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First published online 17 December 2003
doi: 10.1242/dev.00926

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Partial loss-of-function alleles reveal a role for GNOM in auxin transport-related, post-embryonic development of Arabidopsis

Niko Geldner^1,*, Sandra Richter¹, Anne Vieten¹, Sebastian Marquardt¹, Ramon A. Torres-Ruiz², Ulrike Mayer¹ and Gerd Jürgens^1,

¹ ZMBP, Entwicklungsgenetik, Universität Tübingen, Auf der Morgenstelle 3, D-72076 Tübingen, Germany
² Lehrstuhl für Genetik, Technische Universität München, Wissenschaftszentrum Weihenstephan (WZW), Am Hochanger 8, D-85350 Freising, Germany

View larger version (97K): [in a new window]   Fig. 1. GNOM is expressed during embryonic and post-embryonic development. GUS staining of transgenic GNOM-GUS plants. (A) Mature ovule. (B-D) Embryos at (B) dermatogen, (C) heart and (D) torpedo stages. (E-H) Seedling roots: (E) primary root tip; (F) vascular bundle; note strong difference in staining between vascular bundle and endodermis (arrowhead marks border); (G) young lateral root primordium; (H) emerged lateral root primordium. (I) Cotyledon. (J) Undifferentiated vascular strand. (K) Late rosette leaf. (L) Inflorescence stem segment. (M) Young flower buds. (N-Q) Floral organs: (N) sepal; (O) petal; (P) stamen, note weak GUS signal in mature pollen grains (arrowheads); (Q) gynoecium.

View larger version (44K): [in a new window]   Fig. 2. Weak alleles of gnom have a number of post-embryonic growth phenotypes. (A) Diagram of GNOM protein depicting the N-terminal dimerisation and cyclophilin-binding (DCB) domain (Grebe et al., 2000) and the central, catalytic Sec7 domain (Shevell et al., 1994). Small arrowheads indicate positions of premature stop codons leading to strong gnom phenotypes, grey/black double-arrowhead indicates splice-site mutation in gnomS28 (grey), leading to an out-of-frame stop (black). Arrows indicate positions of the two complementing missense mutations (Busch et al., 1996). Large arrowheads indicate mutations leading to premature stops in the weak alleles. gnomSIT4 is a CAA to TAA nonsense mutation of codon 984. Grey/black double arrowhead indicates the AGC to AC frame-shift mutation of codon 1369 leading to an out-of-frame stop (black) in gnomR5. (B) Immunoblot of strong and weak gnom alleles. gnomB4049/emb30-1 (gnomB/E), full-length mutant protein (165 kDa, black arrowhead); gnomS28, negative control. White arrowheads indicate the expected positions of the truncated proteins of gnomR5 (155 kDa) and gnomSIT4 (110 kDa). Four-times as much protein was loaded on the rightmost lane as on the lane to its left. (C) Overview of phenotypic series of 8-day-old seedlings from a strong (emb30-1), a weak (gnomR5) and a very weak gnom line (gnomB/E). (D-G) Cotyledon vasculature of (D) strong, (E) weak, (F) very weak gnom. (G) Vasculature of wild type. (H-K) Rosette stage plants: (H) gnomR5; (I) size comparison between wild-type sister and gnomR5; arrowheads indicate extremely dwarfed gnomR5 plantlets; (J) gnomB/E; (K) wild type. (L-M) Flowering shoots of (L) 7-week and (M) 11-week-old plants of Col and gnomB/E. (N) Plastochrons of Col and gnomB/E. n=31 and n=43 per time-point for Col and gnomB/E, respectively. (O) Comparative histogram between Col and gnomB/E, showing percentages of plants bolting per indicated time period. n=19 and n=18 for Col and gnomB/E, respectively. There was a significant difference between the genotypes (P<0.001). (P) Primary inflorescence height of Col and gnomB/E at maturity. There was a significant difference between the genotypes (P<0.001). (Q) Histogram of percentage of plants with a given number of rosette side branches at several time points. n=47 and n=37 for Col and gnomB/E, respectively. At day 80, there was a significant difference between the genotypes (P<0.001).

