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doi: 10.1242/10.1242/dev.00549

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Arabidopsis CROOKED encodes for the smallest subunit of the ARP2/3 complex and controls cell shape by region specific fine F-actin formation

Botanical Institute III, University of Köln, Gyrhofstrasse 15, Köln, D-50931, Germany

View larger version (167K): [in a new window]   Fig. 1. A comparison of different cell types in Arabidopsis (ecotype Landsberg erecta) wild type and the crooked mutant. (A) Two or three branched, stellate wild-type trichomes. (B) Randomly shaped, distorted trichomes in crooked with unextended branches (arrow) located in aberrant positions. (C) Typically elongated cells occupy the mid-zone of wild-type hypocotyl in 10-day-old seedlings. (D) The hypocotyl mid-zone of 10-day-old crooked seedlings shows radially expanded cells and a lack of the file arrangement observed in wild type. (E) A hypocotyl of 7-day-old wild-type seedling displays clearly differentiated stomatal complexes (arrows). (F) A hypocotyl of a 7-day-old crooked seedling does not possess stomatal complexes in the characteristic regions. (G) The typical epidermal surface of expanding wild-type cotyledons patterned into a jigsaw puzzle shape through cell-lobing. (H) Cotyledon-cells in crooked are less expanded compared with the wild type and are impaired in lobe formation. (I) Hypocotyl of wild-type seedling challenged to elongate rapidly by growing in dark for 7 days show a regular arrangement of cell files with each cell firmly in contact with its neighbors (arrows at cell junction). (J) The hypocotyl of a crooked seedling challenged into rapid elongation became scruffy as cells broke contact with their neighbors along the long axis and curled outwards (arrows) to leave gaps in the epidermis. (K) Wild-type root hairs are straight, tubular and can elongate up to 1 mm. (L) Root hairs in crooked are 1.5- to 2-fold thicker than wild-type hairs, are sinuous (arrowheads) and can display varying diameters along their length. (M) A single trichoblast in crooked showing aberrant development of two root-hair initials (arrowheads). Numbers given below scale bars are in µm.

View larger version (79K): [in a new window]   Fig. 2. GFP-mTalin aided visualization of F-actin organization in trichome and cotyledon-epidermal cells of wild-type Arabidopsis and crooked mutant (n, nucleus; numbers given below scale bars are in µm). (A) A wild-type trichome that has initiated branches and is ready to embark on a rapid expansion phase. Trichomes in crooked exhibit a similar F-actin organization at this stage, with major actin accumulations as shown (arrowheads). (B) Wild-type trichomes where branches have started extending maintain dense F-actin at the tips and junctions of branches (arrowheads), while elongated F-actin cables connecting regions of dense actin start to become prominent. (C) A crooked trichome at a stage comparable with that of wild type in B displays an increase in dense F-actin bundles throughout the cell, resulting in more intracellular areas being covered by dense actin (arrowheads). Note that the cell starts thickening around its mid region while the uppermost branch maintains a rounded tip, suggesting less extension of the flanks. (D) A slightly more advanced stage of development in a crooked trichome shows massive actin bundling and the organization of a characteristic wide-polygonal mesh. Note that in A-D, while dense actin can be easily visualized, areas with very fine and difficult to resolve F-actin meshwork are only visible as a diffuse green fluorescence. (E) A region of a mature wild-type trichome displaying predominantly longitudinally oriented (arrows) F-actin bundles. (F) A mature crooked trichome with predominantly transversely arranged, cross-connected, thick actin bundles appears subdivided into numerous polygonal compartments (arrowhead). Note that the crooked trichome has not been able to expand well and one of the branches `b' is visible as a short unextended spike. (G) Expanding wild-type cotyledon epidermis displaying the typical puzzle-shaped cells with prominent lobes. A fine, diffuse cortical F-actin and thick F-actin cables (e.g. arrowheads) connecting strategic points within each cell are seen. (H) A comparative view of the cotyledon epidermis in crooked shows thickly bundled F-actin (arrowheads) and impaired lobe formation. (I) A higher magnification of wild-type cotyledon epidermal cells shows the occurrence of dense actin specifically at lobe sinuses (e.g. arrowheads), while other areas of the cell possess relatively finer F-actin organization. (J) A cotyledon epidermal cell in crooked with no lobes displays a general distribution of bundled actin. The arrowheads indicate thicker bundles.

