MAP Kinase PrMPK9-1 Contributes to the Self-Incompatibility Response

Abstract

Mitogen-activated protein kinases (MAPKs) form important signaling modules for a variety of cellular responses in eukaryotic cells. In plants, MAPKs play key roles in growth and development as well as in immunity/stress responses. Pollen-pistil interactions are critical early events regulating pollination and fertilization and involve many signaling events. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants and also is known to utilize signaling to achieve incompatible pollen rejection. Although several pollen-expressed MAPKs exist, very little is known about their function. We previously identified a pollen-expressed MAPK (p56) from Papaver rhoeas that was rapidly activated during SI; several studies implicated its role in signaling to SI-induced programmed cell death involving a DEVDase. However, to date, the identity of the MAPK involved has been unknown. Here, we have identified and cloned a pollen-expressed P. rhoeas threonine-aspartate-tyrosine (TDY) MAPK, PrMPK9-1 Rather few data relating to the function of TDY MAPKs in plants currently exist. We provide evidence that PrMPK9-1 has a cell type-specific function, with a distinct role from AtMPK9 To our knowledge, this is the first study implicating a function for a TDY MAPK in pollen. We show that PrMPK9-1 corresponds to p56 and demonstrate that it is functionally involved in mediating SI in P. rhoeas pollen: PrMPK9-1 is a key regulator for SI in pollen and acts upstream of programmed cell death involving actin and activation of a DEVDase. Our study provides an important advance in elucidating functional roles for this class of MAPKs.

Figure 1.

Expression analysis of PrMPK9-1. A, Semiquantitative RT-PCR expression analysis of PrMPK9-1. PrMPK9-1 shows increasing expression during pollen development and low expression in other tissues. I, Immature anthers; −3/−2/−1/+1, stages of anther development (number of days from anthesis); P, hydrated pollen; R, root; L, leaf; S, stigma. GAPD shows control gene expression. B, RT-PCR expression analysis of PrMPK9-1 shows increased expression 10 min after SI induction in incompatible (SI) and compatible (COM) pollen or heat-denatured PrsS (DSI). CK, Control using water as a template; GM, control pollen growing in growth medium (GM). GAPD shows control gene expression.

Figure 2.

Western-blot analysis of PrMPK9-1. A, Expression of full-length PrMPK9-1 recombinant protein was detected with anti-His tag antiserum (1:1,000). B, Anti-AtMPK3 antiserum (1:1,000) cross-reacted with recombinant full-length PrMPK9-1. C, C-terminal His-tagged PrMPK9-1C truncated recombinant protein (∼16 kD) was detected with the anti-His tag antiserum; PrMPK9-1C antiserum was raised against this protein. D, PrMPK9-1C truncated recombinant protein cross-reacted with the PrMPK9-1C antiserum. E, PrMPK9-1C antiserum cross-reacted with the full-length PrMPK9-1 recombinant protein. The smear at the bottom is a likely degradation product. F, PrMPK9-1C antiserum cross-reacted with a protein from P. rhoeas pollen extracts, migrating at 56 kD. G, Preimmune serum did not cross-react with the PrMPK9-1C recombinant protein. The arrows indicate the positions to which full-length PrMPK9-1 and C-terminal truncated PrMPK9-1C migrate.

Figure 3.

PrMPK9-1C antiserum cross-reacts with a 56-kD pollen phosphoprotein. A, Coomassie Blue staining of untreated (UT) and SI-induced (SI) pollen extracts. B, Western blotting of samples in A using anti-pTXpY antiserum revealed the detection of increased levels of a phosphorylated (activated) ∼56-kD phosphoprotein in SI-induced samples (arrow) compared with untreated pollen (left). C, Reprobing this blot with PrMPK9-1C antibody revealed a 56-kD protein of approximately equal intensity in both untreated and SI-induced samples. For an explanation of the ∼72-kD protein detected, see Supplemental Figure S3. D, Coomassie Blue staining of calyculin A-treated (CA) and untreated pollen samples. E and F, Immunoprecipitation of the samples in D using PrMPK9-1C antiserum followed by the detection of immunoprecipitated activated MAPKs using western blotting and probing with the anti-pTXpY antibody revealed that an ∼56-kD protein was immunoprecipitated by PrMPK9-1C antibody specifically in the calyculin A-treated (CA-IP, arrow) sample (E) but not in the untreated (UT-IP) sample (F). G, Immunoprecipitation of SI-induced samples using PrMPK9-1C antiserum revealed an ∼56-kD protein (SI-IP) detected by anti-pTXpY antiserum (arrow). Equivalent untreated samples did not have a detectable ∼56-kD protein. UB, Unbound fractions.

