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Fig. S1. Effects of PIN and PGP overexpression in BY-2 cells. (A-D) DEX-induced expression of PIN4 and PIN6, resulting in similar phenotypes in GVG-PIN4 and GVG-PIN6 BY-2 cells. The phenotype of non-induced GVG-PIN4 (A) and GVG-PIN6 cells (B) after 3-day-long cultivation. DEX induction (1 µM) resulted in the stimulated cell elongation and amyloplasts formation (arrows) in both GVG-PIN4 (C) and GVG-PIN6 (D) cells. Scale bars: 40 µm. (E-G) Induction of BY-2 cell elongation and starch accumulation by auxin starvation. BY-2 cells cultivated in the medium without 2,4-D (E) show defects similar to those after induction of PIN7 or PGP19 expression in GVG-PIN7 and GVG-PGP19-HA lines, namely inhibition of cell division, enhanced elongation and accumulation of starch granules. Non-induced (F) and DEX-induced (G) GVG-PIN7 cells. Identity of the starch granules (marked by arrows) was confirmed by lugol staining. Scale bars: 20 µm. (H) Quantification of increased cell lengths in DEX-induced (+) and non-induced (−) GVG-PIN4, GVG-PIN6 and GVG-PIN7 cell lines (1 µM DEX, 3 days). Similar increase in cell length was observed in cells constitutively overexpressing PIN-GFP in PIN1::PIN1-GFP line at day 3 of the subculture interval. Constitutive expression of PIN1 under natural promoter results in overall prolongation of subculture interval accompanied with decrease in cell division activity (data not shown). Error bars represent standard errors of the mean (n=400). (I) Decreased accumulation of 3HNAA in PIN1::PIN1:GFP cells (Benková et al., 2003; Zazímalová et al., 2007) reflects increased auxin efflux in comparison with control BY-2 cells. Interestingly, decrease in the 3HNAA accumulation by ∼35% corresponds well with the already reported decrease following inducible expression of Arabidopsis PIN 4, 6 and 7 in BY-2 cells (Petrasek et al., 2006). Error bars represent s.e.m. (n=4); where invisible, they are covered by the symbols. (J,K) Quantification of the effect of DEX and DEX/NPA treatment in GVG-PIN7 and GVG-PGP19-HA lines. (J) For better comprehensibility of our results (Fig. 1G), upper limit (90 µm) for the length of all variants were determined. After DEX induction, the percentage of oversized cells increased in both GVG-PIN7 and GVG-PGP19-HA lines. However, the effect of the NPA treatment was clearly different between GVG-PIN7 and GVG-PGP19-HA lines. Although in the GVG-PIN7 line, the number of cells exceeding the determined limits was restored to levels nearly the same as in the non-induced variant, increased length and diameter of GVG-PGP19-HA was not restored with NPA. (K) The table shows the arithmetic means of all measured cell lengths and diameters expressed with standard error (170 cells in each sample).
Fig. S2. Analysis of inducible expression of PGP1-myc and PGP19-HA in GVG-PGP lines. (A-D) Inducibility of PGP1 and PGP19 in GVG-PGP19-HA (A,C) and GVG-PGP1-myc (B,D) lines confirmed by GUS staining of co-regulated UAS::GUS reporter construct (A,B) and by RT-PCR (C,D). Strong GUS staining was observed in seedlings germinated on DEX plates either in the light or in darkness.
Fig. S3. Complementation of the pgp19 and pgp1 mutant phenotypes by the pPGP19::PGP19-GFP and pPGP1::PGP1-GFP constructs. (A-C) Morphological phenotype of pgp19 mutant (epinastic cotyledons and wavy root growth) in seedlings (A); leaf phenotype in adult plants (B) and partially lateral root formation phenotype (C) could be complemented by the pPGP19::PGP19-GFP construct (PGP19-GFP). (D,E) Lateral root initiation phenotype of pgp1 mutant could not be complemented by the pPGP1::PGP1-GFP (PGP1-GFP) construct (D), whereas the hypocotyl elongation phenotype of pgp1 could be complemented by the pPGP1::PGP1-GFP construct (E).
Fig. S4. Opposite effect of pgp1pgp19 and pin2 mutations on root gravitropism. (A) Normal bending and relocation of the DR5::GFP signal in root tip epidermis after 5 hours of gravistimulation in wild type. (B) Enhanced relocation of DR5::GFP signal in pgp1pgp19 reflects the enhanced gravitropic bending. The gravitropism diagram was created as described (Petrásek et al., 2006) (12 hours of gravity stimulation, n=40).
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