View larger version (110K): [in a new window]   Fig. 3. F-actin organization in expanding hypocotyl and root hair cells in wild-type Arabidopsis and crooked mutant. (Numbers given below scale bars are in µm.) (A) Wild-type hypocotyl cells displaying fine cortical F-actin mesh and longitudinally running subcortical F-actin cables connecting the cell ends. (B) Hypocotyl cells in crooked with thick, transversely linked F-actin bundles. (C) Dark grown, elongated wild-type hypocotyl cells with fine F-actin meshwork. (D) Aberrant actin bundling in crooked hypocotyl cells challenged to elongate rapidly. (E) Dark-grown crooked hypocotyl cells, a cell with one end displaying actin bundles (arrowhead). (F) Actin bundles at a crooked cell end mislocalize growth and create less elongating cells with bulged ends. Note that the two contiguous cells now break contact, leaving a gap in the hypocotyl epidermis (double-headed arrow). (G) A single hypocotyl epidermis cell in crooked curls up. Note that the site of dense actin (arrowhead) coincides with a less growing region whereas the outer more expanded curve has a thinner layer of actin. (H) A wild-type root hair with long actin bundles that stop just before the apex. (I,J) Sinuous root hairs from crooked with longitudinally extended actin bundles. Note that the regions where the actin bundle contacts and obscures the plasma membrane do not expand (arrowheads), whereas regions free from the bundle expand more. (K) A swollen root hair from crooked is filled with actin-bundles extending all the way to the tip (arrowhead). (L) A branched crooked root hair creates another region for tip growth. (M) Magnified view of the tip region in L shows dense actin at the tip (arrowhead), suggesting that inhibition of the growth of the original tip region (caused by dense actin) resulted in a new tip being formed.

View larger version (56K): [in a new window]   Fig. 4. Golgi-bodies in wild-type Arabidopsis and crooked cells. (A) Single wild-type and crooked `a' trichomes of the same age, showing the differences in distribution and density of Golgi bodies. (B) Regional variation in distribution of Golgi bodies in a crooked trichome. The tip region has not expanded well but is densely packed with Golgi bodies (arrowheads). (C) Magnified view of a region exhibiting accumulation of Golgi bodies (arrowhead). (D) Co-visualization of F-actin bundles using YFP-mTalin fusion protein (green) and Golgi-bodies using ERD2-GFP label (red) in a crooked trichome shows that multiple Golgi bodies (such as those seen in C) are trapped in regions of dense actin (white arrowheads), whereas more loosely distributed, free Golgi bodies occur in expanded areas (green arrowheads).

View larger version (83K): [in a new window]   Fig. 5. Molecular details of the Arabidopsis CROOKED gene. (A) The exon-intron structure for At4g01710 (CROOKED) as determined by comparing the genomic DNA with RT-PCR-amplified cDNA fragments and RLM-RACE PCR, respectively. (B) The crooked mutant sequence contains a single point mutation (g>a) at the splice donor site, which leads to premature splicing (arrow) and a 35 bp deletion in exon 2 (nucleotides underlined). (C) RT-PCR using wild-type and crooked cDNA shows that the premature splicing produces a smaller transcript of 360 bp (large arrow) and a weak band (small arrow) corresponding to the 399 bp wild-type transcript. (D) The altered splice site predicts a frame shift resulting in a stop codon 129 bp downstream of the start site and a truncated amino acid sequence of only 44 residues (the last 12 shown). (E) Tissue-specific RT-PCR shows CROOKED to be ubiquitously expressed (R, root; S, inflorescence stem; L, leaves; F, flowers; Si, young siliques). The lower lane shows the elongation factor 1 transcript used as a loading control. (F) The predicted amino acid sequence for CROOKED aligned against ARPC5 subunit sequences from other organisms (Ath, A. thaliana; Dic, D. discoideum (31%); Cel, C. elegans (32%); Dan, D. rerio (29%); Hum, H. sapiens (32%); Spo, S. pombe (25%); Sce, S. cerevisiae (23%). Percentages (%) indicate amino acid identity (black); gray areas show similar amino acids.

View larger version (94K): [in a new window]   Fig. 6. A simple actin configuration-based model derived through observations of wild-type Arabidopsis cells depicts how the strategic placement of dense F-actin (red arrowheads/areas) can restrict local cell expansion, whereas vesicles are free to move out into the assimilation zone and produce expansion in other areas (green) where the F-actin meshwork has been loosened through the activity of the ARP2/3 complex (of which CROOKED is an essential component). (A-D) Major locations of dense actin in wild-type Arabidopsis cells. (A) Actin accumulations are seen at branch junctions and branch tips in trichome cells. The non-expanding, dense-actin filled tip is projected through the diffuse expansion growth that takes place in the flanks of the extending branches. Non-expansion of the absolute apex in trichomes produces a fine pointed tip. (B) Dense F-actin is found at lobe junctions and in different strategic locations in multi-lobed pavement cells. Contrary to other cell types, the lobes in pavement cells have to constantly adjust to the growth patterns of neighboring cells on all sides. Thus, they may show fluctuations in their dense actin localization, depending upon their position relative to other cells. (C) The elongate, cylindrical hypocotyl cells appear to have dense actin at their ends. Capping of the ends with dense actin reroutes vesicles into the flanking regions. This results is the formation of long cylindrical cells of varying length whose ends maintain a nearly constant diameter. (D) Side view of a blunt tipped growing root-hair cell. The positioning of dense actin bundles changes constantly in an elongating root hair (data not shown) though the general distance from the absolute tip remains almost the same. A dense-actin cap at the tip frequently indicates cessation of hair growth and may lead to swelling behind the actin-blocked tip region. Note that the formation of a pointed tip, as in trichomes, requires a dense actin cap, whereas a broad growing tip requires placement of dense actin at the sides in order to funnel vesicles into the tip region.

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