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A cotton annexin protein AnxGb6 regulates fiber elongation through its interaction with actin 1.

Huang Y, Wang J, Zhang L, Zuo K - PLoS ONE (2013)

Bottom Line: Here we cloned six Annexin genes (AnxGb) abundantly expressed in fiber from sea-island cotton (G. barbadense). qRT-PCR results indicated that all six G. barbadense annexin genes were expressed in elongating cotton fibers, while only the expression of AnxGb6 was cotton fiber-specific.Analysis of AnxGb6 expression in three cotton genotypes with different fiber length confirmed that AnxGb6 expression was correlated to cotton fiber length, especially fiber elongation rate.Our results demonstrated that AnxGb6 was important for fiber elongation by potentially providing a domain for F-actin organization.

View Article: PubMed Central - PubMed

Affiliation: Plant Biotechnology Research Center, SJTU-Cornell Institute of Sustainable Agriculture and Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Annexins are assumed to be involved in regulating cotton fiber elongation, but direct evidence remains to be presented. Here we cloned six Annexin genes (AnxGb) abundantly expressed in fiber from sea-island cotton (G. barbadense). qRT-PCR results indicated that all six G. barbadense annexin genes were expressed in elongating cotton fibers, while only the expression of AnxGb6 was cotton fiber-specific. Yeast two hybridization and BiFC analysis revealed that AnxGb6 homodimer interacted with a cotton fiber specific actin GbAct1. Ectopic-expressed AnxGb6 in Arabidopsis enhanced its root elongation without increasing the root cell number. Ectopic AnxGb6 expression resulted in more F-actin accumulation in the basal part of the root cell elongation zone. Analysis of AnxGb6 expression in three cotton genotypes with different fiber length confirmed that AnxGb6 expression was correlated to cotton fiber length, especially fiber elongation rate. Our results demonstrated that AnxGb6 was important for fiber elongation by potentially providing a domain for F-actin organization.

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AnxGb6 interacts with GbAct1.A: AnxGb6 binds to GbAct1. Yeast harboring BD-AnxGb6/AD-GbAct1 or BD-GbAct1/AD-AnxGb6 grown on selective plates as indicated. Control medium: (SD/-T-L-H-A) selective medium. The control is yeast transformed with BD-AnxGb6/ADT7 and BD-GbAct1/ADT7. Dilution multiple from left to right is 1 fold, 10 fold, 100 fold. B–C: BiFC of epidermal cells co-expressing split YFP fusions of AnxGb6 and GbAct1 or empty vector controls. Combinations of N- and C-terminal YFP fragments (Yn and Yc, respectively) were infiltrated as vector controls or fused to the N terminus of AnxGb6 and GbAct1 as follows: B: AnxGb6-Yc and GbAct1-Yn, Scale bar: 50 µm; C: vector-Yc and vector-Yn, Scale bar: 100 µm. The interaction and co-localization were observed at the plasma membrane. Right is the corresponding bright-field.
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pone-0066160-g006: AnxGb6 interacts with GbAct1.A: AnxGb6 binds to GbAct1. Yeast harboring BD-AnxGb6/AD-GbAct1 or BD-GbAct1/AD-AnxGb6 grown on selective plates as indicated. Control medium: (SD/-T-L-H-A) selective medium. The control is yeast transformed with BD-AnxGb6/ADT7 and BD-GbAct1/ADT7. Dilution multiple from left to right is 1 fold, 10 fold, 100 fold. B–C: BiFC of epidermal cells co-expressing split YFP fusions of AnxGb6 and GbAct1 or empty vector controls. Combinations of N- and C-terminal YFP fragments (Yn and Yc, respectively) were infiltrated as vector controls or fused to the N terminus of AnxGb6 and GbAct1 as follows: B: AnxGb6-Yc and GbAct1-Yn, Scale bar: 50 µm; C: vector-Yc and vector-Yn, Scale bar: 100 µm. The interaction and co-localization were observed at the plasma membrane. Right is the corresponding bright-field.

Mentions: Recent studies have shown that animal annexins can interact with several protein kinases and F-actin to regulate membrane trafficking and actin reorganization [58], [59]. In plants, protein pull-down analysis showed that a rice annexin protein Os05g31750 probably interacted with Ste20-like kinase Os10g37480, SPK-3 kinase Os01g64970 and casein kinase Os01g28950 [60]. F-actin affinity and chromatography experiments provided evidence that tomato and Mimosa annexins had F-actin binding activity in vitro[61]–[64]. However, systemic investigation of the involvement of annexin proteins in fiber development has not been demonstrated. Since annexins are important regulators of membrane trafficking in animal and are known to influence polar growth in Arabidopsis[59], [65], we predicted that annexins AnxGb6 protein probably had a similar function during fiber elongation in cotton. Therefore, we used AnxGb6 protein as bait to test its interaction with potential protein candidates including 23 CIPKs, 27 CDPKs, wall-associated kinase protein 1 and GbAct1 [10]. Yeast two hybridization results showed that AnxGb6 did not interact with CIPKs, CDPKs, and wall-associated kinase proteins (data not shown). Yeast cells co-transformed with BD-AnxGb6 and AD-GbAct1 could grow on the selective medium SD-Leu-Ade-Trp-His with 2 mM 3-AT, while the control group (AD and BD-AnxGb6) and (AD and BD-GbAct1) could not grow (Figure 6A). These results indicated that AnxGb6 could directly bind to GbAct1 instead of wall-associated kinase1, CIPK and CDPK family proteins in cotton.


