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Arabidopsis Raf-Like Mitogen-Activated Protein Kinase Kinase Kinase Gene Raf43 Is Required for Tolerance to Multiple Abiotic Stresses.

Virk N, Li D, Tian L, Huang L, Hong Y, Li X, Zhang Y, Liu B, Zhang H, Song F - PLoS ONE (2015)

Bottom Line: Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress.Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines.Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, 310058, China.

ABSTRACT
Mitogen-activated protein kinase (MAPK) cascades are critical signaling modules that mediate the transduction of extracellular stimuli into intracellular response. A relatively large number of MAPKKKs have been identified in a variety of plant genomes but only a few of them have been studied for their biological function. In the present study, we identified an Arabidopsis Raf-like MAPKKK gene Raf43 and studied its function in biotic and abiotic stress response using a T-DNA insertion mutant raf43-1 and two Raf43-overexpressing lines Raf43-OE#1 and Raf43-OE#13. Expression of Raf43 was induced by multiple abiotic and biotic stresses including treatments with drought, mannitol and oxidative stress or defense signaling molecule salicylic acid and infection with necrotrophic fungal pathogen Botrytis cinerea. Seed germination and seedling root growth of raf43-1 were significantly inhibited on MS medium containing mannitol, NaCl, H2O2 or methyl viologen (MV) while seed germination and seedling root growth of the Raf43-OE#1 and Raf43-OE#13 lines was similar to wild type Col-0 under the above stress conditions. Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress. Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines. Expression of stress-responsive RD17 and DREB2A genes was significantly down-regulated in raf43-1 plants. However, the raf43-1 and Raf43-overexpressing plants showed similar disease phenotype to the wild type plants after infection with B. cinerea or Pseudomonas syringae pv. tomato DC3000. Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis.

No MeSH data available.


Related in: MedlinePlus

Raf43 is involved in ABA response.(A) Seed germination rates on 1/2 MS supplemented with 0, 1 and 2 μM of ABA. Seed germination was recorded when the radicles emerged completely from seed coat. (B) Root length of seedlings grown on 1/2 MS supplemented with 0 and 1.5 μM of ABA. (C) and (D) Root phenotype of seedlings grown on 1/2 MS supplemented with 0 and 1.5 μM of ABA. (E) Expression of stress-responsive and ABA biosynthesis-related genes in WS-0 and raf43-1 plants. Leaf samples were collected form four-week-old soil-grown plants for analyzing gene expression through qRT-PCR. Expression data were normalized by the transcript level of Ubiquitin. The expression levels of Raf43 in wild type Ws-0 plants was set as 1 and the levels in raf43 mutant plants are shown as folds of the levels in wild type plants. Experiments were repeated three times and data presented in (A), (B) and (E) are the means ± standard errors from three independent experiments. Different letters above the columns indicate significant difference at p = 0.05 between Ws-0 and raf43-1 plants and between Col-0 and Raf43-overexpressing plants.
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pone.0133975.g006: Raf43 is involved in ABA response.(A) Seed germination rates on 1/2 MS supplemented with 0, 1 and 2 μM of ABA. Seed germination was recorded when the radicles emerged completely from seed coat. (B) Root length of seedlings grown on 1/2 MS supplemented with 0 and 1.5 μM of ABA. (C) and (D) Root phenotype of seedlings grown on 1/2 MS supplemented with 0 and 1.5 μM of ABA. (E) Expression of stress-responsive and ABA biosynthesis-related genes in WS-0 and raf43-1 plants. Leaf samples were collected form four-week-old soil-grown plants for analyzing gene expression through qRT-PCR. Expression data were normalized by the transcript level of Ubiquitin. The expression levels of Raf43 in wild type Ws-0 plants was set as 1 and the levels in raf43 mutant plants are shown as folds of the levels in wild type plants. Experiments were repeated three times and data presented in (A), (B) and (E) are the means ± standard errors from three independent experiments. Different letters above the columns indicate significant difference at p = 0.05 between Ws-0 and raf43-1 plants and between Col-0 and Raf43-overexpressing plants.

