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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

Responsiveness of Raf43 to biotic and abiotic stresses.(A) Genevestigator-based analysis of expression profile of Raf43 in response to abiotic stress, pathogen infection and elicitor treatment. Expression data were extracted from Genevestigator microarray datasets obtained from experiments with ecotype Col-0 plants except for the P. syringae pv. tomato experiment, which was conducted with Ws-0 plants. The p values for the expression data between the treated/inoculated and control plants are shown above the columns. B. graminis, Blumeria graminis; P. s. maculicola, Pseudomonas syringae pv. maculicola; P. s. phaseolicola, P. syringae pv. phaseolicola; P. s. tomato, P. syringae pv. tomato DC3000; B. cinerea, Botrytis cinerea; A. brasicicola, Alternaria brassicicola; S. sclerotiorum, Sclerotinia sclerotiorum. (B) qRT-PCR-based analysis of expression patterns of Raf43 in response to treatments with mannitol, drought, methyl viologen or salicylic acid and to infection by B. cinerea. The expression level of Ubiquitin was used to normalize the Raf43 expression data and the relative expression was calculated as folds of expression in treated/inoculated plants vs. those in the control plants. Experiments were repeated with three independent biological samples at each time point and data represented are the means ± standard errors from three independent experiments. * and ** indicate the significant difference at p = 0.05 and p = 0.01, respectively, as compared with those in the controls.
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pone.0133975.g001: Responsiveness of Raf43 to biotic and abiotic stresses.(A) Genevestigator-based analysis of expression profile of Raf43 in response to abiotic stress, pathogen infection and elicitor treatment. Expression data were extracted from Genevestigator microarray datasets obtained from experiments with ecotype Col-0 plants except for the P. syringae pv. tomato experiment, which was conducted with Ws-0 plants. The p values for the expression data between the treated/inoculated and control plants are shown above the columns. B. graminis, Blumeria graminis; P. s. maculicola, Pseudomonas syringae pv. maculicola; P. s. phaseolicola, P. syringae pv. phaseolicola; P. s. tomato, P. syringae pv. tomato DC3000; B. cinerea, Botrytis cinerea; A. brasicicola, Alternaria brassicicola; S. sclerotiorum, Sclerotinia sclerotiorum. (B) qRT-PCR-based analysis of expression patterns of Raf43 in response to treatments with mannitol, drought, methyl viologen or salicylic acid and to infection by B. cinerea. The expression level of Ubiquitin was used to normalize the Raf43 expression data and the relative expression was calculated as folds of expression in treated/inoculated plants vs. those in the control plants. Experiments were repeated with three independent biological samples at each time point and data represented are the means ± standard errors from three independent experiments. * and ** indicate the significant difference at p = 0.05 and p = 0.01, respectively, as compared with those in the controls.

