Figure 1.
Regulation of PIN6 expression during plant development.
Representative transgenic reporter lines harbouring −1794 bp (pPIN6::GUS; A–N) of the PIN6 promoter fused to GUS. GUS activity was observed early during germination in fresh seeds (A), dried seeds (B), the root apical meristem of seedlings (C; 1DAG), regions of asymmetric root growth (D; 2 DAG) and in the SAM (E; 3 DAG). The PIN6 promoter enabled GUS expression during lateral root initiation (F), at the boundaries of developing root primordia (G to J) and in the lateral RAM (K), floral organ boundaries (L, M) and anthers (N). Scale bars for G–K are similar to F. O) to X) Auxin up regulates PIN6 promoter activity. Scale bars for P-S and U–X are similar to O. Auxin treatment (2 hrs) of pPIN6::GUS transgenic lines with 10 µM 2,4-D (T–X) induces GUS expression in the SAM (T) and root (U and V), but not in the lateral root primordia (W and X). Controls (O–S) were treated with ethanol only. Epidermis (Ep), cortex (Co), endodermis (En) and Pericycle (Pe).
Figure 2.
Characterisation of pin6 mutant alleles.
A) Schematic diagram of the PIN6 genomic DNA fragment showing exon/intron structure (ATG and TGA refer to translation start and stop codons, respectively). PIN6 T-DNA insertions and primer positions (See Table S1 for sequences) are marked by grey arrows. The Salk_046393 (pin6-2), Salk_092831 (pin6-4), GABI-KAT_430B01 (pin6-5) and GK711C09 (pin6-6) T-DNA insertions interrupt intron 2, exon 6, exon 3 and intron 6, respectively. B) Primary root length at 9 DAG (n = 25). (C) The development stage distributions of lateral root primordia that have initiated (I–VII) or emerged (Em) were quantified at 9 DAG (n = 10). D) The density (number of primordia per cm of primary root) of lateral root primordia in early (I–IV) and later (V–Em) stages of development, as well as the total number of primordia (n = 10). Similar results were observed in separate experiments. Homogeneity groups according to Student’s t test (p<0.05) are denoted with same letters. Error bars represent standard error.
Figure 3.
PIN6 overexpression affects leaf pigmentation, shoot development and silique number.
A) PIN6 overexpression binary cassette (pMDC32::PIN6). B) PIN6 mRNA abundance in leaf tissues from four representative lines and normalised to wild type (WT). C) Developing rosettes (4 weeks) from wild type and PIN6-OE lines. D) Rosette leaf area was reduced throughout vegetative development. E) Chlorophyll content was enhanced in mature leaf tissues (µg/g fresh weight). Average±SE (n = 8 plants). F) Floral architecture is modified in overexpression lines. G) Floral stem length is reduced in overexpression lines (n = 2–8 plants±SE). (H) PIN6 overexpression enhances the production of siliques (n = 8 plants±SE). These results are representative of 2–3 independent experiments. When necessary homogeneity groups according to Student’s t test (p<0.05) are denoted with same letters. Error bars represent standard error.
Figure 4.
PIN6 overexpression affects organ development and waving in roots.
A). Root architecture of wild type and PIN6-OE (#33 and #1) seedlings (10 DAG). B) Close up of PIN6-OE#1 waving roots. C) PIN6-OE lines show a reduction in root elongation (10 DAG). D) Root biomass of plants growing in soil was quantified 4 weeks after germination. Average±SE (n = 12). E) and F) Wave amplitude and wave length, respectively, for primary roots. Error bars show standard error (n = 9 to 133) from a single experiment representative of three independent experiments. G) Root hair outgrowth (4 DAG) is disrupted by PIN6 overexpression, when compared to wild type. H) Confocal micrographs show the development of trichoblast cells (PIN6-OE#1) or emergence of root hairs in WT. White arrows indicate root hair cells and PIN6-OE#1 is representative of multiple lines examined. I) Cross section of the primary root from wild type and PIN6-OE#14, respectively showing the position of a H (hair) cell in between to two N (no-hair) cells. Similar results were obtained for PIN6-OE#1. J). Development stage distribution of lateral root primordia initiated (I-VI) as well as emerged (Em) (9 DAG; n = 15) representative of multiple experiements. K) Primordium density of lateral root initiation (In) and emergence (Em) per cm of root length (8 DAG; n = 20). O) and P) Cellular patterning of root primordium at different developmental stages from initiation to emergence in PIN6 OE#1 and wild type (WT). All scale bars represent 10 µm and images are representative of multiple primordia examined. When necessary homogeneity groups according to Student’s t test (p<0.05) are denoted with same letters. Error bars represent standard error.
Figure 5.
PIN6 overexpression perturbs ethylene signalling.
A) to B) Root waving was quantified in terms of number of root waves per mm (spatial waving frequency) and number of waves formed per day (temporal waving frequency), in A) and B) respectively (n = 9 to 133±SE). Misexpression of PIN6 reduces lateral root emergence (C), perturbs root elongation (D), enhances adventitious root formation (E) and has not effect upon adventitious root length (F). Average±SE (n = 9–26). For some experiments, seedlings were germinated on media containing concentrations of the ethylene precursor, ACC. G) PIN6-OE lines show a reduction in root elongation as well as reduced sensitivity to ACC. H) to K) Responses of PIN6-OE to ACC show that the root waves became distorted, indicating altered gravitropism and sensitivity to ethylene compared to wild type. When necessary homogeneity groups according to Student’s t test (p<0.05) are denoted with same letters. Error bars represent standard error.
Figure 6.
PIN6 perturbs inter-cellular auxin homeostasis in the stem, roots and cotyledons.
A) Total transport of 3H-IAA through an inflorescence stem section. Total auxin transport was calculated by adding the radioactivity determined for each 3mm segment isolated from a 30mm stem section. Average±SE (n = 4–12 independent stem sections). B) Auxin transport profile showing movement of 3H-IAA through the inflorescence stem segments 3 hours after loading labelled auxin. PIN1-OE refers to a PIN1 overexpressing line. The auxin transport results presented are representative of at least three independent experiments. C) and D) Root growth assays indicate that PIN6-OE#1 is less sensitive to IAA and IAA-alanine when compared to WT (Student’s t test *p<0.05, n = 30 plants, Error bars represent standard deviation). E) to P) PIN6 overexpression enhances auxin accumulation in specific tissues. Histochemical GUS staining of DR5::GUS activity in germinating seeds (E, H), 3 day old seedlings (F, I) and mature primary root tips (G, J). K) to P) GUS staining of auxin treated (200 nM NAA) root tissues from WT (Fig. 5K to 5M) and PIN6-OE#14 (Fig. 5N to 5P) harboring the DR5::GUS reporter. Where appropriate, asterisks indicate a statistically significant difference (Student’s t test p<0.05).