Mitogen-Activated Protein Kinase Cascade MKK7-MPK6 Plays Important Roles in Plant Development and Regulates Shoot Branching by Phosphorylating PIN1 in Arabidopsis

Abstract

Emerging evidences exhibit that mitogen-activated protein kinase (MAPK/MPK) signaling pathways are connected with many aspects of plant development. The complexity of MAPK cascades raises challenges not only to identify the MAPK module in planta but also to define the specific role of an individual module. So far, our knowledge of MAPK signaling has been largely restricted to a small subset of MAPK cascades. Our previous study has characterized an Arabidopsis bushy and dwarf1 (bud1) mutant, in which the MAP Kinase Kinase 7 (MKK7) was constitutively activated, resulting in multiple phenotypic alterations. In this study, we found that MPK3 and MPK6 are the substrates for phosphorylation by MKK7 in planta. Genetic analysis showed that MKK7-MPK6 cascade is specifically responsible for the regulation of shoot branching, hypocotyl gravitropism, filament elongation, and lateral root formation, while MKK7-MPK3 cascade is mainly involved in leaf morphology. We further demonstrated that the MKK7-MPK6 cascade controls shoot branching by phosphorylating Ser 337 on PIN1, which affects the basal localization of PIN1 in xylem parenchyma cells and polar auxin transport in the primary stem. Our results not only specify the functions of the MKK7-MPK6 cascade but also reveal a novel mechanism for PIN1 phosphorylation, establishing a molecular link between the MAPK cascade and auxin-regulated plant development.

Fig 1. MKK7 can phosphorylate MPK3 and MPK6 in vitro and in vivo.

(A) In vitro kinase assays of phosphorylation of MPK3 and MPK6 by constitutively activated MKK7 (cMKK7). The cMKK7 was incubated with MPK3 or MPK6 in the kinase reaction buffer. Aliquots of the samples were separated by SDS-PAGE and subjected to autoradiography. Arrows indicate positions of the detected proteins. (B) Phosphorylation of MPK3 and MPK6 in planta. Samples were prepared from 21-d-old seedlings and subjected to immunoblot analysis with antiphospho-p44/p42 antibody. The Ponceau-stained western blot was used as the loading control.

Fig 2. Genetic and morphological analysis of Col-0, bud1, mpk3, mpk3bud1, mpk6, and mpk6bud1 plants.

(A) Vascular systems of cleared specimens of Col-0, bud1, mpk3, mpk3bud1, mpk6, and mpk6bud1 plants. The seedlings grown on MS plates for 12 d were taken with the same magnification. The upper panel refers to the vascular system of cotyledon and the lower to leaves. (B) The filament elongation of Col-0, bud1, mpk3, mpk3bud1, mpk6, and mpk6bud1 flowers grown under long day conditions. (C) Gravitropic responses of dark-grown seedlings. Seedlings were grown in the dark for 4 d. The plates were reoriented by 90° and photographed after 18 h of gravistimulation. Bars, 0.5 cm. (D) Kinetic analysis of hypocotyl gravitropism. Seedlings were grown in the dark for 3 d on 0.5 × MS plates and reoriented by 90°. The gravitropic curvatures were measured at the time as indicated. Values are means ± SE (n = 20). (E) Statistical analysis of lateral root number. Lateral root numbers were counted at 12 d after germination. The values are means ± SE (n = 19). According to Turkey’s honest significant difference (HSD) test (p < 0.05), means of lateral root number do not differ if they are indicated with the same letter.

Fig 3. The MKK7-MPK6 cascade is involved in shoot branching.

(A) Branching phenotypes of 50-d-old Col-0, bud1, mpk3, mpk3bud1, mpk6, and mpk6bud1 plants grown under the long day condition. Bar, 5 cm. (B) The branch numbers of 50-d-old plant. Each value represents the mean ± SD (n = 15). According to Turkey’s HSD test (p < 0.05), means of branch number do not differ if they are indicated with the same letter.

Fig 4. The MKK7-MPK6 cascade is involved in polar auxin transport.

