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

Bouzroud, Sarah; Gasparini, Karla; Hu, Guojian; Barbosa, Maria Antônia Machado; Rosa, Bruno Luan; Fahr, Mouna; Bendaou, Najib; Bouzayen, Mondher; Zsögön, Agustín; Smouni, Abdelaziz; Zouine, Mohamed

doi:10.3390/genes11030272

Cited by 132 publications

(70 citation statements)

References 80 publications

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“…However, stomatal conductance of the SlARF4-as phenotype did not change significantly under salt or Polyethylene glycol (PEG)-simulated water stress, compared with the control. Nonetheless, the WT showed a normal response to osmotic stress, with a significant decrease in stomatal conductance [ 47 ]. In our study, the epidermis of the upper leaves on arf4 plants showed smaller stomata than that of the WT leaves, and they did not immediately close upon initiation of the water deficit treatment; furthermore, the guard cells still retained their normal plumpness.…”

Section: Discussionmentioning

confidence: 99%

“…The plant #5 bearing the desired mutation of SlARF4 was used for the water deficit study. More information about the SlARF4 CRISPR/Cas 9 lines generation could be found in the paper of Bouzroud et al [ 47 ]. Tomato SlARF4 knockout ( arf4 ) and pARF4::GUS plants were grown in a controlled climate room (25 ± 5 °C) under a 16 h/8 h (light/dark) photoperiod at the South China Agricultural University.…”

Section: Methodsmentioning

confidence: 99%

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Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit

Chen

Zhu

Liu

et al. 2021

IJMS

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Auxin response factors (ARFs) play important roles in various plant physiological processes; however, knowledge of the exact role of ARFs in plant responses to water deficit is limited. In this study, SlARF4, a member of the ARF family, was functionally characterized under water deficit. Real-time fluorescence quantitative polymerase chain reaction (PCR) and β-glucuronidase (GUS) staining showed that water deficit and abscisic acid (ABA) treatment reduced the expression of SlARF4. SlARF4 was expressed in the vascular bundles and guard cells of tomato stomata. Loss of function of SlARF4 (arf4) by using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 (CRISPR/Cas 9) technology enhanced plant resistance to water stress and rehydration ability. The arf4 mutant plants exhibited curly leaves and a thick stem. Malondialdehyde content was significantly lower in arf4 mutants than in wildtype plants under water stress; furthermore, arf4 mutants showed higher content of antioxidant substances, superoxide dismutase, actual photochemical efficiency of photosystem II (PSII), and catalase activities. Stomatal and vascular bundle morphology was changed in arf4 mutants. We identified 628 differentially expressed genes specifically expressed under water deficit in arf4 mutants; six of these genes, including ABA signaling pathway-related genes, were differentially expressed between the wildtype and arf4 mutants under water deficit and unlimited water supply. Auxin responsive element (AuxRE) elements were found in these genes’ promoters indicating that SlARF4 participates in ABA signaling pathways by regulating the expression of SlABI5/ABF and SCL3, thereby influencing stomatal morphology and vascular bundle development and ultimately improving plant resistance to water deficit.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit

Chen

Zhu

Liu

et al. 2021

IJMS

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“…Furthermore, Arabidopsis ARF2 may affect seed size and drought tolerance through regulating ABA signaling [ 52 ]. In addition, transgenic expression of a sweet potato IbARF5 in Arabidopsis and downregulation of a tomato SlARF4 have been found to increase ABA content in the plants [ 53 , 54 ]. From ABA to auxin direction, an ABI3-like factor from the bean is known to bind an auxin-inducible promoter [ 55 ].…”

Section: Discussionmentioning

confidence: 99%

Protein Levels of Several Arabidopsis Auxin Response Factors Are Regulated by Multiple Factors and ABA Promotes ARF6 Protein Ubiquitination

Wang

et al. 2020

IJMS

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The auxin response factor (ARF) transcription factors are a key component in auxin signaling and play diverse functions in plant growth, development, and stress response. ARFs are regulated at the transcript level and posttranslationally by protein modifications. However, relatively little is known regarding the control of ARF protein levels. We expressed five different ARFs with an HA (hemagglutinin) tag and observed that their protein levels under the same promoter varied considerably. Interestingly, their protein levels were affected by several hormonal and environmental conditions, but not by the auxin treatment. ABA (abscisic acid) as well as 4 °C and salt treatments decreased the levels of HA-ARF5, HA-ARF6, and HA-ARF10, but not that of HA-ARF19, while 37 °C treatment increased the levels of the four HA-ARFs, suggesting that the ARF protein levels are regulated by multiple factors. Furthermore, MG132 inhibited the reduction of HA-ARF6 level by ABA and 4 °C treatments, suggesting that these treatments decrease HA-ARF6 level through 26S proteasome-mediated protein degradation. It was also found that ABA treatment drastically increased HA-ARF6 ubiquitination, without strongly affecting the ubiquitination profile of the total proteins. Together, these results reveal another layer of control on ARFs, which could serve to integrate multiple hormonal and environmental signals into the ARF-regulated gene expression.

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“…Auxin response factor (ARF) is one family of functionally distinct DNA-binding transcription factors found widely in all plant species ( Li SB et al, 2016 ). CRISPR/Cas9 knock-out of the arf4 transcription factor gene improved plant tolerance to salinity and osmotic stresses in tomato ( Bouzroud et al, 2020 ). Using the CRISPRa epigenome editor, Roca Paixão et al (2019) successfully fused dCas9 protein with a histone acetyltransferase (AtHAT1) and used this fused CRISPR/dCas9 system to target the abscisic acid (ABA)-responsive element-binding protein 1 (AREB1)/ABRE-binding factor 2 (ABF2).…”

Section: Crispr/cas Is a Robust And Powerful Tool For Precision Brementioning

confidence: 99%

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

Brant

Budak

et al. 2021

J. Zhejiang Univ. Sci. B

108

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Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010s, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has rapidly been developed into a robust, multifunctional genome editing tool with many uses. Following the discovery of the initial CRISPR/Cas-based system, the technology has been advanced to facilitate a multitude of different functions. These include development as a base editor, prime editor, epigenetic editor, and CRISPR interference (CRISPRi) and CRISPR activator (CRISPRa) gene regulators. It can also be used for chromatin and RNA targeting and imaging. Its applications have proved revolutionary across numerous biological fields, especially in biomedical and agricultural improvement. As a diagnostic tool, CRISPR has been developed to aid the detection and screening of both human and plant diseases, and has even been applied during the current coronavirus disease 2019 (COVID-19) pandemic. CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases, including cancers, and has aided drug development. In terms of agricultural breeding, precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins, starch, oil, and other functional components for crop improvement. Adding to this, CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators. Looking to the future, increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology. This review provides an in-depth overview of current CRISPR development, including the advantages and disadvantages of the technology, recent applications, and future considerations.

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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

Cited by 132 publications

References 80 publications

Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit

Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit

Protein Levels of Several Arabidopsis Auxin Response Factors Are Regulated by Multiple Factors and ABA Promotes ARF6 Protein Ubiquitination

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

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