A Receptor-Like Protein That Promotes Polarization of an Asymmetric Cell Division in Maize

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Science  30 Jan 2009:
Vol. 323, Issue 5914, pp. 649-651
DOI: 10.1126/science.1161686


Polarization of cell division is essential for eukaryotic development, but little is known about how this is accomplished in plants. The formation of stomatal complexes in maize involves the polarization of asymmetric subsidiary mother cell (SMC) divisions toward the adjacent guard mother cell (GMC), apparently under the influence of a GMC-derived signal. We found that the maize pan1 gene promotes the premitotic polarization of SMCs and encodes a leucine-rich repeat receptor-like protein that becomes localized in SMCs at sites of GMC contact. PAN1 has an inactive kinase domain but is required for the accumulation of a membrane-associated phosphoprotein, suggesting a function for PAN1 in signal transduction. Our findings implicate PAN1 in the transmission of an extrinsic signal that polarizes asymmetric SMC divisions toward GMCs.

In plants, as in other eukaryotes, asymmetric cell divisions are associated with developmental patterning events, formation of new cell lineages and cell types, and maintenance of stem cell populations (1, 2). In all of these processes, developmental asymmetry is closely tied to division polarity, which is thought to be oriented by either intrinsic or extrinsic cues. However, very little is known about such cues in plants or how cells perceive and respond to them.

Extrinsic cues appear to orient asymmetric cell divisions in maize during the development of epidermal stomatal complexes, each consisting of a pair of guard cells flanked by a pair of subsidiary cells that function in guard cell regulation (Fig. 1A). Stomatal complexes in grasses such as maize develop through an invariant sequence of coordinated asymmetric cell divisions (fig. S1) (3). After an asymmetric division that forms a guard mother cell (GMC), its lateral neighbors, called subsidiary mother cells (SMCs), polarize toward the GMC, positioning their nuclei and forming dense patches of cortical F-actin at the GMC contact site (Fig. 1E) (46). SMCs subsequently divide asymmetrically to form subsidiary cells flanking the GMC (Fig. 1E), and the GMC then divides longitudinally to form a guard cell pair. Analysis of stomatal complex formation in grasses led almost 50 years ago to the proposal that SMC divisions are polarized by a signal emanating from GMCs (3), but the mechanism of interaction between the GMC and SMC has remained unknown.

Fig. 1.

Stomatal complex formation in WT and pan mutant maize leaves. (A to C) Toluidine blue O–stained epidermal peels from adult leaves of WT (A), pan1 (B), and pan2 mutant (C). Guard cells stain blue [black arrow in (A)]; subsidiary cells stain pink [black arrowheads in (A)]. White arrowheads in (B) and (C) indicate abnormal subsidiary cells in pan mutant leaves. Scale bar, 100 μm. (D) Quantitative analysis of abnormal subsidiary cells in mature leaf tissue. Error bars represent SEM (n = 5 plants per genotype; >600 subsidiary cells were analyzed for each genotype). (E to G) F-actin (green) in developing stomata of WT (E), pan1 (F), and pan2 (G) with propidium iodide-stained nuclei in blue (single confocal planes). In (E), arrowheads on GMCs point to normal actin patches in adjacent SMCs, and dashed lines indicate division planes in two divided SMCs. In (F) and (G), arrows indicate delocalized actin patches, and u indicates unpolarized nuclei. Scale bar, 10 μm. (H) Quantitative analysis of SMC polarity defects (P, polarized nucleus; U, unpolarized nucleus). Error bars represent SEM [n = 5 plants per genotype, the same plants analyzed for the graph in (D); >300 SMCs were analyzed for each genotype].

A recessive mutation in maize, pan1, causes defects in the premitotic polarization of SMCs and the formation of abnormal subsidiary cells (5). An ethylmethane sulfonate (EMS)–induced, non-allelic pan2 mutation caused a similar phenotype (Fig. 1, B to D). In wild-type (WT) leaves, 92% of 317 SMCs analyzed in files of developing stomata containing at least one divided SMC had polarized nuclei, and 80% of those had well-focused actin patches (Fig. 1, E and H). In contrast, only 60% of 325 pan1 SMCs and 62% of 392 pan2 SMCs had polarized nuclei, and most of these SMCs either lacked an actin patch or had a delocalized patch (Fig. 1, F to H). The majority of mutant SMCs with unpolarized nuclei lacked actin patches, but many of them had delocalized or normal actin patches (Fig. 1, F to H). Thus, pan mutant SMCs exhibit defects in both nuclear polarization and actin-patch formation, but these defects are not closely correlated, suggesting that they are largely independent effects of the pan mutations on SMC polarity.

