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25 pages, 3904 KiB

Open AccessArticle

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

by Sarah Bouzroud, Karla Gasparini, Guojian Hu, Maria Antonia Machado Barbosa, Bruno Luan Rosa, Mouna Fahr, Najib Bendaou, Mondher Bouzayen, Agustin Zsögön, Abdelaziz Smouni and Mohamed Zouine

Genes 2020, 11(3), 272; https://doi.org/10.3390/genes11030272 - 3 Mar 2020

Cited by 139 | Viewed by 9063

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue TILLING and CRISPR to design the varieties of tomorrow)

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9 pages, 1145 KiB

Open AccessCommunication

TILLING by Sequencing: A Successful Approach to Identify Rare Alleles in Soybean Populations

by Rima Thapa, Militza Carrero-Colón, Katy M. Rainey and Karen Hudson

Genes 2019, 10(12), 1003; https://doi.org/10.3390/genes10121003 - 3 Dec 2019

Cited by 18 | Viewed by 4913

Abstract

Soybean seeds produce valuable protein that is a major component of livestock feed. However, soybean seeds also contain the anti-nutritional raffinose family oligosaccharides (RFOs) raffinose and stachyose, which are not digestible by non-ruminant animals. This requires the proportion of soybean meal in the [...] Read more.

Soybean seeds produce valuable protein that is a major component of livestock feed. However, soybean seeds also contain the anti-nutritional raffinose family oligosaccharides (RFOs) raffinose and stachyose, which are not digestible by non-ruminant animals. This requires the proportion of soybean meal in the feed to be limited, or risk affecting animal growth rate or overall health. While reducing RFOs in soybean seed has been a goal of soybean breeding, efforts are constrained by low genetic variability for carbohydrate traits and the difficulty in identifying these within the soybean germplasm. We used reverse genetics Targeting Induced Local Lesions in Genomes (TILLING)-by-sequencing approach to identify a damaging polymorphism that results in a missense mutation in a conserved region of the RAFFINOSE SYNTHASE3 gene. We demonstrate that this mutation, when combined as a double mutant with a previously characterized mutation in the RAFFINOSE SYNTHASE2 gene, eliminates nearly 90% of the RFOs in soybean seed as a proportion of the total seeds carbohydrates, and results in increased levels of sucrose. This represents a proof of concept for TILLING by sequencing in soybean. Full article

(This article belongs to the Special Issue TILLING and CRISPR to design the varieties of tomorrow)

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14 pages, 1914 KiB

Open AccessArticle

Assessment of Phenotypic Variations and Correlation among Seed Composition Traits in Mutagenized Soybean Populations

by Zhou Zhou, Naoufal Lakhssassi, Mallory A. Cullen, Abdelhalim El Baz, Tri D. Vuong, Henry T. Nguyen and Khalid Meksem

Genes 2019, 10(12), 975; https://doi.org/10.3390/genes10120975 - 27 Nov 2019

Cited by 22 | Viewed by 4442

Abstract

Soybean [Glycine max (L.) Merr.] seed is a valuable source of protein and oil worldwide. Traditionally, the natural variations were heavily used in conventional soybean breeding programs to select desired traits. However, traditional plant breeding is encumbered with low frequencies of spontaneous [...] Read more.

Soybean [Glycine max (L.) Merr.] seed is a valuable source of protein and oil worldwide. Traditionally, the natural variations were heavily used in conventional soybean breeding programs to select desired traits. However, traditional plant breeding is encumbered with low frequencies of spontaneous mutations. In mutation breeding, genetic variations from induced mutations provide abundant sources of alterations in important soybean traits; this facilitated the development of soybean germplasm with modified seed composition traits to meet the different needs of end users. In this study, a total of 2366 ‘Forrest’-derived M2 families were developed for both forward and reverse genetic studies. A subset of 881 M3 families was forward genetically screened to measure the contents of protein, oil, carbohydrates, and fatty acids. A total of 14 mutants were identified to have stable seed composition phenotypes observed in both M3 and M4 generations. Correlation analyses have been conducted among ten seed composition traits and compared to a collection of 103 soybean germplasms. Mainly, ethyl methanesulfonate (EMS) mutagenesis had a strong impact on the seed-composition correlation that was observed among the 103 soybean germplasms, which offers multiple benefits for the soybean farmers and industry to breed for desired multiple seed phenotypes. Full article

(This article belongs to the Special Issue TILLING and CRISPR to design the varieties of tomorrow)

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15 pages, 7230 KiB

Open AccessArticle

Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice

by Haroon Butt, Agnieszka Piatek, Lixin Li, Anireddy S. N. Reddy and Magdy M. Mahfouz

Genes 2019, 10(8), 596; https://doi.org/10.3390/genes10080596 - 7 Aug 2019

Cited by 23 | Viewed by 5901

Abstract

Plant growth responds to various environmental and developmental cues via signaling cascades that influence gene expression at the level of transcription and pre-mRNA splicing. Alternative splicing of pre-mRNA increases the coding potential of the genome from multiexon genes and regulates gene expression through [...] Read more.

