An arabidopsis mitogen-activated protein kinase cascade, MKK9-MPK6, plays a role in leaf senescence

Abstract

Leaf senescence is a developmentally programmed cell death process that constitutes the final step of leaf development, and it can be regulated by multiple environmental cues and endogenous signals. The mitogen-activated protein kinase (MAPK) cascades play diverse roles in intracellular and extracellular signaling in plants. Roles of the MAPK signaling module in leaf senescence are unknown. Here, a MAPK cascade involving MKK9-MPK6 is shown to play an important role in regulating leaf senescence in Arabidopsis (Arabidopsis thaliana). Both MKK9 and MPK6 possess kinase activities, with MPK6 an immediate target of MKK9, as revealed by in vitro, in vivo, and in planta assays. The constitutive and inducible overexpression of MKK9 causes premature senescence in leaves and in whole Arabidopsis plants. The premature senescence phenotype is suppressed when MKK9 is overexpressed in the mpk6 null background. When either MKK9 or MPK6 is knocked out, leaf senescence is delayed.

Figure 1.

MKK9 is up-regulated during leaf senescence in Arabidopsis. RNA gel blot analysis of the expression of MKK9 and SAG12 in leaves at different developmental stages. YL, A young leaf with half the size of a fully expanded leaf; NS, a fully expanded, nonsenescing leaf; ES, an early senescent leaf, with less than 25% leaf area yellowing; LS, a late senescent leaf, with more than 50% leaf area yellowing. [See online article for color version of this figure.]

Figure 2.

MKK9 encodes an active kinase. In vitro kinase activity assay of MKK9. Affinity-purified MBP, MBP-MKK9, or MBP-MKK9KR was incubated in the presence of [γ-³²P]ATP with or without myelin basic protein. After SDS-PAGE, the reaction products were analyzed as indicated. Top, Coomassie Brilliant Blue staining; bottom, autoradiography.

Figure 3.

Delayed senescence in detached leaves of mkk9 and mpk6 mutant plants. A, Phenotypes of detached leaves of wild-type (Col-0), mkk9-1, and mpk6 mutant plants. The seventh and eighth leaves were kept in light (100–120 μmol m⁻² s⁻¹) for 6 d. B and C, F_v/F_m (B) and chlorophyll content (C) of leaves shown in A. Mean values of six samples ± se are shown. Asterisks indicate significant differences between Col-0 and the mutants (Student's t test, P < 0.01). FW, Fresh weight. [See online article for color version of this figure.]

Figure 4.

Complementation of mkk9-1 with MKK9. A, Phenotypes of detached leaves of the wild type (Col-0), the mkk9-1 mutant, and mkk9-1 transformed with MKK9. The seventh and eighth leaves were detached and kept in light for 6 d. B, RT-PCR analysis of the expression of MKK9 of plants described in A. ACT3 served as an internal standard. C, F_v/F_m of leaves shown in A. Mean values of five samples ± se are shown. F_v/F_m was significantly different between the wild type and the mkk9-1 mutant (Student's t test, P < 0.01). The difference between the wild type and the mkk9-1 + MKK9 complementation plants was not significant. [See online article for color version of this figure.]

Figure 5.

Constitutive overexpression of MKK9 in the Col-0 and mpk6-2 backgrounds. A, Phenotypes of 35S:MKK9, 35S:MKK9KR transgenic, and wild-type (Col-0) Arabidopsis plants. Bar = 1 cm. a, b, c, and d are different types of 35S:MKK9 transgenic plants based on their stature and senescence phenotypes as described in the text; e is the wild-type (Col-0) plant; f is a 35S:MKK9KR transgenic plant. B, RNA gel blot analysis of the expression of MKK9 and some senescence marker genes in plants shown in A. C, RNA gel blot analysis of the expression of MKK9 and some senescence marker genes in the mpk6-2 mutant plants that were transformed with 35S:MKK9. c28 is a randomly selected type c plant. Similarly, d19 and d20 are two randomly selected type d plants.

Figure 6.

Inducible overexpression of MKK9 causes precocious leaf senescence. A, Phenotypes of wild-type (Col-0) and transgenic plants harboring different constructs. The photograph was taken 4 d after treatment with 30 μm DEX. B, RNA gel blot analysis of the expression of MKK9 and some SAGs in leaves of plants that were treated with or without DEX. The asterisk indicates that this plant contains a mutated MKK9 that lacks kinase activity. C and D, Chlorophyll levels (C) and F_v/F_m (D) of sixth leaves from different plants shown in A. Mean values of four samples ± se are shown. Lane 1, Col-0; lane 2, DEXⁱⁿ:MKK9 + pTA7001; lane 3, DEXⁱⁿ:MKK9 + Col-0; lane 4, DEXⁱⁿ:MKK9KR + pTA7001; lane 5, DEXⁱⁿ:MKK9KR + Col-0; lane 6, pTA7001. FW, Fresh weight.

Figure 7.

Phosphorylation and activation of MPK6 by MKK9. A, In vitro kinase assay of phosphorylation of MPK6 by MKK9. MBP fusion proteins were affinity purified from E. coli. MKK9 (WT), MKK9EE (EE), or MKK9KR (KR) was incubated with MPK6 or MPK6KR in the kinase reaction mixture. Aliquots of the samples were separated by SDS-PAGE and subjected to autoradiography. The bottom panel is a Coomassie Brilliant Blue (CBB)-stained gel showing MPK6 (black arrowhead) and MKK9 (white arrowheads). B, In vivo assay of phosphorylation of MPK6 by MKK9. Both MKK9 and MPK6 (as GFP fusions) were transferred, individually or together, into mkk9 protoplasts. Protein extracts from the protoplasts were incubated with or without λ-PPase at 30°C for 30 min. Gel mobility shifting was revealed by western-blot analysis. Rabbit anti-GFP serum and goat anti-rabbit IgG were used as primary and secondary antibodies, respectively. C, Activation of MPK6 by MKK9 in planta. Transgenic Arabidopsis plants carrying the inducible overexpression construct of MKK9WT and MKK9EE were treated with DEX and sampled at the indicated times. MKK9 expression levels were determined by RNA gel blot analysis (top). Kinase activities of the anti-MPK6 immune complexes were analyzed by in-gel kinase assay using MBP as a substrate, and MPK6 protein levels were determined by protein gel blot analysis using anti-MPK6 (bottom).