scholarly journals Clarification of the dispensability of PDX1.2 for Arabidopsis viability using CRISPR/Cas9

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Elisa Dell’Aglio ◽  
Ivan Dalvit ◽  
Sylvain Loubéry ◽  
Teresa B. Fitzpatrick
Keyword(s):  
Heat Stress ◽  
Stress Responses ◽  
Gene Editing ◽  
Growth Conditions ◽  
Insertion Mutant ◽  
Wild Type ◽  
Biotic And Abiotic Stress ◽  
Knockout Mutants ◽  
Dna Insertion ◽  
Mutant Lines

Abstract Background PDX1.2 has recently been shown to be a regulator of vitamin B6 biosynthesis in plants and is implicated in biotic and abiotic stress resistance. PDX1.2 expression is strongly and rapidly induced by heat stress. Interestingly, PDX1.2 is restricted to eudicota, wherein it behaves as a non-catalytic pseudoenzyme and is suggested to provide an adaptive advantage to this clade. A first report on an Arabidopsis insertion mutant claims that PDX1.2 is indispensable for viability, being essential for embryogenesis. However, a later study using an independent insertion allele suggests that knockout mutants of pdx1.2 are viable. Therefore, the essentiality of PDX1.2 for Arabidopsis viability is a matter of debate. Given the important implications of PDX1.2 in stress responses, it is imperative to clarify if it is essential for plant viability. Results We have studied the previously reported insertion alleles of PDX1.2, one of which is claimed to be essential for embryogenesis (pdx1.2–1), whereas the other is viable (pdx1.2–2). Our study shows that pdx1.2–1 carries multiple T-DNA insertions, but the T-DNA insertion in PDX1.2 is not responsible for the loss of embryogenesis. By contrast, the pdx1.2–2 allele is an overexpressor of PDX1.2 under standard growth conditions and not a null allele as previously reported. Nonetheless, upregulation of PDX1.2 expression under heat stress is impaired in this mutant line. In wild type Arabidopsis, studies of PDX1.2-YFP fusion proteins show that the protein is enhanced under heat stress conditions. To clarify if PDX1.2 is essential for Arabidopsis viability, we generated several independent mutant lines using the CRISPR-Cas9 gene editing technology. All of these lines are viable and behave similar to wild type under standard growth conditions. Reciprocal crosses of a subset of the CRISPR lines with pdx1.2–1 recovers viability of the latter line and demonstrates that knocking out the functionality of PDX1.2 does not impair embryogenesis. Conclusions Gene editing reveals that PDX1.2 is dispensable for Arabidopsis viability and resolves conflicting reports in the literature on its function.

scholarly journals OsASN1 Plays a Critical Role in Asparagine-Dependent Rice Development

2018 ◽  
Vol 20 (1) ◽  
pp. 130 ◽  
Author(s):  
Le Luo ◽  
Ruyi Qin ◽  
Tao Liu ◽  
Ming Yu ◽  
Tingwen Yang ◽  
...  
Keyword(s):  
Critical Role ◽  
Tiller Number ◽  
Insertion Mutant ◽  
Wild Type ◽  
Long Distance ◽  
Long Distance Transport ◽  
Similar Phenotype ◽  
Dna Insertion ◽  
Mutant Lines ◽  
Distance Transport

Asparagine is one of the important amino acids for long-distance transport of nitrogen (N) in plants. However, little is known about the effect of asparagine on plant development, especially in crops. Here, a new T-DNA insertion mutant, asparagine synthetase 1 (asn1), was isolated and showed a different plant height, root length, and tiller number compared with wild type (WT). In asn1, the amount of asparagine decreased sharply while the total nitrogen (N) absorption was not influenced. In later stages, asn1 showed reduced tiller number, which resulted in suppressed tiller bud outgrowth. The relative expression of many genes involved in the asparagine metabolic pathways declined in accordance with the decreased amino acid concentration. The CRISPR/Cas9 mutant lines of OsASN1 showed similar phenotype with asn1. These results suggest that OsASN1 is involved in the regulation of rice development and is specific for tiller outgrowth.

scholarly journals A unique AtSar1D-AtRabD2a nexus modulates autophagosome biogenesis in Arabidopsis thaliana

