Fig 1.
F-actin organization in type II trichomes at different developmental stages.
(A) The morphology of four types of glandular trichomes (GT). Bar: 50 μm. (B) The morphology of three types of non-glandular trichomes (NGT). Bar: 50 μm. (C, D) Quantification of cell number in type II and type V non-glandular trichomes. The quantification is based on over 200 trichomes for each type. (E) Schematic diagram of the type II trichome with 6 cells. (F-K) F-actin architecture in type II trichomes at different developmental stages. The right image of each trichome shows the cross section of the lined position in the left image. Bar: 20 μm. (L) The frequency of cortical actin filaments with different orientations. The longitudinal, transverse and oblique alignment represent 0–15°, 75–90°and 15–75°related to the axis of trichome growth respectively.
Fig 2.
Visualization of actin organization in type II trichomes using Lifeact-eGFP fusion protein.
(A) A panoramic micrograph of actin organization in the type II trichome cell file. The boxed areas are zoomed in H-L. Bar: 50 μm. (B-G) Zoomed view of actin arrangement in each cell of the type II trichome. The cell order is marked in the individual micrograph. The angle diagram showed in the up right corner of each image represents the average angle of actin filament in each cell. Bar: 10 μm. (H) Zoomed view of the position in the corresponding box in (A). There are transverse actin filament meshes at the end of cell. Bar: 10 μm. (I, J) The cross section of the positions boxed in (H) respectively. The actin filaments are organized in the cortical region. Bar: 10 μm. (K, L) The cross section of the position in the corresponding box in (A). Bar: 10 μm. (M) Projected confocal micrograph of the actin configuration in the top cell of type II trichome. Bar: 10 μm. (N) Zoomed view of the actin arrangement in the dome region of the type II trichome cell file. A double arrowhead dotted line presents the actin filaments loop across the whole dome cap in the top cell. Bar: 10 μm. (O) The cross section of the position in the corresponding box of (M). Bar: 5 μm. (P) Quantification of cortical actin filament orientation in type II trichome cells. The angles are relative to the direction perpendicular to the axis of the growth. (Q) Average actin filament orientation of type II trichome cells measured by FibrilTool.
Fig 3.
Dynamics of actin filament at different developmental stages.
(A-F) The organization of actin filaments during trichome initiation. Bar: 15μm. (B-D) are zoomed view of boxes in (A). Bar: 10μm. (E) The longitudinal section of A. The orange lines show the actin filaments in the cytoplasm. Bar: 10μm. (F) The schematic diagram shows the trichome initiation process. The green lines show the actin filaments; the gray oval shows the nucleus. (G-J) The actin dynamics at the one-cell stage. The red lines show the transverse actin filaments shifting down. The yellow lines show the oblique actin filaments in moving. The arrow indicated the movement direction of the actin filaments. (H and I) are zoomed view of boxed regions in the G. (J) shows the schematic diagram of the one-cell stage trichome and the dynamics of the actin filament. Bar: 5μm. (K-Q) The actin filaments at the four-cell stage. The red dotted lines show the depolymerization of transverse actin filament. The yellow lines show the oblique or helix actin filaments. The green line indicated the actin bundles. (L-P) were zoomed view of boxed region in K. (P) is the schematic diagram of four-cell stage trichome. Bar: 10μm. (Q) The depolymerization rate of the transverse actin alignments (red bar) and the polymerization rate of the longitudinal and oblique actin filaments (yellow bar) in the second and third cell of the trichomes at the four-cell stage.
Fig 4.
Visualization of EB1a-eGFP fusion protein in type II trichomes.
(A)Overall view of the signal of EB1a-eGFP at the one-cell stage. Bar: 5 μm. (B C) Zoomed view of EB1a-eGFP in box regions in A. The zoomed image on the upper right in B shows the corresponding cross-section. The red curved line in B indicated the apical zone of the cell. The red arrows in C show the possible direction of the moving EB1a-GFP. Bar: 2.5 μm. (D) The schematic diagram shows EB1a-eGFP at one-cell stage. The red dots indicate the signal of EB1a-GFP. (E) Overall view of the signal of EB1a-eGFP at the two-cell stage. The white arrow points to the cell division line of the two cells. Bar: 5 μm. (F G) The magnified image of the organization of EB1a-eGFP in the boxed region of E. The zoomed image on the upper right in the (F) shows the corresponding cross-section. The red curved line in the (F) indicated the apical zone of the cell. The red arrows show the possible direction of the moving EB1a-GFP. Bar: 2.5 μm. (H) The schematic diagram shows EB1a-eGFP at two-cell stage. The red dots indicate the signal of EB1a-GFP. (I J) The EB1a-eGFP dynamics of the top cell at the two-cell stage. (J) is zoomed view of boxed region in K. The color asterisks and arrows show the traceable EB1a-eGFP. Among them, the purple arrows show the GFP signal moving to the tip of the cell. Bar:5μm. (K L) The EB1a-eGFP dynamics of the basal cell at the three-cell stage. (L) is zoomed view of boxed region in K. The color asterisks show the traceable EB1a-eGFP. Bar: 5μm. (M) Overall view of the signal of EB1a-eGFP in type II trichomes. Bar: 50 μm. (N-R) zoomed view of the signal of EB1a-eGFP in each cell of type II trichomes. The position of the cell was marked in the bottom left corner of each image. B-G, bar: 10 μm. (S) Immuno-staining image of microtubules in type II trichomes in WT using anti-tubulin. (T) Average direction of EB1a-GFP movement in type II trichomes measured by Image J. The angles are relative to the direction perpendicular to the axis of the growth. (U) Average fibril orientation of EB1a-GFP movement in type II trichomes measured by FibrilTool. (V) Quantitative analyses of the anisotropy of the direction of EB1a-GFP movement by FibrilTool. Anisotropy values range from 0 to 1. 0 indicates pure isotropy, and 1 represents pure anisotropy. (W) The schematic diagram of EB1a-eGFP at the different cell of the type II trichome. the purple dotted line shows the orientation of microtubule.
