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Requirements for Arabidopsis ATARP2 and ATARP3 During Epidermal Development

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Current Biology, Vol. 13, 1341–1347, August 5, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0960-9822(03)00493-7 Requirements for Arabidopsis ATARP2 and ATARP3 during Epidermal Development Jie Le, Salah El-Din El-Assal, Dipanwita Basu, stage-specific swelling and reduced branch elongation Mohamed E. Saad, and Daniel B. Szymanski* defects (data not shown). Because dis1-1 was a pre- Agronomy Department dicted null allele of DIS1 (see below), we used this allele Purdue University for all of our detailed phenotypic characterizations. Mea- Lily Hall surements with scanning electron microscopy (SEM) 915 West State Street demonstrated that 40% (n ϭ 43) of dis1-1 stage 4 tri- West Lafayette, Indiana 47907-2054 chomes had a cell diameter of more than 30 ␮m. In the wild-type, a stage 4 cell diameter exceeding 30 ␮m was not observed in either this (n ϭ 45) or previous studies Summary [4]. During stage 6, papillae or tubercles decorate the surface of highly expanded trichomes. Papillae on the Plant cells employ the actin cytoskeleton to stably stalks of wild-type trichomes were slightly oval and var- ␮ ف ␮ ف position organelles, as tracks for long distance trans- ied in length from 1.5 mto 3 m (Figure 1C). In port, and to reorganize the cytoplasm in response to mature dis1 trichomes, papillae at the tips of abortive developmental and environmental cues [1]. While di- branches were often absent (Figure 1D). In obviously ␮ ف verse classes of actin binding proteins have been im- swollen regions of the stalk, papillae up to 9 min plicated in growth control, the mechanisms of cy- length were common. Equivalently sized papillae were not toskeletal reorganization and the cellular functions of observed in the wild-type. In both mutant and wild-type specific actin filament arrays are unclear. Arabidopsis cells, the long axis of the papillae and the cell were trichome morphogenesis includes distinct require- aligned. Presumably, the orientation of the papillae re- ments for the microtubule and actin filament cytoskel- flected some aspect of polarized growth that was main- etons. It also is a genetically tractable process that is tained in the mutant. Pavement cells are another highly providing new knowledge about cytoskeleton function polarized cell type that appear to utilize both microtu- in plants [2]. The “distorted group” of mutants defines bule and actin-filament structures during morphogene- a class of at least eight genes that are required during sis [6]. At 12 days after germination (DAG), wild-type the actin-dependent phase of trichome growth. Using pavement cells located in the distal 1/3 of the cotyledon map-based cloning and a candidate gene approach, were highly lobed (Figure 1E). Some dis1 cotyledon we identified mutations in ARP3 (ATARP3) and ARP2 pavement cells at comparable positions were highly (ATARP2) genes as the cause of the distorted1 (dis1) lobed (Figure 1F). Other dis1 pavement cells in the same and wurm (wrm) phenotypes, respectively. ARP2 and region of the identical cotyledon had reduced lobe ARP3 are components of the evolutionarily conserved expansion (Figure 1G). dis1-T1 pavement cells displayed ARP2/3 complex that nucleates actin filament poly- similar phenotypes (data not shown). We have no expla- merization [3]. Mutations in DIS1 and WRM caused nation for this patchy phenotype. dis1 pavement cells severe trichome growth defects but had relatively mild consistently failed to adhere normally, and gaps be- effects on shoot development. DIS1 rescued the phe- tween adjacent cells were frequently detected (Figures notype of ⌬arp3 when overexpressed in S. cerevisiae. 