allakhverdiev et al., 2008; Wang et al., 2018). Heat stress significantly impacts nutrient cycling and plant nutrition. High temperatures can impair root function, reducing the plant’s ability to uptake essential nutrients like nitrogen, phosphorus, and potassium. For instance, in tomato plants, heat stress has been found to decrease levels of nutrient‐uptake and ‐assimilation proteins in roots, leading to reduced nutrient absorption (Giri et al., 2017). Additionally, soil warming can accelerate nutrient cycling, resulting in nutrient losses due to faster decomposition rates and reducing nutrient availability in the long term. High temperatures also interfere with plant nutrient translocation, further complicating metabolic processes essential for growth and productivity (Mishra et al., 2023). At the cellular level, heat stress can cause protein denaturation, membrane destabilization, and oxidative stress. Persistent heat stress may result in cell death and tissue necrosis, compromising the overall health and productivity of the plant (Haider et al., 2021). These changes lead to the accumulation of ROS, which can damage cellular components like lipids, proteins, and nucleic acids. Enzymes lose efficiency as temperatures exceed their optimal range; continued stress triggers phytohormonal imbalances, such as altered abscisic acid levels, which affect plant responses. These imbalances activate complex signaling cascades involving calcium, ROS, and other molecules, further challenging the plant’s ability to cope with stress (Potters et al., 2007; Saidi et al., 2009; Devireddy et al., 2021).
1.2.2 Heat Shock Proteins (HSPs) and Their Roles
HSPs are a crucial component of a plant’s response to heat stress, playing key roles in maintaining protein integrity and enabling cellular homeostasis under stressful conditions. These molecular chaperones assist in the folding, refolding, transport, and degradation of proteins, preventing their denaturation and aggregation caused by heat stress (Feder & Hofmann, 1999). They are often regulated by heat shock factors (HSFs), which are activated during extreme temperatures. In wheat, HSFs such as HSFA2 and HSFA6 are activated during heat stress, modulating downstream responses to improve heat tolerance (Xue et al., 2014). Once induced, HSPs bind to misfolded proteins, aid in refolding, and prevent the formation of toxic aggregates, playing a significant role in cellular survival under heat stress (Wu, 1995). Throughout the day, varying temperatures trigger the production and accumulation of different HSPs in plants. In the mid‐morning, HSP20s are synthesized to prevent proteins from misfolding and aggregating before the temperature peaks by noon. Later in the day, as temperatures rise further, the production shifts to HSP60s, HSP70s, HSP90s, and HSP100s, which help resolubilize and reactivate proteins that may have become inactive or misfolded due to heat stress, ensuring their proper functioning (Finka et al., 2016). For instance, overexpressing certain HSPs in some crops like rice (HSP18), barley (HSP70, HSP90, and HSP100), Arabidopsis (HSP101), and cotton (HSP70‐26) improved resilience, making them valuable targets in breeding programs for heat tolerance (Queitsch et al., 2000; Kuang et al., 2017; Chaudhary et al., 2019; Zhiyong et al., 2021). Modifying the expression of HSFs and other directly related noncoding DNA regions that regulate HSPs could be a strategic approach to enhance the overexpression of key HSPs, ultimately developing heat‐resilient plants.
1.2.3 Epigenetic Regulation of Stress Response Pathways (Histone Posttranslational Modifications, DNA Methylation, and ncRNA Expression)
Recent evidence suggests that epigenetic mechanisms of gene expression and regulation are actively involved in thermosensing and heat stress tolerance in plants. Epigenetics, in a broader sense, refers to “chromatin modifications” through chemical modifications of histone proteins or DNA wrapped around them that do not change the base sequence (Deichmann, 2016). The regulation of gene expression occurs through different pathways, including DNA methylation, small RNAs, ATP‐dependent chromatin remodeling, histone variants, histone modifications, histone chaperones, and long noncoding RNAs (lncRNAs). Some of these pathways regulate the expression of high‐temperature‐responsive genes to prevent heat‐related damage and promote subsequent adaptation (reviewed in Perrella et al. (2022)). For example, the expression of many thermoresponsive genes is regulated by the repressive histone variant H2A.Z deposited in nucleosomes of temperature‐regulated loci primarily at the +1 site (Kumar & Wigge, 2010). The deposition and eviction of repressive H2A.Z in exchange for permissive H2A variant are performed by the Snf2 ATPase remodeling complexes, SWR1‐C and INO80‐EIN6 ENHANCER (EEN), respectively (Xue et al., 2021). A component of SWR1‐C, ACTIN‐RELATED PROTEIN 6 (ARP6), is a mediator of temperature responses in Arabidopsis, and arp6 mutants demonstrate elongated hypocotyls already at low temperatures, indicative of a constitutive warm temperature phenotype (Kumar & Wigge, 2010). INO80 and EEN are directly associated with PIF4 in activating the transcription of auxin‐related genes under elevated temperatures through the H2A.Z eviction. The contribution of H2A.Z to stress‐induced gene activation was further supported by the fact that its eviction was compromised in both pif4 and ino80 mutants. Overall, the H2A.Z‐containing nucleosomes are not temperature sensors per se, but rather their presence depends on transcriptional regulators allowing for environment‐dependent chromatin reorganization and release of stress‐responsive gene expression (Cortijo et al., 2017).
