Limits...
Involvement of calmodulin in regulation of primary root elongation by N-3-oxo-hexanoyl homoserine lactone in Arabidopsis thaliana.

Zhao Q, Zhang C, Jia Z, Huang Y, Li H, Song S - Front Plant Sci (2015)

Bottom Line: In this study, we found that the stimulatory effect of N-3-oxo-hexanoyl homoserine lactone (3OC6-HSL) on primary root elongation of Arabidopsis was abolished by the calmodulin (CaM) antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) and trifluoperazine (TFP).Nevertheless, our results suggest that CaM is involved in the bacterial 3OC6-HSL signaling in plant cells.These data offer new insight into the mechanism of plant response to bacterial QS signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Biology Institute, Hebei Academy of Sciences Shijiazhuang, China ; Hebei Engineering and Technology Center of Microbiological Control on Main Crop Disease Shijiazhuang, China.

ABSTRACT
Many bacteria use signal molecules of low molecular weight to monitor their local population density and to coordinate their collective behavior in a process called "quorum sensing" (QS). N-acyl-homoserine lactones (AHLs) are the primary QS signals among Gram-negative bacteria. AHL-mediated QS plays an essential role in diverse bacterial physiological processes. Recent evidence shows that plants are able to sense bacterial AHLs and respond to them appropriately. However, little is known about the mechanism by which plants perceive and transduce the bacterial AHLs within cells. In this study, we found that the stimulatory effect of N-3-oxo-hexanoyl homoserine lactone (3OC6-HSL) on primary root elongation of Arabidopsis was abolished by the calmodulin (CaM) antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) and trifluoperazine (TFP). Western-blot and ELISA analysis revealed that the concentration of CaM protein in Arabidopsis roots increased after treatment with 1 μM 3OC6-HSL. Results from quantitative RT-PCR demonstrated that the transcription of all nine CaM genes in Arabidopsis genome was up-regulated in the plants treated with 3OC6-HSL. The loss-of-function mutants of each AtCaM gene (AtCaM1-9) were insensitive to 3OC6-HSL-stimulation of primary root elongation. On the other hand, the genetic evidence showed that CaM may not participates the inhibition of primary root length caused by application of long-chained AHLs such as C10-HSL and C12-HSL. Nevertheless, our results suggest that CaM is involved in the bacterial 3OC6-HSL signaling in plant cells. These data offer new insight into the mechanism of plant response to bacterial QS signals.

No MeSH data available.


Related in: MedlinePlus

The stimulating effect of 3OC6-HSL on primary root elongation is diminished in loss-of-function mutants of individual AtCaM genes. (A) Images of Arabidopsis wild-type plants and CaM functional-deficiency mutants cam1, cam2, cam3, cam4, cam5, cam6, cam7, cam8, and cam9 grown side by side on vertically oriented plates containing with or without 1 μM 3OC6-HSL after 7 days of cultivation. (B) Plot of effect of 3OC6-HSL on primary root length in wild-type seedlings and cam1-cam9 mutant seedlings. (C) Plot of effect of 3OC6-HSL on fresh weight in wild-type seedlings and cam1-cam9 mutant seedlings. Each experiment included at least 30 seedlings for each genotype and treatment. Data shown are the average of four independent experiments. Entries with p-values < 0.05 shown with asterisk. (D) Identification of CaM  mutants by RT-PCR. Total RNA was extracted from the leaves of one-month-old wild type (Columbia, Col-0) and CaM mutants (cam1-cam9) seedlings and RT-PCR were done to amplify CaM1, CaM2, CaM3, CaM4, CaM5, CaM6, CaM7, CaM8, and CaM9 genes both from wild type RNA and from corresponding CaM mutant RNA. Actin was used as the endogenous reference gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4292405&req=5

Figure 6: The stimulating effect of 3OC6-HSL on primary root elongation is diminished in loss-of-function mutants of individual AtCaM genes. (A) Images of Arabidopsis wild-type plants and CaM functional-deficiency mutants cam1, cam2, cam3, cam4, cam5, cam6, cam7, cam8, and cam9 grown side by side on vertically oriented plates containing with or without 1 μM 3OC6-HSL after 7 days of cultivation. (B) Plot of effect of 3OC6-HSL on primary root length in wild-type seedlings and cam1-cam9 mutant seedlings. (C) Plot of effect of 3OC6-HSL on fresh weight in wild-type seedlings and cam1-cam9 mutant seedlings. Each experiment included at least 30 seedlings for each genotype and treatment. Data shown are the average of four independent experiments. Entries with p-values < 0.05 shown with asterisk. (D) Identification of CaM mutants by RT-PCR. Total RNA was extracted from the leaves of one-month-old wild type (Columbia, Col-0) and CaM mutants (cam1-cam9) seedlings and RT-PCR were done to amplify CaM1, CaM2, CaM3, CaM4, CaM5, CaM6, CaM7, CaM8, and CaM9 genes both from wild type RNA and from corresponding CaM mutant RNA. Actin was used as the endogenous reference gene.

