Figure 1.
pskr1-2 and pskr1-3 seedlings have shorter hypocotyls due to shorter cells.
(A) Arabidopsis seedlings were grown on media containing PSK-α at concentrations between 1 nM and 1 µM or without PSK-α for 5 days at long-day conditions or in the dark. Results are averages (± SE) of a minimum of >35 hypocotyls analyzed per treatment in 3 independent experiments. No PSK-α-dependent growth response was observed (P<0.001, ANOVA, Tukey test). (B) Hypocotyls of representative wt, pskr1-2, pskr1-3, and pskr2-1 seedlings grown for 5 days in the dark. Average (± SE) hypocotyl lengths determined in 3 independent biological experiments. Letters indicate significantly different values (n≥120, P<0.001, ANOVA, Tukey test). (C) Hypocotyl cell lengths of wt, pskr1-2, pskr1-3, and pskr2-1 seedlings were analyzed and plotted against the cell position. On top, cell numbering of a 5-day-old etiolated wt seedling is indicated from base to top. Cell lengths are averages (± SE) of 20 hypocotyls analyzed per genotype in 2 independent experiments. Cell numbers did not differ significantly between genotypes (P<0.05, ANOVA, Tukey test; see also Table S1 in Supporting Information S1).
Figure 2.
Expression analysis of PSK and PSK receptor genes in seedling shoots.
(A) Promoter:GUS analysis of 5-day-old etiolated PSK1:GUS, PSK2:GUS, PSK3:GUS, PSK4:GUS, PSK5:GUS, and PSKR1:GUS seedlings. (B) Cross sections from hypocotyls indicating activity of PSK2:GUS, PSK3:GUS, PSK4:GUS, and PSK5:GUS in 5-day-old etiolated seedlings. (C) RT-PCR analysis of PSKR1 and PSKR2 expression in hypocotyls of 5-day-old etiolated Arabidopsis seedlings. Actin2 cDNA was amplified as a control for RNA input. As a control for contamination with genomic DNA, RNA was added to PCR reactions.
Figure 3.
tpst-1 seedlings have shorter hypocotyls and are responsive to PSK-α.
(A) 5-day-old etiolated wt and tpst-1 seedlings grown without PSK-α. (B) Hypocotyl lengths of etiolated wt and tpst-1 seedlings treated for 5 days with PSK-α at the concentrations indicated. Asterisks indicate significantly different values to the untreated control. Average (±SE) hypocotyl lengths were determined in 2 independent biological experiments with at least 40 seedlings analyzed per data point (P<0.001, 2-sample t-test). (C) Root lengths of etiolated wt and tpst-1 seedlings treated for 5 days with PSK-α at the concentrations indicated. Asterisks indicate significantly different values to the untreated control. Averages (±SE) were determined in 2 independent biological experiments (n≥40; P<0.001, 2-sample t-test). (D) Hypocotyl cell lengths of 5-day-old etiolated wt seedlings treated with or without 1 µM PSK-α were plotted against the cell position from base to top. Results are averages (±SE) of 20 hypocotyls analyzed in 2 independent experiments. Values are not significantly different (P<0.05, 2-sample t-test). (E) Hypocotyl cell lengths of etiolated tpst-1 seedlings that were treated with or without 1 µM PSK-α for 5 days. Results are averages (±SE) of 20 hypocotyls analyzed in 2 independent experiments. Asterisks indicate statistically significant differences (P<0.05, 2-sample t-test).
Figure 4.
Protoplasts from the Arabidopsis hypocotyl expand in response to PSK-α.
