Mutation of the membrane-associated M1 protease APM1 results in distinct embryonic and seedling developmental defects in Arabidopsis

Abstract

Aminopeptidase M1 (APM1), a single copy gene in Arabidopsis thaliana, encodes a metallopeptidase originally identified via its affinity for, and hydrolysis of, the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). Mutations in this gene result in haploinsufficiency. Loss-of-function mutants show irregular, uncoordinated cell divisions throughout embryogenesis, affecting the shape and number of cotyledons and the hypophysis, and is seedling lethal at 5 d after germination due to root growth arrest. Quiescent center and cell cycle markers show no signals in apm1-1 knockdown mutants, and the ground tissue specifiers SHORTROOT and SCARECROW are misexpressed or mislocalized. apm1 mutants have multiple, fused cotyledons and hypocotyls with enlarged epidermal cells with cell adhesion defects. apm1 alleles show defects in gravitropism and auxin transport. Gravistimulation decreases APM1 expression in auxin-accumulating root epidermal cells, and auxin treatment increases expression in the stele. On sucrose gradients, APM1 occurs in unique light membrane fractions. APM1 localizes at the margins of Golgi cisternae, plasma membrane, select multivesicular bodies, tonoplast, dense intravacuolar bodies, and maturing metaxylem cells. APM1 associates with brefeldin A-sensitive endomembrane structures and the plasma membrane in cortical and epidermal cells. The auxin-related phenotypes and mislocalization of auxin efflux proteins in apm1 are consistent with biochemical interactions between APM1 and NPA.

Figure 1.

APM1 Is Strongly Expressed during Embryogenesis, Organogenesis, and in Meristematic Regions. (A) to (H) ProAPM1:GFP expression during Arabidopsis embryogenesis. (A) Early globular stage. (B) Late globular stage. (C) Early heart stage. (D) Late heart stage. (E) Torpedo stage. (F) Bright-field overlay of (E). (G) Mature embryo. (H) Bright-field overlay of (G). (I) to (M) ProAPM1:GUS expression in the seedlings and flowers: root elongation zone (I), root-shoot transition zone (J), shoot apex (K), flowers(L), and anthers and ovules (M). (N) to (R) Confocal laser scanning microscopy images of single sections. ProAPM1:GFP expression in seedlings. (N) GFP autofluorescence control. The same settings were used for all GFP images. (O) Five-day-old root tip. (P) Six-day-old root tip. (Q) Seven-day-old root tip. (R) Four-day-old dark-grown seedling. For these experiments, three GFP lines with ∼50 seedlings per line were examined; nine GUS lines with 50 seedlings per line were examined. These patterns were observed in >90% of the individuals. The GFP lines showed similar expression tissue-specific patterns, including a line that showed nuclear envelope localization, shown in (N) to (R), because the line shows the results more clearly in seedling roots than the other lines. The relative expression is consistent with the microarray data (see Supplemental Figure 1 online). Bars = 100 μm in (A) and (B), 50 μm in (C) to (F), 25 μm in (G) and (H), 100 μm in (I) and (J), 200 μm in (K), 300 μm in (L), 100 μm in (M), and 50 μm in (N) to (R). [See online article for color version of this figure.]

Figure 2.

APM1 Gene and Protein Maps. (A) Map of APM1 gene structure and mutation sites. Black boxes indicate exons, and white boxes indicate introns. The promoter is indicated N-terminal to the start site (ATG and an up arrow). The T-DNA insertion site in the promoter for apm1-1 is indicated with an inverted triangle. The EMS point mutation sites in exon 15 for apm1-2 and apm1-3 are indicated with down arrows. (B) Quantitative real-time PCR expression analysis in apm1 mutants. Means and standard deviations from three independent experiments, true replicates were used, not subsamples (t test,* P < 0.001). (C) Pictographic representation of the mutational sites for apm1-2 and apm1-3 (black lines with arrows) in relation to the catalytic (light-gray boxes with exopeptidase GAMEN and zinc binding HELAH) and protein–protein interaction (dark-gray box) motifs. (D) Top panel: protein gel blot using anti-APM1 in wild-type and apm1 alleles. None of the alleles are knockouts. A faint band can be observed in apm1-1 (−/−) because it has 1 to 3% expression of APM1 compared with the wild type. Two bands can be seen in apm1-2 (+/−), full-length and a truncated protein; the epitope that the antibody recognizes is contained in the truncated protein. apm1-3 contains a point mutation that does not interfere with antibody recognition. The observed ∼72-kD band in apm1-2 heterozygotes is consistent with the predicted length of the truncated protein product based on the position of the introduced stop codon. Therefore, apm1-1 has a very low level of full-length protein, apm1-2 has a truncated protein that contains the catalytic domain but not the protein–protein interaction domains, and apm1-3 has a full-length protein with a point mutation in a protein–protein interaction domain (APM1^A694V). Each lane contains 10 μg of total microsomal protein; an antibody to the C terminus of APM1 was used. Bottom panel: Loading control for the top panel. Each lane contains 10 μg of total microsomal protein; polyclonal antisera to the plasma membrane H⁺ ATPase (PM H⁺ ATPase) was used.

