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First published online 25 February 2004
doi: 10.1242/dev.01028

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Synergistic interaction of three ERECTA-family receptor-like kinases controls Arabidopsis organ growth and flower development by promoting cell proliferation

¹ Department of Biology, University of Washington, Seattle, WA 98195, USA
² CREST, Japan Science and Technology Corporation, Saitama 332-0012, Japan

View larger version (92K): [in a new window]   Fig. 1. ERECTA-LIKE genes ERL1 and ERL2 are immediate, functional paralogs of ERECTA. (A) Amino-acid sequence alignment of ERECTA and its paralogs. The alignment was generated by the CLUSTAL X program. Identical amino acids and similar amino acids are highlighted by black and gray backgrounds, respectively. Arrows indicate the positions of introns. Respective domains are highlighted with colored underlines: N-terminal signal sequence, blue; extracellular paired cysteine regions, red; the LRR-domain, green; the transmembrane domain, black; the juxtamembrane domain, purple; the kinase domain, pink; and a C-terminal tail region, cyan. (B) A phylogenetic tree of ERECTA, ERL1 and ERL2. The single most parsimonious tree generated using heuristic searches (PAUP*) based on the kinase domain sequence is shown. BRI1 and CLV1 were used as outgroups. Branch lengths are proportional to the number of amino-acid substitutions, and the numbers at the branch points indicate bootstrap values of 500 replications. (C) ERL1 and ERL2 genes rescue the erecta phenotype when expressed under the ERECTA promoter and terminator. Shown are the inflorescence apices from wild type, erecta-105, erecta-105 plants expressing ERECTA::ERL1 and erecta-105 plants expressing ERECTA::ERL2. Scale bar: 5 mm.

View larger version (50K): [in a new window]   Fig. 2. Semi-quantitative RT-PCR analysis of ERECTA, ERL1 and ERL2 transcripts in different tissues. The actin fragment was amplified simultaneously as a control. ERECTA, ERL1 and ERL2 were amplified for 32 cycles; the control actin was amplified for 30 cycles.

View larger version (110K): [in a new window]   Fig. 3. ERECTA, ERL1 and ERL2 are expressed in young proliferating tissues in an overlapping but unique manner. Expression of ERECTA::GUS (A,D,G,J,M), ERL1::GUS (B,E,H,K,N) and ERL2::GUS (C,F,I,L,O) reporter genes in: a 14-day-old seedling (A-C); inflorescence apices at lower (D-F) and higher (G-I) magnifications; inflorescence meristem (J-L); and a stage 11 flower (M-O). Scale bars: 3 mm for A-C; 2 mm for D-F; 0.5 mm for G-I; 50 µm for J-L; 0.2 mm for M-O.

View larger version (28K): [in a new window]   Fig. 4. erl1-2 and erl2-1 are null alleles. (A,B) Structure of ERL1 (A) and ERL2 (B) genes and T-DNA insertion sites. The insertion in erl2-1 consists of two T-DNAs with inverse orientation. The sequence of ERL1 and ERL2 adjacent to the insertion is shown in upper case. The T-DNA sequence is in lower case. (C-E) Semi-quantitative RT-PCR analysis of ERECTA, ERL1 and ERL2 transcripts in erl1-2 (C), erl2-1 (D) and erecta-105 (E) mutant backgrounds. erl1-2, erl2-1 and erecta-105 plants do not have detectable levels of ERL1, ERL2 and ERECTA mRNA, respectively. None of these null mutations affect expression levels of other members of ERECTA-family. The actin fragment was amplified simultaneously as a control. ERECTA, ERL1 and ERL2 were amplified for 35 (C,D) or 32 (E) cycles; the control actin was amplified for 30 cycles.