Mentions: Considering that Raf43 is required for multiple abiotic stress tolerance as mentioned above and that ABA is an important stress hormone involved in regulating abiotic stress response [49], we therefore examined the possible involvement of Raf43 in ABA response. For this purpose, seed germination and root growth of the raf43-1 mutant line and the Raf43-OE#1 and Raf43-OE#13 lines in response to exogenous ABA were assayed and compared with their corresponding wild types Ws-0 and Col-0. Germination rates of seeds from the raf43-1 line, the Raf43-OE#1 and Raf43-OE#13 lines and their corresponding wild type Ws-0 and Col-0 were similar when germinated on ABA-free 1/2MS medium but the germination rates decreased significantly on 1/2MS medium supplemented with 1 μM or 2 μM ABA as compared with those on ABA-free medium (Fig 6A). However, germination rates of the raf43-1 mutant seeds markedly decreased on 1/2MS medium supplemented with 1 μM or 2 μM ABA, resulting in 55–60% of reduction as compared with the Ws-0 seeds (Fig 6A). No significant difference in germination rates of seeds from the Raf43-OE#1 and Raf43-OE#13 lines and wild type Col-0 was observed in the presence of ABA (Fig 6A). After germination, root length of the raf43-1 seedlings, the Raf43-OE#1 and Raf43-OE#13 seedlings and their corresponding wild type Ws-0 and Col-0 seedling was similar when grown on ABA-free 1/2MS medium but the root growth was significantly inhibited when grown on 1/2MS medium supplemented with 1.5 μM ABA as compared with those on ABA-free medium (Fig 6B–6D). Root growth of the raf43-1 seedlings was significantly inhibited on 1/2MS medium supplemented with 1.5 μM ABA, leading to a 56% of reduction as compared with the Ws-0 seedlings (Fig 6B and 6C). No significant difference in root length of the Raf43-OE#1 and Raf43-OE#13 seedlings and the wild type Col-0 seedlings was observed (Fig 6B and 6D). These observations indicate that knockout of Raf43 resulted in increased ABA sensitivity but overexpression of Raf43 had no effect on ABA response in Arabidopsis.


Arabidopsis Raf-Like Mitogen-Activated Protein Kinase Kinase Kinase Gene Raf43 Is Required for Tolerance to Multiple Abiotic Stresses.

Virk N, Li D, Tian L, Huang L, Hong Y, Li X, Zhang Y, Liu B, Zhang H, Song F - PLoS ONE (2015)

Raf43 is involved in ABA response.(A) Seed germination rates on 1/2 MS supplemented with 0, 1 and 2 μM of ABA. Seed germination was recorded when the radicles emerged completely from seed coat. (B) Root length of seedlings grown on 1/2 MS supplemented with 0 and 1.5 μM of ABA. (C) and (D) Root phenotype of seedlings grown on 1/2 MS supplemented with 0 and 1.5 μM of ABA. (E) Expression of stress-responsive and ABA biosynthesis-related genes in WS-0 and raf43-1 plants. Leaf samples were collected form four-week-old soil-grown plants for analyzing gene expression through qRT-PCR. Expression data were normalized by the transcript level of Ubiquitin. The expression levels of Raf43 in wild type Ws-0 plants was set as 1 and the levels in raf43 mutant plants are shown as folds of the levels in wild type plants. Experiments were repeated three times and data presented in (A), (B) and (E) are the means ± standard errors from three independent experiments. Different letters above the columns indicate significant difference at p = 0.05 between Ws-0 and raf43-1 plants and between Col-0 and Raf43-overexpressing plants.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519275&req=5