2021 ◽  
Vol 118 (17) ◽  
pp. e2021293118
Author(s):  
Yonglun Zeng ◽  
Baiying Li ◽  
Changyang Ji ◽  
Lei Feng ◽  
Fangfang Niu ◽  
...  
Keyword(s):  
Stress Responses ◽  
Higher Plants ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Secretory Proteins ◽  
Insertion Mutant ◽  
Intermediate Metabolites ◽  
Dna Insertion ◽  
Autophagy Pathway ◽  
Copii Vesicles

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1d-positive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis.

scholarly journals Expression of α-DIOXYGENASE 1 in Tomato and Arabidopsis Contributes to Plant Defenses Against Aphids

2013 ◽  
Vol 26 (8) ◽  
pp. 977-986 ◽  
Author(s):  
Carlos Augusto Avila ◽  
Lirio Milenka Arevalo-Soliz ◽  
Argelia Lorence ◽  
Fiona L. Goggin
Keyword(s):  
Green Peach Aphid ◽  
Macrosiphum Euphorbiae ◽  
Insertion Mutant ◽  
Plant Signaling ◽  
Virus Induced Gene Silencing ◽  
Basal Resistance ◽  
Potato Aphid ◽  
Dna Insertion ◽  
Mutant Lines ◽  
Increased Susceptibility

Plant α-dioxygenases (α-DOX) are fatty acid–hydroperoxidases that contribute to the synthesis of oxylipins, a diverse group of compounds primarily generated through oxidation of linoleic (LA) and linolenic acid (LNA). Oxylipins are implicated in plant signaling against biotic and abiotic stresses. We report here that the potato aphid (Macrosiphum euphorbiae) induces Slα-DOX1 but not Slα-DOX2 expression in tomato (Solanum lycopersicum). Slα-DOX1 upregulation by aphids does not require either jasmonic acid (JA) or salicylic acid (SA) accumulation, since tomato mutants deficient in JA (spr2, acx1) or SA accumulation (NahG) still show Slα-DOX1 induction. Virus-induced gene silencing of Slα-DOX1 enhanced aphid population growth in wild-type (WT) plants, revealing that Slα-DOX1 contributes to basal resistance to aphids. Moreover, an even higher percent increase in aphid numbers occurred when Slα-DOX1 was silenced in spr2, a mutant line characterized by elevated LA levels, decreased LNA, and enhanced aphid resistance as compared with WT. These results suggest that aphid reproduction is influenced by oxylipins synthesized from LA by Slα-DOX1. In agreement with our experiments in tomato, two independent α-dox1 T-DNA insertion mutant lines in Arabidopsis thaliana also showed increased susceptibility to the green peach aphid (Myzus persicae), indicating that the role α-DOX is conserved in other plant-aphid interactions.

scholarly journals Gibberellin 20-Oxidase Gene OsGA20ox3 Regulates Plant Stature and Disease Development in Rice

2013 ◽  
Vol 26 (2) ◽  
pp. 227-239 ◽  
Author(s):  
Xue Qin ◽  
Jun Hua Liu ◽  
Wen Sheng Zhao ◽  
Xu Jun Chen ◽  
Ze Jian Guo ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Critical Role ◽  
Insertion Site ◽  
Insertion Mutant ◽  
Wild Type Plant ◽  
Wild Type ◽  
Oxidase Gene ◽  
Dna Insertion ◽  
Flanking Sequences ◽  
Plant Stature

Gibberellin (GA) 20-oxidase (GA20ox) catalyses consecutive steps of oxidation in the late part of the GA biosynthetic pathway. A T-DNA insertion mutant (17S-14) in rice, with an elongated phenotype, was isolated. Analysis of the flanking sequences of the T-DNA insertion site revealed that an incomplete T-DNA integration resulted in enhanced constitutively expression of downstream OsGA20ox3 in the mutant. The accumulation of bioactive GA1 and GA4 were increased in the mutant in comparison with the wild-type plant. Transgenic plants overexpressing OsGA20ox3 showed phenotypes similar to those of the 17S-14 mutant, and the RNA interference (RNAi) lines that had decreased OsGA20ox3 expression exhibited a semidwarf phenotype. Expression of OsGA20ox3 was detected in the leaves and roots of young seedlings, immature panicles, anthers, and pollens, based on β-glucuronidase (GUS) activity staining in transgenic plants expressing the OsGA20ox3 promoter fused to the GUS gene. The OsGA20ox3 RNAi lines showed enhanced resistance against rice pathogens Magnaporthe oryzae (causing rice blast) and Xanthomonas oryzae pv. oryzae (causing bacterial blight) and increased expression of defense-related genes. Conversely, OsGA20ox3-overexpressing plants were more susceptible to these pathogens comparing with the wild-type plants. The susceptibility of wild-type plants to X. oryzae pv. oryzae was increased by exogenous application of GA3 and decreased by S-3307 treatment. Together, the results provide direct evidence for a critical role of OsGA20ox3 in regulating not only plant stature but also disease resistance in rice.