View larger version (99K): [in a new window]   Fig. 3. Weak alleles are defective in meristem maintenance. (A-L) Primary root meristems of gnomR5, gnomB/E and wild-type at (A-F) day 7 and (G-L) day 15. (A,D,G,J) gnomR5, (B,E,H,K) gnomB/E, (C,F,I,L) wild type. (D-F,K,L) Higher magnifications of A-C,H,I, respectively. Arrowheads in A-C,H,I indicate approximate position of the onset of cell elongation. (G,J) Two examples of collapsed gnomR5 root meristems at day 15 (arrows, vascular strands; asterisks, bloated root hairs). (M) Root lengths of gnomB/E and gnomR5 relative to wild type. Both genotypes are significantly different from wild-type control (P<0.001). (N) Lateral root density of gnomB/E and gnomR5 relative to wild type. Note that in gnomR5, lateral roots were never observed. Both genotypes are significantly different from wild-type control (P<0.001). (O) Gravitropic growth response of gnomB/E as compared to wild type. Seedlings grown upright were turned by 135° and re-alignment to the gravity vector was recorded after 36 hours. Each root was assigned one of twelve 30° sectors. Genotypes were significantly different (P<0.001).

View larger version (99K): [in a new window]   Fig. 4. Auxin response gradients in gnomR5 root tips break down upon auxin treatment. DR5::GUS signals in (A-D) wild type, (E-H) gnomR5. (A,E) Untreated, (B,F) 0.1 µM NAA, (C,G) 1 µM NAA, (D,H) 10 µM NAA. Treatment was done for 24 hours. Note that DR5::GUS response gradients are severely affected at 1 µM NAA in gnomR5 (G), whereas in wild type a comparable change can only be observed at tenfold higher concentration (D).

View larger version (45K): [in a new window]   Fig. 5. Root growth phenotypes of gnomR5 roots are auxin-mediated. (A,B) Local application of NPA at the hypocotyl-root junction of wild-type roots severely reduces lateral root formation (A) and decreases primary root elongation (B). Plants were grown for 15 days. Differences between treatments were significant (P<0.001). (C-H) Comparison of (C-E) root tips and (F-H) rosette leaves after local application of NPA. (C,F) Untreated gnomR5, (D,G) NPA-treated wild type, (E,H) DMSO-treated wild type (control). (I) gnomR5 root meristem collapse can be reduced by transferring seedlings to plates supplemented with NAA. Observed numbers were significantly different from each other (P<0.01). (J-L) Root regeneration of seedlings. (J) Comparison of wild-type (left) and gnomR5 (right), 9 days after cutting. (K,L) Magnification of regeneration zone of (K) wild type and (L) gnomR5.

View larger version (135K): [in a new window]   Fig. 6. Auxin induces disorganised pericycle divisions in gnomR5. Lateral root primordia of (A-C) wild type and (D-F) gnomR5, after 48 hours of treatment. (A,D) Control, (B,E) 0.1 µM NAA, (C,F) 0.1 µM 2,4-D.

View larger version (135K): [in a new window]   Fig. 7. gnomR5 defects in lateral root formation correlate with an inability to establish transport-dependent auxin-response gradients. (A-H) Different stages of wild-type lateral root development. (A-D) PIN1 signals, (E-H) DR5::GUS signals. (A,E) stage I, (B,F) stages III-IV, (C,G) stage VI, (D,H) emerged lateral root. Arrows indicate borders of peripheral cells without PIN1. Staging according to Malamy and Benfey (Malamy and Benfey, 1997). (I-L) Wild-type treated with 0.1 µM 2,4-D. (I,J) PIN1 signals, (K,L) DR5::GUS signals. (I,K) One- or two-layered dividing pericycle, (J,L) multi-layered division zone. (M-P) gnomR5 treated with 0.1 µM NAA. (M,N) PIN1 signals, (O,P) DR5::GUS signals. (M,O) One- or two-layered dividing pericycle, (N,P) multi-layered division zone.

View larger version (53K): [in a new window]   Fig. 8. A speculative model for GNOM action – canalising auxin fluxes. (A) Heart-stage embryos of wild-type (top) and strong gnom allele (bottom). Yellow lines delineate the apical (a), central (c) and basal (b) regions of the embryos and their relation to the body pattern of the seedling (right). Black arrows indicate auxin flow from sources in the apical part of the embryo to the sink in the basal part. Presumed auxin gradients are shown at the left. (B) Relationship between localisation of PIN1 efflux carrier (red) and auxin-response gradients (blue) in lateral root primordium development. Arrows indicate auxin canalisation by gradual re-orientation of individual transport polarities of cells. Red stubs touching a given cell boundary mark the cell to which the respective PIN1 label is thought to belong. (C) Presumptive critical step for the canalisation of auxin flow during lateral root formation. Stage II lateral root primordium immediately after division is shown at the left, with the two daughter cell layers displaying opposite polarities. Gradual, GNOM-dependent, relocalisation of efflux carriers might be guided by weak polarising cues from adjacent tissues, supplying more auxin to the inner layer, which then imposes its auxin transport polarity on the outer layer. Arrows indicate direction of auxin flux; auxin efflux carriers (PIN1; in red); GN, GNOM-positive endosomes involved in recycling auxin carriers.

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