Mentions: In our bioinformatics analyses of expression profiles of putative stress-responsive MAPKKKs, we found that a Raf-like MAPK kinase kinase gene, designated as Raf43, was highly induced by abiotic and biotic stresses. To analyze in detail the expression profile of Raf43 under stress conditions, we extracted the expression data of Raf43 (At3g46930) from public microarray databases using GENEVESTIGATOR platfrom. In our analyses, only expression changes of Raf43 in stress/elicitor-treated or pathogen-infected wild type Col-0 plants (except the Pst DC3000 experiment which used wild type Ws-0 plants) >2 folds with p values <0.05 were used to generate the expression profile of Raf43 in response to abiotic stress, pathogen infection and elicitor treatments (Fig 1A). Under abiotic stress conditions such as cold, drought, oxidative, osmotic and salt treatments, the expression levels of Raf43 in Col-0 plants were significantly increased by 2.26~4.23 folds over those in the controls (Fig 1A). In response to infection by different types of pathogens, e.g. biotrophic/hemibiotrophic (e.g. B. graminis and P. syringae) vs. necrotrophic (e.g. B. cinerea, A. brassicicola and S. sclerotiorum) and fungal (e.g. B. graminis, B. cinerea, A. brassicicola and S. sclerotiorum) vs. bacterial (e. g. P. syringae pvs. maculicola, phaseolicola and tomato) pathogens, the expression of Raf43 was significanlty induced in Col-0 and Ws-0 (only for P. syringae pv. tomato DC3000) plants, leading to 2.26~8.91 folds of increase over those in mock-inoculated plants (Fig 1A). In particular, relatively high levels of induction of Raf43 expression was observed after infections by bacterial pathogens P. syringae pv. maculicola and P. syringae pv. tomato DC3000 and necrotrophic fungal pathogen S. sclerotiorum, showing 8.91, 6.95 and 7.64 folds of increase, respectively (Fig 1A). Similarly, several well-known pathogen-associated molecular patterns such as fungal chitin and bacterial EF-Tu (elf18 and elf26) and FLG22, bacterial effectors such as HrpZ, Arabidopsis Pep2 protein and well-known defense signaling hormone SA, which are capable of activating both innate and inducible immune responses in Arabidopsis, induced significantly the expression of Raf43 in Col-0 plants, giving 2.16~8.48 folds of increase over those in the untreated plants (Fig 1A). Notably, the induced expression of Raf43 occurred rapidly in response to elicitor treatment; for example, significant induction of Raf43 expression was detected within one hour after treatment with EF-Tu and FLG22 (Fig 1A). To further confirm the expression profile of Raf43 in response to stress, we analyzed by qRT-PCR the expression patterns of Raf43 in Col-0 plants after treatment with mannitol, drought, MV and SA and infection with B. cinerea. The expression levels of Raf43 in mannitol- and MV-treated plants peaked with approximately 3 folds of increase over those in untreated plants at 3 hr and then decreased, whereas the expression level of Raf43 in drought stress-treated plants markedly increased with 6 folds over that in untreated control plants at 1 hr and continuously increased during a period of 12 hr (Fig 1B). When treated with 1 mM SA, the expression level of Raf43 increased at 3 hr, peaked with 2.35 folds of increase over that in untreated plants at 12 hr and then decreased (Fig 1B). Upon infection by B. cinerea, the expression level of Raf43 continuously increased during a period of 48 hr after inoculation (Fig 1B). These results confirmed the inducible expression feature of Raf43 revealed from analyses of public microarray data. It is worthy to note that the expression levels of Raf43 induced by SA treatment and by infection of B. cinerea in the qRT-PCR experiments were comparable to the levels observed from analyses of the public microarray data (Fig 1A and 1B). Taken together, these data indicate that Raf43 is responsive to multiple abiotic and biotic stresses and this stress-inducbile expression feature imply the involvement of Raf43 in abiotic and biotic stress response.


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)

Responsiveness of Raf43 to biotic and abiotic stresses.(A) Genevestigator-based analysis of expression profile of Raf43 in response to abiotic stress, pathogen infection and elicitor treatment. Expression data were extracted from Genevestigator microarray datasets obtained from experiments with ecotype Col-0 plants except for the P. syringae pv. tomato experiment, which was conducted with Ws-0 plants. The p values for the expression data between the treated/inoculated and control plants are shown above the columns. B. graminis, Blumeria graminis; P. s. maculicola, Pseudomonas syringae pv. maculicola; P. s. phaseolicola, P. syringae pv. phaseolicola; P. s. tomato, P. syringae pv. tomato DC3000; B. cinerea, Botrytis cinerea; A. brasicicola, Alternaria brassicicola; S. sclerotiorum, Sclerotinia sclerotiorum. (B) qRT-PCR-based analysis of expression patterns of Raf43 in response to treatments with mannitol, drought, methyl viologen or salicylic acid and to infection by B. cinerea. The expression level of Ubiquitin was used to normalize the Raf43 expression data and the relative expression was calculated as folds of expression in treated/inoculated plants vs. those in the control plants. Experiments were repeated with three independent biological samples at each time point and data represented are the means ± standard errors from three independent experiments. * and ** indicate the significant difference at p = 0.05 and p = 0.01, respectively, as compared with those in the controls.
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Related In: Results  -  Collection