pone.0133975.g006: Raf43 is involved in ABA response.(A) Seed germination rates on 1/2 MS supplemented with 0, 1 and 2 μM of ABA. Seed germination was recorded when the radicles emerged completely from seed coat. (B) Root length of seedlings grown on 1/2 MS supplemented with 0 and 1.5 μM of ABA. (C) and (D) Root phenotype of seedlings grown on 1/2 MS supplemented with 0 and 1.5 μM of ABA. (E) Expression of stress-responsive and ABA biosynthesis-related genes in WS-0 and raf43-1 plants. Leaf samples were collected form four-week-old soil-grown plants for analyzing gene expression through qRT-PCR. Expression data were normalized by the transcript level of Ubiquitin. The expression levels of Raf43 in wild type Ws-0 plants was set as 1 and the levels in raf43 mutant plants are shown as folds of the levels in wild type plants. Experiments were repeated three times and data presented in (A), (B) and (E) are the means ± standard errors from three independent experiments. Different letters above the columns indicate significant difference at p = 0.05 between Ws-0 and raf43-1 plants and between Col-0 and Raf43-overexpressing plants.
Mentions: Considering that Raf43 is required for multiple abiotic stress tolerance as mentioned above and that ABA is an important stress hormone involved in regulating abiotic stress response [49], we therefore examined the possible involvement of Raf43 in ABA response. For this purpose, seed germination and root growth of the raf43-1 mutant line and the Raf43-OE#1 and Raf43-OE#13 lines in response to exogenous ABA were assayed and compared with their corresponding wild types Ws-0 and Col-0. Germination rates of seeds from the raf43-1 line, the Raf43-OE#1 and Raf43-OE#13 lines and their corresponding wild type Ws-0 and Col-0 were similar when germinated on ABA-free 1/2MS medium but the germination rates decreased significantly on 1/2MS medium supplemented with 1 μM or 2 μM ABA as compared with those on ABA-free medium (Fig 6A). However, germination rates of the raf43-1 mutant seeds markedly decreased on 1/2MS medium supplemented with 1 μM or 2 μM ABA, resulting in 55–60% of reduction as compared with the Ws-0 seeds (Fig 6A). No significant difference in germination rates of seeds from the Raf43-OE#1 and Raf43-OE#13 lines and wild type Col-0 was observed in the presence of ABA (Fig 6A). After germination, root length of the raf43-1 seedlings, the Raf43-OE#1 and Raf43-OE#13 seedlings and their corresponding wild type Ws-0 and Col-0 seedling was similar when grown on ABA-free 1/2MS medium but the root growth was significantly inhibited when grown on 1/2MS medium supplemented with 1.5 μM ABA as compared with those on ABA-free medium (Fig 6B–6D). Root growth of the raf43-1 seedlings was significantly inhibited on 1/2MS medium supplemented with 1.5 μM ABA, leading to a 56% of reduction as compared with the Ws-0 seedlings (Fig 6B and 6C). No significant difference in root length of the Raf43-OE#1 and Raf43-OE#13 seedlings and the wild type Col-0 seedlings was observed (Fig 6B and 6D). These observations indicate that knockout of Raf43 resulted in increased ABA sensitivity but overexpression of Raf43 had no effect on ABA response in Arabidopsis.

Bottom Line: Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress.Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines.Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, 310058, China.

ABSTRACT
Mitogen-activated protein kinase (MAPK) cascades are critical signaling modules that mediate the transduction of extracellular stimuli into intracellular response. A relatively large number of MAPKKKs have been identified in a variety of plant genomes but only a few of them have been studied for their biological function. In the present study, we identified an Arabidopsis Raf-like MAPKKK gene Raf43 and studied its function in biotic and abiotic stress response using a T-DNA insertion mutant raf43-1 and two Raf43-overexpressing lines Raf43-OE#1 and Raf43-OE#13. Expression of Raf43 was induced by multiple abiotic and biotic stresses including treatments with drought, mannitol and oxidative stress or defense signaling molecule salicylic acid and infection with necrotrophic fungal pathogen Botrytis cinerea. Seed germination and seedling root growth of raf43-1 were significantly inhibited on MS medium containing mannitol, NaCl, H2O2 or methyl viologen (MV) while seed germination and seedling root growth of the Raf43-OE#1 and Raf43-OE#13 lines was similar to wild type Col-0 under the above stress conditions. Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress. Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines. Expression of stress-responsive RD17 and DREB2A genes was significantly down-regulated in raf43-1 plants. However, the raf43-1 and Raf43-overexpressing plants showed similar disease phenotype to the wild type plants after infection with B. cinerea or Pseudomonas syringae pv. tomato DC3000. Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis.

No MeSH data available.


Related in: MedlinePlus