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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 oxidative stress tolerance.(A) Germination rates of seeds on 1/2 MS supplemented with 20 mM H2O2 or 10 μM methyl viologen (MV). Seed germination was recorded when the radicles emerged completely from seed coat. (B) and (C) Phenotype and chlorophyll contents in leaf tissues of the wild type Ws-0 and mutant raf43-1 plants after treatment with 50 μM MV. Representative leaves showing typical yellowish and necrotic symptom from MV-treated Ws-0 and raf43-1 plants were also shown. Three-week-old soil-grown plants were treated by foliar spraying with 50 μM MV or water as a control and allowed to grow for 7 days. Photos were taken at 5 days after treatment and leaf samples were collected for analyzing chlorophyll contents. Experiments were repeated three times and data presented in (A) and (C) 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.g005: Raf43 is required for oxidative stress tolerance.(A) Germination rates of seeds on 1/2 MS supplemented with 20 mM H2O2 or 10 μM methyl viologen (MV). Seed germination was recorded when the radicles emerged completely from seed coat. (B) and (C) Phenotype and chlorophyll contents in leaf tissues of the wild type Ws-0 and mutant raf43-1 plants after treatment with 50 μM MV. Representative leaves showing typical yellowish and necrotic symptom from MV-treated Ws-0 and raf43-1 plants were also shown. Three-week-old soil-grown plants were treated by foliar spraying with 50 μM MV or water as a control and allowed to grow for 7 days. Photos were taken at 5 days after treatment and leaf samples were collected for analyzing chlorophyll contents. Experiments were repeated three times and data presented in (A) and (C) 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: Because expression of Raf43 was induced by oxidative stress (Fig 1), we thus examined whether mutation or overexpression of Raf43 would affect tolerance to oxidative stress during seed germination or in mature Arabidopsis leaves. In 1/2MS medium without supplementation of MV or H2O2, seeds of the raf43-1 mutant and the Raf43-OE#1 and Raf43-OE#13 lines showed similar germination rates to those of the corresponding wild type Ws-0 and Col-0 seeds (Fig 5A). In 1/2MS medium supplemented with 20 mM H2O2 or 10 μM MV, germination rates of seeds from the raf43-1 mutant line, the Raf43-OE#1 and Raf43-OE#13 lines and the wild types Ws-0 and Col-0 lines were decreased significantly, but seeds of the raf43-1 mutant line showed a lower germination rate than the wild type Ws-0 seeds (Fig 5A). At 6 days, only 20% and 10% of the raf43-1 mutant seeds had germinated in the presence of 20 mM H2O2 or 10 mM MV, respectively, while 40–50% of wild type Ws-0 seeds had germinated (Fig 5A). By contrast, germination rates for seeds of the Raf43-OE#1 and Raf43-OE#13 lines in the presence of 20 mM H2O2 or 10 μM MV were comparable to those of the wild type Col-0 seeds (Fig 5A). We further assessed the oxidative stress tolerance in mature leaves of the raf43-1 and wild type Ws-0 plants to MV. Without treatment with MV, both the raf43-1 and wild type Ws-0 plants grew well; however, exogenous application of 50 μM MV by foliar spraying caused significant necrosis or bleaching, indicative of oxidative damage, on leaves of soil-grown four-week-old raf43-1 and Ws-0 plants (Fig 5B). Oxidative damage caused by exogenously applied MV on leaves of the raf43-1 plants was much more pronounced than on leaves of the wild type Ws-0 plants (Fig 5B). MV-induced oxidative damage in leaves of the raf43-1 and wild type Ws-0 plants resulted in significant reduction of the chlorophyll contents as compared with those in untreated control plants (Fig 5C). The reduction of chlorophyll contents in the MV-treated leaves of the raf43-1 plants was much evident than that in the wild type Ws-0 plants (Fig 5C). Chlorophyll content in MV-treated leaves of the raf43-1 plants decreased to 39% of that in leaves without MV treatment, whereas chlorophyll content in MV-treated leaves of the wild type Ws-0 was about 71% of that in leaves without MV treatment (Fig 5C). These results indicate that knockout of Raf43 could weaken oxidative stress tolerance, leading to an increased level of cellular damage under oxidative stress conditions but overexpression of Raf43 did not affect the oxidative stress tolerance 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 oxidative stress tolerance.