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Arabidopsis AtPLC2 Is a Primary Phosphoinositide-Specific Phospholipase C in Phosphoinositide Metabolism and the Endoplasmic Reticulum Stress Response.

Kanehara K, Yu CY, Cho Y, Cheong WF, Torta F, Shui G, Wenk MR, Nakamura Y - PLoS Genet. (2015)

Bottom Line: The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth.Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles.Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan; Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan; Graduate Institute of Biotechnology and Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan; Muroran Institute of Technology, Muroran, Japan.

ABSTRACT
Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response.

No MeSH data available.


Enhanced susceptibility of the plc2-1 mutant to the ER stress.(A) The wild type, plc2-1 (Col) and plc2-1 (Col) ProPLC2:PLC2 (line #33) were grown on MS media for 7 days, and were grown on MS media containing 0.3 μg/ml of tunicamycin (TM) for additional 6 days. The representative seedlings were photographed on 13th day (the lower panel). The seedlings without TM treatment were shown in the upper panel as control. (B) Root length measurement of (A). (C) Fresh weight of a plant shown in (A). In (B) and (C), data are mean±SD of 30 seedlings and three biologically independent experiments were performed with similar results. ****P<0.0001 (Student’s t-test). (D) qRT-PCR analysis of binding protein 3 (BiP3) transcripts in the wild-type and the plc2-1 (Col) plants in response to the ER stress. RNA was extracted from the 7-day-old plants treated with 5 μg/ml TM at each time point (0, 2, 5 hours of the treatment). The expression level of the wild type at the time zero was set to 1. Data at time 0 were magnified at the inset panel. (E) qRT-PCR analysis of calnexin (CNX) transcripts in the wild-type and the plc2-1 (Col) plants in response to the ER stress as described in (D). (F) qRT-PCR analysis of calreticulin (CRT) transcripts in the wild-type and the plc2-1 (Col) plants in response to the ER stress as described in (D). In (D), (E), and (F), Student’s t-test was performed between the wild-type and plc2-1 (Col) plants at each time point. *P<0.05, **P<0.01, ****P<0.0001.
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pgen.1005511.g007: Enhanced susceptibility of the plc2-1 mutant to the ER stress.(A) The wild type, plc2-1 (Col) and plc2-1 (Col) ProPLC2:PLC2 (line #33) were grown on MS media for 7 days, and were grown on MS media containing 0.3 μg/ml of tunicamycin (TM) for additional 6 days. The representative seedlings were photographed on 13th day (the lower panel). The seedlings without TM treatment were shown in the upper panel as control. (B) Root length measurement of (A). (C) Fresh weight of a plant shown in (A). In (B) and (C), data are mean±SD of 30 seedlings and three biologically independent experiments were performed with similar results. ****P<0.0001 (Student’s t-test). (D) qRT-PCR analysis of binding protein 3 (BiP3) transcripts in the wild-type and the plc2-1 (Col) plants in response to the ER stress. RNA was extracted from the 7-day-old plants treated with 5 μg/ml TM at each time point (0, 2, 5 hours of the treatment). The expression level of the wild type at the time zero was set to 1. Data at time 0 were magnified at the inset panel. (E) qRT-PCR analysis of calnexin (CNX) transcripts in the wild-type and the plc2-1 (Col) plants in response to the ER stress as described in (D). (F) qRT-PCR analysis of calreticulin (CRT) transcripts in the wild-type and the plc2-1 (Col) plants in response to the ER stress as described in (D). In (D), (E), and (F), Student’s t-test was performed between the wild-type and plc2-1 (Col) plants at each time point. *P<0.05, **P<0.01, ****P<0.0001.

Mentions: The seedlings of plc2-1 cultured on solid Murashige and Skoog (MS) media showed growth retardation both in shoots and roots (Fig 6A). The overall root length of 15-day-old seedlings was significantly reduced as compared with that of the wild-type plants in Wassilewskija (Ws) ecotype (Fig 6B). The fresh weight and dry weight were also reduced by nearly 70% and 80%, respectively, compared with that of the wild-type plants (Fig 6C and 6D). These growth retardations of the plc2-1 mutant were fully complemented in two independent lines harboring a genomic sequence of AtPLC2 (ProPLC2:PLC2) in the plc2-1 mutant (plc2-1 ProPLC2:PLC2 #1 and #3) in root length, fresh weight and dry weight (Fig 6A–6D). To exclude a possibility that this growth defect is ecotype-specific, the plc2-1 mutant was introgressed into Columbia-0 (Col) ecotype by backcrossing six times. In consistent with that in the Ws background, the plc2-1 (Col) mutant showed growth retardation both in shoots and roots (Fig 6E and 6F). Moreover, this growth defect was also fully complemented by heterologous expression of AtPLC2 in the plc2-1 (Col) [plc2-1 (Col) ProPLC2:PLC2] as shown in Fig 7A–7C. These observations indicate that the growth defect in the plc2-1 mutant is due to knocking out of AtPLC2.