1F and 1G). Developing dis1 trichomes had defects in cytoplasmic With the exception of the striking trichome phenotype, actin bundle organization and reduced relative amounts the whole plant architecture of dis1 and wrm plants was of cytoplasmic actin filaments in developing branches. very similar to that of the wild-type (Figures 1H–1J). However, shoot fresh weight accumulation of dis1-1 and phenotypically wild-type siblings differed at 15 DAG. Results and Discussion In one representative experiment, the mean dis1 shoot weight (75 Ϯ 9mg[nϭ 6]) was reduced by 35% com- Branch position, shape, and number are tightly regu- pared to the wild-type (117 Ϯ 9mg[nϭ 6]). The pheno- lated during wild-type trichome development [4]. Wild- types are consistent with the detection of DIS1 transcript type trichomes in the process of branch formation (stage in roots and leaves [7]. Despite the clear actin-depen- 3) and one that recently completed branching (stage 4) dent growth of root hairs [8], dis1 root hairs were very are labeled in Figure 1A. Most of the cell volume is similar to those of the wild-type (data not shown). Pollen generated during a massive cell expansion phase (stage germination and tip growth did not require DIS1 or WRM, 5) that occurs after branch initiation is complete. Tri- because all of the alleles examined in this study were chome shape on dis1 leaves was indistinguishable from efficiently transmitted through pollen and dis1 and wrm that of the wild-type through stage 3, but subsequently, plants yielded thousands of seeds. However, these re- the trichomes were swollen, twisted, and often had a sults do not preclude a subtle or conditional effect of reduced branch length (Figure 1B). Recessive dis1, wrm, DIS1 or WRM on pollen development. and gnarled (grl) mutations cause indistinguishable To identify the affected genes, we initiated map-based stage-specific swelling [4, 5], and each of the dis1 and cloning of DIS1. Consistent with its published position wrm alleles described in this paper also had the same on the classical map [9], dis1 mapped between T28P6 and F9L1 on the upper arm of chromosome 1 (Figure *Correspondence: [email protected] 2A). Unfortunately, in mapping populations that em- Current Biology 1342 Figure 2. Mapping and Molecular Characterization of DIS1 and WRM Mutant Alleles (A) The mapping interval of the DIS1 gene. The boxes indicate the molecular marker, and the oval represents DIS1-containing BAC (F3F19). The recombinant frequency for each marker is given. (B) The physical structure of the DIS1 gene. Exons are represented by closed rectangles, and introns are represented by lines. F1, R2, and R9 are PCR primers. The dis1-1, dis1-2, and dis1-T1 alleles are Figure 1. Trichome, Pavement Cell, and Shoot Phenotypes of Wild- defined. Type, dis, and wrm Seedlings (C) The physical structure of the WRM gene. The wrm-1, wrm-2, (A) The upper surface of developing leaves of wild-type. Inset, higher and wrm-T1 alleles are defined. magnification view of a stage 4 trichome. (D and E) RT-PCR analysis of the DIS1 mRNA in wild-type and (B) The upper surface of developing leaves of dis1. Inset, higher mutant backgrounds. (D) RT-PCR using primers F1 and R2. (E) RT- magnification view of a stage 4 trichome. PCR using primers F1 and R9. Lane 1, no (Ϫ) reverse transcriptase (C) Papillae on mature wild-type trichomes. (RT) wild-type; lane 2, Plus (ϩ) RT wild-type; lane 3, dis1-1 (ϩRT); (D) Papillae on mature dis1 trichomes; middle panel, higher magnifi- lane 4, dis1-2 (ϩRT); lane 5, dis1-T1 (ϩRT). cations of regions from (C) and (D). (E) The upper surface of 12 DAG wild-type cotyledon pavements cells. distorted trichome phenotype. Salk_010045 contained (F and G) The upper surface of 12 DAG dis1 cotyledon pavements an insertion in the ATARP3 gene, and lines homozygous cells. (H) A wild-type seedling (11 DAG). for the insertion displayed