(A) Germination rates of seeds on 1/2 MS supplemented with 20 mM H2O2 or 10 μM methyl viologen (MV). Seed germination was recorded when the radicles emerged completely from seed coat. (B) and (C) Phenotype and chlorophyll contents in leaf tissues of the wild type Ws-0 and mutant raf43-1 plants after treatment with 50 μM MV. Representative leaves showing typical yellowish and necrotic symptom from MV-treated Ws-0 and raf43-1 plants were also shown. Three-week-old soil-grown plants were treated by foliar spraying with 50 μM MV or water as a control and allowed to grow for 7 days. Photos were taken at 5 days after treatment and leaf samples were collected for analyzing chlorophyll contents. Experiments were repeated three times and data presented in (A) and (C) 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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Show All Figures
getmorefigures.php?uid=PMC4519275&req=5

pone.0133975.g005: Raf43 is required for oxidative stress tolerance.(A) Germination rates of seeds on 1/2 MS supplemented with 20 mM H2O2 or 10 μM methyl viologen (MV). Seed germination was recorded when the radicles emerged completely from seed coat. (B) and (C) Phenotype and chlorophyll contents in leaf tissues of the wild type Ws-0 and mutant raf43-1 plants after treatment with 50 μM MV. Representative leaves showing typical yellowish and necrotic symptom from MV-treated Ws-0 and raf43-1 plants were also shown. Three-week-old soil-grown plants were treated by foliar spraying with 50 μM MV or water as a control and allowed to grow for 7 days. Photos were taken at 5 days after treatment and leaf samples were collected for analyzing chlorophyll contents. Experiments were repeated three times and data presented in (A) and (C) 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: Because expression of Raf43 was induced by oxidative stress (Fig 1), we thus examined whether mutation or overexpression of Raf43 would affect tolerance to oxidative stress during seed germination or in mature Arabidopsis leaves. In 1/2MS medium without supplementation of MV or H2O2, seeds of the raf43-1 mutant and the Raf43-OE#1 and Raf43-OE#13 lines showed similar germination rates to those of the corresponding wild type Ws-0 and Col-0 seeds (Fig 5A). In 1/2MS medium supplemented with 20 mM H2O2 or 10 μM MV, germination rates of seeds from the raf43-1 mutant line, the Raf43-OE#1 and Raf43-OE#13 lines and the wild types Ws-0 and Col-0 lines were decreased significantly, but seeds of the raf43-1 mutant line showed a lower germination rate than the wild type Ws-0 seeds (Fig 5A). At 6 days, only 20% and 10% of the raf43-1 mutant seeds had germinated in the presence of 20 mM H2O2 or 10 mM MV, respectively, while 40–50% of wild type Ws-0 seeds had germinated (Fig 5A). By contrast, germination rates for seeds of the Raf43-OE#1 and Raf43-OE#13 lines in the presence of 20 mM H2O2 or 10 μM MV were comparable to those of the wild type Col-0 seeds (Fig 5A). We further assessed the oxidative stress tolerance in mature leaves of the raf43-1 and wild type Ws-0 plants to MV. Without treatment with MV, both the raf43-1 and wild type Ws-0 plants grew well; however, exogenous application of 50 μM MV by foliar spraying caused significant necrosis or bleaching, indicative of oxidative damage, on leaves of soil-grown four-week-old raf43-1 and Ws-0 plants (Fig 5B). Oxidative damage caused by exogenously applied MV on leaves of the raf43-1 plants was much more pronounced than on leaves of the wild type Ws-0 plants (Fig 5B). MV-induced oxidative damage in leaves of the raf43-1 and wild type Ws-0 plants resulted in significant reduction of the chlorophyll contents as compared with those in untreated control plants (Fig 5C). The reduction of chlorophyll contents in the MV-treated leaves of the raf43-1 plants was much evident than that in the wild type Ws-0 plants (Fig 5C). Chlorophyll content in MV-treated leaves of the raf43-1 plants decreased to 39% of that in leaves without MV treatment, whereas chlorophyll content in MV-treated leaves of the wild type Ws-0 was about 71% of that in leaves without MV treatment (Fig 5C). These results indicate that knockout of Raf43 could weaken oxidative stress tolerance, leading to an increased level of cellular damage under oxidative stress conditions but overexpression of Raf43 did not affect the oxidative stress tolerance 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