Fig 5.
Phenotype of distorted trichome (dt) mutants.
(A, E, I, M, Q, U) WT; (B, F, J, N, R, V) dt-1 mutant; (C, G, K, O, S, W) dt-2 mutant; (D, H, L, P, T, X) dt-3 mutant. Bar: 1mm. (A-D) Phenotypes of WT and dt mutants. Bar: 1cm. (E-L) Stereoscopic microscopes of trichomes on the stem (E-H) and the edge of the leaf (I-L). Bar: 1 mm; I-L, Bar: 250 μm. (M-X) Trichome phenotype of WT and dt mutants under SEM. Type II, Type III and Type V trichomes are marked. The anisotropic expanding cells are marked by asterisks. The dotted lines outline bending cells. Bar: 100 μm.
Fig 6.
Quantitative comparison of trichome phenotypes between WT and dt mutants.
(A, B) Schematic illustration of type II in WT (A) and dt mutants (B). (C, D) Schematic illustration of type V trichomes (C) in dt mutants (D). (E) Schematic illustration of the cell length and width measured in this analysis. (F, H) Cell length and width of type II trichomes of WT and dt mutants. (**P<0.01) (G, I) Cell length and width of type V trichomes of WT and dt mutants. (**P<0.01).
Fig 7.
Mapping and verification of the mutation in dt mutants.
(A-C) Mapping of dt-1 (A), dt-2 (B) and dt-3 (C) by BSA-seq analysis. Triangles show the causal mutations with high frequency (Ideally 100% having the variant allele). (D-F) The location of causal mutations in SlSCAR2 of dt-1 mutant (D), SlARPC4 of dt-2 mutant (E), and SlARPC1B of dt-3 mutant (F). (G-N) Complementary verification of SlSCAR2. Over-expression of SlSCAR2 in dt-2 mutant rescued the distorted trichome phenotype (G-J), and knockout of SlSCAR2 by CRISPR-cas9 in WT resulted in distorted trichomes (K-N).
Fig 8.
Visualization of actin organization in type II trichomes in dt-1 mutant.
(A) Overall view of actin organization in type II trichomes in dt-1 mutant. Most of actin filaments orientated randomly. Bar: 20 μm. (B) Overall view of actin organization in type III trichomes in dt-1 mutant. The alignment of actin filaments derived from the swollen cell. The yellow arrow indicates the major swelling direction; white arrow shows the main orientation of actin filaments. White Triangles point to dense actin bundles. Bar: 20 μm. (C-E) Actin configuration in swollen trichome cells. (E) shows the cross section of the boxed region in (D). Bar: 20 μm. (F, G) Actin organization in the curved cell of the box of (A). Bar: 20 μm. (G) is the amplification of the boxed region in (F). White Triangles point to dense actin bundles. Bar: 10 μm. (H, I) Actin organization in the curved cell locating on the top of type II trichomes. Bar: 20 μm. (I) is the amplification of the boxed region in (H). Triangles point to dense actin bundles. Bar: 10 μm. (J) Quantitative analyses of the anisotropy of actin filaments by FibrilTool. Anisotropy values range from 0 to 1. 0 indicates pure isotropy, and 1 represents pure anisotropy. (*P<0.1 and**P<0.01).
Fig 9.
Effect of pharmacological disruption of microtubules and actin filaments on trichomes.
(A) The seedling phenotype after four-day treatment with Oryzalin (Ory) and Latrunculin B (Lat B). (B) Disruption of microtubules and actin filaments dramatically changed the trichome morphology but did not inhibit trichome initiation (Statistic analysis of the trichomes density of over 100 hundred epidermal cells for each treatment). (C-E) Trichome morphology and Lifeact-eGFP in WT trichomes. (F-I) Treatment with Ory resulted in isotropically expanded cells (G, H) and horizontal actin bundles (I). (J-M) Treatment with Lat B caused the entire depolymerization of actin filaments (M) and decreased cell elongation (K, L). (D, G, J) are SEM micrographs and (E H K) are confocal images. A, Bar: 1 cm. C and I, Bar: 500 μm. F, Bar: 250 μm. D, G and J, Bar: 500 μm. E, H and K, Bar: 50 μm. H and L, Bar: 100 μm.
Fig 10.
Schematic illustration of the role of actin filaments (AFs) and micro tubules (MTs) in multicellular trichome development.
(A) The morphogenesis of the multicellular trichomes. The first step is the outgrowth of the cell. At this stage, the nucleus enters into the bulging site. The second step is the cell division and cell expansion. The grey oval shows the nucleus. (B) The organization of Liftact decorated AFs and EB1 decorated MTs during trichome cell morphogenesis. The green lines show the organization of Liftact decorated AFs. The red dots show the signal of EB1a. In the apical cell, the orientation of actin filaments is longitudinal which may promote the polar growth of the cell. In the middle and basal cells, actin filaments are spirally organized. The green angles mean that the alignment of AFs gradually changes from transverse and spiral to longitudinal in cells from the basal to the top. (C D) The organization of actin filaments in WT and dt-2 mutant. The mutation of SCAR2 leads to misshaped cells. (C) In the top bent cells, actin cables align longitudinally in WT while gathered on adaxial side of the cell (pointed by black triangle) in scar. (D) In the swollen cell in scar-2, aggregated actin bundles were often observed in the cortical region of the cell (black triangle), and thick actin cables in the cytoplasm was more frequent than WT.