We used a mutant allele containing a Mutator1 (Mu1) transposon insertion to clone the pan1 gene (7). In this allele, Mu1 is inserted into the codon encoding amino acid 135 of a 662–amino acid leucine-rich repeat receptor-like kinase (LRR-RLK) belonging to the LRRIII subfamily (8). This protein consists of a predicted extracellular domain containing five LRR motifs, a transmembrane domain, and an intracellular serine/threonine kinase domain. In plants carrying an EMS-induced pan1 mutation (pan1-EMS), a premature stop codon in the first exon truncates this protein at amino acid 47, confirming the identity of this gene as pan1. Other LRR-RLKs function as receptors or coreceptors in diverse aspects of plant development and defense (9, 10). Extracellular LRR motifs are implicated in ligand binding; intracellular kinase domains are implicated in signal transduction via autophosphorylation and phosphorylation of target proteins.

To further investigate PAN1 function, we raised a polyclonal antibody that recognized a protein of around 75 kD by immunoblotting, which is close to the predicted molecular mass of PAN1 (68.4 kD). This protein was depleted in extracts of both pan1-Mu and pan1-EMS mutants, confirming its identity as PAN1 (Fig. 2). This protein was found in the microsomal fraction of extracts from the division zone of WT maize leaves (Fig. 2), consistent with the expected membrane localization for PAN1. Immunoblot analysis indicated that in developing leaves, PAN1 was most abundant in the division zone, but the protein was also found in dividing regions of other tissues that lack stomata (fig. S2), suggesting that PAN1 may function in other processes besides stomatal development. However, pan1 mutants do not have obvious developmental defects affecting other tissues or cell types.

Fig. 2.

Immunoblot of proteins extracted from the division zone of leaves of the indicated genotypes probed with anti-PAN1 antibody. Extracts were separated into 110,000g supernatants (S) and pellets representing the microsomal fraction (P). Coomassie staining of the equivalent region of a duplicate gel demonstrates equal loading.

In WT leaves, whole mount immunofluorescence revealed PAN1 only in SMCs and newly formed subsidiary cells at sites of contact with GMCs (Fig. 3A). These “PAN1 patches” were absent in pan1 mutants (Fig. 3B), confirming the specificity of immunolabeling for PAN1. Specific labeling with the PAN1-specific antibody to (anti-PAN1) was observed only at the cell periphery, consistent with localization to the plasma membrane. Alternatively, although the cortical endoplasmic reticulum (ER) itself was not specifically enriched in SMCs at GMC contact sites (fig. S3), some or all of the PAN1 protein may be ER-localized. PAN1 protein was present in pan2 mutants at reduced levels and remained associated with the microsomal fraction (Fig. 2), but no specific PAN1 staining was observed in pan2 mutants (Fig. 3C).

Fig. 3.

PAN1 and actin localization in developing stomata. White arrowheads on GMCs indicate PAN1 and actin patches in adjacent SMCs. SMC nuclei are identified as unpolarized (u), partially polarized (pp), polarized (p), or divided (d) and are numbered in (D) and (G) for reference. (A to C) PAN1 staining in green and propidium iodide-stained nuclei in blue (single confocal planes). (D to L) Double staining of PAN1 [monochrome in (D), (G), and (J); green in (F), (I), and (L)] and actin [monochrome in (E), (H), and (K); red in (F), (I), and (L)] in WT leaves (Z projections of confocal stacks). In (D) to (I), SMCs 1, 2, and 6 have polarized nuclei and both PAN1 and actin patches; the remaining SMCs have unpolarized or partially polarized nuclei with both PAN1 and actin patches (SMC3), a PAN1 patch only (SMC5), or neither PAN1 nor actin patches (SMC4). Scale bar, 10 μm. (M) Lengths of GMCs adjacent to SMCs with nuclei that were unpolarized (U, n = 119), polarized (P, n = 168), or divided (D, n = 36). (N) Lengths of GMCs adjacent to SMCs with unpolarized nuclei having neither PAN1 nor actin patches (n = 32), PAN1 patches only (n = 39), or both PAN1 and actin patches (n = 48). In M and N, error bars indicate SEM; all differences shown are significant (P < 0.01) by Student's t test.

Double labeling of PAN1 and actin was employed to investigate the timing of PAN1 patch formation relative to nuclear polarization and actin-patch formation in WT cells. 92% of 168 SMCs with fully polarized nuclei had both PAN1 and actin patches (Fig. 3, D to I). Among 119 SMCs with unpolarized or partially polarized nuclei (presumably in the process of becoming polarized), 27% had neither PAN1 nor actin patches, 33% had only PAN1 patches, and 40% had both PAN1 and actin patches (Fig. 3, D to I). None of these SMCs had actin patches only. GMC length provides a measure of developmental age, increasing from inception, when flanking SMCs have unpolarized nuclei, through nuclear polarization and SMC division (Fig. 3M). The average lengths of adjacent GMCs were lowest for the SMC class lacking both PAN1 and actin patches, intermediate for the PAN1+ actin class, and highest for the PAN1+ actin+ class (Fig. 3N). Thus, PAN1 and actin patches both appeared after GMC formation and before nuclear polarization, but PAN1 patches were detectable before actin patches. The presence of PAN1 and actin patches exclusively at sites of contact with GMCs supports the conclusion that these patches are induced by GMCs rather than by intrinsic cues. Further supporting this conclusion, in unusual cases where a single SMC flanks two GMCs, PAN1 and actin patches were observed adjacent to both GMCs (n = 14) (Fig. 3, J to L).

