T-DNA as an Insertional Mutagen in Arabidopsis

Krysan, Patrick J.; Young, Jeffery C.; Sussman, Michael R.

doi:10.1105/tpc.11.12.2283

Cited by 626 publications

(113 citation statements)

References 27 publications

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“…In the Atpub46-2 and Atpub48-1 mutants, the T-DNA is inserted in the 5' UTR. The T-DNA insertions in the 5’ UTRs have been proposed to result from reduced gene expression [54] and also significantly affect protein translation [55]. Thus, Atpub46-2 and Atpub48-1 can be regarded as knockdown mutants [56].…”

Section: Resultsmentioning

confidence: 99%

The Arabidopsis paralogs, PUB46 and PUB48, encoding U-box E3 ubiquitin ligases, are essential for plant response to drought stress

et al. 2017

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BackgroundPlants respond to abiotic stress on physiological, biochemical and molecular levels. This includes a global change in their cellular proteome achieved by changes in the pattern of their protein synthesis and degradation. The ubiquitin-proteasome system (UPS) is a key player in protein degradation in eukaryotes. Proteins are marked for degradation by the proteasome by coupling short chains of ubiquitin polypeptides in a three-step pathway. The last and regulatory stage is catalyzed by a member of a large family of substrate-specific ubiquitin ligases.ResultsWe have identified AtPUB46 and AtPUB48—two paralogous genes that encode ubiquitin ligases (E3s)—to have a role in the plant environmental response. The AtPUB46, −47, and −48 appear as tandem gene copies on chromosome 5, and we present a phylogenetic analysis that traces their evolution from an ancestral PUB-ARM gene. Single homozygous T-DNA insertion mutants of AtPUB46 and AtPUB48 displayed hypersensitivity to water stress; this was not observed for similar mutants of AtPUB47. Although the three genes show a similar spatial expression pattern, the steady state levels of their transcripts are differentially affected by abiotic stresses and plant hormones.Conclusions AtPUB46 and AtPUB48 encode plant U-Box E3s and are involved in the response to water stress. Our data suggest that despite encoding highly homologous proteins, AtPUB46 and AtPUB48 biological activity does not fully overlap.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0963-5) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

The Arabidopsis paralogs, PUB46 and PUB48, encoding U-box E3 ubiquitin ligases, are essential for plant response to drought stress

et al. 2017

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“…With A. thaliana , knockout mutants are available for essentially every gene of the plant. This includes characterized classical mutants generated by the mutagen ethyl methane sulfonate (EMS) (http://www.arabidopsis.org/) and T-DNA insertion lines (Krysan et al, 1999). However, ultimately, the target site must be validated by showing that it binds the phytotoxin and is inhibited in vitro .…”

Section: Validationmentioning

confidence: 99%

Omics Methods for Probing the Mode of Action of Natural and Synthetic Phytotoxins

2013

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For a little over a decade, omics methods (transcriptomics, proteomics, metabolomics, and physionomics) have been used to discover and probe the mode of action of both synthetic and natural phytotoxins. For mode of action discovery, the strategy for each of these approaches is to generate an omics profile for phytotoxins with known molecular targets and to compare this library of responses to the responses of compounds with unknown modes of action. Using more than one omics approach enhances the probability of success. Generally, compounds with the same mode of action generate similar responses with a particular omics method. Stress and detoxification responses to phytotoxins can be much clearer than effects directly related to the target site. Clues to new modes of action must be validated with in vitro enzyme effects or genetic approaches. Thus far, the only new phytotoxin target site discovered with omics approaches (metabolomics and physionomics) is that of cinmethylin and structurally related 5-benzyloxymethyl-1,2-isoxazolines. These omics approaches pointed to tyrosine amino-transferase as the target, which was verified by enzyme assays and genetic methods. In addition to being a useful tool of mode of action discovery, omics methods provide detailed information on genetic and biochemical impacts of phytotoxins. Such information can be useful in understanding the full impact of natural phytotoxins in both agricultural and natural ecosystems.

