Expression levels of flowering control genes after complementation of the <i>pp2a-b’γ</i> mutant.

<p>Shoots were harvested ten days after germination, 12 h into the 16 h photoperiod. Gene expression was tested in WT, and the mutants, <i>pp2a-b’γ, pp2a-b’γ-complemented</i>, and e<i>lf6</i> (control). Genes tested were: <b>a</b>) <i>FLC</i>, <b>b</b>) <i>SOC1</i>, and <b>c</b>) <i>FT</i>. The <i>pp2a-b’γ</i> mutant showed normal WT expression levels when complemented with the <i>35S-PP2A-B’γ</i> gene construct. Data presented are means of three independent experiments for <i>FLC</i> expression and two for <i>SOC1</i> and <i>FT</i>. Each sample contained 50 plants and was assayed in triplicate. Vertical bars indicate the standard error of the mean.</p

A schematic model for involvement of PP2A in flowering time pathways in <i>Arabidopsis</i>.

<p>Expression of key genes of each pathway, <i>CO</i>, <i>FLC</i>, and <i>MYB33</i> as well as <i>FT</i> and <i>SOC1</i> of the floral integrator were tested. <i>SPL3</i> and <i>miR156</i>, which can influence flowering by an endogenous pathway acting downstream of the floral integrator were also tested. <i>ELF6</i> and <i>EDM2</i> genes are known to delay and advance flowering, respectively. Mutants (knockout) lines of <i>elf6</i> and <i>edm2</i> were included as control lines. Genes (transcripts) tested in this work are shown in blue. Genes mutated in <i>Arabidopsis</i> lines tested in this work are shown in red. The work showed that <i>PP2A-B55</i> was a negative regulator of flowering time possibly acting downstream of/at the floral integrator, whereas <i>PP2A-B’γ</i> was a positive component in flower induction acting through modulation of <i>FLC</i> expression.</p

Expression levels of genes important in different flowering time controlling pathways and the floral integrator.

<p>Shoots were harvested ten days after germination, 12 h into the 16 h photoperiod. Gene expression was tested in WT and the mutants <i>pp2a-bα SALK_09504</i>, <i>pp2a-bβ SALK_062614</i>, <i>pp2a-b’γ</i>, e<i>lf6</i> (early flowering control) and <i>edm2</i> (<i>late flowering control</i>). Genes tested were: <b>a</b>) <i>FLC</i>, <b>b</b>) <i>CO</i>, <b>c</b>) <i>MYB33</i>, <b>d</b>) <i>SOC1</i>, <b>e</b>) <i>FT</i> and <b>f</b>) <i>SPL3</i>. Expression of established flowering pathway genes are modulated in <i>pp2a-b’γ</i> consistent with this mutant being late flowering, whereas <i>pp2a-b55</i> mutants show only minor changes in transcript levels and may act on flowering time through an unknown pathway. Data presented are means of three (except for <i>SPL3</i>, which had two) independent experiments of samples each containing 50 plants and assayed in triplicate. Vertical bars indicate the standard error of the mean.</p

Visible phenotypes of representative <i>pp2a</i> mutants.

<p><b>a</b>) Representative plants of WT and early flowering mutant lines <i>pp2a-bα</i> (SALK_09504) and <i>pp2a-bβ</i> (SALK_062614). <b>b</b>) Mutant line <i>pp2a-b’γ</i> complemented with 35S::PP2A-B’γ showing flowering time as WT, late flowering line <i>b’γ</i> (SALK_039172), and WT plants. Plants were grown in 16 h days.</p

Growth of WT, <i>PP2A-b55α</i>, and <i>PP2A -b55β</i>.

<p>Number of leaves per plant 16 days after germination, and mean leaf fresh weight 21 days after germination. For each genotype and treatment 45 plants were scored. SE is given.</p

Flowering time for WT, <i>pp2a-bα</i>, <i>pp2a-bβ</i> and <i>pp2a-b’γ</i> lines grown in 8, 12, 16 or 24 h days.

<p><b>a</b>) Days until first bud (green columns) and first flower (white columns) for WT, <i>pp2a-bα</i> (SALK_09504), <i>pp2a-bβ</i> (SALK_062614) in 12 h and 24 h days. <b>b</b>) Days until first bud (green columns) or first flower (white columns) for WT, <i>pp2a-bα</i> (SALK_032080), <i>pp2a-bβ</i> (GK_ 290G04) in 12 h and 24 h days. <b>c</b>) Numbers of leaves at first flower (dark green columns) and number of days to first flower (white columns) for WT, <i>pp2a-bα</i> (SALK_09504), <i>pp2a-bβ</i> (SALK_062614) in 8 h and 16 h days. <b>d</b>) Numbers of leaves at first flower (dark green columns) and number of days to first flower (white columns) for WT, and <i>pp2a-b’γ</i> (SALK_039172) in 8 h and 16 h days. The data show that the four <i>pp2a-b55</i> mutant lines tested were early flowering, and the <i>pp2a-b’γ</i> mutant line was late flowering under all conditions tested. For each genotype and treatment 30 plants were scored. SE is given. Columns marked with one or two stars are significantly different from WT at p < 0.05 and p < 0.01, respectively (student’s t-test).</p

A conserved glycine is important for MPK4 and MPK12 function.