In addition to chromatin remodeling, histone methylation and histone acetylation marks have been implemented to regulate the gene expression of growth‐promoting genes in response to heat stress. For example, histone H3K4me2 (an activating mark) is demethylated in response to the binding of FLOWERING CONTROL LOCUS A (FCA) to PIF4‐activated growth‐promoting genes, like YUC8, therefore eventually suppressing high‐temperature‐induced hypocotyl elongation (Lee et al., 2014). Therefore, fca mutants with a continuous expression of the YUC8 gene display hyperelongated hypocotyls when exposed to 28 °C. At the same time, the H3K4me3 (considered an activating mark) demethylation of double mutant Jumonji C (JmjC) JMJ14 with its cofactor‐producing enzyme cytosolic isocitrate dehydrogenases (cICDHs) showed suppression of several auxin‐related genes, including YUC8, and resulted in reduced thermomorphogenesis capacity (Cui et al., 2021). These results suggest that distinctive histone demethylases can have either negative (FCA) or positive (JMJ14, JMJ15, and JMJ18) effects on the genes involved in thermomorphogenesis. Histone methylation can also affect alternative splicing (AS), and H3K36me3 enrichment was associated with differentially spliced (DiS) events upon temperature changes (Pajoro et al., 2017). Mutants of the histone H3K36 methyltransferases SET DOMAIN‐CONTAINING GROUP 8 (SDG8) and SDG26 had reduced DiS events, suggesting that H3K36me3 is indeed required for AS (Sidaway‐Lee et al., 2014). Histone acetylation affects gene expression through “opening” (acetylation) and “closing” (deacetylation) of specific chromatin regions in response to environmental stimuli (Asensi‐Fabado et al., 2017). Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze this process, which ultimately modulate chromatin accessibility for TFs, other regulatory proteins, and the transcription machinery (Asensi‐Fabado et al., 2017). Histone deacetylation is involved in H2A.Z eviction since the HISTONE DEACETYLASE 9 (hda9) mutant displayed high levels of H2A.Z occupancy at the YUC8 locus and a reduced ability for PIF4 binding at warm temperatures (van der Woude et al., 2019). This translates into an impaired response of the hda9 mutants to hypocotyl elongation and a compact rosette morphology in response to elevated temperatures (van der Woude et al., 2019). Nevertheless, the role of other HDACs in the heat stress response is more elaborate since hda15 mutant seedlings displayed longer hypocotyls, while hda9 and hda19 hypocotyls were shorter than the wild type at 27 °C (Shen et al., 2019). It remains to be shown whether HDA15 and HDA19 also regulate gene expression through H2A.Z occupancy.
Overall, histone and chromatin‐modifying enzymes are guided to specific regions of the chromatin by heat‐responsive factors such as TFs and lncRNAs, which act as a molecular signal to direct the epigenetic machinery to genes or genomic regions (Deng et al., 2018; Ueda & Seki, 2020). The interplay between these epigenetic regulators and their recruiters forms a sophisticated system for adapting to high‐temperature conditions. For instance, in rice, the histone modification H3K9me2 (H3K9 methylation 2) on the Oryza sativa FERTILIZATION‐INDEPENDENT ENDOSPERM1 (OsFIE1) gene is sensitive to moderate heat stress (34 °C). This modification appears to regulate OsFIE1 expression, which is important for rice seed development under elevated temperatures (Folsom et al., 2014), highlighting how histone modifications can directly impact important agronomic traits in response to heat stress. By altering the accessibility of DNA to TFs and other regulatory proteins, these epigenetics mechanisms provide a dynamic way for plants to rapidly adjust their gene expression profiles in response to heat stress.
