The ESCRT-related CHMP1A and B proteins mediate multivesicular body sorting of auxin carriers in Arabidopsis and are required for plant development

Abstract

Plasma membrane proteins internalized by endocytosis and targeted for degradation are sorted into lumenal vesicles of multivesicular bodies (MVBs) by the endosomal sorting complexes required for transport (ESCRT) machinery. Here, we show that the Arabidopsis thaliana ESCRT-related CHARGED MULTIVESICULAR BODY PROTEIN/CHROMATIN MODIFYING PROTEIN1A (CHMP1A) and CHMP1B proteins are essential for embryo and seedling development. Double homozygous chmp1a chmp1b mutant embryos showed limited polar differentiation and failed to establish bilateral symmetry. Mutant seedlings show disorganized apical meristems and rudimentary true leaves with clustered stomata and abnormal vein patterns. Mutant embryos failed to establish normal auxin gradients. Three proteins involved in auxin transport, PINFORMED1 (PIN1), PIN2, and AUXIN-RESISTANT1 (AUX1) mislocalized to the vacuolar membrane of the mutant. PIN1 was detected in MVB lumenal vesicles of control cells but remained in the limiting membrane of chmp1a chmp1b MVBs. The chmp1a chmp1b mutant forms significantly fewer MVB lumenal vesicles than the wild type. Furthermore, CHMP1A interacts in vitro with the ESCRT-related proteins At SKD1 and At LIP5. Thus, Arabidopsis CHMP1A and B are ESCRT-related proteins with conserved endosomal functions, and the auxin carriers PIN1, PIN2, and AUX1 are ESCRT cargo proteins in the MVB sorting pathway.

Figure 1.

Characterization of Arabidopsis CHMP1 Protein Structure and Phylogeny of CHMP1 Proteins in Eukaryotes. (A) Phylogenetic analysis of CHMP1-related proteins from plants, animals, fungi, and other organisms using RAxML. The bootstrap values are shown above each branch. The accession numbers and gene identifiers for the sequences used in this analysis are provided in Methods. Scale indicates 0.1 amino acid substitutions per site. (B) Schematic representation of Arabidopsis CHMP1A and B proteins. CHMP1A and B differ in 10 amino acid residues from each other. The asymmetric amino acid charge distribution of CHMP1 proteins is indicated by + and − for predominantly basic and acidic amino acid residues, respectively. NLS, nuclear localization signal. Coiled-coil domains were identified with the algorithm from Lupas et al. (1991). (C) Amino acid alignment of Arabidopsis CHMP1A and B, human CHMP1A and B, and yeast Did2p. Black indicates identical residues, and gray represents similar residues. Asterisks indicate conserved leucine residues in the MIM domain.

Figure 2.

Interaction Analysis between CHMP1 and ESCRT-Related Proteins and Characterization of Arabidopsis chmp1 Mutant Alleles. (A) and (B) In vitro pull-down assays confirmed the interaction between CHMP1A and SKD1 (A) and CHMP1A and LIP5 (B). Protein gel blots of in vitro glutathione agarose pull-down show that 6xHis tagged At-SKD1 interacts with GST-At-CHMP1A but not with GST alone and 6xHis tagged At-CHMP1a interacts with GST-At-LIP5 but not with GST alone. All the recombinant proteins were detected using either anti-GST (bottom panels) or anti-His (top panels) antibodies. (C) Schematic representation of distribution of exons (black) and introns (white) in CHMP1A and B. Inverted wedges indicate the T-DNA insertions in the first exon of CHMP1A and CHMP1B. (D) RT-PCR from RNA extracts of the chmp1a and chmp1b single mutants. Two biological replicates were performed. (E) Seeds from wild-type and chmp1a/CHMP1A chmp1b/chmp1b plants. Asterisks indicate double mutant seeds. (F) Detail of seeds dissected from one single mutant silique showing double mutant and wild type–looking (control) seeds containing two or more chmp1 mutant alelles. (G) Protein gel blot of total protein extracts from wild-type and chmp1a chmp1b mutant embryos. CHMP1 proteins were detected with a polyclonal antibody raised against the full-length maize SAL1/CHMP1 protein (Tian et al., 2007). Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was used as loading control. Bars = 1 mm.

