A Cotton Annexin Protein AnxGb6 Regulates Fiber Elongation through Its Interaction with Actin 1

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

Induced <i>GbSBT1</i> expression following <i>V</i>. <i>dahliae</i> V991 infection.

<p>(A) Expression analysis of the <i>GbSBT1</i> gene in vegetative tissues (roots, stems, and leaves) via qPCR. The <i>Ubiquitin</i> gene is used as an internal control. At least three biological replicates were performed. (B) Expression analysis of <i>GbSBT1</i> in roots following <i>V</i>. <i>dahliae</i> strain V991 infection. The comparative CT method is adopted, and the expression is normalized. Each sample was repeated at least thrice. Error bars represent SE. Double asterisks represent a significant expression change of <i>GbSBT1</i> compared to uninfected condition (P < 0.01) in t-test.</p

Multiple sequence alignment analysis of GbSBT1 and other plant subtilases.

<p>GbSBT1 (KT336228, <i>Gossypium barbadense</i>), subtilase-like (XP_007017870, <i>Theobroma cacao</i>), SBT5.2 (NP_564107, <i>Arabidopsis thaliana</i>), SBT-like (AAM65424, <i>A</i>. <i>thaliana</i>). SP: signal peptide; Inhibitor I9: serine protease inhibitor domain; black triangles indicate conserved active sites; blank triangles indicate catalytic triads of serine protease; and blank box A indicates PA/protease or protease-like domain interface.</p

Comparison of F-actin organization in fiber cells between Pima-90, Coker312 and T586 Plants.

<p>A-C: Fiber cells at +3 DPA. A: Pima-90, B: Coker312, and C: T586. Note the length of fiber is different in the three varieties at the same stages. D: Fiber cells at +6 DPA and E: Fiber cells at +9 DPA. Bars: 10 µm in D and E.</p

Interaction between GbSBT1 and prohibitin secreted by <i>Verticillium dahliae</i>.

<p>(A) The yeast cells co-expressing GbSBT1 and benzodiazepine receptor family protein or peptidyl-tRNA hydrolase could not grow on the selective medium SD/-T-L-H, whereas the yeast cells co-transformed with GbSBT1 and prohibitin could grow on the selective medium SD/-T-L-H; (B-C) BiFC of epidermal cells co-expressing split YFP fusions of <i>GbSBT1</i> and prohibitin 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 <i>GbSBT1</i> and prohibitin as follows: B: <i>GbSBT1</i>-Yc and prohibitin -Yn, Scale bar: 25 μm; C: vector-Yc and vector-Yn, Scale bar: 10 μm. The interaction and co-localization were observed at the plasma membrane. Right is the corresponding bright-field</p

AnxGb6 interacts with GbAct1.

<p>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 (<i>Yn</i> and <i>Yc</i>, respectively) were infiltrated as vector controls or fused to the N terminus of AnxGb6 and GbAct1 as follows: B: AnxGb6-<i>Yc</i> and GbAct1-<i>Yn</i>, Scale bar: 50 µm; C: vector-<i>Yc</i> and vector-<i>Yn</i>, Scale bar: 100 µm. The interaction and co-localization were observed at the plasma membrane. Right is the corresponding bright-field.</p

Fiber length and <i>AnxGb6</i> gene expression pattern in Pima-90, Coker312 and T586 cotton seeds.

<p>A: Fiber length of Pima-90, Coker312 and T586 cotton seeds at +3, +6, +9 and +12 DPA. Ovules were sectioned and the length of 400 fiber cells was measured under a microscope for each type. Data was processed with Microsoft Excel. Error bars represent standard errors. B: Real-time quantitative PCR analysis of the <i>AnxGb6</i> gene and its alleles in Pima-90, Coker312 and T586. Expression analysis of <i>AnxGb6</i> gene and its alleles in Pima-90 (<i>G. barbadense</i> L.), Coker312 (<i>G. hirsutum</i> L.) and T586 (<i>G. hirsutum</i> L.). 3DPA: ovules in +3 DPA, 6DPA: ovules in +6 DPA, 9DPA: ovules in +9 DPA, 12DPA: ovules in +12DPA. The comparative C_T method was adopted and the expression was normalized to the levels of Pima-90, Coker312 and T586. Error bars represent standard errors.</p

GbST1-interacting proteins secreted from <i>V</i>. <i>dahliae</i> V991.

<p>GbST1-interacting proteins secreted from <i>V</i>. <i>dahliae</i> V991.</p

Extracellular localization of the GbSBT1 protein in tobacco leaves determined through confocal microscopy.

<p>(A) Localization of GbSBT1-YFP in tobacco leaves; localization of GbSBT1 (without signal peptide)-YFP in tobacco leaves; localization of GbSBT1-YFP in tobacco leaves under JA treatment; localization of GbSBT1-YFP in tobacco leaves under ethylene treatment. Arrows indicate the difference in protein localization between normal and phytohormone treatments; (B) GbSBT1 protein co-localized with plasma membrane integral protein PIP1 on the cell membrane using protoplast and tobacco lead epidermal cells as the protein expressing materials. Upper panels: protoplast; lower panels: tobacco leaf epidermal cells.</p

Enhanced disease resistance of <i>Arabidopsis</i> against <i>Fusarium oxysporum</i> after <i>GbSBT1</i> overexpression.

<p>Wild-type (WT): <i>Arabidopsis</i> Columbia type Col-0; OEX: transgenic <i>GbSBT1</i> lines. Left panels: un-inoculated plants; right panels: plants 2 days after <i>F</i>. <i>oxysporum</i> inoculation. (A–B) WT plants 0 and 2 days after ddH₂O inoculation; (C–D) WT plants 0 and 2 days after <i>F</i>. <i>oxysporum</i> inoculation; (E–F) transgenic <i>GbSBT1</i> plants 0 and 2 days after inoculation with ddH₂O containing 0.2% Tween-20; (G–H) transgenic <i>GbSBT1</i> plants 0 and 2 days after <i>F</i>. <i>oxysporum</i> inoculation. At least three biological replicates were performed in the <i>F</i>. <i>oxysporum</i> resistance analysis.</p