DNA methylation is a dynamic process that regulates gene expression in response to environmental stress (Boyko et al., 2010; Li et al., 2016). DNA methylation involves the addition of methyl groups to cytosine bases in DNA, forming CG, CHG, and CHH patterns (where H represents A, T, or C), which play a crucial role in regulating plant responses to heat stress (Cokus et al., 2008; Popova et al., 2013; Liu et al., 2015, 2017; Talarico et al., 2024). Arabidopsis plants exposed to heat shock (HS) (42 °C for different durations – 6, 12, or 24 hours) demonstrated global DNA demethylation with many specific genes affected primarily related to stress response (e.g. HSFs, HSPs, fructose‐bisphosphate aldolase 6, and 60S RIBOSOMAL PROTEIN L4‐1) (Korotko et al., 2021). Similarly, heat‐sensitive cotton also showed global DNA demethylation in response to heat stress (Min et al., 2014). In contrast, the genome‐wide methylation greatly increases in heat‐sensitive Brassica napus under extreme heat exposure (Gao et al., 2014). Further research on DNA methylation‐deficient mutants revealed the importance of the RNA‐directed DNA methylation (RdDM) pathway in plant heat tolerance (Popova et al., 2013). In Arabidopsis, different components of this pathway have varying effects on heat sensitivity. Plants lacking certain methyltransferases, such as domains rearranged methyltransferase 1/2 (DRM1/DRM2) and chromomethylase 3 (CMT3), exhibit reduced heat sensitivity. However, mutants deficient in other RdDM pathway components such as nuclear RNA polymerase D 2 (nrpd2), Dicer‐like 3 (dcl3), RNA‐dependent RNA polymerase 2 (rdr2), and argonaute 4 (ago4) lead to increased heat sensitivity. This suggests a complex relationship between DNA methylation and heat stress responses. Similarly, a copia‐type, heat‐responsive retrotransposon ONSEN has been found to influence the heat responsiveness of nearby genes when it inserts into new locations (Ito et al., 2011). Additionally, the expression of certain genes, such as CALMODULIN‐LIKE 41 (CML41, At3g50770), is enhanced by heat stress and shows reduced methylation near transposable elements (TEs) (Naydenov et al., 2015). These findings suggest that the RdDM pathway modulates heat stress responses by regulating DNA methylation patterns near transposons and TEs, thereby affecting the expression of adjacent genes. Interestingly, plants lacking CMT2, which is involved in a specific type of DNA methylation (CHH), show improved heat tolerance, indicating that different methylation pathways may have distinct roles in heat stress responses (Shen et al., 2014).
Noncoding RNAs (ncRNAs), including micro RNAs (miRNAs), small‐interfering RNAs (siRNAs), and lncRNAs, play crucial roles in regulating gene expression under heat stress, especially miRNAs and siRNAs. NcRNAs are functional RNA molecules transcribed from DNA but not translated into proteins. These ncRNAs form complex regulatory networks that enable plants to fine‐tune their gene expression in response to environmental stresses, including heat. They act at various levels of gene regulation, from transcriptional control to posttranscriptional modification, allowing for rapid and flexible responses to changing conditions (Hirayama and Shinozaki, 2010; de Lima et al., 2012; Khraiwesh et al., 2012; Jha et al., 2023).
MiRNAs are short, typically 21–25 nucleotides long, ncRNAs that regulate gene expression posttranscriptionally (Voinnet, 2009). miRNAs are typically generated through a process involving the RNase III enzyme (Dicer‐like [DCL] protein), which cleaves the miRNA primary transcripts into the precursor molecules, characterized by a distinctive stem‐loop structure (de Lima et al., 2012). Once formed, mature miRNAs, along with protein factors, make an RNA‐induced silencing complex (RISC) to target mRNAs to modulate the expression of the target genes posttranscriptionally. They function by base pairing with complementary sequences on target mRNAs, usually leading to mRNA degradation or translational repression (Baulcombe, 2004; Voinnet, 2009). This posttranscriptional regulation allows miRNAs to adjust gene expression in response to various cellular needs, including development, differentiation, stress responses, and environmental cues (Rogers & Chen, 2013). For instance, miR398 is a well‐studied example of rapidly induced by heat stress in Arabidopsis and other plants. It targets genes encoding copper/zinc superoxide dismutases (CSD1 and CSD2) and their copper chaperone (CCS), which are involved in ROS scavenging. Downregulation of these targets by miR398 enhances heat tolerance (Zhu et al., 2011; Guan et al., 2013). DNA methylation can affect the expression of the miRNA‐coded genes, which can regulate the genes involved in abiotic stress response. For example, methylation‐sensitive amplification polymorphisms analysis of differentially methylated regions in response to temperature stress in Populus simonii identified seven miRNA‐coded genes significantly affected by stress application (Ci et al., 2015). Three of those miRNAs, miR156i/j, miR390c, and miR396e/g, target the products of ACYL‐COA OXIDASE (ACOX1 and ACOX3), PHOSPHOLIPID/GLYCEROL ACYLTRANSFERASE FAMILY PROTEINS (LPCAT1 and LPCAT2), and ISOCITRATE DEHYDROGENASE (IDH), which are involved in lipid metabolism and peroxisome biogenesis.