<p>(A) Inhibition of HT1 kinase activity in vitro by MPK4 and MPK4 G55R. Upper panel: autoradiography of the SDS PAGE gel; lower panel: Coomassie-stained SDS PAGE. Reaction mixture was incubated for 30 min. (B) Whole protein (left) and close-up (right) view of the superposition of models for MPK12 wild-type (secondary structure and surface in white) and MPK12 G53R (secondary structure in green). There is a close structural similarity between the structures except where the arginine at position 53 protrudes from the mutant protein surface and changes the loop region for the mutant. (C) Whole protein (left) and close-up (right) view of the superposition of models for MPK4 wild-type (secondary structure and surface in white) and MPK4 G55R (secondary structure in yellow). Similar to MPK12 G53R, the arginine at position 55 in MPK4 protrudes from the mutant protein surface and changes the loop region.</p

Stomatal conductance of the NIL Col-S2, <i>mpk12</i> mutants, and complementation lines.

<p>(A) Diurnal pattern of stomatal conductance with 12 h/12 h light–dark periods (<i>n</i> = 13–16). (B) Instantaneous water use efficiency (WUE) measured as an average of daytime light period from 09:00 to 17:00 (<i>n</i> = 13–16). (C) Stomatal conductance of Cvi-0 transformed with Col-0 <i>MPK12</i> driven by its native promoter in T2 generation (<i>n</i> = 9). (D) Stomatal conductance of Col-S2 complementation line in T2 generation transformed with Col-0 <i>MPK12</i>, driven by its native promoter (<i>n</i> = 5–8). (E) Stomatal conductance of T3 transformants in the <i>mpk12-4</i> background transformed with either the Col-0 or Cvi-0 version of <i>MPK12</i>, driven by its respective native promoter (<i>n</i> = 5–6). All graphs present mean ± SEM. Small letters denote statistically significant differences according to one-way ANOVA with Tukey HSD post hoc test for either unequal (B, D, E) or equal sample size (C). The raw data for panels A–E can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000322#pbio.2000322.s011" target="_blank">S1 Data</a>.</p

MPK12 interacts with HT1.

<p>(A) Split-ubiquitin yeast two-hybrid assay on the SD-LeuTrp plate (left and middle panels) indicates the presence of both bait and prey plasmids; X-gal overlay assay (middle) and growth assay on the SD-LeuTrpHisAde plate (right) show HT1 interaction with MPK12 that is similar to the positive control (pAI-Alg5). Only weak or no interaction was detected with MPK12 G53R and MPK11, similar to the negative control (pDL2-Alg5). (B) Quantitative β-galactosidase assay from pools of ten colonies each. Activities are shown as the percentage of the positive control (± SEM; <i>n</i> = 3). (C) High-magnification (63x objective) BiFC images from a single infiltrated <i>N</i>. <i>benthamiana</i> leaf with identical confocal microscopy acquisition settings. Scale bar = 50 μm. (D) Ratiometric BiFC shows weaker interaction of MPK12 G53R than MPK12 with HT1, while MPK11 exhibits a weak interaction with HT1. The plasma membrane–localized SLAC1-CFP was used as an internal control. Eighteen images (from three leaves) of each construct set were analyzed. (E) Western blot together with Coomassie staining of proteins extracted from BiFC samples used for confocal imaging and controls with single construct shows expression of all fusion proteins. (F) Steady-state stomatal conductance of Col-S2 <i>ht1-2</i>, <i>mpk12-4 ht1-2</i>, and Col-S2 <i>abi1-1</i> (<i>ABA insensitive 1–1</i>) double mutants (mean ± SEM, <i>n</i> = 11–13). Experiments were repeated at least three times. Letters in B, D, and F denote statistically significant differences with one-way ANOVA and Tukey HSD post hoc test for equal B, D, or unequal F sample size. The raw data for panels B, D, and F can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000322#pbio.2000322.s011" target="_blank">S1 Data</a>.</p

Regulation of HT1 by MPK12.

<p>(A) Inhibition of HT1 kinase activity in vitro by different versions of MPK12 (MPK12 G53R—Cvi-0 version of MPK12; MPK12 K70R—inactive kinase; MPK12 Y122C—hyperactive kinase). Upper panel: autoradiography of the SDS PAGE gel; lower panel: Coomassie-stained SDS PAGE. Reaction mixture was incubated for 30 min. (B) Casein phosphorylation by HT1 with different MPK12 concentrations (mean ± SEM; <i>n</i> = 3). The raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000322#pbio.2000322.s011" target="_blank">S1 Data</a>. (C) Kinase-dead HT1 K113M was not in vitro phosphorylated by different versions of MPK12, and only MPK12 and MPK12 (Y122C) display clear autophosphorylation activities.</p