View larger version (76K): [in a new window]   Fig. 5. ERL1 and ERL2 genes are partially redundant and show synergistic interaction with ERECTA. (A-C) Morphometric analysis of fully-grown eight-week-old wild-type, erl1-2, erl2-1, erl1-2 erl2-1, erecta-105, erecta-105 erl1-2, erecta-105 erl2-1 and erecta-105 erl1-2 erl2-1 plants. (A) Plant (inflorescence) height (n=20). (B,C) Lengths of mature pedicels (B) and siliques (C) on the main stem (n=40; eight measurements per stem). Bars represent the average; error bars represent s.d. (D) Mature siliques and attached pedicels of wild-type, erl1-2, erl2-1, erl1-2 erl2-1, erecta-105, erecta-105 erl1-2 and erecta-105 erl2-1. Scale bars: 5 mm. (E) Six-week-old plants of erecta-105, erecta-105 erl1-2, erecta-105 erl2-1 and erecta-105 erl1-2 erl2-1. Scale bars: 3 cm. The top right insert is an image of an erecta-105 erl1-2 erl2-1 plant at higher magnification (scale bar: 5 mm). (F) Scanning electron micrographs of silique tips from wild type, erecta-105, erecta-105 erl1-2 and erecta-105 erl2-1, and a silique from erecta-105 erl1-2 erl2-1. Scale bar: 100 µm

View larger version (88K): [in a new window]   Fig. 6. erecta-105 erl1-2 erl2-1 triple mutations lead to dramatically reduced plant growth, aberrant flower development, and reduced cell numbers in petals and pedicels. (A) 12-day-old seedlings of wild type and erecta-105 erl1-2 erl2-1. Scale bars: 3 mm. (B) 24-day-old plants of erecta-105 and erecta-105 erl1-2 erl2-1. Scale bars: 1 cm. (C) Phenotypic variation of seven-week-old erecta-105 erl1-2 erl2-1 plants. Although the main inflorescence stem (arrowheads) always exhibits severe elongation defects, axillary branches occasionally show various degrees of phenotypic rescue. Scale bars: 5 mm. (D) Scanning electron micrographs of erecta-105 and erecta-105 erl1-2 erl2-1 flowers at stage 9. Note that one sepal was removed from the erecta-105 flower and several sepals were removed from erecta-105 erl1-2 erl2-1. Anther development and polar expansion of petals (arrowhead) are severely defective in the erecta-105 erl1-2 erl2-1 flower. Scale bars: 40 µm. (E) Flowers of erecta-105 and erecta-105 erl1-2 erl2-1 with mild phenotype. One sepal and two petals were removed from the erecta-105 flower to expose the inner organs. Note that none of the floral organs were removed from the erecta-105 erl1-2 erl2-1 flowers. The triple mutant flowers have reduced numbers of organs and develop characteristic needle-like petals. Scale bars: 0.5 mm. (F) Scanning electron micrographs of petal abaxial epidermis cells of erecta-105 and erecta-105 erl1-2 erl2-1. The images are taken at the same magnifications. Scale bars: 10 µm. (G) Longitudinal sections of mature pedicels from wild type, erecta-105, erecta-105 erl1-2, erecta-105 erl2-1 and erecta-105 erl1-2 erl2-1 plants. All combinations of mutants shown have defects in shape, size and number of cells in the cortex cell file (asterisks). Scale bars: 50 µm. (H) Longitudinal section of the mature flower of erecta-105 and erecta-105 erl1-2 erl2-1. The lack of proper ovule and anther (asterisk) differentiation is visible in erecta-105 erl1-2 erl2-1. The images are taken under the same magnifications. Scale bars: 0.2 mm. (I) Number of cells in the longitudinal cortex file of a mature pedicel of wild type, erecta-105, erecta-105 erl1-2, erecta-105 erl2-1 and erecta-105 erl1-2 erl2-1 plants. (J) Effect of erecta-105 and erecta-105 erl1-2 erl2-1 mutations on RNA levels of genes regulating meristem (STM, WUS) or organ (ANT, KNAT1, D-type cyclins) growth. Semi-quantative RT-PCR analysis was performed. An actin fragment was amplified simultaneously as a control. The number of PCR cycles were as follows: ANT, 28 cycles; STM, 28 cycles; WUS, 29 cycles; KNAT1, 32 cycles; CYCD2;1, 32 cycles; CYCD3;1, 29 cycles; actin, 24 cycles.