Activation of Arabidopsis seed hair development by cotton fiber-related genes

Abstract

Each cotton fiber is a single-celled seed trichome or hair, and over 20,000 fibers may develop semi-synchronously on each seed. The molecular basis for seed hair development is unknown but is likely to share many similarities with leaf trichome development in Arabidopsis. Leaf trichome initiation in Arabidopsis thaliana is activated by GLABROUS1 (GL1) that is negatively regulated by TRIPTYCHON (TRY). Using laser capture microdissection and microarray analysis, we found that many putative MYB transcription factor and structural protein genes were differentially expressed in fiber and non-fiber tissues. Gossypium hirsutum MYB2 (GhMYB2), a putative GL1 homolog, and its downstream gene, GhRDL1, were highly expressed during fiber cell initiation. GhRDL1, a fiber-related gene with unknown function, was predominately localized around cell walls in stems, sepals, seed coats, and pollen grains. GFP:GhRDL1 and GhMYB2:YFP were co-localized in the nuclei of ectopic trichomes in siliques. Overexpressing GhRDL1 or GhMYB2 in A. thaliana Columbia-0 (Col-0) activated fiber-like hair production in 4-6% of seeds and had on obvious effects on trichome development in leaves or siliques. Co-overexpressing GhRDL1 and GhMYB2 in A. thaliana Col-0 plants increased hair formation in ∼8% of seeds. Overexpressing both GhRDL1 and GhMYB2 in A. thaliana Col-0 try mutant plants produced seed hair in ∼10% of seeds as well as dense trichomes inside and outside siliques, suggesting synergistic effects of GhRDL1 and GhMYB2 with try on development of trichomes inside and outside of siliques and seed hair in A. thaliana. These data suggest that a different combination of factors is required for the full development of trichomes (hairs) in leaves, siliques, and seeds. A. thaliana can be developed as a model a system for discovering additional genes that control seed hair development in general and cotton fiber in particular.

Figure 1. Comparative and K-means analyses of differentially expressed genes detected by microarrays.

A heat map of K-means cluster analysis of differentially expressed genes in protodermal cells (−2 DPA), fiber cell initials (0 and +2 DPA), and fibers (+7 DPA). Changes of gene expression in six Gene Ontology (GO) categories are shown with the expression patterns of three representative genes shown below each cluster graph. Cluster 1: kinase activity; 2: hydrolase activity; 3: transferase activity; 4: transcription factor; 5: DNA/RNA binding activity; and 6: structural molecule. Expression of representative genes was validated by qRT-PC. ID in parenthesis is updated from ICG10.

Figure 2. GhRDL1 expression patterns in cotton and GFP:GhRDL1 and GhMYB2:YFP transgene expression in A. thaliana Col and try mutant transgenic plants.

(A) Quantitative RT PCR (qRT-PCR) analysis of GhRDL1 expression in ovules, fibers, and non-fiber tissues of TM-1 and ovules (0 DPA) of N1N1. (B–C) RNA in situ hybridization in cotton ovules (0 DPA) using sense (B) and anti-sense (C) probes showing accumulation of GhRDL1 transcripts in fiber cell initials (C). (D) RT-PCR analysis of transgene expression in 35S:GFP:GhRDL1 transgenics, 35S:GhMYB2:YFP transgenics, and 35S:GFP:GhRDL1 and 35S:GhMYB2:YFP double transgenics. Amplification of Tublin (beta 6) gene (TUB) was used as an RNA loading control. (E) RT-PCR analysis of the transgene expression as in (D), except that the transgenic plants were in the try mutant background.

Figure 3. Localization of 35S:GFP:GhRDL1 in A. thaliana transgenic plants.

