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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

Structures of the Raf43 gene/protein and characterization of the raf43-1 mutant and the Raf43-overexpressing lines.(A) Diagram showing the structure of Raf43 and the T-DNA location in raf43-1 mutant. Black boxes, black lines and red line indicate the exons, introns and putative promoter region, respectively. Sizes (bp) of the exons and introns are indicated above and below the diagram, respectively. The location and orientation of the T-DNA inserted in raf43-1 mutant line are shown. The start (ATG) and stop (TGA) codons are also indicated. (B) Diagram showing the structure of the Raf43 protein. The red box indicates the conserved protein kinase (PK) catalytic domain in Raf43 protein. Signature motif and its position in Raf43 protein and consensus of the signature motif commonly present in Raf-like MAPKKKs are shown. The amino acid positions are also indicated above the diagram. (C) Levels of the Raf43 transcripts in raf43-1 mutant and Raf43-overxpressing lines Raf43-OE#1 and Raf43-OE#13 plants. The transcript levels of Raf43 in four-week-old plants grown under normal conditions were analyzed by qRT-PCR using Raf43-specific primers and data were normalized by the transcript level of Ubiquitin. The transcript levels of Raf43 in wild type (Ws-0 and Col-0) plants were set as 1 and the levels in raf43 mutant and Raf43-overxpressing are shown as folds of the levels in wild type plants. Data represented 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. (D) Morphological and growth phenotypes of four-week-old plants of wild type (Ws-0 and Col-0), raf43-1 mutant and Raf43-overexpressing lines OE#1 and OE#13.
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pone.0133975.g002: Structures of the Raf43 gene/protein and characterization of the raf43-1 mutant and the Raf43-overexpressing lines.(A) Diagram showing the structure of Raf43 and the T-DNA location in raf43-1 mutant. Black boxes, black lines and red line indicate the exons, introns and putative promoter region, respectively. Sizes (bp) of the exons and introns are indicated above and below the diagram, respectively. The location and orientation of the T-DNA inserted in raf43-1 mutant line are shown. The start (ATG) and stop (TGA) codons are also indicated. (B) Diagram showing the structure of the Raf43 protein. The red box indicates the conserved protein kinase (PK) catalytic domain in Raf43 protein. Signature motif and its position in Raf43 protein and consensus of the signature motif commonly present in Raf-like MAPKKKs are shown. The amino acid positions are also indicated above the diagram. (C) Levels of the Raf43 transcripts in raf43-1 mutant and Raf43-overxpressing lines Raf43-OE#1 and Raf43-OE#13 plants. The transcript levels of Raf43 in four-week-old plants grown under normal conditions were analyzed by qRT-PCR using Raf43-specific primers and data were normalized by the transcript level of Ubiquitin. The transcript levels of Raf43 in wild type (Ws-0 and Col-0) plants were set as 1 and the levels in raf43 mutant and Raf43-overxpressing are shown as folds of the levels in wild type plants. Data represented 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. (D) Morphological and growth phenotypes of four-week-old plants of wild type (Ws-0 and Col-0), raf43-1 mutant and Raf43-overexpressing lines OE#1 and OE#13.

Mentions: Raf43 is encoded by At3g46930 and the predicted ORF of Raf43 contains 4 exons and 3 introns (Fig 2A). Our cloning and sequencing confirmed the sequence of the predicted ORF of Raf43. The Raf43 gene encodes a 475 aa protein, which contains a typical protein kinase catalytic domain (170–416 aa) and a signature motif (322–330 aa) commonly present in Raf-like MAPKKKs [10] (Fig 2B). The protein structure feature indicates that Raf43 is a Raf-like MAPKKK and belongs to Group C of plants MAPKKKs [8]. To assess the biological function of Raf43, one T-DNA insertion line FLAG_505A06 in ecotype Ws-0 background [48] was analyzed. The T-DNA was inversely inserted in the last exon of the Raf43 gene (Fig 2A). PCR-based genotyping using a pair of gene-specific primers and a T-DNA primer identified individual plants that were homozygous at the T-DNA insertion site in the FLAG_505A06 line and this homozygous T-DNA insertion line was designated as raf43-1. qRT-PCR analysis revealed that the transcript level of Raf43 in raf43-1 plants was significantly reduced as compared with its wild type Ws-0 plants (Fig 2C), indicating that raf43-1 is a mutant for Raf43. Meanwhile, we cloned the Raf43 ORF and inserted into pCAMBIA-991 vector under the control of CaMV 35S promoter to make a Raf43-overexpressing (OE) construct. The Raf43-OE construct was introduced into wild type Col-0 plants through floral dip transformation procedure and Raf43-overexpressing transgenic lines were obtained. Homozygous T3 lines with a single copy of the transgene were screened based on a 3:1 Hgr-resistant/Hgr-sensitive segregation ratio on selective medium and two independent lines, named Raf43-overexpressing OE#1 and Raf43-overexpressing OE#13 (thereafter referred as to Raf43-OE#1 and Raf43-OE#13), were chosen for further studies. qRT-PCR analysis showed that the transcript levels of Raf43 in Raf43-OE#1 and Raf43-OE#13 plants were significantly increased, leading to 3–4 folds of increase as compared with wild type Col-0 (Fig 2C). During our experiments, no any defect in growth and development was observed for the raf43-1 mutant plants and for the Raf43-OE#1 and Raf43-OE#13 plants when compared with their corresponding wild type Ws-0 and Col-0 plants, respectively (Fig 2D).