Plant growth responds to various environmental and developmental cues via signaling cascades that influence gene expression at the level of transcription and pre-mRNA splicing. Alternative splicing of pre-mRNA increases the coding potential of the genome from multiexon genes and regulates gene expression through multiple mechanisms. Serine/arginine-rich (SR) proteins, a conserved family of splicing factors, are the key players of alternative splicing and regulate pre-mRNA splicing under stress conditions. The rice (Oryza sativa) genome encodes 22 SR proteins categorized into six subfamilies. Three of the subfamilies are plant-specific with no mammalian orthologues, and the functions of these SR proteins are not well known. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a genome engineering tool that cleaves the target DNA at specific locations directed by a guide RNA (gRNA). Recent advances in CRISPR/Cas9-mediated plant genome engineering make it possible to generate single and multiple functional knockout mutants in diverse plant species. In this study, we targeted each rice SR locus and produced single knockouts. To overcome the functional redundancy within each subfamily of SR genes, we utilized a polycistronic tRNA-gRNA multiplex targeting system and targeted all loci of each subfamily. Sanger sequencing results indicated that most of the targeted loci had knockout mutations. This study provides useful resource materials for understanding the molecular role of SR proteins in plant development and biotic and abiotic stress responses. Full article

(This article belongs to the Special Issue TILLING and CRISPR to design the varieties of tomorrow)

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17 pages, 3762 KiB

Open AccessArticle

Identification and Characterization of a Thermotolerant TILLING Allele of Heat Shock Binding Protein 1 in Tomato

by Dominik Marko, Asmaa El-shershaby, Filomena Carriero, Stephan Summerer, Angelo Petrozza, Rina Iannacone, Enrico Schleiff and Sotirios Fragkostefanakis

Genes 2019, 10(7), 516; https://doi.org/10.3390/genes10070516 - 7 Jul 2019

Cited by 24 | Viewed by 5007

Abstract

The identification of heat stress (HS)-resilient germplasm is important to ensure food security under less favorable environmental conditions. For that, germplasm with an altered activity of factors regulating the HS response is an important genetic tool for crop improvement. Heat shock binding protein [...] Read more.

The identification of heat stress (HS)-resilient germplasm is important to ensure food security under less favorable environmental conditions. For that, germplasm with an altered activity of factors regulating the HS response is an important genetic tool for crop improvement. Heat shock binding protein (HSBP) is one of the main negative regulators of HS response, acting as a repressor of the activity of HS transcription factors. We identified a TILLING allele of Solanum lycopersicum (tomato) HSBP1. We examined the effects of the mutation on the functionality of the protein in tomato protoplasts, and compared the thermotolerance capacity of lines carrying the wild-type and mutant alleles of HSBP1. The methionine-to-isoleucine mutation in the central heptad repeats of HSBP1 leads to a partial loss of protein function, thereby reducing the inhibitory effect on Hsf activity. Mutant seedlings show enhanced basal thermotolerance, while mature plants exhibit increased resilience in repeated HS treatments, as shown by several physiological parameters. Importantly, plants that are homozygous for the wild-type or mutant HSBP1 alleles showed no significant differences under non-stressed conditions. Altogether, these results indicate that the identified mutant HSBP1 allele can be used as a genetic tool in breeding, aiming to improve the thermotolerance of tomato varieties. Full article

(This article belongs to the Special Issue TILLING and CRISPR to design the varieties of tomorrow)

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13 pages, 1296 KiB

Open AccessArticle

EcoTILLING Reveals Natural Allelic Variations in Starch Synthesis Key Gene TaSSIV and Its Haplotypes Associated with Higher Thousand Grain Weight

by Ahsan Irshad, Huijun Guo, Shunlin Zhang, Jiayu Gu, Linshu Zhao, Yongdun Xie, Hongchun Xiong, Shirong Zhao, Yuping Ding, Youzhi Ma and Luxiang Liu

Genes 2019, 10(4), 307; https://doi.org/10.3390/genes10040307 - 18 Apr 2019

Cited by 16 | Viewed by 3738

Abstract

Wheat is a staple food commodity grown worldwide, and wheat starch is a valuable source of energy and carbon that constitutes 80% of the grain weight. Manipulation of genes involved in starch synthesis significantly affects wheat grain weight and yield. TaSSIV plays an [...] Read more.