Figure 3.

Loss of APM1 Function Results in Embryonic Defects. Representative images for each stage in embryogenesis are shown to emphasize that the described defects are common among all three independent alleles; for brevity, panels of each allele at each stage are not shown. All alleles were backcrossed at least five times (see Methods); therefore, it is unlikely that these phenotypes described below are related to an unlinked gene in each independent allele and because of the consistency of the defects observed among all the alleles. (A) to (C) First zygotic division. (A) Wild-type embryo with apical cell and basal cell. (B) apm1-1 missing the apical cell. (C) apm1-1 with poorly formed apical cell and basal cell with early division. (D) to (G) Two-cell embryo proper. (D) Wild-type embryo with symmetric radial division in apical cell. (E) apm1-3 with anticlinal division in suspensor below apical embryo. (F) apm1-2 with asymmetric radial division in apical embryo. (G) apm1-3 with asymmetric radial division in apical embryo and anticlinal division in the adjacent suspensor cell. (H) to (J) Late globular embryo. (H) Wild-type embryo. (I) apm1-1 embryo has asymmetric anticlinal division in the hypophysis and an additional division periclinal to the suspensor. (J) apm1-3 embryo has asymmetric divisions in the hypophysis, adjacent suspensor and adjacent epidermal cells. (K) and (L) Heart-stage embryo. (K) Wild-type embryo shows bilateral symmetry and cotyledon primordia. (L) and (M) apm1-2 shows asymmetric cotyledon primordial and poorly defined root primordia. Bars = 5 μm (A) to (J) and 50 μm in (K) to (M). Arrowheads indicate aberrant planes of cell division.

Figure 4.

Loss of APM1 Function in Seedlings and Adults. (A) Wild-type and apm1 heterozygous and homozygous mutants and 5-d-old seedlings. (B) apm1-1 (−/−) seedling. (C) apm1-1 (−/−) seedling. (D) apm1-2 (−/−) seedling. (E) apm1-2 (−/−) seedling. (F) Wild-type and apm1 alleles, 4-week-old plants, and siliques. (G) Wild type, apm-1-1, and apm1-1 transformed with either ProAPM1:YFP-APM1 or ProAPM1:APM1-YFP at 5-d, showing complemented root phenotype. (H) Wild type, apm-1- 1, and apm1-1 with either ProAPM1:YFP-APM1 or ProAPM1:APM1-YFP at 2 weeks. apm1-1 and apm1-3 transformed with ProAPM1:YFP-APM1 or ProAPM1:APM1-YFP displayed a wild-type phenotype, including secondary root formation. apm1-2 (containing a truncated protein) did not display full complementation when transformed with either ProAPM1:YFP-APM1 or ProAPM1:APM1-YFP. Bars = 5 mm in (A), 200 μm in (B) to (E), and 3 cm in (F) to (H). [See online article for color version of this figure.]

Figure 5.