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pone.0133975.g001: Responsiveness of Raf43 to biotic and abiotic stresses.(A) Genevestigator-based analysis of expression profile of Raf43 in response to abiotic stress, pathogen infection and elicitor treatment. Expression data were extracted from Genevestigator microarray datasets obtained from experiments with ecotype Col-0 plants except for the P. syringae pv. tomato experiment, which was conducted with Ws-0 plants. The p values for the expression data between the treated/inoculated and control plants are shown above the columns. B. graminis, Blumeria graminis; P. s. maculicola, Pseudomonas syringae pv. maculicola; P. s. phaseolicola, P. syringae pv. phaseolicola; P. s. tomato, P. syringae pv. tomato DC3000; B. cinerea, Botrytis cinerea; A. brasicicola, Alternaria brassicicola; S. sclerotiorum, Sclerotinia sclerotiorum. (B) qRT-PCR-based analysis of expression patterns of Raf43 in response to treatments with mannitol, drought, methyl viologen or salicylic acid and to infection by B. cinerea. The expression level of Ubiquitin was used to normalize the Raf43 expression data and the relative expression was calculated as folds of expression in treated/inoculated plants vs. those in the control plants. Experiments were repeated with three independent biological samples at each time point and data represented are the means ± standard errors from three independent experiments. * and ** indicate the significant difference at p = 0.05 and p = 0.01, respectively, as compared with those in the controls.
Mentions: In our bioinformatics analyses of expression profiles of putative stress-responsive MAPKKKs, we found that a Raf-like MAPK kinase kinase gene, designated as Raf43, was highly induced by abiotic and biotic stresses. To analyze in detail the expression profile of Raf43 under stress conditions, we extracted the expression data of Raf43 (At3g46930) from public microarray databases using GENEVESTIGATOR platfrom. In our analyses, only expression changes of Raf43 in stress/elicitor-treated or pathogen-infected wild type Col-0 plants (except the Pst DC3000 experiment which used wild type Ws-0 plants) >2 folds with p values <0.05 were used to generate the expression profile of Raf43 in response to abiotic stress, pathogen infection and elicitor treatments (Fig 1A). Under abiotic stress conditions such as cold, drought, oxidative, osmotic and salt treatments, the expression levels of Raf43 in Col-0 plants were significantly increased by 2.26~4.23 folds over those in the controls (Fig 1A). In response to infection by different types of pathogens, e.g. biotrophic/hemibiotrophic (e.g. B. graminis and P. syringae) vs. necrotrophic (e.g. B. cinerea, A. brassicicola and S. sclerotiorum) and fungal (e.g. B. graminis, B. cinerea, A. brassicicola and S. sclerotiorum) vs. bacterial (e. g. P. syringae pvs. maculicola, phaseolicola and tomato) pathogens, the expression of Raf43 was significanlty induced in Col-0 and Ws-0 (only for P. syringae pv. tomato DC3000) plants, leading to 2.26~8.91 folds of increase over those in mock-inoculated plants (Fig 1A). In particular, relatively high levels of induction of Raf43 expression was observed after infections by bacterial pathogens P. syringae pv. maculicola and P. syringae pv. tomato DC3000 and necrotrophic fungal pathogen S. sclerotiorum, showing 8.91, 6.95 and 7.64 folds of increase, respectively (Fig 1A). Similarly, several well-known pathogen-associated molecular patterns such as fungal chitin and bacterial EF-Tu (elf18 and elf26) and FLG22, bacterial effectors such as HrpZ, Arabidopsis Pep2 protein and well-known defense signaling hormone SA, which are capable of activating both innate and inducible immune responses in Arabidopsis, induced significantly the expression of Raf43 in Col-0 plants, giving 2.16~8.48 folds of increase over those in the untreated plants (Fig 1A). Notably, the induced expression of Raf43 occurred rapidly in response to elicitor treatment; for example, significant induction of Raf43 expression was detected within one hour after treatment with EF-Tu and FLG22 (Fig 1A). To further confirm the expression profile of Raf43 in response to stress, we analyzed by qRT-PCR the expression patterns of Raf43 in Col-0 plants after treatment with mannitol, drought, MV and SA and infection with B. cinerea. The expression levels of Raf43 in mannitol- and MV-treated plants peaked with approximately 3 folds of increase over those in untreated plants at 3 hr and then decreased, whereas the expression level of Raf43 in drought stress-treated plants markedly increased with 6 folds over that in untreated control plants at 1 hr and continuously increased during a period of 12 hr (Fig 1B). When treated with 1 mM SA, the expression level of Raf43 increased at 3 hr, peaked with 2.35 folds of increase over that in untreated plants at 12 hr and then decreased (Fig 1B). Upon infection by B. cinerea, the expression level of Raf43 continuously increased during a period of 48 hr after inoculation (Fig 1B). These results confirmed the inducible expression feature of Raf43 revealed from analyses of public microarray data. It is worthy to note that the expression levels of Raf43 induced by SA treatment and by infection of B. cinerea in the qRT-PCR experiments were comparable to the levels observed from analyses of the public microarray data (Fig 1A and 1B). Taken together, these data indicate that Raf43 is responsive to multiple abiotic and biotic stresses and this stress-inducbile expression feature imply the involvement of Raf43 in abiotic and biotic stress response.

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