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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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AnxGb5 and 6 interact with each other and self-associate.A: AnxGb6 binds AnxGb5 or itself. Yeast harbouring BD-AnxGb6/AD-AnxGb5, BD-AnxGb5/AD-AnxGb6, BD-AnxGb6/AD-AnxGb6 and BD-AnxGb5/AD-AnxGb5 grown on selective plates as indicated. Control medium: (SD/-T-L-H-A) selective medium. The control yeast transformed with BD-AnxGb6/ADT7 and BD-AnxGb5/ADT7. Dilution multiple from left to right is 1 fold, 10 fold, 100 fold. B-E: BiFC of epidermal cells co-expressing split YFP fusions of AnxGb6 and AnxGb5 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 AnxGb5 as follows: B: AnxGb6-Yc and AnxGb5-Yn, Scale bar: 50 µm; C: AnxGb6-Yc and AnxGb6-Yn, Scale bar: 25 µm; D: AnxGb5-Yc and AnxGb5-Yn, Scale bar: 25 µm; E: vector-Yc and vector-Yn, Scale bar: 50 µm. The interaction and co-localization was observed in the plasma membrane. Right is the corresponding bright-field.
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pone-0066160-g007: AnxGb5 and 6 interact with each other and self-associate.A: AnxGb6 binds AnxGb5 or itself. Yeast harbouring BD-AnxGb6/AD-AnxGb5, BD-AnxGb5/AD-AnxGb6, BD-AnxGb6/AD-AnxGb6 and BD-AnxGb5/AD-AnxGb5 grown on selective plates as indicated. Control medium: (SD/-T-L-H-A) selective medium. The control yeast transformed with BD-AnxGb6/ADT7 and BD-AnxGb5/ADT7. Dilution multiple from left to right is 1 fold, 10 fold, 100 fold. B-E: BiFC of epidermal cells co-expressing split YFP fusions of AnxGb6 and AnxGb5 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 AnxGb5 as follows: B: AnxGb6-Yc and AnxGb5-Yn, Scale bar: 50 µm; C: AnxGb6-Yc and AnxGb6-Yn, Scale bar: 25 µm; D: AnxGb5-Yc and AnxGb5-Yn, Scale bar: 25 µm; E: vector-Yc and vector-Yn, Scale bar: 50 µm. The interaction and co-localization was observed in the plasma membrane. Right is the corresponding bright-field.

Mentions: In animals, annexin-annexin protein interactions are able to provide a stable cytoskeleton with bisphosphate protein, resulting in the formation of a protein scaffold with subsequent F-actin recruitment [67]. Thus we hypothesized that AnxGb5 and 6 could interact with GbAct1, and also interact with themselves to generate a protein complex scaffold. In order to validate this speculation, yeast two hybridization and BiFC were performed. As shown in Figure 7, both AnxGb5 and AnxGb6 could interact with themselves to form a homodimer complex (AnxGb5-AnxGb5, AnxGb6-AnxGb6) (Figure 7A, C, D). AnxGb5 was also found to be capable of binding with AnxGb6 to form a heterodimer complex (Figure 7A, B). Unlike AnxGb6, AnxGb5 could not directly bind with GbAct1 protein in vitro (data not shown). In conclusion, the annexin proteins in subgroup III could assemble a membrane targeting protein complex, which provided a domain for AnxGb6 homodimer to directly bind with GbAct1 protein for actin polymerization.


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)

AnxGb5 and 6 interact with each other and self-associate.A: AnxGb6 binds AnxGb5 or itself. Yeast harbouring BD-AnxGb6/AD-AnxGb5, BD-AnxGb5/AD-AnxGb6, BD-AnxGb6/AD-AnxGb6 and BD-AnxGb5/AD-AnxGb5 grown on selective plates as indicated. Control medium: (SD/-T-L-H-A) selective medium. The control yeast transformed with BD-AnxGb6/ADT7 and BD-AnxGb5/ADT7. Dilution multiple from left to right is 1 fold, 10 fold, 100 fold. B-E: BiFC of epidermal cells co-expressing split YFP fusions of AnxGb6 and AnxGb5 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 AnxGb5 as follows: B: AnxGb6-Yc and AnxGb5-Yn, Scale bar: 50 µm; C: AnxGb6-Yc and AnxGb6-Yn, Scale bar: 25 µm; D: AnxGb5-Yc and AnxGb5-Yn, Scale bar: 25 µm; E: vector-Yc and vector-Yn, Scale bar: 50 µm. The interaction and co-localization was observed in the plasma membrane. Right is the corresponding bright-field.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672135&req=5

pone-0066160-g007: AnxGb5 and 6 interact with each other and self-associate.A: AnxGb6 binds AnxGb5 or itself. Yeast harbouring BD-AnxGb6/AD-AnxGb5, BD-AnxGb5/AD-AnxGb6, BD-AnxGb6/AD-AnxGb6 and BD-AnxGb5/AD-AnxGb5 grown on selective plates as indicated. Control medium: (SD/-T-L-H-A) selective medium. The control yeast transformed with BD-AnxGb6/ADT7 and BD-AnxGb5/ADT7. Dilution multiple from left to right is 1 fold, 10 fold, 100 fold. B-E: BiFC of epidermal cells co-expressing split YFP fusions of AnxGb6 and AnxGb5 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 AnxGb5 as follows: B: AnxGb6-Yc and AnxGb5-Yn, Scale bar: 50 µm; C: AnxGb6-Yc and AnxGb6-Yn, Scale bar: 25 µm; D: AnxGb5-Yc and AnxGb5-Yn, Scale bar: 25 µm; E: vector-Yc and vector-Yn, Scale bar: 50 µm. The interaction and co-localization was observed in the plasma membrane. Right is the corresponding bright-field.
Mentions: In animals, annexin-annexin protein interactions are able to provide a stable cytoskeleton with bisphosphate protein, resulting in the formation of a protein scaffold with subsequent F-actin recruitment [67]. Thus we hypothesized that AnxGb5 and 6 could interact with GbAct1, and also interact with themselves to generate a protein complex scaffold. In order to validate this speculation, yeast two hybridization and BiFC were performed. As shown in Figure 7, both AnxGb5 and AnxGb6 could interact with themselves to form a homodimer complex (AnxGb5-AnxGb5, AnxGb6-AnxGb6) (Figure 7A, C, D). AnxGb5 was also found to be capable of binding with AnxGb6 to form a heterodimer complex (Figure 7A, B). Unlike AnxGb6, AnxGb5 could not directly bind with GbAct1 protein in vitro (data not shown). In conclusion, the annexin proteins in subgroup III could assemble a membrane targeting protein complex, which provided a domain for AnxGb6 homodimer to directly bind with GbAct1 protein for actin polymerization.

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