Although the PAN1 kinase domain is well conserved with enzymatically active serine/threonine kinases, it lacks several amino acid residues conserved in active kinases, including a lysine residue in subdomain II that is critical for kinase activity (11) (fig. S4), suggesting that it is not an active kinase. Consistent with this prediction, the kinase domain of PAN1 did not detectably phosphorylate itself or the artificial substrate myelin basic protein (MBP) in vitro under conditions where the kinase domain of the LRR-RLK BRI1 phosphorylated both substrates robustly (Fig. 4A). Some LRR-containing transmembrane proteins lack intracellular kinase domains altogether and are thought to function in signal transduction processes by associating with an active kinase (9). To investigate whether PAN1 might function similarly, microsomal fractions of extracts from WT, pan1, and pan2 mutants were probed on immunoblots with anti-phosphoamino acid antibodies. No differences between wild type and mutant were observed with anti-phosphoserine or anti-phosphotyrosine, but a 52-kD protein (smaller than PAN1) identified by anti-phosphothreonine was almost undetectable in both pan1-Mu and pan1-EMS mutants (Fig. 4B). This protein was depleted in pan2 mutants and in WT extracts treated with lambda phosphatase (Fig. 4B and fig. S5). Thus, it appears that phosphorylation of a 52-kD membrane protein of unknown identity depends on PAN1, suggesting a role for PAN1 in signal transduction. Alternatively, the 52-kD phosphoprotein may depend on PAN1 and PAN2 for its synthesis or stability rather than its phosphorylation. As demonstrated or proposed for other LRR-RLKs with conserved but enzymatically inactive kinase domains (12, 13), the PAN1 kinase domain may mediate interaction(s) with other proteins that function in concert with or downstream of PAN1.

Fig. 4.

Analysis of PAN1-dependent protein phosphorylation. (A) In vitro kinase assays. Lanes 1 and 2 show Coomassie staining of the proteins used in each reaction: MBP and the kinase domains of BRI1 and PAN1 fused to glutathione S-transferase. Autoradiography in lanes 3 and 4 reveals that only the BRI1 kinase domain phosphorylates itself (19) and MBP. (B) Immunoblot of microsomal fractions of extracts from the division zones of leaves of the indicated genotypes was probed with anti-phosphothreonine antibody. A duplicate blot was probed with anti-actin antibody to demonstrate equal loading.

Our results implicate PAN1 as a receptor or co-receptor of GMC-derived signals that promote the polarization of SMCs in preparation for their asymmetric division. Because SMC polarization is not completely abolished in pan1 null mutants, it is clear that PAN1 is not solely responsible for the reception of polarizing cues from GMCs. Thus, parallel or cooperating pathways may work together with the PAN1 pathway to promote SMC polarization. The reduction in PAN1 protein levels and lack of detectable PAN1 patches in pan2 mutants suggest that PAN2 functions in the PAN1 pathway, but the identity of PAN2 and its functional relationship to PAN1 remains to be determined. If PAN1 relays polarity cues from GMCs, why is it already localized at sites of GMC contact at its earliest appearance? PAN1 may be initially distributed uniformly at the SMC surface but detectable by immunolocalization only after becoming locally concentrated as a result of binding to a GMC-derived ligand. Alternatively, PAN1 may be recruited directly to GMC contact sites in response to an upstream sensor of GMC position, functioning there as a secondary sensor. The localization of PAN1 at a specific site on the cell surface, its enzymatically inactive kinase domain, and its cellular function differentiate it from two other receptor-like proteins previously shown to play a role in polarized cell division or growth in plants. The Arabidopsis TMM protein, which contains LRR and transmembrane domains but lacks a kinase domain, functions in a well-characterized signal transduction pathway that controls the entry of epidermal cells into the stomatal lineage and the orientation of asymmetric, stomate-forming divisions (14). However TMM does not promote the polarization of these divisions, nor is it asymmetrically localized (15). Tomato LRR-RLK proteins LePRK1 and LePRK2 promote the polarized growth of pollen tubes (16). They have active kinase domains and are localized uniformly at the pollen tube surface but appear to interact with rho of plants (ROP) guanine nucleotide exchange factors only at the tube tip to stimulate localized actin assembly and polarized growth (17, 18).

Supporting Online Material

Materials and Methods

Figs. S1 to S5


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