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First published online 11 September 2008
doi: 10.1242/dev.021071

Development 135, 3345-3354 (2008)
Published by The Company of Biologists 2008
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Interaction of PIN and PGP transport mechanisms in auxin distribution-dependent development

Jozef Mravec1,2, Martin Kubes3,4, Agnieszka Bielach1,2, Vassilena Gaykova2, Jan Petrásek3,4, Petr Skupa3,4, Suresh Chand2, Eva Benková1,2, Eva Zazímalová3 and Jirí Friml1,2,*

1 Department of Plant Systems Biology, VIB, and Department of Molecular Genetics, Ghent University, 9052 Gent, Belgium.
2 Center for Plant Molecular Biology (ZMBP), University of Tübingen, D-72076 Tübingen, Germany.
3 Institute for Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Praha 6, Czech Republic.
4 Department of Plant Physiology, Faculty of Science, Charles University, Vinicná 5, 128 44 Praha 2, Czech Republic.


Figure 1
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  		Fig. 1. Identical phenotypes indicative of auxin starvation in DEX-induced expression of PIN7 or PGP19 in BY-2 cells. (A,B,D,E) Effect of DEX induction in GVG-PIN7 and GVG-PGP19-HA tobacco cell lines. Non-induced GVG-PIN7 (A) and GVG-PGP19-HA line (D). GVG-PIN7 (B) and GVG-PGP19-HA (E) cells after 3 days of cultivation with DEX, showing decrease in cell division, increase in cell elongation and formation of starch-containing amyloplasts (arrows in B,E). (C,F) Chemical inhibition of auxin transport (10 µM NPA) reversing these defects in DEX-induced GVG-PIN7 cells (C) but not in DEX-induced GVG-PGP19-HA cells (F). Scale bars: 20 µm. (G) Depiction (reciprocal plots) of the cell size distribution (cell length and cell diameter) after NPA treatment (10 µM, 3 days) in DEX-induced GVG-PIN7 and GVG-PGP19-HA cells scored at day 3 after inoculation. Non-induced GVG-PIN7 and GVG-PGP19-HA cells were used as a control.

 

Figure 2
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  		Fig. 2. Differential effect of PIN1 and PGP19 overexpression in Arabidopsis seedlings. (A,B) Immunolocalization of PIN1 in the XVE-PIN1 line without (A) and with estradiol (B) induction. Ectopically expressed PIN1 localizes to the basal (lower) side of epidermal cells (arrowheads). (C,D) Immunolocalization of DEX-induced PGP19-HA in GVG-PGP19-HA (C) and PGP1-myc in GVG-PGP1-myc (D) lines show non-polar localization in epidermal cells. (E-H) Differential effects of PIN1, PGP19-HA and PGP1-myc overexpression on seedling development. Non-induced control (E); reduced root length and gravitropic response by induced PIN1 expression (F); no dramatic phenotypes caused by induced PGP19-HA (G) and PGP1-myc (H) expression, apart from a reduction in cotyledon outgrowth in the DEX-treated GVG-PGP19-HA line (G). (I-K) PIN1 overexpression phenotypes in dark-grown seedlings: straight hypocotyls in untreated controls (I); hypocotyl twists in estradiol-treated seedlings (J); this phenotype is almost completely reversed by the auxin transport inhibitor NPA (K). (L-O) Changes in the DR5::GUS auxin response reporter expression after PIN1 overexpression: DR5::GUS is weakly and equally expressed in hypocotyls of dark-grown seedlings (L), but shows randomly distributed local maxima that correlate with unequal cell elongation after PIN1 induction (M); GUS signal in the root tip is confined to the columella in the non-induced control (N), but increases and extends to the lateral root cup after PIN1 induction (O). (P,Q) Reduction of DR5rev::GFP signal in the columella after PGP19-HA expression (Q) when compared with untreated controls (P). (R) Concentration-dependent effect of estradiol-induced PIN1 overexpression on root elongation and gravitropism (calculated as vertical growth index (VGI) (Grabov et al., 2005Go). (S) Hypocotyl twisting and inhibition of root length following PIN overexpression can be reversed by NPA. (T) PGP1-myc and PGP19-HA overexpression have no pronounced effects on root growth, hypocotyl growth in the dark or sensitivity to NPA. Scale bars: 3 mm. Error bars represent s.e.m., n=20.

 

Figure 3
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  		Fig. 3. Expression and localization of PGP1 and PGP19 during Arabidopsis embryogenesis. (A,B) Immunolocalization of PGP1-myc in Arabidopsis embryos (PGP1 in red, DAPI in blue). Expression of PGP1-myc in all cells and non-polar localization to the plasma membrane at octant (o) (A) and mid-globular (mg) (B) stages. Inset shows staining in the suspensor (s). (C,D) PGP19-GFP localization during early embryogenesis. PGP19-GFP localizes apolarly to the plasma membrane in derivatives of the basal cells at the octant stage (C) and in the suspensor and lower tier cells at the dermatogens (d) stage (D). (E-J) Restriction of the expression of PGP19-GFP at later stages of embryogenesis to protoderm and cells surrounding the vascular primordium, which is mainly complementary to PIN1 expression. Immunolocalization at late-globular (lg) (E-G) and mid-heart (mh) stages (H-J) of PGP19-GFP (green) (E,H), PIN1 (red) (F,I). (G,J) Overlay of PIN1, PGP19 and DAPI (blue).

