Table 1.
Oligonucleotides used for qRT-PCR in this work.
Figure 1.
Plasma membrane localization of PCC1 protein.
(A) Bioinformatic prediction of transmembrane potential for PCC1 using TMPred tool. The C-terminal domain with positive potential is written in blue in the amino acid sequence below the plot. (B) Extraction of PCC1 protein from Nicotiana benthamina leaves transiently transformed with 35S::PCC1-GFP construct was assessed by using TBS buffer supplemented or not with different detergents as indicated, and further detected by Western blot with anti-GFP antibodies. Ponceau S-stained Rubisco is shown as loading control. (C) Expression of GFP-tagged versions of PCC1 in its C- (PCC1-GFP) and N-terminus (GFP-PCC1) as wells as the free GFP control (GFP-stop-PCC1) was analyzed by Western blot with anti-GFP antibodies and confocal microscopy. (D) Expression of a GFP-tagged truncated PCC1 version (Δ177-PCC1-GFP) without the potential C-terminal membrane-associated domain leads to cytoplasmic localization instead of the membrane localization for the whole GFP-PCC1 protein. (E) Isolation of protoplasts from transiently transformed Nicotiana benthamina leaves confirmed the plasma membrane association of PCC1 and allowed to rule out cell wall localization.
Figure 2.
Plasma membrane localization of PCC1 in transgenic 35S::PCC1-GFP Arabidopsis plants.
(A) Levels of PCC1-GFP protein in three independent homozygous lines and control C non-transformed plants were analyzed by Western blot with anti-GFP antibodies. PonceauS-stained Rubisco is shown as loading control. Transgenic lines 1.6 and 3.8 with maximal PCC1 expression showed GFP-associated fluorescence by confocal microscopy in the plasma membrane. (B) Membrane-associated localization of PCC1-GFP contrasts with Δ177PCC1-GFP that localizes in both the cytoplasm and nucleus. The second row of images for every genotype shows magnification of stomata guard cells.
Figure 3.
(A) The fusion of PCC1 with the activation domain (AD) of GAL4 is expressed in Mav203 yeast strain as shown by Western blot with anti-AD antibodies. A negative control E and the non-transformed yeast are also shown (top panel). Growth of yeasts co-transformed with AD-PCC1 and PCC1 fused to the DNA binding domain of GAL4 (BD-PCC1) but not with AD-PCC1 and the empty BD vector in minimal media –Leu – Trp –His is indicative of self-interaction of PCC1 in yeast two-hybrid. (B) Bimolecular fluorescence complementation (BiFC)-based demonstration of PCC1-homodimerization and localization of dimers in the plasma membrane of Nicotiana benthamiana leaves transiently co-transformed with the indicated constructs. (C) Confirmation of PCC1 homodimerization by pull-down assays in Nicotiana benthamiana leaves transiently co-transformed with PCC1-HA and GFP- and c-myc-tagged versions of PCC1 as indicated. Immunoprecipitation (IP) was performed with anti-HA and pulled-down proteins detected by Western blot with the polyclonal antibodies indicated. (D) Homodimerization of PCC1 required the transmembrane C-terminal domain as demonstrated by pull-down assays in Nicotiana benthamiana leaves transiently co-transformed with PCC1-HA and GFP-tagged versions of complete and truncated PCC1 molecules. IPs using anti-HA and anti-GFP followed by WB using the indicated antibodies are shown at the left.
Figure 4.
Spatial pattern of PCC1 expression analyzed with pPCC1::GUS transgenic plants.
GUS-stained seedlings of (A) 4 days after sowing (d.a.s.); (B) 6 d.a.s.; (C) 8 d.a.s.; (D) 10 d.a.s.; (E) 14 d.a.s. (F) Detail of GUS-stained guard cells of seedling shown in (A). (G) Leaf showing showing staining from the petiole to the distal parts. (H) and (I) Control untreated and 0.1 mM SA-treated 14-day old seedlings, respectively. (J) Vascular tissue stained in the upper part of roots. (K) Absence of GUS staining in the elongation zone and tip of roots. (L) Detail of stained calyptra. (M) and (N) Absence of expression in flower and siliques, respectively. (O) Time-course of GUS staining in cotyledons at different times after germination showing the stomata- and vascular tissue-associated patterns. The generation of pPCC1::GUS transgenic lines and the protocols used for GUS staining were previously reported [33].
Figure 5.
PCC1 does not interfere with photoperiod dependent floral transition pathway nor interacts with its regulatory components.
(A) PCC1, CO and FT transcript levels in wild type, double transgenics iPCC1/35S::CO-GR and their parental plants in the absence and presence of 10 µM of the GR ligand dexamethasone (DEX). (B) Flowering time quantified by counting total (rosette plus cauline) leaves in long day-grown plants of the genotypes described in (A) treated or not with DEX. * represents statistically significant (p<0.05 in Students t-test) different values in DEX-treated compared to untreated (Mock) seedlings. (C) Analysis of potential interactions between PCC1 and CO or FT by BiFC. The interaction between FT and FD in nuclei is shown as positive control.
Figure 6.
Light- and gibberellin-related hypocotyl phenotypes of iPCC1 plants.
Hypocotyl length of seedlings grown under (A) 10 µmole m−2 s−1 of red light (RL), 30 µmole m−2 s−1 of blue light (BL), or 5 µmole m−2 s−1 of far-red light (FRL) for 4 days, and (D) the same light conditions as in (A) but treated as indicated with the gibberellin synthesis inhibitor paclobutrazol (PAC). (B) PCC1 transcript levels were quantified by qRT-PCR in wild type Col-0 and the indicated photoreceptor mutant seedlings. Values are the mean of three independent biological replicates ± SD and are expressed as relative levels to those detected in wild type seedlings. (C) Hypocotyl length of seedlings grown under 60 µmole m−2 s−1 of white light (WL), with the indicated µM concentrations of GA3 (GA). Values are the mean of 20 hypocotyls per genotype and condition ± SD. * and ** represents statistically significant (p<0.05 or p<0.01, respectively, in Students t-test) different values in iPCC1 seedlings when compared to wild type seedlings under the same condition. (E) Transcript levels of the indicated genes involved in the perception and signaling of gibberellins were analyzed by qRT-PCR in Col-0 and iPCC1 seedlings grown under 16 h light/8 h darkness photoperiodic white light conditions for 14 days. Values are the mean of three independent biological replicates ± SD and are expressed as relative levels to those detected in wild type seedlings. * represents statistically significant (p<0.05 in Students t-test) different values in iPCC1 seedlings when compared to wild type seedlings.
Figure 7.
Functional interaction between PCC1 and the subunit 5 of the COP9 signalosome.
(A) Growth in -Leu-Trp-His media of yeasts co-transformed with clone A8 corresponding to CSN5B subunit of COP9 signalosome fused to the GAL4 activation domain (pB66-A8) and either PCC1 fused to the GAL4 DNA binding domain (pP6-PCC1) or the empty vector (pP6). Growth was tested in increasing concentration of 3-aminotriazol (3-AT). (B) Subcellular localization of CSN5B in cytoplasm and nucleus of Nicotiana benthamiana leaves transformed with 35S::GFP-CSN5B. (C) Interaction of PCC1 with CSN5A and CSN5B in the plasma membrane as demonstrated by BiFC in Nicotiana benthamiana leaves transiently co-transformed with the indicated constructs. (D) Levels of free and rubylated (RUB-) forms of CUL 1 in wt and iPCC1 plants as shown by Western blot with anti-CUL1 polyclonal antibodies. Ponceau S-stained Rubisco is shown as loading control.