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)

Structures of the Raf43 gene/protein and characterization of the raf43-1 mutant and the Raf43-overexpressing lines.(A) Diagram showing the structure of Raf43 and the T-DNA location in raf43-1 mutant. Black boxes, black lines and red line indicate the exons, introns and putative promoter region, respectively. Sizes (bp) of the exons and introns are indicated above and below the diagram, respectively. The location and orientation of the T-DNA inserted in raf43-1 mutant line are shown. The start (ATG) and stop (TGA) codons are also indicated. (B) Diagram showing the structure of the Raf43 protein. The red box indicates the conserved protein kinase (PK) catalytic domain in Raf43 protein. Signature motif and its position in Raf43 protein and consensus of the signature motif commonly present in Raf-like MAPKKKs are shown. The amino acid positions are also indicated above the diagram. (C) Levels of the Raf43 transcripts in raf43-1 mutant and Raf43-overxpressing lines Raf43-OE#1 and Raf43-OE#13 plants. The transcript levels of Raf43 in four-week-old plants grown under normal conditions were analyzed by qRT-PCR using Raf43-specific primers and data were normalized by the transcript level of Ubiquitin. The transcript levels of Raf43 in wild type (Ws-0 and Col-0) plants were set as 1 and the levels in raf43 mutant and Raf43-overxpressing are shown as folds of the levels in wild type plants. Data represented 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. (D) Morphological and growth phenotypes of four-week-old plants of wild type (Ws-0 and Col-0), raf43-1 mutant and Raf43-overexpressing lines OE#1 and OE#13.
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Related In: Results  -  Collection

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Show All Figures
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pone.0133975.g002: Structures of the Raf43 gene/protein and characterization of the raf43-1 mutant and the Raf43-overexpressing lines.(A) Diagram showing the structure of Raf43 and the T-DNA location in raf43-1 mutant. Black boxes, black lines and red line indicate the exons, introns and putative promoter region, respectively. Sizes (bp) of the exons and introns are indicated above and below the diagram, respectively. The location and orientation of the T-DNA inserted in raf43-1 mutant line are shown. The start (ATG) and stop (TGA) codons are also indicated. (B) Diagram showing the structure of the Raf43 protein. The red box indicates the conserved protein kinase (PK) catalytic domain in Raf43 protein. Signature motif and its position in Raf43 protein and consensus of the signature motif commonly present in Raf-like MAPKKKs are shown. The amino acid positions are also indicated above the diagram. (C) Levels of the Raf43 transcripts in raf43-1 mutant and Raf43-overxpressing lines Raf43-OE#1 and Raf43-OE#13 plants. The transcript levels of Raf43 in four-week-old plants grown under normal conditions were analyzed by qRT-PCR using Raf43-specific primers and data were normalized by the transcript level of Ubiquitin. The transcript levels of Raf43 in wild type (Ws-0 and Col-0) plants were set as 1 and the levels in raf43 mutant and Raf43-overxpressing are shown as folds of the levels in wild type plants. Data represented 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. (D) Morphological and growth phenotypes of four-week-old plants of wild type (Ws-0 and Col-0), raf43-1 mutant and Raf43-overexpressing lines OE#1 and OE#13.
Mentions: Raf43 is encoded by At3g46930 and the predicted ORF of Raf43 contains 4 exons and 3 introns (Fig 2A). Our cloning and sequencing confirmed the sequence of the predicted ORF of Raf43. The Raf43 gene encodes a 475 aa protein, which contains a typical protein kinase catalytic domain (170–416 aa) and a signature motif (322–330 aa) commonly present in Raf-like MAPKKKs [10] (Fig 2B). The protein structure feature indicates that Raf43 is a Raf-like MAPKKK and belongs to Group C of plants MAPKKKs [8]. To assess the biological function of Raf43, one T-DNA insertion line FLAG_505A06 in ecotype Ws-0 background [48] was analyzed. The T-DNA was inversely inserted in the last exon of the Raf43 gene (Fig 2A). PCR-based genotyping using a pair of gene-specific primers and a T-DNA primer identified individual plants that were homozygous at the T-DNA insertion site in the FLAG_505A06 line and this homozygous T-DNA insertion line was designated as raf43-1. qRT-PCR analysis revealed that the transcript level of Raf43 in raf43-1 plants was significantly reduced as compared with its wild type Ws-0 plants (Fig 2C), indicating that raf43-1 is a mutant for Raf43. Meanwhile, we cloned the Raf43 ORF and inserted into pCAMBIA-991 vector under the control of CaMV 35S promoter to make a Raf43-overexpressing (OE) construct. The Raf43-OE construct was introduced into wild type Col-0 plants through floral dip transformation procedure and Raf43-overexpressing transgenic lines were obtained. Homozygous T3 lines with a single copy of the transgene were screened based on a 3:1 Hgr-resistant/Hgr-sensitive segregation ratio on selective medium and two independent lines, named Raf43-overexpressing OE#1 and Raf43-overexpressing OE#13 (thereafter referred as to Raf43-OE#1 and Raf43-OE#13), were chosen for further studies. qRT-PCR analysis showed that the transcript levels of Raf43 in Raf43-OE#1 and Raf43-OE#13 plants were significantly increased, leading to 3–4 folds of increase as compared with wild type Col-0 (Fig 2C). During our experiments, no any defect in growth and development was observed for the raf43-1 mutant plants and for the Raf43-OE#1 and Raf43-OE#13 plants when compared with their corresponding wild type Ws-0 and Col-0 plants, respectively (Fig 2D).

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