scholarly journals Gibberellins Inhibit Flavonoid Biosynthesis and Promote Nitrogen Metabolism in Medicago truncatula

2021 ◽  
Vol 22 (17) ◽  
pp. 9291
Author(s):  
Hao Sun ◽  
Huiting Cui ◽  
Jiaju Zhang ◽  
Junmei Kang ◽  
Zhen Wang ◽  
...  
Keyword(s):  
Nitrogen Metabolism ◽  
Medicago Truncatula ◽  
Flavonoid Biosynthesis ◽  
Insertion Mutant ◽  
Wild Type ◽  
Dwarf Phenotype ◽  
Retrotransposon Insertion ◽  
Prohexadione Calcium ◽  
Mutant Lines ◽  
Tnt1 Retrotransposon

Bioactive gibberellic acids (GAs) are diterpenoid plant hormones that are biosynthesized through complex pathways and control various aspects of growth and development. Although GA biosynthesis has been intensively studied, the downstream metabolic pathways regulated by GAs have remained largely unexplored. We investigated Tnt1 retrotransposon insertion mutant lines of Medicago truncatula with a dwarf phenotype by forward and reverse genetics screening and phylogenetic, molecular, biochemical, proteomic and metabolomic analyses. Three Tnt1 retrotransposon insertion mutant lines of the gibberellin 3-beta-dioxygenase 1 gene (GA3ox1) with a dwarf phenotype were identified, in which the synthesis of GAs (GA3 and GA4) was inhibited. Phenotypic analysis revealed that plant height, root and petiole length of ga3ox1 mutants were shorter than those of the wild type (Medicago truncatula ecotype R108). Leaf size was also much smaller in ga3ox1 mutants than that in wild-type R108, which is probably due to cell-size diminution instead of a decrease in cell number. Proteomic and metabolomic analyses of ga3ox1/R108 leaves revealed that in the ga3ox1 mutant, flavonoid isoflavonoid biosynthesis was significantly up-regulated, while nitrogen metabolism was down-regulated. Additionally, we further demonstrated that flavonoid and isoflavonoid biosynthesis was induced by prohexadione calcium, an inhibitor of GA3ox enzyme, and inhibited by exogenous GA3. In contrast, nitrogen metabolism was promoted by exogenous GA3 but inhibited by prohexadione calcium. The results of this study further demonstrated that GAs play critical roles in positively regulating nitrogen metabolism and transport and negatively regulating flavonoid biosynthesis through GA-mediated signaling pathways in leaves.

scholarly journals Analysis of the Involvement of an Inducible Arabidopsis RNA-Dependent RNA Polymerase in Antiviral Defense

2003 ◽  
Vol 16 (3) ◽  
pp. 206-216 ◽  
Author(s):  
Diqiu Yu ◽  
Baofang Fan ◽  
Stuart A. MacFarlane ◽  
Zhixiang Chen
Keyword(s):  
Viral Vector ◽  
Potato Virus X ◽  
Insertion Mutant ◽  
Antiviral Defense ◽  
Wild Type ◽  
Knockout Mutant ◽  
General Role ◽  
Viral Rnas ◽  
Posttranscriptional Gene Silencing ◽  
Dna Insertion