 

Figure 4
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  		Fig. 4. Genetic interaction of PGPs with PIN1 during embryonic leaf formation. (A-D) Synergistic interaction of pgp1pgp19 and pin1 during cotyledon formation. Typical defects in cotyledon formation during embryogenesis and their postgermination appearance are shown: wild type (A), pin1 (B), pgp1pgp19 (C) and pin1pgp1pgp19 (D). (E) Cup-shaped cotyledons of the pin1pgp1pgp19 seedling that are rarely seen in the pin1 mutant. (F) Strong enhancement of the pin1 phenotype in post-embryonal development by the pgp1pgp19 mutation. An adult, 5-week-old, plant with extremely dwarf appearance, reduced leaf number and apical dominance is shown. (G) Quantification of frequencies of cotyledon defects in different mutants and their combinations (n=200). Scale bars: 1 mm in A-D; 5 mm E,F.

 

Figure 5
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  		Fig. 5. Genetic interaction of PGPs with RCN1. (A-D) Aberrant cell divisions of the hypophysis at the globular stage in rcn1pgp1pgp19 mutant embryos. The wild-type hypophysis divides into two derivatives: the smaller lens-shaped cells and bigger basal cells (A). Different aberrations in the cell division of the rcn1pgp1pgp19 mutant (B-D). (E,F) Rootless (E) and cotyledon patterning (F) defects in rcn1pgp1pgp19 seedlings (n=97). (G) Enhanced defects in root elongation and gravitropism in 10-day-old seedlings of rcn1pgp1pgp19 as compared with controls. (H,I) Defects in root tip organization, visualized by a lugol staining in rcn1pgp1pgp19 (I) when compared with wild type (H). (J) Quantification of root length and gravitropism phenotypes of the rcn1pgp1pgp19 mutant. For comparison, pin2 and pin2pgp1pgp19 data are also included (n=25). Scale bars: 2 mm. Error bars represent s.e.m.

 

Figure 6
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  		Fig. 6. Post-embryonic expression and the role of PGP1/PGP19 in lateral root development. (A,B) Expression and localization of PGP1-GFP and PGP19-GFP in root tips of 5-day-old seedlings. PGP1-GFP is expressed in all cells, except the columella (A); PGP19-GFP expression is more restricted to endodermal and pericycle cells (B). (C-F) Expression of PGP1-GFP and PGP19-GFP in hypocotyls and main root. PGP1-GFP is expressed in all cells of hypocotyls (C) and main root (D), whereas PGP19-GFP expression is more restricted to cells surrounding vascular tissues in hypocotyls (E) and main root (F). h, hypocotyl; r, root. (G,H) Expression of PGP1-GFP and PGP19-GFP during lateral root development. PGP1-GFP expression is detected in all cells during all stages (G) (indicated) and that of PGP19-GFP is more confined at later stages (indicated) to the new forming endodermal and pericycle cells (H). Arrowheads indicate the localization of PGP1/PGP19-GFP on anticlinal membranes at stage I. (I) Initiation and (J) emergence phenotypes of pgp1, pgp19, pin1, pgp1pgp19 and pin1pgp1pgp19 mutants (n=40).

 

Figure 7
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  		Fig. 7. Role of PGPs and PINs in the regulation of the spatial distribution of the auxin response. (A-H) Roles of PGP1/PGP19 and PIN1 in auxin response distribution (as visualized by DR5rev::GFP) in heart-stage embryos (A-D) and root tips (E-H). Wild type (A,E). Increased signal in pgp1pgp19 (B,F), decreased signal in pin1 (C,G) and pronounced defects in the distribution of the DR5 signal in the pin1pgp1pgp19 embryos (D) are seen, but restoration occurs in roots (H). At least five roots or embryos from all mutant combination were simultaneously analysed in two independent experiments (for pin1 and pin1pgp1pgp19 mutants, only embryos with visible phenotypes were analysed). Arrowheads in A-D indicate auxin maxima in cotyledon primordia.

 

Figure 8
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  		Fig. 8. Model for interaction of PGPs and PINs in the local auxin distribution in meristematic tissues. (A) Enhanced effects (~20%) of estradiol-induced PIN1 overexpression on root length and hypocotyl twisting in the pgp1pgp19 mutant when compared with wild type, confirming the antagonistic roles of PIN1 and PGP1/PGP19 in seedling development. Error bars represent s.e.m., n=20. (B) Immunolocalization of PIN2 and PGP19-HA. Polar and non-polar localization of PIN2 and PGP19-HA in the root epidermis, respectively. Expression of PGP19 is higher in the endodermis and the pericycle that form the border between acropetal and basipetal auxin streams. (C) Model of PIN and PGP interaction. PGPs and PINs interact intermoleculary at the PIN-containing polar domain, possibly regulating the PIN stability in the plasma membrane. The PGPs remaining in these cells control the cellular auxin pool available for the PIN transport. In pgp1pgp19, the cellular auxin concentration is increased and, therefore, the PIN transport is enhanced but less focused.

 

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© The Company of Biologists Ltd 2008