Mutations in actin-related proteins 2 and 3 affect cell shape development in Arabidopsis

Abstract

ACTIN-RELATED PROTEINS 2 and 3 form the major subunits of the ARP2/3 complex, which is known as an important regulator of actin organization in diverse organisms. Here, we report that two genes, WURM and DISTORTED1, which are important for cell shape control in Arabidopsis, encode the plant ARP2 and ARP3 orthologs, respectively. Mutations in these genes result in misdirected expansion of various cell types: trichome expansion is randomized, pavement cells fail to produce lobes, hypocotyl cells curl out of the normal epidermal plane, and root hairs are sinuous. At the subcellular level, cell shape changes are linked to severe filamentous actin aggregation and compromised vacuole fusion. Because all seven subunits of the ARP2/3 complex are present in plants, our data indicate that this complex may play a pivotal role during plant cell morphogenesis.

Figure 1.

Molecular Organization and Homology between WRM (Arabidopsis ARP2) and DIS1 (Arabidopsis ARP3). (A) and (B) Schemes of the genomic organization and mutations (arrows) in WRM (A) and DIS1 (B) in Arabidopsis. The positions of mutations are indicated relative to the A of the start codon. wrm 1-1 and dis1-1 are EMS-generated alleles, whereas wrm1-2 and dis1-2 are T-DNA insertion alleles. Gray boxes represent exons, and black lines represent introns. (C) Amino acid sequence alignment of WRM and DIS1 using the CLUSTAL W program. Black-shaded areas indicate amino acid identity, and gray-shaded areas indicate sequence similarity. Mutations indicated by asterisks in the two genes are predicted to truncate the protein at amino acid position 14 in wrm1-2 (SALK_003448), at amino acid 97 in dis1-1, and at amino acid position 290 in dis1-2 (SALK_010045), whereas a G-to-A change in wrm1-1 changes a conserved Gly (G) at position 151 (θ) to Asp (D).

Figure 2.

Representative Images Comparing Salient Phenotypic Characters of wrm and dis1 Mutants with Those of Wild-Type Arabidopsis (ecotype Landsberg erecta). (A) Typical two- to three-branched trichomes on wild-type leaves. Inset (a) shows a single, mature wild-type trichome. (B) Short, nearly supine, distorted trichomes on dis1-1 mutant leaves. Inset (b) shows a single wrm1-1 trichome for comparison with inset (a). (C) Epidermal cells in wild-type cotyledons arranged in the characteristic jigsaw-puzzle pattern with prominent lobes. (D) Randomly shaped, less lobed, and relatively unexpanded cotyledon epidermal cells seen in both wrm and dis1 mutants. (E) Elongated hypocotyl cells in light-grown wild-type plants show rows of stomatal complexes (arrowheads). (F) A dis1-1 hypocotyl shows that cells are short and broad and stomatal rows often are absent in the mutants. (G) and (H) Dark-grown wild-type hypocotyl cells are elongated and maintain firm contact with each other (arrowhead) at their ends (G), whereas contiguous hypocotyl cells developing under the same conditions in both wrm and dis1 mutants lose contact with their neighbors along the long axis and curl out of the epidermal plane (arrowheads) (H). (I) Wild-type root hairs are straight and up to 1 mm long. (J) and (K) Root hairs of dis1-1 representing the relatively short, stubby cells (J) and cells with varying degrees of waviness (K) (arrowheads) seen in the mutants. Bars = 500 μm in (A) and (B), 50 μm in (C), (D), (G), (H), and (K), 100 μm in (E) and (F), and 200 μm in (I) and (J).

Figure 3.

F-Actin Organization in Different Cell Types in Wild-Type Arabidopsis and wrm/dis1 Mutants. Localized increase in F-actin aggregation is a feature observed in both mutants. (A) A wild-type trichome with extending branches organizes long F-actin cables (arrow) while maintaining a dense actin cap at the tips of branches (arrowheads). (B) A dis1-1 trichome at approximately the same stage as that in (A) displaying abnormal accumulation of F-actin (arrowheads) and a conspicuous absence of long actin cables. Note that one branch (b) remains relatively unextended compared with the other branch. (C) A developing wrm1-2 trichome showing increased F-actin aggregation in reticulate cytoplasmic networks containing brightly fluorescent actin foci (arrowheads). Similar cytoplasmic and F-actin organization is not observed in wild-type trichomes. Note that the lower portion of the trichome (arrow) corresponding to the stalk region is more radially expanded, whereas a small branch (b) remains unextended. (D) Portion of a wild-type trichome showing a well-extended branch. Note that the F-actin organization consists predominantly of longitudinally oriented subcortical cables and a finer reticulum of cortical F-actin visible in the area shown by the arrow. (E) The tip region of a wild-type trichome branch contains fine, longitudinally oriented F-actin strands (arrow) that appear to meet at the apex. (F) An abnormally expanded tip region in a dis1-2 trichome cell representing the reticulate actin-filled cytoplasm in nonexpanding branch tips in mutant trichomes (arrow), clearly different from the wild-type situation seen in (E). (G) A wrm1-2 trichome representing the characteristic actin organization seen in mature mutant trichomes. The abundance of intersecting, actin-rich cytoplasmic strands produces a loose polygonal mesh (arrow) that is clearly different from the longitudinally oriented cytoplasmic strands containing relatively thin, long, extended F-actin cables typical of mature wild-type trichomes (D). Note that the mutant branches (arrowheads) remain unextended as short spikes. (H) and (I) Comparison of F-actin organization between wild-type and mutant trichome cells based on the fluorescence levels of the F-actin binding GFP-mTalin probe. Comparison of the wild-type image (H) and the dis1-2 mutant cell image (I) demonstrates the relative difference in fluorescence levels. The large areas covered with red in (I) have only small counterparts (arrowheads) in (H). Insets (h) and (i) are histograms that differentiate between the red and black levels to accentuate the relative differences in (H) and (I). Small arrows in histogram (h) indicate the minute red regions in (H). Although the images shown are of mature trichomes, the yellow circles in (H) and (I) indicate representative areas that were used to obtain the gray-scale readings reported in Table 2. Circle 1, very fine, diffuse F-actin; circle 2, defined F-actin strand; circle 3, brightest fluorescent spots in an image; circle 4, background.