RNA-dependent RNA polymerases (RdRPs) have been implicated in posttranscriptional gene silencing (PTGS) and antiviral defense. An Arabidopsis RdRP (SDE1/SGS2) has been previously shown to be required for transgene-induced PTGS but has no general role in antiviral defense. On the other hand, we have recently shown that transgenic tobacco deficient in an inducible RdRP (NtRdRP1) activity became more susceptible to both Tobacco mosaic virus and Potato virus X. Thus, different RdRPs may have distinct roles in closely related PTGS and antiviral defense. In the present study, we analyzed roles of a newly identified Arabidopsis RdRP gene (AtRdRP1) in plant antiviral defense. AtRdRP1 encodes an RdRP closely related structurally to NtRdRP1 and is also induced by salicylic acid treatment and virus infection. A T-DNA insertion mutant for AtRdRP1 has been isolated and analyzed for possible alterations in response to viral infection. When infected by a to-bamovirus and a tobravirus, the knockout mutant accumulated higher and more persistent levels of viral RNAs in both the lower, inoculated and in upper, systemically infected leaves than did wild-type plants. These results suggest that the inducible AtRdRP1 is the Arabidopsis ortholog of NtRdRP1 and plays a role in antiviral defense. Examination of short viral RNAs and silencing studies using a viral vector harboring an endogenous plant gene suggest that, while not required for virus-induced PTGS, AtRdRP1 can apparently promote turnover of viral RNAs in infected plants.

scholarly journals Using CRISPR-Cas9 to generate semi-dwarf rice lines in elite landraces

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Xingming Hu ◽  
Yongtao Cui ◽  
Guojun Dong ◽  
Anhui Feng ◽  
Danying Wang ◽  
...  
Keyword(s):  
Gene Editing ◽  
Agronomic Traits ◽  
Genetic Erosion ◽  
Field Trials ◽  
Wild Type ◽  
Biotic And Abiotic Stresses ◽  
Rice Varieties ◽  
Broad Spectrum Resistance ◽  
Mutant Lines ◽  
Better Than

AbstractGenetic erosion refers to the loss of genetic variation in a crop. In China, only a few original landraces of rice (Oryza sativa) were used in breeding and these became the primary genetic background of modern varieties. Expanding the genetic diversity among Chinese rice varieties and cultivating high-yielding and high-quality varieties with resistance to different biotic and abiotic stresses is critical. Here, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9(Cas9) genome editing system to edit Semi-Dwarf1 (SD1) in the elite landraces Kasalath and TeTePu (TTP), which contain many desired agronomic traits such as tolerance to low phosphorous and broad-spectrum resistance to several diseases and insects. Mutations of SD1 confer shorter plant height for better resistance to lodging. Field trials demonstrated that the yield of the new Kasalath and TTP mutant lines was better than that of the wild type under modern cultivation and that the lines maintained the same desirable agronomic characteristics as their wild-type progenitors. Our results showed that breeding using available landraces in combination with genomic data of different landraces and gene-editing techniques is an effective way to relieve genetic erosion in modern rice varieties.

scholarly journals Melatonin Deficiency Confers Tolerance to Multiple Abiotic Stresses in Rice via Decreased Brassinosteroid Levels

2019 ◽  
Vol 20 (20) ◽  
pp. 5173 ◽  
Author(s):  
Ok Jin Hwang ◽  
Kyoungwhan Back
Keyword(s):  
Heat Stress ◽  
Abiotic Stress ◽  
Stress Tolerance ◽  
Stress Responses ◽  
Abiotic Stress Tolerance ◽  
Grain Morphology ◽  
Wild Type ◽  
Gene Encoding ◽  
Adverse Conditions ◽  
Leaf Phenotype

Melatonin has long been recognized as a positive signaling molecule and potent antioxidant in plants, which alleviates damage caused by adverse conditions such as salt, cold, and heat stress. In this study, we found a paradoxical role for melatonin in abiotic stress responses. Suppression of the serotonin N-acetyltransferase 2 (snat2) gene encoding the penultimate enzyme in melatonin biosynthesis led to simultaneous decreases in both melatonin and brassinosteroid (BR) levels, causing a semi-dwarf with erect leaf phenotype, typical of BR deficiency. Here, we further characterized snat2 rice in terms of grain morphology and abiotic stress tolerance, to determine whether snat2 rice exhibited characteristics similar to those of BR-deficient rice. As expected, the snat2 rice exhibited tolerance to multiple stress conditions including cadmium, salt, cold, and heat, as evidenced by decreased malondialdehyde (MDA) levels and increased chlorophyll levels, in contrast with SNAT2 overexpression lines, which were less tolerant to stress than wild type plants. In addition, the length and width of grain from snat2 plants were reduced relative to the wild type, which is reminiscent of BR deficiency in rice. Other melatonin-deficient mutant rice lines with suppressed BR synthesis (i.e., comt and t5h) also showed tolerance to salt and heat stress, whereas melatonin-deficient rice seedlings without decreased BR levels (i.e., tdc) failed to exhibit increased stress tolerance, suggesting that stress tolerance was increased not by melatonin deficiency alone, but by a melatonin deficiency-mediated decrease in BR.

