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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 required for osmotic and drought stress tolerance.(A)–(D) Seed germination and root growth of seedlings grown on 1/2 MS supplemented with different concentrations of mannitol. (A) Seed germination rates on 1/2 MS supplemented with 0, 250 and 400 mM of mannitol. 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 350 mM of mannitol. (C) and (D) Growth phenotype of seedlings grown on 1/2 MS supplemented with 0 and 350 mM mannitol. (E)-(G) Phenotype, relative water content and survival rates of soil-grown plants after drought stress treatment. (E) Phenotype of the wild type Ws-0 and mutant raf43-1 plants under drought stress condition. Three-week-old soil-grown plants were withheld from watering for drought stress treatment and allowed to grow for two weeks. Left photo shows the growth status of the plants before drought stress treatment while right photo shows phenotype of the plants at 7 days after drought stress treatment. (F) Relative water contents in leaf tissues of wild type Ws-0 and mutant raf43-1 plants grown under normal and drought stress condition at 7 days after treatment. (G) Survival rates of wild type Ws-0 and mutant raf43-1 plants grown under normal and drought stress condition at 14 days after treatment. Experiments were repeated three times and data presented in (A), (B), (F) and (G) 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.g003: Raf43 is required for osmotic and drought stress tolerance.(A)–(D) Seed germination and root growth of seedlings grown on 1/2 MS supplemented with different concentrations of mannitol. (A) Seed germination rates on 1/2 MS supplemented with 0, 250 and 400 mM of mannitol. 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 350 mM of mannitol. (C) and (D) Growth phenotype of seedlings grown on 1/2 MS supplemented with 0 and 350 mM mannitol. (E)-(G) Phenotype, relative water content and survival rates of soil-grown plants after drought stress treatment. (E) Phenotype of the wild type Ws-0 and mutant raf43-1 plants under drought stress condition. Three-week-old soil-grown plants were withheld from watering for drought stress treatment and allowed to grow for two weeks. Left photo shows the growth status of the plants before drought stress treatment while right photo shows phenotype of the plants at 7 days after drought stress treatment. (F) Relative water contents in leaf tissues of wild type Ws-0 and mutant raf43-1 plants grown under normal and drought stress condition at 7 days after treatment. (G) Survival rates of wild type Ws-0 and mutant raf43-1 plants grown under normal and drought stress condition at 14 days after treatment. Experiments were repeated three times and data presented in (A), (B), (F) and (G) 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: To explore the possible role of Raf43 in abiotic stress responses, we first examined and compared the seed germination and seedling root growth of the raf43-1 mutant line and the Raf43-OE#1 and Raf43-OE#13 lines with their corresponding wild types Ws-0 and Col-0 under osmotic stress condition mimicked by mannitol treatment. Germination rates of seeds from the raf43-1 mutant line, the Raf43-OE#1 and Raf43-OE#13 lines and their corresponding wild type Ws-0 and Col-0 was similar on mannitol-free 1/2MS medium but the germination rates of the raf43-1, Raf43-OE#1, Raf43-OE#13 and wild type seeds significantly decreased on 1/2MS medium supplemented with 200 mM or 400 mM mannitol as compared with those on mannitol-free medium (Fig 3A). In contrast, germination rates of the raf43-1 mutant seeds markedly decreased on 1/2MS medium supplemented with 200 mM or 400 mM mannitol, resulting in 70–75% of reduction as compared with the Ws-0 seeds (Fig 3A). 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 (Fig 3A). Similarly, after germination, root length of seedlings from the raf43-1 mutant line, the Raf43-OE#1 and Raf43-OE#13 lines and their corresponding wild types Ws-0 and Col-0 was similar when grown on mannitol-free 1/2MS medium but the root growth of the raf43-1, Raf43-OE#1, Raf43-OE#13 and wild type seedlings was significantly inhibited when grown on 1/2MS medium supplemented with 350 mM mannitol as compared with those on mannitol-free medium (Fig 3B–3D). Root growth of the raf43-1 seedlings was significantly inhibited on 1/2MS medium supplemented with 350 mM mannitol, leading to a 55% reduction in root length as compared with the Ws-0 seedlings (Fig 3B and 3C). No significant difference in root length of the Raf43-OE#1 and Raf43-OE#13 seedlings and the Col-0 seedlings was observed (Fig 3B and 3D). These observations indicate that knockout of Raf43 attenuated the osmotic stress tolerance but overexpression of Raf43 did not affect the osmotic stress tolerance, demonstrating the requirement of Raf43 in tolerance to osmotic stress 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 required for osmotic and drought stress tolerance.