a completely penetrant, dis- (I) A dis1 seedling (11 DAG). torted trichome phenotype that was indistinguishable (J) A wrm seedling (11 DAG). from dis1-1. Progeny from hemizygous Salk_010045 The scale bars represent 50 ␮m in (A) and (B), 10 ␮m in the insets plants segregated for the trichome phenotype. All F1 ␮ ␮ of (A) and (B), 10 m in (C) and (D), 2 m in the insets of (C) and plants derived from crosses between dis1-1 (n ϭ 24), (D), 50 ␮m in (E)–(G), and 2 mm in (H)–(J). The stage of an individual dis1-2 (n ϭ 29), and Salk_010045 were mutant. Therefore trichome is labeled with a number. The arrowheads indicate the gaps between adjacent pavement cells. Salk_010045 was allelic to dis1-1 and dis1-2 and was renamed dis1-T1. We wanted to determine if the ATARP3 gene sequence was altered in dis1-1 and ployed the dis1-1 allele, we failed to detect recombina- dis1-2. dis1-1 was originally isolated in the Landsberg tion within a 500 kb interval that was expected to contain erecta (Ler) background, and dis1-2 was originally iso- DIS1 (0 recombinants/1012 chromosomes). ATARP3 [7] lated in Columbia-O (Col). Compared to the Ler se- is located within our mapping interval on chromosome quence, dis1-1 had a single nucleotide change that intro- 1. wrm also mapped to a region of chromosome 3 that duced a nonsense mutation at amino acid 98 (W98*) contains ATARP2 ([5], D.B.S., unpublished data). There- (Figures 2B and S1 [in the Supplemental Data available fore, we tested the ability of publicly available Salk with this article online]). The dis1-2 allele eliminated a T-DNA insertions into ATARP3 and ATARP2 to cause a consensus splice acceptor sequence of intron 1 (Figure DIS1 and WRM Encode ARP2/3 Complex Homologs 1343 Table 1. Amino Acid Sequence Comparisons between Predicted Arabidopsis ARP2/3 Complex Components and Homologs in Human and S. cerevisiae Arabidopsis Human: Ident. % Yeast: Ident. % Genea (Sim. %) (Sim. %) ATARP2b 61 (79) 56 (74) ATARP3b 56 (71) 51 (63) ATARPC1Ab 42 (59) 34 (51) ATARPC1Bb 42 (61) 34 (51) ATARPC2Ab 26 (46) 28 (47) ATARPC2Bb 29 (47) 23 (44) ATARPC3b 47 (66) 39 (58) ATARPC4 65 (81) 59 (74) ATARPC5b 32 (53) 23 (45) Figure 3.
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  • Primepcr™Assay Validation Report

    Primepcr™Assay Validation Report

    PrimePCR™Assay Validation Report Gene Information Gene Name actin related protein 2/3 complex, subunit 4, 20kDa Gene Symbol ARPC4 Organism Human Gene Summary This gene encodes one of seven subunits of the human Arp2/3 protein complex. This complex controls actin polymerization in cells and has been conserved throughout eukaryotic evolution. This gene encodes the p20 subunit which is necessary for actin nucleation and high-affinity binding to F-actin. Alternative splicing results in multiple transcript variants. Naturally occurring read-through transcription exists between this gene and the downstream tubulin tyrosine ligase-like family member 3 (TTLL3) which results in the production of a fusion protein. Gene Aliases ARC20, MGC13544, P20-ARC RefSeq Accession No. NC_000003.11, NT_022517.18 UniGene ID Hs.323342 Ensembl Gene ID ENSG00000241553 Entrez Gene ID 10093 Assay Information Unique Assay ID qHsaCED0002460 Assay Type SYBR® Green Detected Coding Transcript(s) ENST00000498623, ENST00000397261, ENST00000433034, ENST00000287613 Amplicon Context Sequence TTTTAAACCATCTGGCTGGATCTCGTGGCCTTCCCCCTCAGACTACCCATGTCTC CACGAAGGCGTCCTGGAGTCACTCCCCGAGCAGCG Amplicon Length (bp) 60 Chromosome Location 3:9847894-9847983 Assay Design Exonic Purification Desalted Validation Results Efficiency (%) 104 R2 0.9987 cDNA Cq 19.57 cDNA Tm (Celsius) 81.5 Page 1/5 PrimePCR™Assay Validation Report gDNA Cq 24.52 Specificity (%) 100 Information to assist with data interpretation is provided at the end of this report. Page 2/5 PrimePCR™Assay Validation Report ARPC4,