Root Phenotypes of APM1 Loss-of-Function Mutants. (A) to (D) Starch granules in 5-d-old root stained with Lugol's. Wild type (A), apm1-1 (+/−) (B), apm1-2 (+/−) (C), and apm1-3 (−/−) (D). The columella cells are disorganized in apm1 mutants. (E) ProCyclinB1;1:GUS root. (F) ProCyclinB1;1:GUS in apm1-1 (+/−) root. (G) ProCyclinB1;1:GUS in apm1-1 (−/−) root. CyclinB is a marker for cell division, and cell division appears to cease in apm1 mutants, consistent with arrested root growth. (H) Wild type root stained with propidium iodide. (I) apm1-1 (+/−) root stained with propidium iodide. Planes of cell division are altered in apm1 seedlings as in apm1 embryos. (J) QC-104 shows GUS staining in the quiescent center (QC). (K) QC-104 in apm1-1 (+/−) shows faint staining in the QC. (L) QC-104 in apm1-1 (−/−) shows no staining in the QC. (M) QC-25 shows GUS staining in the QC. (N) QC-25 in apm1-1 (−/−) shows no staining in the QC. The QC appears to be inactive in apm1 mutants. (O) ProSHR:GFP. (P) ProSHR:GFP in apm1-1 (+/−) shows restricted expression near the meristematic regions. (Q) ProSHR:SHR-GFP is expressed in the stele. (R) ProSHR:SHR-GFP in apm1-1 (+/−) shows no signal. (S) ProSCR:GFP signal in vascular strands. (T) ProSCR:GFP in apm1-1 (−/−) shows no signal in the vascular tissue. (U) Ectopic expression of ProSCR:GFP in apm1-1 (+/−) in epidermal cells. This is also observed in apm1-1 (−/−) (see Supplemental Figure 8 online). The ground tissue specifiers SCR and SHR are misexpressed/mislocalized in apm1 mutants. (V) ProBXL1-1:GUS shows staining in the xylem parenchyma. (W) ProBXL1-1:GUS in apm1-1 (+/−) shows no staining, indicating that these cells have altered cell identity. (X) Root cross section stained with toluidine blue; arrows point to xylem poles. apm1-1 (+/−), apm1-2 (+/−), and apm1-3 (−/−). The xylem poles are not opposite each other in apm1 mutants, the vascular tissue appears to have a greater number of smaller cells than does the wild type, and additional cortical cells are observed. For the ProcylcinB1;1:GUS reporter, 100 seedlings were observed, with the results presented observed in all seedlings. For the QC:GUS and ProBXL1-1:GUS reporters, 50 seedlings were observed, with the results presented observed in all seedlings. For the GFP reporters and fusions, 30 seedlings were observed, with the results presented observed in all seedlings. Bars = 100 μm in (A) to (V) and 50 μm in (X).

Figure 6.

Shoot Phenotypes of APM1 Loss-of-Function Mutants. (A) ProCyclinB1;1:GUS in shoot tip of 5-d-old seedling. (B) ProCyclinB1;1:GUS in apm1-1 (+/−) shows no staining in 5-d-old shoot tip. (C) Wild-type trichomes. (D) apm1-1 (+/−) trichomes show additional branching and hooking at the tips. (E) Wild-type hypocotyls. (F) apm1-1 (+/−) hypocotyl epidermal cells show cell adhesion and cell expansion defects. Bars = 200 μm in (A) and (B), 100 μm in (C) and (D), and 200 μm in (E) and (F). [See online article for color version of this figure.]

Figure 7.

Inducible Silencing and Expression of APM1. Inducible silencing of APM1 phenocopies apm1.Wild type transformed with the pOpOff artificial microRNA APM1 construct. (A) and (B) Dosage effect of silencing APM1 in 5-d-old seedling (A) and silique (B) development. (C) Seeds germinated on plates without 10 μM Dex were subsequently transferred to plates supplemented with 10 μM Dex for 5, 4, 3, 2, or 1 d to induce APM1 silencing. (D) The reciprocal experiment was also performed whereby plants were first germinated on 10 μM Dex for 1, 2, 3, 4, or 5 d and then transferred to plates without the induction medium. Inducible expression of APM1 complements apm1. apm1-1 (+/−) transformed with the pOpOn APM1 construct (E) and (F) Dosage effect on expressing APM1 in 5-d-old seedling (E) and silique (F) development. (G) Seeds were germinated on plates without Dex and then transferred to plates supplemented with 10 μM Dex for 5, 4, 3, 2, or 1 d to induce APM1 expression. (H) The reciprocal experiment was also performed whereby plants were first germinated on 10 μM Dex for 1, 2, 3, 4, or 5 d prior to transfer to plates without Dex. (I) Wild type and apm1-1 (+/−) without and with 10 μM Dex for 5 d. (J) Wild type, apm1-1 (+/−), and wild type transformed with pOpOff GFP grown on with 10 μM Dex for 5 d. (K) Wild type, apm1-1 (+/−), and apm1-1 (+/−) transformed with pOpOn GFP with 10 μM Dex for 5 d. Bars = 5 mm in (A), (C) to (E), and (G) to (K) and 3 cm in (B) and (F). [See online article for color version of this figure.]

Figure 8.