(A–D) Localization of 35S:GFP:GhRDL1 in stem (A), sepal cell walls (B), stamen (C) and pollen grains (D). (E) 35S:GFP alone in the seed coat at 9 days after pollination (DAP). (F–H) Localization of 35S:GFP:GhRDL1 in cell walls of seed coat in A. thaliana: 35S:GFP:GhRDL1 at 9 DAP (F), 11 DAP (G), and longitudinal section of seed coat at 13 DAP (H). (I) Merged image of 35S:GFP:GhRDL1 and propidium iodide (PI, red) at 13 DAP. (J–L) Enlarged views of a section in (I) stained with GFP:GhRDL1 (J), PI (K), and merged (L). (M–P) Unstained (M), GFP:GhRDL1 (N), PI (O), and merged images (P) of 35:GFP:GhRDL in columella cells of seed coat. (Q–T) Enlarged views of the selected areas (boxed in I) showing unstained (Q), GFP:GhRDL1 (R), PI (S), and merged (T) of a fiber-like cell initial (arrowheads). Scale bars represent 50 mm.

Figure 4. Seed hair production in the transgenic plants overexpressing GhRDL1 alone or GhRDL1 and GhMYB2 together.

(A–D) Initiation of seed hair in the try transgenic plants (seeds) expressing 35S:GFP:GhRDL1 alone. Scanning electron microscope (SEM) images of developing seeds at 13 days after pollination (DAP) (A), 15 DAP (B), 17 DAP (C), and 21 DAP (D). Hair-like initial was observed as early as 11 DAP. The boxed areas were zoomed in (E–H). Scale bar represents 100 µm. (E–H) Enlarged SEM images of corresponding boxed regions in (A–D). Scale bar represents 10 µm. (I–J) A single seed hair in a mature seed observed using light microscope (I) and SEM (J). (K) Multiple seed hairs (two shown) produced in a seed of the transgenic plants overexpressing both GhRDL1 and GhMYB2. (L–M) Seed hair without staining (L) and PI staining (M) in GhRDL1 transgenic plants. PI stained cell walls of seed coat and seed hair. Scale bars in (I–M) represent 50 µm. Arrows in (E–M) indicate seed hair or fiber.

Figure 5. Ectopic trichome production in the transgenic plants overexpressing GhRDL1, GhMYB2, or GhRDL1 and GhMYB2 together.

(A) Flowers of A. thaliana Col (WT, left) and transgenic plants overexpressing GhRDL1, GhMYB2, and both (right). (B) Flowers of A. thaliana try mutant (left) and transgenic plants overexpressing GhRDL1, GhMYB2, and both (right). Arrows indicate ectopic trichomes. (C) Ectopic trchomes outside siliques produced in three try transgenic plants (T2-1 to T2-3) overexpressing both GhRDL1 and GhMYB2. (D) No ectopic silique trichome was observed in the try mutant. (E–F) Ectopic trichomes produced outside (E) and inside (F) siliques in the try transgenic plants ovexpressing GhRDL1 and GhMYB2. Scale bars (D–F) represent 300 µm.

Figure 6. Trichome production inside siliques and cotton bolls.

(A) An SEM image showing clustered silique trichomes in try transgenic plants overexpressing GhRDL1 and GhMYB2. (B) An SEM image of branched and unbranched trichomes inside siliques of the transgenic plants. Scale bars in (A and B) represent 100 µm. (C) qRT-PCR analysis of a putative cotton cpc gene (DW512016) in cotton tissues (n = 3). (D) Unbranched trichomes (white) produced inside cotton bolls. (E) A model of leaf trichome development. (F) A model of ectopic trichome production in Arabidopsis silique and seed coat. (G) A model of seed hair production in cotton. See text for detailed explanations.

Figure 7. Subcellular localization of GFP:GhRDL1 and GhMYB2:YFP.

(A–B) Localization of GFP:GhRDL1 (A) and YFP:GhMYB2 (B) in silique trichomes. (C–F) Silique trichomes in the try transgenic plants overexpressing GhRDL1 and GhMYB2 stained with GFP (C), YFP (D), PI (E, red), and merged (F). (G–I) Diffusion of GFP in leaf trichomes (G), epidermal cells (H), and siliques (I) in 35S:GFP transgenic plants. GFP donuts in (I) indicate guard cells of stomata.