Figure 3.

Phenotypic Analysis of chmp1a chmp1b Double Mutant Embryos and Seeds. (A) to (F) Developmental stages of dissected embryos from chmp1a/chmp1a CHMP1B/chmp1b plants. Wild type–looking embryos shown on the left side and double mutant embryos (arrowheads) on the right side of panels. (G) and (H) Confocal images of control and mutant embryos. The mutant embryo is seen from a top view showing the presence of four rudimentary cotyledons (arrowheads). (I) to (L) Longitudinal sections of seeds produced by chmp1a/chmp1a CHMP1B/chmp1b plants. (I) and (J) Wild type–looking seed used as control. (J) Detail of the root and procambial strand (indicated by brackets) of the embryo shown in (I). (K) and (L) chmp1a chmp1b double mutant embryo. (L) Detail of the root pole of the mutant embryo shown in (K). Note the excentrically located procambial strand (indicated by brackets). Bars = 50 μm.

Figure 4.

Phenotype of chmp1a chmp1b Double Mutant Seedlings. (A) to (G) Seedlings derived from chmp1a/chmp1a CHMP1B/chmp1b plants. Note multiple cotyledons of mutant seedlings in (B) to (D). (E) to (G) Shoot apical regions in control (E) and chmp1a chmp1b mutant seedlings ([F] and [G]). (H) and (I) Cotyledons stained with 4',6-diamidino-2-phenylindole showing the distribution of stomata in control (H) and mutant seedlings (I). Note the clustered stomata in mutant cotyeldons (arrows in [I]). (J) and (K) Seedlings stained with 4',6-diamidino-2-phenylindole showing the venation pattern in cotyledons. Whereas the lateral veins in the control seedlings are fused close to the cotyledon margin (arrow in [J]), lateral veins in the mutant cotyledons end freely (arrows in [K]). SAM, shoot apical meristem. (L) and (M) Root architecture in control (L) and chmp1a chmp1b mutant seedlings (M) stained with propidium iodide. Note the enlarged root epidermal cells in the mutant (arrow). Bars = 5 mm in (A) to (D), 2 mm in (E) to (G), 50 μm in (H) and (I), 500 μm in (J) and (K), and 20 μm in (L) and (M).

Figure 5.

DR5rev_pro:GFP Expression in Control and chmp1a chmp1b Mutant Embryos. (A) Overview of wild type–looking (control) and chmp1a chmp1b mutant embryos (arrows) expressing the DR5rev_pro:GFP reporter. Chlorophyll autofluorescence (red) was used to visualize the embryos. (B) and (C) Control mature embryos. GFP signal (arrowheads) was detected in the tips of cotyledons (C) and in the root pole. (C) shows detail of apical view of cotyledon. (D) to (F) Optical cross sections through the apical region of double mutant embryos showing GFP signal (arrowheads) in the tip of rudimentary cotyledons (C). (G) and (H) Wild-type embryos with undetectable GFP signal in the procambial strand region (brackets) of cotyledons (G) and axis (H). (I) Double mutant embryo showing strong GFP signal in procambial strands (arrowheads). Bars = 50 μm.

Figure 6.

PIN1-GFP Expression and Localization in Control and chmp1a chmp1b Mutant Embryos. (Embryos were stained with FM4-64 [red] to visualize the cell outlines.) (A) to (C) Control embryos. Note PIN1-GFP expression at apical/central region (arrow) of a globular stage embryo (A), tips of developing cotyledon (arrows) in heart stage embryo (B), and procambial strands (arrows) of torpedo stage embryo (C). (D) to (F) chmp1a chmp1b mutant embryos with altered PIN1-GFP expression pattern. Arrows indicate the areas with higher PIN1-GFP expression. (G) Control heart stage embryo. PIN1-GFP localizes to the plasma membrane in the emerging cotyledons, predominantly to the apical side of the cells (toward the tip of the cotyledons; arrowheads). (H) and (I) chmp1a chmp1b mutant embryo dissected from the same silique used in (G). Note the substantial PIN1-GFP signal from vacuolar membranes and FM4-64–stained compartments. V, vacuole. Bars = 20 μm.