Research has revealed complex interactions between heat stress responses and phytohormone signaling pathways in plants, mediated by various miRNAs. For example, microRNA miR167, which regulates AUXIN RESPONSE FACTOR 8, ARF8 (a gene involved in the development of floral organs, gynoecium and stamen), shows a peculiar response to high temperatures. While miR167 decreases under heat stress, one specific variant, miR167h, exhibits a significant increase (Wu et al., 2006; Kruszka et al., 2014). Furthermore, other auxin signaling‐related miRNAs such as miR390 and miR393 have been implicated in heat stress responses, indicating a broader involvement of auxin‐related miRNAs in plant‐ thermotolerance mechanisms (Vidal et al., 2010; Xin et al., 2010; Guan et al., 2013; Hivrale et al., 2016).
Studies have shown that some miRNAs, typically associated with regulating plant developmental processes, also have important functions in how plants respond to heat stress. For instance, miR171 is a key regulator of gene expression for certain members of the GRAS family, specifically SCARECROW‐LIKE6‐III (SCL6‐III) and SCL6‐IV. These genes play crucial roles in various plant developmental processes, including forming radial patterns in root and shoot structures (Kamiya et al., 2003). By modulating the expression of these GRAS family genes, miR171 indirectly influences important aspects of plant architecture and growth. In Arabidopsis, heat stress increased miR171 levels, resulting in a greater suppression of GRAS gene expression (Mahale et al., 2014). Interestingly, a contrasting pattern was observed in Populus species, where pto‐miR171 and ptc‐miR171 showed reduced expression under heat stress conditions (Lu et al., 2008; Chen et al., 2012). This difference highlights the species‐specific nature of miRNA responses to environmental stressors like heat. Further, wheat research has revealed that an additional target for miR164 is the heat shock protein HSP17. Under heat stress conditions, the expression of HSP17 increased, suggesting a complex interplay between miR164 and its targets in response to elevated temperatures (Kumar et al., 2015). Furthermore, miR396 and its target gene HaWRKY6, as well as miR397 and its target gene LACCASE, have been implicated in heat stress adaptation (McCaig et al., 2005; Giacomelli et al., 2012).
siRNAs and lncRNAs are important ncRNAs that play crucial roles in plant gene regulation and stress responses. siRNAs are typically 21–24 nucleotides long and are derived from double‐stranded RNA (dsRNA) precursors. The DCL enzymes cleave these precursors into siRNAs, which then associate with the RISC to target complementary mRNA for degradation or inhibit translation, playing a key role in regulating gene expression (Axtell, 2013). They can be classified into trans‐acting siRNAs (ta‐siRNAs), natural antisense transcript siRNAs (nat‐siRNAs), and heterochromatic siRNAs. siRNAs are involved in various abiotic stress responses, including heat stress, where they regulate gene expression through mRNA degradation or translational repression (Sunkar & Zhu, 2007; Axtell, 2013). For example, three siRNAs were found to be downregulated by heat stress and upregulated by cold stress in wheat (Yao et al., 2010) and an miR173‐cleaved ta‐siRNA (TAS1) targets HEAT‐INDUCED TAS1 TARGET1 (HTT1) and HTT2, involved in thermotolerance in Arabidopsis (Khraiwesh et al., 2012; Li et al., 2014).
