Figure 7.
Responses of TRS120 phosphovariants to osmotic or additive stress. Phosphovariants of PTRS120::TRS120:GFP (Rybak et al., 2014) were in trs120-4 homozygous backgrounds with the exception of the TRS120-SαβγD variant in A–C, which was in a hemizygous trs120-4/+ background. (A–C) Germination of TRS120 phosphovariants under osmotic stress. Effect of (A) 0 mM, (B) 200 mM, and (C) 400 mM mannitol on seed germination of the TRS120-SαβγA, the TRS120-SαβγD mutants, and the following controls: Col-0 wild type; the ABA deficient mutant aba2-1 and the ABA PP2C coreceptor triple knock-out line hab1-1 abi1-2 pp2ca-1. The non-phosphorylatable TRS120-SαβγA mutation increased seed germination on higher mannitol concentrations (on day 4, we observed 70% germination in TRS120-SαβγA versus 35% germination for the control on 400 mM mannitol, C), whereas the phosphomimetic TRS120-SαβγD decreased seed germination. Shown are the means ± SD of three technical replicates. Similar results were obtained in three independent biological replicates. Stars indicate statistical significance compared with the wild-type Col-0 calculated with a two-tailed Student’s t test (*: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001). DAS: days after stratification. (D–F) Response quotients (RQ) of (D) the hypocotyl, (E) the root, and (F) the hypocotyl/root ratio of the non-phosphorylatable TRS120-SαβγA and the phosphomimetic TRS120-SαβγD mutants for dark-to-dark with water stress (−0.4 MPa, darkW) adaptation. The divergent hypocotyl and root responses of TRS120-SαβγA and TRS120-SαβγD mutants resulted in a strong hypocotyl/root ratio phenotype under darkW conditions compared with the TRS120-WT control. TRS120-SαβγA showed an enhanced ratio adaption, whereas TRS120-SαβγD mutants had an attenuated ratio response. RQ response quotients are normalized to the rescue mutant TRS120-WT (PTRS120::TRS120:GFP in trs120-4/trs120-4). A value of 1 (vertical red line) corresponds to an identical adaptation to darkW conditions as the rescue mutant. Note that due to the opposite adaptations of the hypocotyl and root under the additive stress conditions (decrease in hypocotyl and increase in root length from dark-to-darkW), the respective thresholds for attenuated and enhanced responses are opposite. Dots represent biological replicates, of which there were a total of four primary transformants per line. Mean RQ values are given on the right. Related to Figs. S12 and S13.

Responses of TRS120 phosphovariants to osmotic or additive stress. Phosphovariants of PTRS120::TRS120:GFP (Rybak et al., 2014) were in trs120-4 homozygous backgrounds with the exception of the TRS120-SαβγD variant in A–C, which was in a hemizygous trs120-4/+ background. (A–C) Germination of TRS120 phosphovariants under osmotic stress. Effect of (A) 0 mM, (B) 200 mM, and (C) 400 mM mannitol on seed germination of the TRS120-SαβγA, the TRS120-SαβγD mutants, and the following controls: Col-0 wild type; the ABA deficient mutant aba2-1 and the ABA PP2C coreceptor triple knock-out line hab1-1 abi1-2 pp2ca-1. The non-phosphorylatable TRS120-SαβγA mutation increased seed germination on higher mannitol concentrations (on day 4, we observed 70% germination in TRS120-SαβγA versus 35% germination for the control on 400 mM mannitol, C), whereas the phosphomimetic TRS120-SαβγD decreased seed germination. Shown are the means ± SD of three technical replicates. Similar results were obtained in three independent biological replicates. Stars indicate statistical significance compared with the wild-type Col-0 calculated with a two-tailed Student’s t test (*: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001). DAS: days after stratification. (D–F) Response quotients (RQ) of (D) the hypocotyl, (E) the root, and (F) the hypocotyl/root ratio of the non-phosphorylatable TRS120-SαβγA and the phosphomimetic TRS120-SαβγD mutants for dark-to-dark with water stress (−0.4 MPa, darkW) adaptation. The divergent hypocotyl and root responses of TRS120-SαβγA and TRS120-SαβγD mutants resulted in a strong hypocotyl/root ratio phenotype under darkW conditions compared with the TRS120-WT control. TRS120-SαβγA showed an enhanced ratio adaption, whereas TRS120-SαβγD mutants had an attenuated ratio response. RQ response quotients are normalized to the rescue mutant TRS120-WT (PTRS120::TRS120:GFP in trs120-4/trs120-4). A value of 1 (vertical red line) corresponds to an identical adaptation to darkW conditions as the rescue mutant. Note that due to the opposite adaptations of the hypocotyl and root under the additive stress conditions (decrease in hypocotyl and increase in root length from dark-to-darkW), the respective thresholds for attenuated and enhanced responses are opposite. Dots represent biological replicates, of which there were a total of four primary transformants per line. Mean RQ values are given on the right. Related to Figs. S12 and S13.

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