(A)–(D) Seed germination and root growth of seedlings grown on 1/2 MS supplemented with different concentrations of mannitol. (A) Seed germination rates on 1/2 MS supplemented with 0, 250 and 400 mM of mannitol. 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 350 mM of mannitol. (C) and (D) Growth phenotype of seedlings grown on 1/2 MS supplemented with 0 and 350 mM mannitol. (E)-(G) Phenotype, relative water content and survival rates of soil-grown plants after drought stress treatment. (E) Phenotype of the wild type Ws-0 and mutant raf43-1 plants under drought stress condition. Three-week-old soil-grown plants were withheld from watering for drought stress treatment and allowed to grow for two weeks. Left photo shows the growth status of the plants before drought stress treatment while right photo shows phenotype of the plants at 7 days after drought stress treatment. (F) Relative water contents in leaf tissues of wild type Ws-0 and mutant raf43-1 plants grown under normal and drought stress condition at 7 days after treatment. (G) Survival rates of wild type Ws-0 and mutant raf43-1 plants grown under normal and drought stress condition at 14 days after treatment. Experiments were repeated three times and data presented in (A), (B), (F) and (G) 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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Related In: Results  -  Collection

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pone.0133975.g003: Raf43 is required for osmotic and drought stress tolerance.(A)–(D) Seed germination and root growth of seedlings grown on 1/2 MS supplemented with different concentrations of mannitol. (A) Seed germination rates on 1/2 MS supplemented with 0, 250 and 400 mM of mannitol. 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 350 mM of mannitol. (C) and (D) Growth phenotype of seedlings grown on 1/2 MS supplemented with 0 and 350 mM mannitol. (E)-(G) Phenotype, relative water content and survival rates of soil-grown plants after drought stress treatment. (E) Phenotype of the wild type Ws-0 and mutant raf43-1 plants under drought stress condition. Three-week-old soil-grown plants were withheld from watering for drought stress treatment and allowed to grow for two weeks. Left photo shows the growth status of the plants before drought stress treatment while right photo shows phenotype of the plants at 7 days after drought stress treatment. (F) Relative water contents in leaf tissues of wild type Ws-0 and mutant raf43-1 plants grown under normal and drought stress condition at 7 days after treatment. (G) Survival rates of wild type Ws-0 and mutant raf43-1 plants grown under normal and drought stress condition at 14 days after treatment. Experiments were repeated three times and data presented in (A), (B), (F) and (G) 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: To explore the possible role of Raf43 in abiotic stress responses, we first examined and compared the seed germination and seedling root growth of the raf43-1 mutant line and the Raf43-OE#1 and Raf43-OE#13 lines with their corresponding wild types Ws-0 and Col-0 under osmotic stress condition mimicked by mannitol treatment. Germination rates of seeds from the raf43-1 mutant line, the Raf43-OE#1 and Raf43-OE#13 lines and their corresponding wild type Ws-0 and Col-0 was similar on mannitol-free 1/2MS medium but the germination rates of the raf43-1, Raf43-OE#1, Raf43-OE#13 and wild type seeds significantly decreased on 1/2MS medium supplemented with 200 mM or 400 mM mannitol as compared with those on mannitol-free medium (Fig 3A). In contrast, germination rates of the raf43-1 mutant seeds markedly decreased on 1/2MS medium supplemented with 200 mM or 400 mM mannitol, resulting in 70–75% of reduction as compared with the Ws-0 seeds (Fig 3A). 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 (Fig 3A). Similarly, after germination, root length of seedlings from the raf43-1 mutant line, the Raf43-OE#1 and Raf43-OE#13 lines and their corresponding wild types Ws-0 and Col-0 was similar when grown on mannitol-free 1/2MS medium but the root growth of the raf43-1, Raf43-OE#1, Raf43-OE#13 and wild type seedlings was significantly inhibited when grown on 1/2MS medium supplemented with 350 mM mannitol as compared with those on mannitol-free medium (Fig 3B–3D). Root growth of the raf43-1 seedlings was significantly inhibited on 1/2MS medium supplemented with 350 mM mannitol, leading to a 55% reduction in root length as compared with the Ws-0 seedlings (Fig 3B and 3C). No significant difference in root length of the Raf43-OE#1 and Raf43-OE#13 seedlings and the Col-0 seedlings was observed (Fig 3B and 3D). These observations indicate that knockout of Raf43 attenuated the osmotic stress tolerance but overexpression of Raf43 did not affect the osmotic stress tolerance, demonstrating the requirement of Raf43 in tolerance to osmotic stress 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