Auxin-Induced APM1 Expression and Responsive Reporters in apm1 Mutants. (A) to (C) APM1 expression is auxin responsive. (A) ProAPM1:GFP signal is observed in 4-d-old root tip. (B) ProAPM1:GFP signal after 100 nM IAA treatment for 2 h is strongly enhanced in the stele but reduced in the epidermis. Arrowheads in (A) and (B) point to the region where the signal is observed in (A) but absent in (B). See Figure 9C for comparison following gravity stimulus. (C) ProAPM1:GFP signal after 1 μM 2,4-D treatment for 2 h. A change seen in the subcellular pattern of the ProAPM1:GFP signal after 2,4-D treatment is an apparent result of altered reticulation of the endoplasmic reticulum after 2,4-D treatment. (D) to (I) Auxin accumulation in 5-d-old wild-type and apm1 seedlings. (D) ProIAA2:GUS activity in control cotyledon. (E) ProIAA2:GUS activity is absent in apm1-1 (+/−) cotyledon. (F) ProDR5:GUS activity in 5-d-old control root. (G) ProDR5:GUS activity in the apm1-2 (+/−) root is greater than in the control. (H) ProDR5:GFP signal in 5-d-old control root. (I) ProDR5:GFP signal is greater in the 5-d-old apm1-1 (+/−) root than in the control. Bars = 100 μm in (A) to (C) and (F) to (I) and 200 μm in (D) to (E).

Figure 9.

Auxin Transport and Gravitropism in apm1 Mutants. (A) Basipetal auxin transport is significantly reduced in the first 2 mm from the tip in apm1-3 (−/−). This region coincides with the columella region, which exhibits the cell differentiation defects, and where the DR5 reporter shows auxin accumulation. Basipetal auxin transport from the shoot apex of apm1-3 (−/−) is enhanced compared with that of the wild type. Means and standard deviation from three independent experiments are shown (t test, *P < 0.01). It was not possible to reproducibly measure shoot basipetal auxin transport in 5-d-old apm1-1 (+/−) or apm1-2 (+/−) seedlings. (B) Gravitropic response of apm1-1 (+/−), apm1-2 (+/−), and apm1-3 (−/−) mutants compared with that of the wild type. Angle of curvature is reduced in the apm1-1 and apm1-2 mutant compared with the wild type. Means and standard deviation from two independent experiments are shown (n = 50; t test, *P < 0.01). (C) ProAPM1:GFP signal is not observed on the auxin-accumulating side of the root after 30 min of gravity stimulus. Bar = 50 μm. [See online article for color version of this figure.]

Figure 10.

Analyses of APM1 Subcellular Localization by Electron Microscopy. Immunogold localization of APM1 in high-pressure frozen/freeze-substituted 5-d-old seedlings. Arrows point to immunogold labeling of APM1. The experiment was repeated twice with each antibody. See Supplemental Figure 6 online for preimmune controls. Bars = in 500 nm in (A) to (F) and 1 μm in (G) to (I). (B) and (D) to (G) C terminus antibody. (A), (C), (H), and (I) Peptide antibody. (A) and (B) Labeling on Golgi stacks (G) and flanking cytoplasm in root cortical cells. (C) Labeled multivesicular bodies (MVBs) in root cortical cells. (D) to (F) Labeling on tonoplast, plasma membrane (PM), and cell walls (CW) of perivascular root cells. C, cytoplasm, V, vacuole. (G) to (I) Labeling in metaxylem cells.

Figure 11.

