QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online September 28, 2005
doi: 10.1242/10.1242/dev.02027

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vieten, A. Articles by Friml, J. Search for Related Content PubMed PubMed Citation Articles by Vieten, A. Articles by Friml, J.

Functional redundancy of PIN proteins is accompanied by auxin-dependent cross-regulation of PIN expression

Anne Vieten¹, Steffen Vanneste², Justyna Winiewska^1,3, Eva Benková¹, René Benjamins⁴, Tom Beeckman², Christian Luschnig⁴ and Jií Friml^1,*

¹ Centre for Molecular Biology of Plants, University Tübingen, Auf der Morgenstelle 3, 72076 Tübingen, Germany
² Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Gent University, Technologiepark 927, B-9052 Gent, Belgium
³ Department of Biotechnology, Institute of General and Molecular Biology, 87-100 Toru, Poland
⁴ Institute for Applied Genetics and Cell Biology, University of Applied Life Sciences and Natural Resources, Muthgasse 18, A-1190 Vienna, Austria

View larger version (109K): [in a new window]   Fig. 1. Cross-regulation of PIN expression and function in embryo development. (A-C) Novel embryo phenotypes in pin1,3,4,7 multiple mutants (C) compared with wild-type (A) and pin1,7 mutant (B) embryos. (D-G) Immunostaining showing that PIN4 is ectopically expressed in the suspensor of the pin7 preglobular embryo (G) in a pattern similar to that of PIN7 expression in wild type (F). No PIN4 expression in wild type at this stage (D) and expression restricted to root meristem precursors at the later stages (E). For the embryo stages the numbers indicate the developmental stage according to the actual number of pro-embryo cells of the corresponding wild-type stage. G, globular; H, heart; l, late; T, Torpedo; Tr, triangular; y, young.

View larger version (69K): [in a new window]   Fig. 2. Cross-regulation of PIN gene expression in root. (A) Symbolic depiction of PIN gene expression and polar localization in preglobular, early heart embryos and in seedling root. Arrows indicate presumed directions of auxin flow based on subcellular PIN polarity. (B) Comparison of root and meristem length between pin1 and pin2 single mutants and pin1,2 double mutants. Standard deviations are depicted. (C) Seedling phenotypes of wild type, pin2 single mutant and pin1,2 double mutants. (D-L) Immunostaining showing the cross-regulation of PIN expression. PIN1 is upregulated in the epidermis of pin2 root (E) compared with wild type (D). Detail showing polar PIN1 localization in epidermis of pin2 root (F). PIN2 is ectopically expressed in the stele of pin1 root (H) compared with wild type (G). Detail showing polar PIN2 localization in pin1 stele (I). PIN4 is upregulated in the stele of pin1 root (K,L) compared with wild type (J). Arrows indicate corresponding expression domains; arrowheads polarity of PIN localization.

View larger version (67K): [in a new window]   Fig. 3. Manipulation of auxin homeostasis leads to ectopic PIN gene expression. (A-H) Inhibition of auxin transport by NPA (50 µmol/l for 24 hours) leads to upregulation of PIN1::PIN1:GFP in epidermis and cortex (B); PIN2::PIN2:HA (D), PIN4::PIN4:GFP (F) and PIN4::GUS (H) in the stele compared with untreated controls (A,C,E,G). (I-L) Treatment for 24 hours with biologically active auxins such as IAA (50 µmol/l, I), 2,4-D (0.1 µmol/l, J) and NAA (10 µmol/l, K) or for 5 days with auxin precursor sirtinol (20 µmol/l, L) leads to an upregulation of PIN4::GUS expression compared with control (G). (M,N) Upregulation of PIN2::GUS expression in root following treatment with 50 µmol/l NAA for 24 hours (N) compared with untreated control (M). (O,P) Treatment for 5 days with auxin precursor sirtinol (20 µmol/l) leads to upregulation of PIN1::PIN1:GFP in epidermis and cortex cells (P) compared with control (O).

View larger version (77K): [in a new window]   Fig. 4. Tissue-specific regulation of PIN gene expression by auxin. (A) Quantitative RT-PCR showing upregulation of PIN gene expression in cotyledons, hypocotyls and roots following auxin treatment (10 µmol/l NAA for 3 hours). Induction of PIN gene expression is depicted relative to the non-induced controls. (B-D) Induction of PIN1::GUS (1 µmol/l, B), PIN3::GUS (0.5 µmol/l, C) and PIN4::GUS (0.5 µmol/l, D) expression in cotyledons, hypocotyls and roots after growing the plants for 4 days on medium containing NAA.

View larger version (94K): [in a new window]   Fig. 5. Time- and concentration-dependence of auxin-regulated PIN gene expression. (A-D) Upregulation of PIN1::GUS (A), PIN3::GUS (B), PIN4::GUS (C) and PIN7::GUS (D) expression after different times and different concentrations of 2,4-D incubation. Inset (D) shows untreated PIN7::GUS roots after prolonged GUS staining. (E) Downregulation of PIN7:GUS fusion protein abundance in PIN7::PIN7:GUS seedlings.

View larger version (61K): [in a new window]   Fig. 6. Quantitative evaluation of auxin-regulated PIN gene expression. (A,B) Quantitative RT-PCRs showing time-dependence (A) and concentration-dependence (B) of the effect of auxin (1 µmol/l 2,4-D in A) on PIN expression. Insets show higher magnifications of early time points (A) and low concentrations (B). The legend in B also applies to A. (C,D) Auxin (1 µmol/l 2,4-D) does not induce PIN expression in slr-1 mutants (C) or after induction of axr3 expression in HS::axr3-1 lines (D), as shown by quantitative RT-PCR. Cycloheximide alone induces PIN expression (C). (E,F) Northern blot (E) and quantitative GUS assays (F) show the time-dependence of auxin (10 µmol/l NAA) effect on PIN2 expression. (G) An expression profiling experiment shows auxin-dependent upregulation of PIN genes, PID and selected primary auxin response genes in differentiated parts of the root grown on NPA. The auxin-dependent upregulation of PIN gene expression is abolished in the slr1 mutant. Induction of PIN gene expression is depicted relative to the non-induced controls.

View larger version (159K): [in a new window]   Fig. 7. Increased auxin levels lead to a decrease in PIN levels in PIN::PIN:GFP roots. (A-C) The PIN7:GFP (A), PIN2:GFP (B) and PIN1:GFP (C) protein levels decrease at higher auxin concentrations. Four-day-old roots were treated with different concentrations of 2,4-D for 24 hours.