Mentions: The available pharmacological and molecular evidence showed that CaM might be involved in primary root elongation regulated by bacterial AHL, but direct genetic evidence for the participation of CaM is still missing. To solve this question and pursue the specificity of different AtCaM isoform in participation in AHL signaling in plant cells, we compared the primary root elongation in response to 3OC6-HSL between wild-type and the T-DNA insertional mutant lines of individual AtCaM gene. All mutants for all nine AtCaM genes are confirmed to be homozigous lines by antibiotic resistance screening and RT-PCR (Figure 6D) and exhibit no observed phenotypic differences compared with wild-type plants under normal growth conditions (Figure 6A). As shown in Figure 6, the observed increased root elongation upon 3OC6-HSL exposure in wild-type plant was impaired in all mutation lines of nine AtCaM genes. The length of primary roots in cam1, cam2, cam4, cam5, cam6, cam7, and cam9 after treatment with 3OC6-HSL was similar to that of untreated plants of respective mutants (Figures 6A,B). The primary root in cam3 and cam8 after contact to 3OC6-HSL exhibited even slightly shorter than that of untreated respective mutants, however no significant difference was found between treatment and untreatment with 3OC6-HSL after statistical analysis (Figure 6B). Additionaly, we measured the effects of 3OC6-HSL on the fresh weight of seedlings of wild-type and the individual AtCaM mutants. An obvious increase in the fresh weight of wild type seedlings was observed after treatment with 3OC6-HSL, whereas the fresh weight of seedlings of all nine AtCaM mutants was similar between treated and untreated plants (Figure 6C). Although the conclusion for role of AtCaM in AHL signaling at genetic level needs to be reinforced by complementary and over-expression analysis, our data provide preliminary evidence that all nine AtCaM genes might be involved in plant response to 3OC6-HSL with respect to primary root elongation.


Involvement of calmodulin in regulation of primary root elongation by N-3-oxo-hexanoyl homoserine lactone in Arabidopsis thaliana.

Zhao Q, Zhang C, Jia Z, Huang Y, Li H, Song S - Front Plant Sci (2015)

The stimulating effect of 3OC6-HSL on primary root elongation is diminished in loss-of-function mutants of individual AtCaM genes. (A) Images of Arabidopsis wild-type plants and CaM functional-deficiency mutants cam1, cam2, cam3, cam4, cam5, cam6, cam7, cam8, and cam9 grown side by side on vertically oriented plates containing with or without 1 μM 3OC6-HSL after 7 days of cultivation. (B) Plot of effect of 3OC6-HSL on primary root length in wild-type seedlings and cam1-cam9 mutant seedlings. (C) Plot of effect of 3OC6-HSL on fresh weight in wild-type seedlings and cam1-cam9 mutant seedlings. Each experiment included at least 30 seedlings for each genotype and treatment. Data shown are the average of four independent experiments. Entries with p-values < 0.05 shown with asterisk. (D) Identification of CaM  mutants by RT-PCR. Total RNA was extracted from the leaves of one-month-old wild type (Columbia, Col-0) and CaM mutants (cam1-cam9) seedlings and RT-PCR were done to amplify CaM1, CaM2, CaM3, CaM4, CaM5, CaM6, CaM7, CaM8, and CaM9 genes both from wild type RNA and from corresponding CaM mutant RNA. Actin was used as the endogenous reference gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4292405&req=5