Figure 4.

Immunolocalization of PrMPK9-1 in poppy pollen. A to C, Representative examples of the localization of PrMPK9-1 in a typical normally growing pollen tube. A, PrMPK9-1 localization using PrMPK9-1C antiserum and anti-rabbit IgG-FITC secondary antibody using fluorescence microscopy with an FITC filter. PrMPK9-1 appears to be a largely cytosolic protein localized throughout the pollen tube. B, 4′,6-Diamino-phenylindole (DAPI) staining showing the pollen tube generative cell (GC) and vegetative nucleus (VN). C, Bright-field (bf) image. D to F, Representative examples of the localization of PrMPK9-1 at 30 min after SI induction. D, PrMPK9-1 localization using PrMPK9-1C antiserum and anti-rabbit IgG-FITC secondary antibody using fluorescence microscopy with an FITC filter. PrMPK9-1 retains its largely cytosolic localization. E, 4′,6-Diamino-phenylindole staining showing the pollen tube generative cell and vegetative nucleus. F, Bright-field image. Bar = 10 µm.

Figure 5.

PrMPK9-1 antisense oligonucleotides attenuate SI specifically in incompatible pollen tubes. Mean pollen tube length is shown for incompatible (black bar) and compatible (gray bar) pollen after treatment with antisense (as-ODN; diagonals) or sense (s-ODN; dotted) oligonucleotides prior to SI treatment. With SI treatment, incompatible (SI) pollen tubes are inhibited (black bar); inhibition of growth is alleviated by the PrMPK9-1 as-ODNs, while compatible (SC) pollen treated with the same recombinant PrsS proteins (PrsS₁ and PrsS₃) grow normally (gray bar). Untreated controls (UT; white bars) are unaffected by PrMPK9-1 ODNs. Four independent data sets were used, totaling n = 200 for each treatment; error bars indicate se.

Figure 6.

PrMPK9-1 antisense oligonucleotides prevent the formation of SI-induced actin punctate foci. Pollen tubes were pretreated for 45 min with antisense (as-ODN) or sense (s-ODN) oligonucleotides, and SI was induced for 2 h. Typical representative images of actin configuration in pollen tubes are shown. A, Two hours after SI treatment, the pollen tube exhibits punctate actin foci (scored as phenotype foci in F). B, An untreated (UT) pollen tube control (scored as phenotype filaments in F). C, Actin configuration in an as-ODN-treated SI pollen tube showing normal actin filament organization. D, Actin configuration in an s-ODN-treated SI pollen tube showing punctate actin foci. E, Actin configuration in an as-ODN control untreated pollen tube showing filaments. Bar = 10 µm. F, Quantification of the effect of pretreatment of pollen with antisense (as-ODN) or sense (s-ODN) oligonucleotides: untreated or SI treated. Four independent experiments scored 50 pollen tubes each for actin phenotype (foci or filaments). Error bars indicate se.

Figure 7.

Antisense oligonucleotides block the SI-induced DEVDase/caspase-3-like activity. A, Representative image of an untreated (UT) pollen tube with no DEVDase/caspase 3-like activity. B, Corresponding bright-field image. C, Representative image of an SI-induced pollen tube after 4 h, using live-cell caspase 3/7 activity probe and an FITC filter set, showing fluorescence. D, Corresponding bright-field image. E, Representative image of an SI-induced pollen tube with antisense PrMPK9-1 oligonucleotide pretreatment (SI + as-ODN) after 4 h, using live-cell caspase 3/7 activity probe and an FITC filter set, with no detectable FITC signal. F, Corresponding bright-field image. G, Representative image of an SI-induced pollen tube with sense PrMPK9-1 oligonucleotide pretreatment (SI + s-ODN) after 4 h, using live-cell caspase 3/7 activity probe and an FITC filter set, with fluorescence detected. H, Corresponding bright-field image. Bar = 10 µm. I, Quantification of the effect of oligonucleotide pretreatment of pollen tubes (as-ODN or s-ODN) on DEVDase activity. Five independent experiments scored 50 pollen tubes in total for each treatment, and the percentage of pollen tubes in each category is indicated. Error bars indicate se.