scholarly journals Down Regulation and Loss of Auxin Response Factor 4 Function Using CRISPR/Cas9 Alters Plant Growth, Stomatal Function and Improves Tomato Tolerance to Salinity and Osmotic Stress

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 272 ◽  
Author(s):  
Sarah Bouzroud ◽  
Karla Gasparini ◽  
Guojian Hu ◽  
Maria Antonia Machado Barbosa ◽  
Bruno Luan Rosa ◽  
...  

Auxin controls multiple aspects of plant growth and development. However, its role in stress responses remains poorly understood. Auxin acts on the transcriptional regulation of target genes, mainly through Auxin Response Factors (ARF). This study focuses on the involvement of SlARF4 in tomato tolerance to salinity and osmotic stress. Using a reverse genetic approach, we found that the antisense down-regulation of SlARF4 promotes root development and density, increases soluble sugars content and maintains chlorophyll content at high levels under stress conditions. Furthermore, ARF4-as displayed higher tolerance to salt and osmotic stress through reduced stomatal conductance coupled with increased leaf relative water content and Abscisic acid (ABA) content under normal and stressful conditions. This increase in ABA content was correlated with the activation of ABA biosynthesis genes and the repression of ABA catabolism genes. Cu/ZnSOD and mdhar genes were up-regulated in ARF4-as plants which can result in a better tolerance to salt and osmotic stress. A CRISPR/Cas9 induced SlARF4 mutant showed similar growth and stomatal responses as ARF4-as plants, which suggest that arf4-cr can tolerate salt and osmotic stresses. Our data support the involvement of ARF4 as a key factor in tomato tolerance to salt and osmotic stresses and confirm the use of CRISPR technology as an efficient tool for functional reverse genetics studies.

2019 ◽  
Author(s):  
Sarah Bouzroud ◽  
Karla Gasparini ◽  
Guojian Hu ◽  
Maria Antonia Machado Barbosa ◽  
Bruno Luan Rosa ◽  
...  

AbstractAuxin controls multiple aspects of plant growth and development. However, its role in stress responses remains poorly understood. Auxin acts on the transcriptional regulation of target genes, mainly through Auxin Response Factors (ARF). This study focuses on the involvement of SlARF4 in tomato tolerance to salinity and osmotic stress. Using a reverse genetic approach, we found that the antisense down-regulation of SlARF4 promotes root development and density, increases soluble sugars content and maintains chlorophyll content at high levels under stress conditions. Furthermore, ARF4-as displayed higher tolerance to salt and osmotic stress through reduced stomatal conductance coupled with increased leaf relative water content and ABA content under normal and stressful conditions. This increase in ABA content was correlated with the activation of ABA biosynthesis genes and the repression of ABA catabolism genes. cat1, Cu/ZnSOD and mdhar genes were up-regulated in ARF4-as plants which can result in a better tolerance to salt and osmotic stress. A CRISPR/Cas9 induced SlARF4 mutant showed similar growth and stomatal responses as ARF4-as plants, which suggest that arf4-cr can tolerate salt and osmotic stresses. Our data support the involvement of ARF4 as a key factor in tomato tolerance to salt and osmotic stresses and confirm the use of CRISPR technology as an efficient tool for functional reverse genetics studies.


2020 ◽  
Vol 71 (5) ◽  
pp. 1706-1722 ◽  
Author(s):  
Marieke Dubois ◽  
Dirk Inzé

Abstract Drought stress forms a major environmental constraint during the life cycle of plants, often decreasing plant yield and in extreme cases threatening survival. The molecular and physiological responses induced by drought have been the topic of extensive research during the past decades. Because soil-based approaches to studying drought responses are often challenging due to low throughput and insufficient control of the conditions, osmotic stress assays in plates were developed to mimic drought. Addition of compounds such as polyethylene glycol, mannitol, sorbitol, or NaCl to controlled growth media has become increasingly popular since it offers the advantage of accurate control of stress level and onset. These osmotic stress assays enabled the discovery of very early stress responses, occurring within seconds or minutes following osmotic stress exposure. In this review, we construct a detailed timeline of early responses to osmotic stress, with a focus on how they initiate plant growth arrest. We further discuss the specific responses triggered by different types and severities of osmotic stress. Finally, we compare short-term plant responses under osmotic stress versus in-soil drought and discuss the advantages, disadvantages, and future of these plate-based proxies for drought.


2019 ◽  
Vol 145 ◽  
pp. 43-51 ◽  
Author(s):  
Jinhui Lin ◽  
Fengfeng Dang ◽  
Yongping Chen ◽  
Deyi Guan ◽  
Shuilin He

2021 ◽  
Vol 22 (24) ◽  
pp. 13535
Author(s):  
Rui Ma ◽  
Weigang Liu ◽  
Shigui Li ◽  
Xi Zhu ◽  
Jiangwei Yang ◽  
...  