A cotton annexin protein AnxGb6 regulates fiber elongation through its interaction with actin 1.

Huang Y, Wang J, Zhang L, Zuo K - PLoS ONE (2013)

AnxGb6 interacts with GbAct1.A: AnxGb6 binds to GbAct1. Yeast harboring BD-AnxGb6/AD-GbAct1 or BD-GbAct1/AD-AnxGb6 grown on selective plates as indicated. Control medium: (SD/-T-L-H-A) selective medium. The control is yeast transformed with BD-AnxGb6/ADT7 and BD-GbAct1/ADT7. Dilution multiple from left to right is 1 fold, 10 fold, 100 fold. B–C: BiFC of epidermal cells co-expressing split YFP fusions of AnxGb6 and GbAct1 or empty vector controls. Combinations of N- and C-terminal YFP fragments (Yn and Yc, respectively) were infiltrated as vector controls or fused to the N terminus of AnxGb6 and GbAct1 as follows: B: AnxGb6-Yc and GbAct1-Yn, Scale bar: 50 µm; C: vector-Yc and vector-Yn, Scale bar: 100 µm. The interaction and co-localization were observed at the plasma membrane. Right is the corresponding bright-field.
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Related In: Results  -  Collection

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pone-0066160-g006: AnxGb6 interacts with GbAct1.A: AnxGb6 binds to GbAct1. Yeast harboring BD-AnxGb6/AD-GbAct1 or BD-GbAct1/AD-AnxGb6 grown on selective plates as indicated. Control medium: (SD/-T-L-H-A) selective medium. The control is yeast transformed with BD-AnxGb6/ADT7 and BD-GbAct1/ADT7. Dilution multiple from left to right is 1 fold, 10 fold, 100 fold. B–C: BiFC of epidermal cells co-expressing split YFP fusions of AnxGb6 and GbAct1 or empty vector controls. Combinations of N- and C-terminal YFP fragments (Yn and Yc, respectively) were infiltrated as vector controls or fused to the N terminus of AnxGb6 and GbAct1 as follows: B: AnxGb6-Yc and GbAct1-Yn, Scale bar: 50 µm; C: vector-Yc and vector-Yn, Scale bar: 100 µm. The interaction and co-localization were observed at the plasma membrane. Right is the corresponding bright-field.
Mentions: Recent studies have shown that animal annexins can interact with several protein kinases and F-actin to regulate membrane trafficking and actin reorganization [58], [59]. In plants, protein pull-down analysis showed that a rice annexin protein Os05g31750 probably interacted with Ste20-like kinase Os10g37480, SPK-3 kinase Os01g64970 and casein kinase Os01g28950 [60]. F-actin affinity and chromatography experiments provided evidence that tomato and Mimosa annexins had F-actin binding activity in vitro[61]–[64]. However, systemic investigation of the involvement of annexin proteins in fiber development has not been demonstrated. Since annexins are important regulators of membrane trafficking in animal and are known to influence polar growth in Arabidopsis[59], [65], we predicted that annexins AnxGb6 protein probably had a similar function during fiber elongation in cotton. Therefore, we used AnxGb6 protein as bait to test its interaction with potential protein candidates including 23 CIPKs, 27 CDPKs, wall-associated kinase protein 1 and GbAct1 [10]. Yeast two hybridization results showed that AnxGb6 did not interact with CIPKs, CDPKs, and wall-associated kinase proteins (data not shown). Yeast cells co-transformed with BD-AnxGb6 and AD-GbAct1 could grow on the selective medium SD-Leu-Ade-Trp-His with 2 mM 3-AT, while the control group (AD and BD-AnxGb6) and (AD and BD-GbAct1) could not grow (Figure 6A). These results indicated that AnxGb6 could directly bind to GbAct1 instead of wall-associated kinase1, CIPK and CDPK family proteins in cotton.

Bottom Line: Here we cloned six Annexin genes (AnxGb) abundantly expressed in fiber from sea-island cotton (G. barbadense). qRT-PCR results indicated that all six G. barbadense annexin genes were expressed in elongating cotton fibers, while only the expression of AnxGb6 was cotton fiber-specific.Analysis of AnxGb6 expression in three cotton genotypes with different fiber length confirmed that AnxGb6 expression was correlated to cotton fiber length, especially fiber elongation rate.Our results demonstrated that AnxGb6 was important for fiber elongation by potentially providing a domain for F-actin organization.

View Article: PubMed Central - PubMed

Affiliation: Plant Biotechnology Research Center, SJTU-Cornell Institute of Sustainable Agriculture and Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Annexins are assumed to be involved in regulating cotton fiber elongation, but direct evidence remains to be presented. Here we cloned six Annexin genes (AnxGb) abundantly expressed in fiber from sea-island cotton (G. barbadense). qRT-PCR results indicated that all six G. barbadense annexin genes were expressed in elongating cotton fibers, while only the expression of AnxGb6 was cotton fiber-specific. Yeast two hybridization and BiFC analysis revealed that AnxGb6 homodimer interacted with a cotton fiber specific actin GbAct1. Ectopic-expressed AnxGb6 in Arabidopsis enhanced its root elongation without increasing the root cell number. Ectopic AnxGb6 expression resulted in more F-actin accumulation in the basal part of the root cell elongation zone. Analysis of AnxGb6 expression in three cotton genotypes with different fiber length confirmed that AnxGb6 expression was correlated to cotton fiber length, especially fiber elongation rate. Our results demonstrated that AnxGb6 was important for fiber elongation by potentially providing a domain for F-actin organization.

Show MeSH
Related in: MedlinePlus