Figure 1.
PLAT1 expression is induced by salt stress conditions.
Relative PLAT1 and PLAT2 expression in 14-d-old Col-0 seedlings following transfer to salt stress medium compared to control conditions. PLAT1 black bars, PLAT2 grey bars. Values are means of 3 replicates ± standard deviation. n≥10 per replicate. ** or * indicate statistical significance calculated using the unpaired Student's t-test at p<0.01 or p<0.05, respectively.
Figure 2.
PLAT1 loss-of-function reduces abiotic stress tolerance.
(A) Salt stress tolerance in wild-type (Col-0) and plat1-1 seedlings irrigated with 200 mM NaCl for 14 d. n≥10 (B) Drought stress tolerance in wild-type and plat1-1 seedlings, following 14 d without watering. n≥10 (C) Cold stress tolerance in 7-d-old wild-type, plat1-1 and plat1-2 seedlings following 14 d of incubation at 8°C. n = 14. Scale bar = 1 cm.
Figure 3.
PLAT1 promotes tolerance towards salt stress conditions.
Survival, expressed as the percentage of the seedlings transferred at the 6-d-old stage that developed (pale) green leaves during 4 d of salt stress conditions for the different mutant and transgenic lines (grey bars, n≥26 per replicate) compared to Col-0 control plants (black bars, n≥12 per replicate) that were grown on the same plates. Values are means of 3 replicates ± standard deviation. ***, ** or * indicate statistical significance calculated using the unpaired Student's t-test at p<0.001, p<0.01 or p<0.05, respectively.
Figure 4.
PLAT1 loss-of-function reduces ABA sensitivity during seed germination.
(A) Seed germination of plat1-1 and wild-type (Col-0). (B) Seed germination of plat1-1 and wild-type on 300 mM mannitol (osmotic stress). (C) Seed germination of plat1-1 and wild-type on 200 mM NaCl (salt stress). (D) Seed germination of plat1-1 and wild-type on 1.5 µM ABA. Values are means of 3 replicates ± standard deviation. n≥100 per replicate.
Figure 5.
Phenotypes of plants from control medium including 5 µM DEX from the salt stress experiment shown in Figure 3. (A) Wild-type. (B) plat1-1. (C-E) Three independent PLAT1 ectopic overexpression lines (OX). Scale bar = 1 cm. (F) Shoot biomass production (weight per 5 shoots) for the different PLAT1 lines. Values are means of 8 replicates ± standard deviation. ***, ** or * indicate statistical significance calculated using the unpaired Student's t-test at p<0.001, p<0.01 or p<0.05, respectively.
Figure 6.
PLAT1 is expressed in the vasculature, hydathodes and stomata of aerial organs.
PLAT1 expression is reflected by GUS activity in plat1-1 mutant plants rescued by the PLAT1:PLAT1-GUS rescue/reporter construct (line GUS3-5). The PLAT1 promoter confers expression in the leaf vasculature (A, B, E), hydathodes (B, F, G), floral organs (C), stomata (D) and the primary root tip and root pericycle cells (H). PLAT1 expression in primary root tips, 24 h following transfer to control (I), 200 mM NaCl (J) or 1.5 µM ABA plates (K). PLAT1 expression in lateral root tips, 24 h following transfer to control (L), 200 mM NaCl (M) or 1.5 µM ABA plates (N). PLAT1 expression in fully expanded rosette leaf from 4-w-old seedling, control (O), following 24 h (P), or 48 h (Q) of treatment with 1.5 µM ABA and following 24 h (R), or 48 h (S) watering with 200 mM NaCl. Detail of rosette leaf showing expansion of expression domain in leaf mesophyll following 24 h ABA treatment (T), or 48 h watering with NaCl (U). Scale bar = 1 cm (A-C) and (O-S), 1 mm (D-H), (T), (U), 0.1 mm (I-N). n≥10.
Figure 7.
PLAT1 is localised to the ER in Arabidopsis and Nicotiana benthamiana.
Transient transformation of the PLAT1:PLAT1-YFP construct to N. benthamiana. YFP channel showing PLAT1-YFP expression (A), RFP channel showing ER-rk CD3-959 mCherry marker expression (B), co-localisation of PLAT1-YFP with the ER-rk CD3-959 marker (C). PLAT1 expression in stable plat1-1 transgenics (line YFP13-1) rescued by the PLAT1:PLAT1-YFP rescue/reporter construct (D-I) 48 h following transfer of 3-d-old seedlings to control, NaCl or ABA plates, with YFP channel (D-F) and merged YFP and bright field channels (G-I). PLAT1 expression following transfer to control medium (D, G), following transfer to 200 mM NaCl (E, H), and expression following transfer to 1.5 µM ABA (F, I). Hypocotyl section from line YFP13-1 with YFP channel showing PLAT1:PLAT1-YFP reporter activity (J), red autofluorescence of chloroplasts (K), merged image of bright field, YFP and red autofluorescence (L). (M) Detail of (L), showing PLAT1 localisation to putative ER bodies (arrows) and the ER (arrow heads). Scale bar = 10 µm (A-C), 0.1 mm (D-L), 0.05 mm (M). n≥10.
Figure 8.
The PLAT1 promoter is activated by the ABF transcription factors.
GUS activity in 8-w-old N. benthamiana leaves infiltrated with A. tumefaciens harbouring the PLAT1:PLAT1-GUS reporter (pGUS) or an empty vector (EV) as negative control, compared to the simultaneous infiltration of the 35S:ABF1–4 and PLAT1:PLAT1-GUS constructs. Values are means of 3 replicates ± standard deviation. *** or ** indicate statistical significance calculated using the unpaired Student's t-test at p<0.001 or p<0.01, respectively.
Figure 9.
PLAT1 promotes tolerance towards tunicamycin elicited ER stress.
Survival is expressed as the percentage of the plated seeds that developed (pale) green seedlings. Control conditions black bars, ER stress (0.05 µg l−1 TM) grey bars. Values are means of 3 replicates ± standard deviation. n≥70 per replicate. *** or ** indicate statistical significance calculated using the unpaired Student's t-test at p<0.001 or p<0.01, respectively.