Wheat is a staple food commodity grown worldwide, and wheat starch is a valuable source of energy and carbon that constitutes 80% of the grain weight. Manipulation of genes involved in starch synthesis significantly affects wheat grain weight and yield. TaSSIV plays an important role in starch synthesis and its main function is granule formation. To mine and stack more favorable alleles, single nucleotide polymorphisms (SNPs) of TaSSIV-A, B, and D were investigated across 362 wheat accessions by Ecotype-Targeting Induced Local Lesions IN Genome (EcoTILLING). As a result, a total of 38 SNPs in the amplified regions of three TaSSIV genes were identified, of which 10, 15, and 13 were in TaSSIV-A, B, and D, respectively. These 38 SNPs were evaluated by using KASP and six SNPs showed an allele frequency >5% whereas the rest were <5%, i.e., considered to be minor alleles. In the Chinese mini core collection, three haplotypes were detected for TaSSIV–A and three for TaSSIV–B. The results of an association study in the Chinese mini core collection with thousand grain weight (TGW) and spike length (SPL) showed that Hap-2-1A was significantly associated with TGW and Hap-3-1B with SPL. Allelic frequency and geographic distribution indicated that the favored haplotype (Hap-2-1A) has been positively selected in Chinese wheat breeding. These results suggested that the Kompetitive Allele Specific PCR (KASP) markers can be applied in starch improvement to ultimately improve wheat yield by marker assisted selection in wheat breeding. Full article

(This article belongs to the Special Issue TILLING and CRISPR to design the varieties of tomorrow)

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Review

Jump to: Research

23 pages, 401 KiB

Open AccessReview

Phenotyping in Arabidopsis and Crops—Are We Addressing the Same Traits? A Case Study in Tomato

by Paolo Korwin Krukowski, Jan Ellenberger, Simone Röhlen-Schmittgen, Andrea Schubert and Francesca Cardinale

Genes 2020, 11(9), 1011; https://doi.org/10.3390/genes11091011 - 27 Aug 2020

Cited by 6 | Viewed by 4454

Abstract

The convenient model Arabidopsis thaliana has allowed tremendous advances in plant genetics and physiology, in spite of only being a weed. It has also unveiled the main molecular networks governing, among others, abiotic stress responses. Through the use of the latest genomic tools, [...] Read more.

The convenient model Arabidopsis thaliana has allowed tremendous advances in plant genetics and physiology, in spite of only being a weed. It has also unveiled the main molecular networks governing, among others, abiotic stress responses. Through the use of the latest genomic tools, Arabidopsis research is nowadays being translated to agronomically interesting crop models such as tomato, but at a lagging pace. Knowledge transfer has been hindered by invariable differences in plant architecture and behaviour, as well as the divergent direct objectives of research in Arabidopsis vs. crops compromise transferability. In this sense, phenotype translation is still a very complex matter. Here, we point out the challenges of “translational phenotyping” in the case study of drought stress phenotyping in Arabidopsis and tomato. After briefly defining and describing drought stress and survival strategies, we compare drought stress protocols and phenotyping techniques most commonly used in the two species, and discuss their potential to gain insights, which are truly transferable between species. This review is intended to be a starting point for discussion about translational phenotyping approaches among plant scientists, and provides a useful compendium of methods and techniques used in modern phenotyping for this specific plant pair as a case study. Full article

(This article belongs to the Special Issue TILLING and CRISPR to design the varieties of tomorrow)

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Graphical abstract

15 pages, 2029 KiB

Open AccessReview

Base Editing: The Ever Expanding Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Tool Kit for Precise Genome Editing in Plants

by Mahmuda Binte Monsur, Gaoneng Shao, Yusong Lv, Shakeel Ahmad, Xiangjin Wei, Peisong Hu and Shaoqing Tang

Genes 2020, 11(4), 466; https://doi.org/10.3390/genes11040466 - 24 Apr 2020

Cited by 41 | Viewed by 7698

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), a newly developed genome-editing tool, has revolutionized animal and plant genetics by facilitating modification of target genes. This simple, convenient base-editing technology was developed to improve the precision of genome editing. Base [...] Read more.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), a newly developed genome-editing tool, has revolutionized animal and plant genetics by facilitating modification of target genes. This simple, convenient base-editing technology was developed to improve the precision of genome editing. Base editors generate precise point mutations by permanent base conversion at a specific point, with very low levels of insertions and deletions. Different plant base editors have been established by fusing various nucleobase deaminases with Cas9, Cas13, or Cas12a (Cpf1), proteins. Adenine base editors can efficiently convert adenine (A) to guanine (G), whereas cytosine base editors can convert cytosine (C) to thymine (T) in the target region. RNA base editors can induce a base substitution of A to inosine (I) or C to uracil (U). In this review, we describe the precision of base editing systems and their revolutionary applications in plant science; we also discuss the limitations and future perspectives of this approach. Full article

(This article belongs to the Special Issue TILLING and CRISPR to design the varieties of tomorrow)

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