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“…T-DNA inserted mutational approaches have been used to determine the function of several genes (Krysan et al 1999;Zou et al 2013;Ding et al 2013). Zou et al (2013) used this approach to discover that the GSA-1/ARG1 gene functions in root gravitropism in Arabidopsis thaliana under ammonium stress.…”

Section: Introductionmentioning

confidence: 99%

Morphological and gene expression pattern changes in wrky46 mutant Arabidopsis thaliana under altered gravity conditions

Soh

Srikanth

Whang

et al. 2015

Botany

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Arabidopsis roots are important organs for gravity sensing and gravitropic response. Long-term exposure of Arabidopsis to three-dimensional (3D) clinorotation was found to result in increased expression of WRKY46. In this study, the response of wrky46 mutants, constructed by T-DNA insertion, to simulated microgravity and gravistimulation by reorientation was studied. Microgravity conditions were simulated using a 3D clinostat, which simulates weightlessness. Morphological changes, such as root coiling, waving, and multidirectional growth patterns, were observed in the wild type (WT) seedlings after only 4 days of 3D clinorotation treatment, while in the wrky46 mutant seedlings, the changes observed were minimal compared with those of WT seedlings. Polar auxin transport played an important role in the observed morphological changes. Therefore, expression of the polar auxin transport genes AUX1, PIN2, -3, -4, and -7, and ARG1 was analyzed. Their expression was found to be higher in wrky46 seedlings than in WT seedlings under 3D clinorotation. Gravistimulation by reorientation was achieved by horizontally reorienting the seedlings. Root tip bending to 90°was observed in all WT seedlings within 18-20 h, whereas in mutant seedlings, bending of the root tip to a right angle took more than 20 h. AXR expression is known to directly correlate with root tip bending. The expression of AXR1, -2, -3, -4 was found to be higher in WT seedlings relative to mutant seedlings under gravistimulation by reorientation. However, their expression was relatively low in both WT and wrky46 plants under 3D clinorotation. These results suggest that WRKY46 plays an important role in the root gravitropic response, and in the reduction of polar auxin transport that is necessary for decreasing the effect of gravity due to clinorotation. Résumé :Les racines d'Arabidopsis sont des organes importants pour la détection de la gravité et la réponse géotropique. Une exposition à long terme d'Arabidopsis à une clinorotation en 3D s'est avérée résulter en une augmentation de l'expression de WKRY46. Dans cette étude, la réponse de mutants wkry46, construits au moyen d'une insertion par un ADN-T, à une microgravité simulée et à une gravistimulation par réorientation a été étudiée. Les conditions de microgravité ont été simulées à l'aide d'un clinostat qui simule l'apesanteur. Des changements morphologiques comme l'enracinement en vrille, l'ondulation et les patrons de croissance multidirectionnelle ont été observés chez les pousses sauvages après seulement 4 jours de traitement en clinorotation en 3D, alors que chez les pousses des mutants wrky46, les changements observés étaient minimes comparativement aux pousses sauvages. Le transport polaire de l'auxine jouait un rôle important dans les changements morphologiques observés. Ainsi, l'expression des gènes de transport de l'auxine AUX1, PIN2, -3, -4 et -7, et ARG1 a été analysée. Leur expression s'est révélée plus élevée chez les pousses wrky46 que chez les pousses sauvages soumises à la clinorotation en 3...

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T-DNA as an Insertional Mutagen in Arabidopsis

Cited by 626 publications

References 27 publications

The Arabidopsis paralogs, PUB46 and PUB48, encoding U-box E3 ubiquitin ligases, are essential for plant response to drought stress

The Arabidopsis paralogs, PUB46 and PUB48, encoding U-box E3 ubiquitin ligases, are essential for plant response to drought stress

Omics Methods for Probing the Mode of Action of Natural and Synthetic Phytotoxins

Morphological and gene expression pattern changes in wrky46 mutant Arabidopsis thaliana under altered gravity conditions

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