LncRNAs, longer than 200 nucleotides, can be categorized as antisense or intronic based on their genomic locations (Wierzbicki, 2012). They regulate gene expression at various levels, including chromatin modification, where they can recruit chromatin‐modifying complexes to specific genomic loci to alter the epigenetic state of chromatin. During transcription, lncRNAs can interact with TFs or the transcriptional machinery to enhance or repress transcription. In posttranscriptional processing, lncRNAs can influence mRNA splicing, stability, and translation. LncRNAs have been implicated in plant development and stress responses, including heat stress (Budak et al., 2020; Xin et al., 2011). For example, Xin et al. (2011) characterized long‐nonprotein‐coding RNAs (npcRNAs) in wheat with powdery mildew infection and heat stress, four of which were identified as miRNA precursors (TalnRNA5, TapmlnRNA8, TapmlnRNA19, and TahlnRNA27), in which TahlnRNA27 was upregulated under heat stress. LncRNAs have been found to be differentially expressed under heat stress conditions in various plant species, suggesting their important roles in plant thermotolerance. However, while their involvement in heat stress responses is evident, a complete and detailed understanding of the specific mechanisms by which lncRNAs contribute to plant thermotolerance is still lacking. Further comprehensive studies are needed to fully unravel the complex functions of these ncRNAs in plant heat stress response pathways (Zhao et al., 2020).
Overall, the mechanisms mentioned earlier can lead to an epigenetic memory, which refers to the ability of plants to retain information about previous stress exposures and pass it on to subsequent generations. This mechanism plays a crucial role in plant adaptation, including heat stress (Kinoshita & Seki, 2014; Crisp et al., 2016; He & Li, 2018; Ramakrishnan et al., 2022).
1.2.4 Other Protective Mechanisms and Molecular Responses
Transcriptomic and proteomic adjustments are also prevalent responses of plants to heat stress exposure. AS is a widespread mechanism in plants that enhances the diversity of both transcripts and proteins (Syed et al., 2012). Research has demonstrated that numerous genes involved in plant stress responses undergo AS, which allows plants to adjust their gene expression in response to various environmental challenges (Qin et al., 2007; Matsukura et al., 2010; Guerra et al., 2015; Tognacca et al., 2022). For instance, in maize, the expression of DEHYDRATION‐RESPONSIVE ELEMENT BINDING 2B (DREB2B), a key gene in stress response pathways, is regulated through AS when the plant experiences heat stress (Qin et al., 2007), and in rice, one of the three isoforms of gene OsHSFA2d, OsHSFA2dI, is specifically induced by HS. The gene is involved in the heat stress‐induced unfolded protein response (Cheng et al., 2015), highlighting how plants can use AS to modulate their molecular responses to elevated temperatures.
Ubiquitination, also known as ubiquitylation, is a form of posttranslational modification. It involves the attachment of a small protein called ubiquitin to a target protein within the cell. This process requires three types of enzymes working in sequence: ubiquitin‐activating enzyme (E1), ubiquitin‐conjugating enzyme (E2), and ubiquitin ligase (E3) (Vierstra, 2009). The E3 enzymes play a crucial role in determining which proteins are targeted for ubiquitination, thus providing specificity to the process. Proteins that undergo ubiquitination can experience various outcomes. Ubiquitination is often associated with protein degradation, but it can also serve nonproteolytic functions (Hershko & Ciechanover, 1998). For example, in rice, the rice RING domain E3 ligase, HEAT AND COLD INDUCED 1 (OsHCI1) has been identified as a key player in heat response. When exposed to high temperatures, OsHCI1 adds a single ubiquitin molecule to the target proteins, known as monoubiquitination. This monoubiquitination triggers the movement of these proteins from the nucleus to the cytoplasm, which is thought to contribute to the plant’s ability to withstand heat stress. Furthermore, Arabidopsis plants with overexpression of OsHCI1‐enhenced yellow fluorescent protein (EYFP) displayed improved resistance to high temperatures (Lim et al., 2013).
1.3 Strategies for Mitigating Heat Stress Response Through Gene Editing and Derivative Technologies
1.3.1 Overview of Noncoding DNA
Noncoding DNA refers to genome regions that do not code for proteins. While once considered “junk DNA,” it is now understood that noncoding DNA plays crucial roles in regulating gene expression and maintaining cellular function. Noncoding DNA includes sequences that control the activity of genes, influence chromatin structure, and regulate various cellular processes (Simna & Han, 2022). There is a growing consensus that the two key factors determining gene expression level are cis‐regulatory modules (CRMs) and trans‐acting factors (TAFs) (Schmitz et al., 2022