(A) Induction of hypocotyl elongation by high temperature. Wild-type and mutant seedlings were grown on 0.5 × MS solid media at 20°C and 29°C, respectively, and photographed at 9 d after germination. Bars, 5 mm. (B) Statistical analysis of high temperature-induced hypocotyl elongation. Values are means ± SE (n = 20). (C) Polar auxin transport assays of seedlings. Values are means ± SE of three independent assays. The difference significance was determined with Turkey’s HSD test (p < 0.05). (D) Polar auxin transport assays of inflorescence stems. Values are means ± SE of ten independent assays. The difference significance was determined with Turkey’s HSD test (p < 0.05).

Fig 5. The MKK7-MPK6 cascade regulates PIN1 polar localization in shoot stem.

Localization of PIN1-GFP in longitudinal hand sections of 35-d-old basal inflorescence stems of Col-0 (A), bud1 (B), mpk3 (C), mpk3bud1 (D), mpk6 (E), and mpk6bud1 (F). Sections were mounted in water, and the GFP signal was examined under a confocal microscope at an excitation wavelength of 488 nm. Red arrows indicate PIN1-GFP localization. Bars, 50 μm.

Fig 6. In vitro phosphorylation assay of PIN1HL by the MKK7-MPK6 cascade.

(A) In vitro assay of phosphorylation by the MKK7-MPK6 cascade using wild-type GST-PIN1HL or site-mutated PIN1Hl, in which the indicated residues were replaced with Ala (A) residues, respectively. The positions of GST-PIN1HL are indicated in the autoradiograph (top panel) and the Coomassie-stained gel (bottom panel). The Coomassie blue-stained gel was used as a control for protein loading. (B) Spectra for representative identified phosphopeptides S337 and T340. Asterisk represents the phosphate moiety.

Fig 7. The S337 phosphorylation status is responsible for PIN1 polar localization and regulation of shoot branching.

(A) PIN1-GFP localization in inflorescence stems of 35S::PIN1^WT-GFP, 35S::PIN1^S337A-GFP, and 35S::PIN1^S337D-GFP transgenic plants. Red arrows indicate PIN1-GFP localization. Bars, 50 μm. (B) Comparison of branch number among wild-type, bud1, 35S::PIN1^WT-GFP, 35S::PIN1^S337A-GFP, and 35S::PIN1^S337D-GFP transgenic plants. Primary rosette-leaf branch (RI), secondary rosette-leaf branch (RII), primary cauline-leaf branch (CI), and secondary cauline-leaf branch (CII) were counted at 60 d. Data are shown as mean ± SE (n ≥ 17). The difference significance was determined with Turkey’s HSD (p < 0.01). (C–G) Expression of phospho-mimicking PIN1^S337D confers branching phenotype (G), whereas the expression of nonphosphorylatable PIN1^S337A (F) or wild-type PIN1 (E) results in normal phenotype. Pictures of representative transgenic lines, along with Col-0 and bud1, grown under long day conditions, were taken at 60 d. Bars, 5 cm. (H) Branching phenotypes of 50-d-old Col-0, bud1, and 35S:PIN1^S337A/bud1 plants grown under the long day condition. Bar, 4 cm. (I) Branch number of 50-d-old plant. Data are shown as mean ± SE (n = 15). The difference significance was determined with Turkey’s HSD test (p < 0.01).

Fig 8. A proposed working model of the MKK7-MPK6/3 cascade involved in plant development in Arabidopsis.

The MKK7-MPK6 cascade plays predominant roles in diverse developmental processes including leaf venation architecture, filament elongation, lateral root formation, and shoot branching. MKK7-MPK3 and MKK7-MPK6 cascades function redundantly in leaf morphology; the MKK7-MPK6 signaling pathway regulates PAT through phosphorylating PIN1. In the wild type, PIN1 basal localization is controlled by reversible phosphorylation of S337 site by the MKK7-MPK6 cascade. In the bud1 plants, constitutively activated MKK7-MPK6 signaling leads to sustained phosphorylation of the PIN1 S337 site, which leads to PIN1 apolar localization and results in branching phenotype.