scholarly journals c-di-AMP-Regulated K+ Importer KtrAB Affects Biofilm Formation, Stress Response, and SpeB Expression in Streptococcus pyogenes

2021 ◽  
Vol 89 (4) ◽  
Author(s):  
Sabrina Faozia ◽  
Tazin Fahmi ◽  
Gary C. Port ◽  
Kyu Hong Cho
Keyword(s):  
Stress Response ◽  
Streptococcus Pyogenes ◽  
Stress Responses ◽  
Biofilm Formation ◽  
Growth Conditions ◽  
Infection Model ◽  
Wild Type ◽  
Content Type ◽  
Double Deletion ◽  
A Subunit

ABSTRACT The second messenger cyclic di-AMP (c-di-AMP) controls biofilm formation, stress response, and virulence in Streptococcus pyogenes. The deletion of the c-di-AMP synthase gene, dacA, results in pleiotropic effects including reduced expression of the secreted protease SpeB. Here, we report a role for K+ transport in c-di-AMP-mediated SpeB expression. The deletion of ktrB in the ΔdacA mutant restores SpeB expression. KtrB is a subunit of the K+ transport system KtrAB that forms a putative high-affinity K+ importer. KtrB forms a membrane K+ channel, and KtrA acts as a cytosolic gating protein that controls the transport capacity of the system by binding ligands including c-di-AMP. SpeB induction in the ΔdacA mutant by K+ specific ionophore treatment also supports the importance of cellular K+ balance in SpeB production. The ΔdacA ΔktrB double deletion mutant not only produces wild-type levels of SpeB but also partially or fully reverts the defective ΔdacA phenotypes of biofilm formation and stress responses, suggesting that many ΔdacA phenotypes are due to cellular K+ imbalance. However, the null pathogenicity of the ΔdacA mutant in a murine subcutaneous infection model is not restored by ktrB deletion, suggesting that c-di-AMP controls not only cellular K+ balance but also other metabolic and/or virulence pathways. The deletion of other putative K+ importer genes, kup and kimA, does not phenocopy the deletion of ktrB regarding SpeB induction in the ΔdacA mutant, suggesting that KtrAB is the primary K+ importer that is responsible for controlling cellular K+ levels under laboratory growth conditions.

scholarly journals Total reducing capacity in Arabidopsis thaliana cat2cat3 knockout mutants under heat stress

2017 ◽  
Vol 15 (1) ◽  
pp. 47-51
Author(s):  
I. I. Panchuk ◽  
I. M. Buzduga ◽  
R. A. Volkov
Keyword(s):  
Molecular Weight ◽  
Arabidopsis Thaliana ◽  
Heat Stress ◽  
Water Soluble ◽  
Antioxidant Defenses ◽  
Low Molecular Weight ◽  
Wild Type ◽  
Knock Out ◽  
Knockout Mutants ◽  
Reducing Capacity

Aim. It was investigated whether the simultaneous loss of the two catalase isoforms CAT2 and CAT3 can be compensated by the increase in content of low-molecular weight antioxidants. To clarify this question, the total reducing capacity in Arabidopsis wild type and cat2cat3 knockout mutants was evaluated under optimal growth conditions and after heat stress. Methods. Leaves of Arabidopsis thaliana wild type and cat2cat3 knockout mutants were exposed to high temperatures. The content of water-soluble low molecular weight antioxidants was evaluated by determining the total reducing capacity using iodometry. Results. In intact cat2cat3 mutants there is an 1.7 times increase in the content of low-molecular weight antioxidants compared to wild type plants. A high content of these compounds in knockout plants was also observed upon heat stress. Patterns of changes in total reducing capacity differ between wild type and knockout lines. Conclusion. The loss of activity of the catalase isoforms CAT2 and CAT3 in knock-out mutants of Arabidopsis results in activation of non-enzymatic antioxidant defenses. The increase of the content of low-molecular weight antioxidants is one of the mechanisms that provide protection of mutant plants from chronic oxidative stress, both under optimal cultivation conditions and under the influence of elevated temperatures.Keywords: multigenic family, heat shock, total reducing capacity, knockout mutants, Arabidopsis thaliana.

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