Figure 6: The stimulating effect of 3OC6-HSL on primary root elongation is diminished in loss-of-function mutants of individual AtCaM genes. (A) Images of Arabidopsis wild-type plants and CaM functional-deficiency mutants cam1, cam2, cam3, cam4, cam5, cam6, cam7, cam8, and cam9 grown side by side on vertically oriented plates containing with or without 1 μM 3OC6-HSL after 7 days of cultivation. (B) Plot of effect of 3OC6-HSL on primary root length in wild-type seedlings and cam1-cam9 mutant seedlings. (C) Plot of effect of 3OC6-HSL on fresh weight in wild-type seedlings and cam1-cam9 mutant seedlings. Each experiment included at least 30 seedlings for each genotype and treatment. Data shown are the average of four independent experiments. Entries with p-values < 0.05 shown with asterisk. (D) Identification of CaM mutants by RT-PCR. Total RNA was extracted from the leaves of one-month-old wild type (Columbia, Col-0) and CaM mutants (cam1-cam9) seedlings and RT-PCR were done to amplify CaM1, CaM2, CaM3, CaM4, CaM5, CaM6, CaM7, CaM8, and CaM9 genes both from wild type RNA and from corresponding CaM mutant RNA. Actin was used as the endogenous reference gene.
Mentions: The available pharmacological and molecular evidence showed that CaM might be involved in primary root elongation regulated by bacterial AHL, but direct genetic evidence for the participation of CaM is still missing. To solve this question and pursue the specificity of different AtCaM isoform in participation in AHL signaling in plant cells, we compared the primary root elongation in response to 3OC6-HSL between wild-type and the T-DNA insertional mutant lines of individual AtCaM gene. All mutants for all nine AtCaM genes are confirmed to be homozigous lines by antibiotic resistance screening and RT-PCR (Figure 6D) and exhibit no observed phenotypic differences compared with wild-type plants under normal growth conditions (Figure 6A). As shown in Figure 6, the observed increased root elongation upon 3OC6-HSL exposure in wild-type plant was impaired in all mutation lines of nine AtCaM genes. The length of primary roots in cam1, cam2, cam4, cam5, cam6, cam7, and cam9 after treatment with 3OC6-HSL was similar to that of untreated plants of respective mutants (Figures 6A,B). The primary root in cam3 and cam8 after contact to 3OC6-HSL exhibited even slightly shorter than that of untreated respective mutants, however no significant difference was found between treatment and untreatment with 3OC6-HSL after statistical analysis (Figure 6B). Additionaly, we measured the effects of 3OC6-HSL on the fresh weight of seedlings of wild-type and the individual AtCaM mutants. An obvious increase in the fresh weight of wild type seedlings was observed after treatment with 3OC6-HSL, whereas the fresh weight of seedlings of all nine AtCaM mutants was similar between treated and untreated plants (Figure 6C). Although the conclusion for role of AtCaM in AHL signaling at genetic level needs to be reinforced by complementary and over-expression analysis, our data provide preliminary evidence that all nine AtCaM genes might be involved in plant response to 3OC6-HSL with respect to primary root elongation.

Bottom Line: In this study, we found that the stimulatory effect of N-3-oxo-hexanoyl homoserine lactone (3OC6-HSL) on primary root elongation of Arabidopsis was abolished by the calmodulin (CaM) antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) and trifluoperazine (TFP).Nevertheless, our results suggest that CaM is involved in the bacterial 3OC6-HSL signaling in plant cells.These data offer new insight into the mechanism of plant response to bacterial QS signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Biology Institute, Hebei Academy of Sciences Shijiazhuang, China ; Hebei Engineering and Technology Center of Microbiological Control on Main Crop Disease Shijiazhuang, China.

ABSTRACT
Many bacteria use signal molecules of low molecular weight to monitor their local population density and to coordinate their collective behavior in a process called "quorum sensing" (QS). N-acyl-homoserine lactones (AHLs) are the primary QS signals among Gram-negative bacteria. AHL-mediated QS plays an essential role in diverse bacterial physiological processes. Recent evidence shows that plants are able to sense bacterial AHLs and respond to them appropriately. However, little is known about the mechanism by which plants perceive and transduce the bacterial AHLs within cells. In this study, we found that the stimulatory effect of N-3-oxo-hexanoyl homoserine lactone (3OC6-HSL) on primary root elongation of Arabidopsis was abolished by the calmodulin (CaM) antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) and trifluoperazine (TFP). Western-blot and ELISA analysis revealed that the concentration of CaM protein in Arabidopsis roots increased after treatment with 1 μM 3OC6-HSL. Results from quantitative RT-PCR demonstrated that the transcription of all nine CaM genes in Arabidopsis genome was up-regulated in the plants treated with 3OC6-HSL. The loss-of-function mutants of each AtCaM gene (AtCaM1-9) were insensitive to 3OC6-HSL-stimulation of primary root elongation. On the other hand, the genetic evidence showed that CaM may not participates the inhibition of primary root length caused by application of long-chained AHLs such as C10-HSL and C12-HSL. Nevertheless, our results suggest that CaM is involved in the bacterial 3OC6-HSL signaling in plant cells. These data offer new insight into the mechanism of plant response to bacterial QS signals.

No MeSH data available.


Related in: MedlinePlus