APM1 Subcellular Localization by Confocal Laser Scanning Microscopy and Colocalization with Compartmental Markers in Sucrose Density Gradients. APM1 was immunolocalized using purified antisera generated against the C-terminal 30 kD of the protein (Murphy et al., 2002) or against a unique peptide (E₁₂₄-C₁₄₅) derived from the APM1 sequence adjacent to the hydrophobic membrane interaction domain (see Methods). The two antisera produced identical localization results. In addition, transformants were generated with ProAPM1:YFP-APM1 and ProAPM1:APM1-YFP constructs that complemented the apm1-1 loss-of-function mutants (see Methods; Figures 4G and 4H; see Supplemental Figure 3 online). APM1 shows plasma membrane, endomembrane, and cytosolic localizations. Bars = 100 μm in (A), (H), (J), and (K), 50 μm in (B), (C), (G), and (I), 10 μm in (D), and 20 μm in (E) and (F). (A) to (L) Subcellular localization of APM1 in 5-d-old seedlings using purified APM1 antisera ([A], [D], and [I]) and ProAPM1:YFP-APM1 ([B], [C], [E] to [H], and [J] to [L]). (A) Immunolocalization of APM1 in root tips using the C terminus antibody. (B) ProAPM1:YFP-APM1 in root tips. (C) Differential interference contrast (DIC) overlay of (B). (D) Immunolocalization of APM1 in root tips using the C terminus antibody showing subcellular localization. (E) Subcellular localization of ProAPM1:YFP-APM1 in root tips. (F) DIC overlay of (E). (G) ProAPM1:YFP-APM1 localization at the newly forming cell plate (left) and DIC overlay (right). (H) Localization of ProAPM1:YFP-APM1 in the root tip and metaxylem (top) and DIC overlay (bottom). (I) Immunolocalization of APM1 in the shoot apical meristem using the C terminus antibody. (J) ProAPM1:YFP-APM1 localization in the shoot apical meristem and epidermal cells of the hypocotyl. Inset: primary leaf. (K) ProAPM1:YFP-APM1 localization in the shoot apical meristem of a 3-week-old plant. (L) ProAPM1:YFP-APM1 localization in a lateral root of a 3-week-old plant. For these experiments, 50 seedlings were observed for immunolocalization studies, experiments were repeated five times, using both antibodies, and the C terminus antibody is shown here. Approximately fifty seedlings from each of seven YFP lines were examined. These localizations were observed in >90% of the individuals. Eight ProAPM1:APM1-YFP lines were also examined; however, YFP fluorescence was not observed, except in one line, which had a cytosolic signal. (M) Protein gel blot analysis of sucrose density fractionation of Arabidopsis microsomal membranes probed with antisera to APM1, PEP12/SYP21, AHA-2, SYP22, SEC12, and SYP-41. The membranes were fractionated with a 14 to 55% continuous sucrose (w/w) gradient, and the sucrose concentration of each fraction was measured by its refractive index. A total of 25 μL of each fraction was loaded per lane. This experiment was repeated four times using both antibodies (two times each) with similar results. The representative western shown here was with the C terminus antibody.

Figure 12.

NPA Treatment Produces No Additional Root Phenotypes in apm1-1 and Results in Loss of APM1 Signal. (A) Five-day-old wild-type root tip. (B) Wild type treated with a high concentration of NPA (30 μM) can phenocopy apm1 roots. (C) Five-day-old apm1-1 (+/−) seedling showing abnormal primary root. (D) apm1-1 (+/−) seedling grown on 30 μM NPA. (E) ProAPM1:YFP-APM1 signal is observed after 30 min 30 μM NPA treatment. (F) ProAPM1:YFP-APM1 signal is not observed after 60 min 30 μM NPA treatment. Bars = 100 μm in (A) to (D) and 50 μm in (E) and (F).

Figure 13.

Localization of Auxin Transporters Are Altered in APM1 Loss-of-Function Mutants. (A) to (C) PIN1 immunolocalization. (F) and (G) PIN2 immunolocalization. (K) to (M) ABCB19 immunolocalization. (D), (I), and (N) Functional fluorescent protein fusion controls in the respective mutant background. (A) PIN1 immunolocalization signal in the wild type. (B) PIN1 immunolocalization signal in apm1-1 (+/−). (C) PIN1 immunolocalization signal in apm1-2 (+/−) (D) PIN1-GFP signal in pin1 transformed with ProPIN1:PIN1-GFP. (E) PIN1-GFP signal in apm1-1 (+/−). Restricted PIN1 subcellular and tissue-specific localization is consistent with altered auxin levels, an indirect effect of apm1. (F) PIN2 immunolocalization in the wild type. (G) PIN2 immunolocalization in apm1-1 (+/−). (H) PIN2 immunolocalization in apm1-2 (+/−). (I) PIN2-GFP signal in eir1 transformed with ProPIN2:PIN1-GFP. (J) PIN2-GFP signal in apm1-1 (+/−). PIN2 localization is diffuse in apm1 mutants. (K) ABCB19 localization in the wild type. (L) ABCB19 localization in apm1-1 (+/−). (M) ABCB19 localization apm1-2 (+/−). (N) ABCB19-GFP signal in abcb19 transformed with ProABCB19:ABCB19-GFP. (O) ABCB19-GFP signal in apm1-1 (+/−). ABCB19 localization is diffuse in apm1 mutants. (P) to (S) FM4-64 signal in the wild type (P), apm1-1 (+/−) (Q), apm1-2 (+/−) (R), and apm1-3 (−/−) (S). Bars = 50 μm. [See online article for color version of this figure.]