Figure 4.

Defects in F-Actin Organization Precede Morphological Changes in Mutant Cells. Elongating mutant hypocotyl epidermal cells of the dis1-1 mutant were categorized into different sequential stages of expansion that lead ultimately to contact breakage between contiguous cells. This categorization formed the basis for construing the sequence of changes in F-actin organization that occur in these cells. Hypocotyl cells in GFP-mTalin transgenic plants (Landsberg erecta ecotype) were used as wild-type controls. Bars = 40 μm. (A) Portion of the hypocotyl in a dis1-1 seedling carrying a GFP-mTalin transgene challenged to elongate rapidly by growing for 8 days in the dark shows cells (labeled 1 to 4) in different stages of expansion. (1) Normally expanding cells in firm contact with their neighbors along the long axis. (2) A bulging cell end signifying the initiation of abnormal expansion; such cells later would break contact with their neighbors along their end walls. (3) Cells with gaps between them resulting from different elongation rates. (4) Cells that have broken contact with their neighbors along the long axis and whose ends have curled out of the epidermal plane. (B) The actin cytoskeleton at cell ends in control wild-type hypocotyl cells showing the cortical organization (box) and a subcortical, longitudinally oriented F-actin cable (arrow). Nonresponding and stage-1 mutant hypocotyl cells exhibit a similar organization. (C) The complete F-actin organization of a stage-2 mutant cell depicted by compression of a stack of 30 confocal images shows increased actin accumulation at one end of the cell (box), whereas the opposite side remains relatively clear. (D) A single median-longitudinal confocal section through two contiguous epidermal cells (stage 3) that have broken contact with each other along the longitudinal growth direction (double-headed arrow). The boxed area shows brightly fluorescent spots indicative of dense F-actin aggregation at the cell end. (E) Mutant hypocotyl cells in the final stage (stage 4) of development. After breaking contact, the gaps have further widened between cells (double-headed arrow) and many cell ends have curled out. Note the presence of thick F-actin bundles in the boxed area, the occurrence of long F-actin cables (arrowheads), and the nearly normal F-actin organization in an underlying hypocotyl cell (arrow).

Figure 5.

The Intracellular Organization in wrm/dis1 Trichomes Resembles That Achieved upon Lat-B Treatment of Wild-Type Trichome Cells. (A) Actin cytoskeleton in an untreated mature wild-type trichome (carrying a GFP-mTalin transgene for F-actin visualization) serving as a control in the Lat-B treatment experiments displays the normal long F-actin cables (arrowhead). (B) A mature wild-type trichome treated with 5 nM Lat-B for 2 h displaying a general increase in cytoplasmic granularity (arrow) and thick F-actin bundling. Inset (b) shows a magnified view of the region indicated by the arrowhead. (C) and (D) Basal portion of a trichome cell treated with Lat-B for 2 h and then stained simultaneously with FDA (C) and FM4-64 (D). FDA is excluded from the miniature vacuoles, whereas FM4-64 staining labels vacuolar membranes specifically. (E) An FDA-stained wild-type trichome shows a large central vacuole (arrow). (F) An FDA-stained wrm1-2 trichome at a developmental stage comparable to that of the wild-type trichome in (E) shows many small vacuoles (arrowheads) of varying size in the vicinity of larger vacuoles. (G) The base of an FDA-stained trichome that developed on medium containing 5 nM Lat-B showing numerous apparently unfused vacuoles of varying size (cf. with [E] for the absence of a large central vacuole and with [F] for the presence of small vacuoles). Bars = 80 μm in (A), (B), and (E) to (G) and 40 μm in inset (b), (C), and (D).