Figure 7.

Immunogold Localization of PIN1-GFP. Immunogold labeling was performed on high-pressure frozen/freeze-substituted wild type–looking (control) and chmp1a chmp1b mutant embryos from self-pollinated CHMP1A/chmp1a chmp1b/chmp1b/ PIN1_pro:PIN1-GFP/PIN1_pro:PIN1-GFP plants using polyclonal anti-GFP antibodies. Bars = 500 nm. (A) Control embryo. GFP signal is detected at the plasma membrane (white arrowheads). (B) chmp1a chmp1b mutant embryos. White arrowheads indicate gold labeling on the plasma membrane and black arrowheads on the vacuolar membrane. CW, cell wall; V, vacuole.

Figure 8.

Immunogold Detection of PIN1-GFP and the Endosomal Marker RHA1/RabF2A in Control and Mutant MVBs. (A) to (G) Immunolabeling of GFP in high-pressure frozen/freeze-substituted WT-looking (control) and chmp1a chmp1b mutant embryos expressing PIN1-GFP. CW, cell wall; TGN, trans Golgi network. (A) Overview of a control embryo cell showing PIN1-GFP signal on the trans Golgi network, MVBs, and plasma membrane (white arrowheads). (B) to (D) Detail of control MVBs with gold labeling on MVB lumenal vesicles (black arrowheads). (E) to (G) Detail of chmp1a chmp1b mutant MVBs. Most of the gold labeling is on the MVB limiting membrane (black arrowheads) and not on MVB lumenal vesicles. (H) to (J) Immunolabeling of RHA1/RABF2A on control (H) and chmp1a chmp1b mutant MVBs ([I] and [J]). Bars = 200 nm.

Figure 9.

Expression Pattern and Subcellular Localization of AUX1-YFP and PIN2-GFP in Control and chmp1a chmp1b Mutant Embryos. (A) and (B) Superimposed transmission and confocal images of control and chmp1a chmp1b mutant embryos expressing AUX1_pro:AUX1-YFP. (A) Control embryos at the early torpedo stage expressed AUX1-YFP at the procambial strand and root pole, whereas the chmp1a chmp1b mutant embryos (arrows) do not express detectable levels of AUX1-GFP. (B) Mature control and chmp1a chmp1b mutant embryos expressing AUX1-YFP at the root pole. (C) and (D) Detail of control and chmp1a chmp1b mutant roots expressing AUX1-YFP. (E) and (F) Control and chmp1a chmp1b embryo cells expressing AUX1-YFP at the root pole. Note the AUX1-YFP signal from the vacuolar membrane in mutant cells. V, vacuole. (G) to (N) Expression of PIN2-GFP in epidermal and cortical cells in roots of control and chmp1a chmp1b mutant seedlings stained with FM4-64. (I) to (K) Polarized localization of PIN2-GFP in the plasma membrane of epidermal cells in control roots. (L) to (N) Localization of PIN2-GFP in chmp1a chmp1b root epidermal cells. Note the partial loss of polarized localization and the strong PIN2-GFP signal from vacuolar membranes (arrowheads). Bars = 100 μm in (A) and (B) and 20 μm in (C) to (N).

Figure 10.

Model of CHMP1 Function in ESCRT-Mediated Sorting of Auxin Carriers. AUX1, PIN1, and PIN2 cycle between endosomes and the plasma membrane by distinct mechanisms (Geldner et al., 2003; Kleine-Vehn et al., 2006; Robert et al., 2008). CHMP1 and ESCRT proteins mediate the degradation of PIN1, PIN2, and AUX1 in the vacuolar lumen in wild-type cells by sorting these proteins into MVB lumenal vesicles. In chmp1a chmp1b mutant cells, the sorting of PIN1, PIN2, and AUX1 and the formation of lumenal vesicles is compromised. As a result of MVB sorting defects, PIN1, PIN2, and AUX1 remain in the MVB limiting membrane and accumulate in the vacuolar membrane upon MVB vacuole fusion.