Protoplasts were isolated from etiolated hypocotyls and their volume was determined at 5 min intervals. After 30 min, at t = 0 min, effectors were added and protoplast volumes were recorded for another 35 min. (A) Addition of 0.1 nM or 1 µM PSK-α caused a rapid and continuous increase in protoplast volume whereas unsulfated PSK peptide (usPSK) or 100 nM of the sulfated peptide CCK8 did not (n = 3–5, P<0.05, 2-sample t-test). (B) Dose-response curve of protoplast expansion in wt and pskr1-3 at PSK-α concentrations between 0.01 nM and 1 µM. The net volume change was determined 30 min after addition of PSK-α. Results are averages (±SE) of 3 to 7 protoplasts analyzed per treatment and genotype. Different letters indicate significantly different values (P<0.05, ANOVA, Tukey test). (C) Protoplasts were pre-treated with 50 µM cycloheximide (CHX) for 1 h prior to the addition of 1 µM fusicoccin (FC) or 1 nM PSK-α at t = 0 in addition to CHX. As a control, protoplasts were treated with CHX only. CHX did not inhibit protoplast expansion induced by FC or PSK-α (n = 4–5, P<0.05, 2-sample t-test). (D) Protoplasts from wt, pskr1-2, pskr1-3, and pskr2-1 were treated with 1 nM PSK-α. Protoplasts from wt and pskr2-1, but not from pskr1-2 or pskr1-3 seedlings expanded. Results are averages (±SE) of 3 to 7 protoplasts analyzed per treatment and genotype. Rates of net volume change of wt and pskr2-1 are not significantly different (P<0.05, 2-sample t-test). Expansion of pskr1-2 or pskr1-3 protoplasts is significantly different to wt and pskr2-1 (P<0.05, 2-sample t-test).
Figure 5.
PSK-α induced protoplast swelling is not inhibited by ortho-vanadate.
(A) Protoplasts from wt hypocotyls were left untreated (control), or were treated with 1 nM PSK-α, 1 µM FC, or 1 nM PSK-α+1 µM FC at t = 0. Results are averages (±SE) from 4 to 8 protoplasts analyzed per genotype or treatment. Protoplast expansion was not significantly different between effector treatments (P<0.05, 2-sample t-test). (B) WT protoplasts were pre-treated with 0.5 mM ortho-vanadate for 1 h. At t = 0 min 1 nM PSK-α, 1 µM FC, or 1 nM PSK-α+1 µM FC were added, or protoplasts remained untreated (control). Results are averages (±SE) of 3 or 5 protoplasts analyzed per treatment and genotype. (C) Protoplasts from pskr1-3 hypocotyls were treated with or without 1 µM FC at t = 0. Protoplasts showed a wt swelling response (see A; P<0.05, 2-sample t-test).
Figure 6.
PSK-α does not cause acidification.
(A) pH changes in media containing Arabidopsis hypocotyls. At t = 0 hypocotyls were treated with 10 nM PSK-α or remained untreated (control). After hypocotyls were added, the pH of the buffer dropped from pH 5.7 to pH 5.4 within 130 min due to the pH equilibration between tissue and buffer. Subsequently, PSK-α was applied, or hypocotyls remained untreated (arrow). (B) The ΔpH between control and PSK treated hypocotyls was determined after 0, 30, 60, and 90 min. Results are averages of 5 (controls) or 6 (10 nM PSK-α) experiments with 60 hypocotyls each. The increase in pH was not significant at any time point (P<0.001, 2-sample t-test). In addition, pH changes were determined in media containing 8 coleoptiles from etiolated maize seedlings (n = 3; n.d. - not determined).
Figure 7.
Maize protoplasts expand in a K+-dependent manner.
(A) Protoplasts were isolated from maize coleoptiles and treated with PSK-α at concentrations between 0.05 nM and 10 nM. The dose-response curve was highly similar to that observed with Arabidopsis hypocotyl protoplasts with a maximal response at 0.1 nM PSK-α. Results are averages (±SE) from 3 to 5 protoplasts analyzed per treatment. Letters indicate significantly different values (P<0.001, ANOVA, Tukey test). (B) Maize coleoptile protoplasts were incubated in media containing varying concentrations of K+. After addition of 0.1 nM PSK-α at t = 0 min, protoplast swelling was recorded for another 100 min. Maximal protoplast expansion was observed at 10 mM K+ whereas a weaker response was observed at 1 mM K+. Without K+ in the media, protoplasts did not expand (P<0.001, ANOVA, Tukey test).