The potato (Solanum tuberosum L.), one of the most important food crops worldwide, is sensitive to environmental stresses. Sensor–responder complexes comprising calcineurin B-like (CBL) proteins and CBL-interacting protein kinases (CIPKs) not only modulate plant growth and development but also mediate numerous stress responses. Here, using a Hidden Markov Model and BLAST searches, 27 CIPK genes were identified in potato and divided into five groups by phylogenetic analysis and into two clades (intron-poor and intron-rich) by gene structure analysis. Quantitative reverse-transcription PCR (qRT-PCR) assays revealed that StCIPK genes play important roles in plant growth, development and abiotic stress tolerance. Up-regulated expression of StCIPK10 was significantly induced by drought, PEG6000 and ABA. StCIPK10 enhances both the ability of potato to scavenge reactive oxygen species and the content of corresponding osmoregulation substances, thereby strengthening tolerance to drought and osmotic stress. StCIPK10 is located at the intersection between the abscisic acid and abiotic stress signaling pathways, which control both root growth and stomatal closure in potato. In addition, StCIPK10 interacts with StCBL1, StCBL4, StCBL6, StCBL7, StCBL8, StCBL11 and StCBL12, and is specifically recruited to the plasma membrane by StCBL11.


Gene ◽  
2010 ◽  
Vol 457 (1-2) ◽  
pp. 1-12 ◽  
Author(s):  
Qiang Zhu ◽  
Jiantao Zhang ◽  
Xiaoshu Gao ◽  
Jianhua Tong ◽  
Langtao Xiao ◽  
...  

2019 ◽  
Vol 6 (1) ◽  
pp. 180735 ◽  
Author(s):  
Songtao Jiu ◽  
Xiangpeng Leng ◽  
Muhammad Salman Haider ◽  
Tianyu Dong ◽  
Le Guan ◽  
...  

MicroRNAs (miRNAs) are a class of single-stranded non-coding small RNAs (sRNAs) that are 20–24 nucleotides (nt) in length. Extensive studies have indicated that miRNAs play important roles in plant growth, development and stress responses. With more copper (Cu) and copper containing compounds used as bactericides and fungicides in plants, Cu stress has become one of the serious environmental problems that affect plant growth and development. In order to uncover the hidden response mechanisms of Cu stress, two small RNA libraries were constructed from Cu-treated and water-treated (Control) leaves of ‘Summer Black’ grapevine. Following high-throughput sequencing and filtering, a total of 158 known and 98 putative novel miRNAs were identified in the two libraries. Among these, 100 known and 47 novel miRNAs were identified as differentially expressed under Cu stress. Subsequently, the expression patterns of nine Cu-responsive miRNAs were validated by quantitative real-time PCR (qRT-PCR). There existed some consistency in expression levels of Cu-responsive miRNAs between high throughput sequencing and qRT-PCR assays. The targets prediction of miRNAs indicates that miRNA may regulate some transcription factors, including AP2, SBP, NAC, MYB and ARF during Cu stress. The target genes for two known and two novel miRNAs showed specific cleavage sites at the 10th and/or 11th nucleotide from the 5′-end of the miRNA corresponding to their miRNA complementary sequences. The findings will lay the foundation for exploring the role of the regulation of miRNAs in response to Cu stress and provide valuable gene information for breeding some Cu-tolerant grapevine cultivars.


2015 ◽  
Vol 34 (7) ◽  
pp. 1127-1138 ◽  
Author(s):  
Vaishali N. Tarte ◽  
Hye-Yeon Seok ◽  
Dong-Hyuk Woo ◽  
Dinh Huan Le ◽  
Huong T. Tran ◽  
...  

2020 ◽  
Author(s):  
Huisen Zhu ◽  
Rong Jia ◽  
Jiali Tao ◽  
Junbing Jiang ◽  
Kuanhu Dong

Abstract Background Alfalfa (Medicago sativa cv. Pianguan) is one of the most widely cultivated perennial leguminous forage. Drought is one of the major abiotic factors that affect alfalfa productivity. MicroRNAs (miRNAs) have been proved playing important roles in plant growth, development and stress response. Results In this study, High-throughput sequencing (HTSeq) was used to identify the miRNAs and their target related to drought stress from polyethylene glycol treated and control alfalfa samples, differential expression analysis showed that a large number of miRNAs were down-regulated or up-regulated as a result of PEG-6000 stress. qRT-PCR and sequencing analysis results showed that the relative expression of 9 miRNAs and their target genes related to drought stress, 3 miRNAs were considered potential drought-responsive miRNAs, including miR159b, miR397-5p and unconservative_6_4438. The recombinant plasmid vector pBWA(V)KS-miR397 was constructed and introduced into tobacco successfully. 31 positive seedlings were obtained through the process of infection, co-culture, differentiation and rooting. The physiologieal variation of transgenic tobacco under drought stress was researched. Finally, a new tobacco variety with high drought resistance was obtained. Conclusion These finding although need further studies to confirm, these data provide a useful evidence for the possible involvement of miRNAs in the process of drought response in the alfalfa plant which could help explain the drought stress responses and to alleviate the adverse effects of drought stress on plant growth and development.


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