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The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport

Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, Leiden, The Netherlands

View larger version (63K): [in a new window]   Fig. 1. The PINOID gene encodes a plant-specific protein-serine/threonine kinase with a role in organ development. (A) Schematic presentation of the PINOID protein showing the positions of the 12 conserved protein kinase subdomains, the newly identified AEP domain characteristic for ACG-group VIII members, the amino acid residues that are conserved in 95% of all protein kinases and the positions of the mutations in respectively pid-2 (codon 380), pid::En197 (codon 197) and pid::En310 (codon 310). (B) PID is expressed at low levels, but expression is upregulated 4 hours after treatment with IAA and/or cycloheximide (CHX), as shown by an autoradiograph of a RNA gel blot containing poly(A) enriched mRNA from roots after hybridization with a genomic fragment containing the second exon of the PID gene. The ethidium bromide-stained gel in the lower panel shows that equal amounts of RNA were loaded. Flowers of pid::En310 (C,D), pid-2 (F) and wild-type Arabidopsis (G). (E) The three cotyledon phenotype observed in the loss-of-function mutants pid::En197 and pid::En310. Scale bars, 2.5 mm (C,E-G) and 1.5 mm (D).

View larger version (106K): [in a new window]   Fig. 2. Histochemical staining of plant tissues of line PID:GUS-18 for GUS activity shows that in 7-day-old seedlings PID:GUS is mainly localised in the vascular tissue proximal to the root meristem (A), the shoot apical meristem (E) or lateral root primordia (C). Expression is also present in developing cotyledons and leaves (E) and is significantly increased when the seedlings are incubated overnight in the presence of 5 µM IAA (B,D,F). (G) A transverse section through the hypocotyl, just below the shoot apex, shows that PID:GUS is localised in the cytoplasm (see detail in H) of xylem parenchyma (xp) and endodermis (e) cells but not in the xylem plate (x) or in cells of the cortex (c) or the phloem (ph). Histochemical staining of (I) an inflorescence stem segment with a bract and a secondary inflorescence and (J-M) of flower buds in progressive stages of development shows that PID:GUS is transiently expressed in anther primordia (J-L) and in the vascular tissue of young pedicels and bracts (I) and of developing sepals (M). In the pistil, expression is detected in the vascular tissue of the style and septum (M) and in the integument and funiculus of fertilised ovules (N). During embryogenesis PID:GUS expression is localised to the cotyledon primordia (O). Scale bars, 30 µm (A-D), 80 µm (E), 60 µm (F,N), 15 µm (G,O), 5 µm (H) and 0.3 mm (I-M).

View larger version (118K): [in a new window]   Fig. 3. Localisation of PID mRNA by whole-mount in situ hybridisation in roots (A,C) and in the shoot apex (D,E) of 4-day-old seedlings. (A,D) Anti-sense PID probe. (C,E) Sense PID probe. (B) Root of a seedling of line PID:GUS-18 stained for GUS activity.

View larger version (53K): [in a new window]   Fig. 4. CaMV 35S promoter-controlled expression of the PID cDNA in Arabidopsis ecotypes C24 and Columbia (Col). (A) Autoradiograph of an RNA gel blot containing total RNA from 7-day-old seedlings of wild-type (C24 and Col) and 35S::PID after hybridization with the PID cDNA probe. (B) Ethidium bromide-stained gel. (C) Root and hypocotyl lengths and gravitropy, as measured in 7-day-old seedlings. Histogram bars indicate average lengths of 20 roots or hypocotyls. The bars marked with an asterisk differ significantly from the wild-type control (Student’s t-test: P<0.05). The gravitropic response is depicted as the percentage of hypocotyls or root tips that was classified into each of twelve 30º sectors on a circle. The number of seedlings scored per line is indicated in the middle of each circle.

View larger version (92K): [in a new window]   Fig. 5. High levels of 35S::PID expression result in frequent disintegration of the primary root meristem and reduced apical dominance. (A) CLSM optical section of propidium iodide-stained collapsed primary root tip of a 4-day-old 35S::PID Col-21 seedling. No cells with columella identity are detected after staining for starch granules (B), whereas columella cells are clearly stained in a root meristem of a 6-day-old wild-type seedling (C). (D) Lateral roots have emerged from the primary root of a 10-day-old 35S::PID Col-21 seedling following collapse of the root meristem (white arrow). (E) CLSM optical section of a propidium iodide-stained lateral root meristem from a seedling as in D. (F,G) Wild-type and 35S::PID plants photographed 5 and 12 days after bolting, respectively. The white arrows indicate the positions where lateral branches emerge on the primary inflorescence. Scale bars, 25 µm (A,E) and 35 µm (B,C).

View larger version (48K): [in a new window]   Fig. 6. Rescue of primary root growth of 35S::PID seedlings by polar auxin transport inhibitors. Seedlings of Columbia wild type (A,B) and line 35S::PID Col-21 (C,D) 7 days after germination (dag) on vertical plates. CLSM optical sections of propidium iodide-stained root tips of wild-type (E,F) and 35S::PID Col-21 (G,H) seedlings, 7 dag. Root tips of 4-day-old wild-type (I,J) or 35S::PID Col-10 (K,L) seedlings containing the DR5::GUS reporter gene after staining for GUS activity. (B,D,F,H,J,L) On 0.3 µM NPA. (M) Timing and frequency of primary root meristem collapse in 35S::PID lines Col-10 and Col-21. Frequencies are averages from counting phenotypes in three independent populations of 20 seedlings. (N) Length and number of lateral roots on primary roots of 14-day-old wild-type, 35S::PID Col-10 and 35S::PID Col-21 seedlings when rescued with 0.3 µM NPA. Mean values are based on at least 15 independent roots. Error bars indicate the standard error of the mean. Scale bars, 50 µm.

View larger version (78K): [in a new window]   Fig. 7. Expression pattern of the epidermis-specific LTP1 promoter in an F2 seedling from the cross ACT-LTP1 x EF-PID after histochemical staining for activity of the co-expressed GFP:GUS gene. The reporter gene is predominantly expressed in the shoot (A). Expression is also observed in the young epidermis of lateral root primordia, but only after a stage VI primordium has been formed. Stage V (B) and stage VI (C) lateral root primordia of a GUS-positive F2 seedling.

View larger version (14K): [in a new window]   Fig. 8. A schematic model to explain the observed seedling phenotypes caused by ectopic PID expression. In wild-type seedlings auxin is transported from its location of synthesis in the shoot downward to the root. Lateral transport of auxin is essential for hypocotyl elongation and tropic growth (a) and lateral root formation (b). Redistribution of auxin at the root tip by basipetal transport toward the elongation zone is essential for growth and gravitropy of the root (c). 35S::PID expression preferentially stimulates downward directed PAT to the root apex, thereby reducing lateral transport. From the root tip auxin is rapidly transported to a location where it is metabolised or secreted into the medium. This deprives the peripheral cell layers and root apex of auxin and results in reduced gravitropy and growth of hypocotyl and root and reduced lateral root formation. Removal of auxin from the root apex eventually leads to collapse () of the primary root meristem. LTP1::PID expression only enhances PAT from the shoot into the root, thereby increasing lateral root formation. The directions and level of PAT is indicated by black arrows. Elongation growth is indicated by black arrows that start with a dot.

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