Arabidopsis AtPLC2 Is a Primary Phosphoinositide-Specific Phospholipase C in Phosphoinositide Metabolism and the Endoplasmic Reticulum Stress Response.

Kanehara K, Yu CY, Cho Y, Cheong WF, Torta F, Shui G, Wenk MR, Nakamura Y - PLoS Genet. (2015)

Enhanced susceptibility of the plc2-1 mutant to the ER stress.(A) The wild type, plc2-1 (Col) and plc2-1 (Col) ProPLC2:PLC2 (line #33) were grown on MS media for 7 days, and were grown on MS media containing 0.3 μg/ml of tunicamycin (TM) for additional 6 days. The representative seedlings were photographed on 13th day (the lower panel). The seedlings without TM treatment were shown in the upper panel as control. (B) Root length measurement of (A). (C) Fresh weight of a plant shown in (A). In (B) and (C), data are mean±SD of 30 seedlings and three biologically independent experiments were performed with similar results. ****P<0.0001 (Student’s t-test). (D) qRT-PCR analysis of binding protein 3 (BiP3) transcripts in the wild-type and the plc2-1 (Col) plants in response to the ER stress. RNA was extracted from the 7-day-old plants treated with 5 μg/ml TM at each time point (0, 2, 5 hours of the treatment). The expression level of the wild type at the time zero was set to 1. Data at time 0 were magnified at the inset panel. (E) qRT-PCR analysis of calnexin (CNX) transcripts in the wild-type and the plc2-1 (Col) plants in response to the ER stress as described in (D). (F) qRT-PCR analysis of calreticulin (CRT) transcripts in the wild-type and the plc2-1 (Col) plants in response to the ER stress as described in (D). In (D), (E), and (F), Student’s t-test was performed between the wild-type and plc2-1 (Col) plants at each time point. *P<0.05, **P<0.01, ****P<0.0001.
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pgen.1005511.g007: Enhanced susceptibility of the plc2-1 mutant to the ER stress.(A) The wild type, plc2-1 (Col) and plc2-1 (Col) ProPLC2:PLC2 (line #33) were grown on MS media for 7 days, and were grown on MS media containing 0.3 μg/ml of tunicamycin (TM) for additional 6 days. The representative seedlings were photographed on 13th day (the lower panel). The seedlings without TM treatment were shown in the upper panel as control. (B) Root length measurement of (A). (C) Fresh weight of a plant shown in (A). In (B) and (C), data are mean±SD of 30 seedlings and three biologically independent experiments were performed with similar results. ****P<0.0001 (Student’s t-test). (D) qRT-PCR analysis of binding protein 3 (BiP3) transcripts in the wild-type and the plc2-1 (Col) plants in response to the ER stress. RNA was extracted from the 7-day-old plants treated with 5 μg/ml TM at each time point (0, 2, 5 hours of the treatment). The expression level of the wild type at the time zero was set to 1. Data at time 0 were magnified at the inset panel. (E) qRT-PCR analysis of calnexin (CNX) transcripts in the wild-type and the plc2-1 (Col) plants in response to the ER stress as described in (D). (F) qRT-PCR analysis of calreticulin (CRT) transcripts in the wild-type and the plc2-1 (Col) plants in response to the ER stress as described in (D). In (D), (E), and (F), Student’s t-test was performed between the wild-type and plc2-1 (Col) plants at each time point. *P<0.05, **P<0.01, ****P<0.0001.
Mentions: The seedlings of plc2-1 cultured on solid Murashige and Skoog (MS) media showed growth retardation both in shoots and roots (Fig 6A). The overall root length of 15-day-old seedlings was significantly reduced as compared with that of the wild-type plants in Wassilewskija (Ws) ecotype (Fig 6B). The fresh weight and dry weight were also reduced by nearly 70% and 80%, respectively, compared with that of the wild-type plants (Fig 6C and 6D). These growth retardations of the plc2-1 mutant were fully complemented in two independent lines harboring a genomic sequence of AtPLC2 (ProPLC2:PLC2) in the plc2-1 mutant (plc2-1 ProPLC2:PLC2 #1 and #3) in root length, fresh weight and dry weight (Fig 6A–6D). To exclude a possibility that this growth defect is ecotype-specific, the plc2-1 mutant was introgressed into Columbia-0 (Col) ecotype by backcrossing six times. In consistent with that in the Ws background, the plc2-1 (Col) mutant showed growth retardation both in shoots and roots (Fig 6E and 6F). Moreover, this growth defect was also fully complemented by heterologous expression of AtPLC2 in the plc2-1 (Col) [plc2-1 (Col) ProPLC2:PLC2] as shown in Fig 7A–7C. These observations indicate that the growth defect in the plc2-1 mutant is due to knocking out of AtPLC2.

Bottom Line: The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth.Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles.Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan; Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan; Graduate Institute of Biotechnology and Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan; Muroran Institute of Technology, Muroran, Japan.

ABSTRACT
Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response.

No MeSH data available.