Clathrin mediates endocytosis and polar distribution of PIN auxin transporters in Arabidopsis

Abstract

Endocytosis is a crucial mechanism by which eukaryotic cells internalize extracellular and plasma membrane material, and it is required for a multitude of cellular and developmental processes in unicellular and multicellular organisms. In animals and yeast, the best characterized pathway for endocytosis depends on the function of the vesicle coat protein clathrin. Clathrin-mediated endocytosis has recently been demonstrated also in plant cells, but its physiological and developmental roles remain unclear. Here, we assessed the roles of the clathrin-mediated mechanism of endocytosis in plants by genetic means. We interfered with clathrin heavy chain (CHC) function through mutants and dominant-negative approaches in Arabidopsis thaliana and established tools to manipulate clathrin function in a cell type-specific manner. The chc2 single mutants and dominant-negative CHC1 (HUB) transgenic lines were defective in bulk endocytosis as well as in internalization of prominent plasma membrane proteins. Interference with clathrin-mediated endocytosis led to defects in constitutive endocytic recycling of PIN auxin transporters and their polar distribution in embryos and roots. Consistent with this, these lines had altered auxin distribution patterns and associated auxin transport-related phenotypes, such as aberrant embryo patterning, imperfect cotyledon specification, agravitropic growth, and impaired lateral root organogenesis. Together, these data demonstrate a fundamental role for clathrin function in cell polarity, growth, patterning, and organogenesis in plants.

Figure 1.

Requirement of Clathrin Function for Endocytosis. (A) to (D) Uptake of endocytic tracer dye FM4-64 (2 μM) after 8 min in root meristem epidermal cells of the INTAM driver line ([A] and [C]) and INTAM>>RFP-HUB1 ([B] and [D]) induced for 1 ([A] and [B]) or 2 ([C] and [D]) d with 2 μM 4-hydroxytamoxifen. (E) to (J) Immunolocalization of PIN1 and PIN2 (red signal; median section) ([E] and [F]), PM-ATPase (green signal; epidermis) ([G] and [H]), and ARF1 (red signal; epidermis) ([I] and [J]) after 1 h of BFA (50 μM) treatment on seedlings of the INTAM driver line ([E], [G], and [I]) and INTAM>>RFP-HUB1 ([F], [H], and [J]) germinated on 2 μM 4-hydroxytamoxifen. Arrowheads highlight BFA bodies. (K) Immunolocalization of PIN1 and PIN2 in J0571>>RFP-HUB1 after 1 h of BFA (50 μM) treatment (median section). En, endodermis; C, cortex; Ep, epidermis. Right-hand panels indicate J0571>>mGFP5-ER (green), and J0571>>RFP-HUB1 (red) expression in cortex and endodermis prior to immunolocalization.

Figure 2.

Effect of HUB Induction on Seedling Growth. (A) and (B) Seedlings grown for 3 d in the presence of 4-hydroxytamoxifen of the INTAM driver control (A) and INTAM>>RFP-HUB1 (B). (C) and (D) Etiolated seedlings, germinated in the presence of 4-hydroxytamoxifen of the INTAM driver control (C) and INTAM>>RFP-HUB1 (D). (E) and (F) Root growth of seedlings grown for 2 d on half-strength Murashige and Skoog medium followed by transfer to 4-hydroxytamoxifen for 2 d of INTAM driver control (E) and INTAM>>RFP-HUB1 (F). (G) and (H) Auxin-induced lateral roots of INTAM driver control (G) and INTAM>>RFP-HUB1 (H), transferred for 2 d to 4-hydroxytamoxifen and 10 μM 1-naphthaleneacetic acid.

Figure 3.

Changes in Auxin Distribution Caused by HUB Induction. (A) to (D) DR5rev:GFP activity in root tips of control (A) and INTAM>>RFP-HUB1 (B) after 24 h of induction with 4-hydroxytamoxifen. DR5rev:GFP activity in gravistimulated (5 h) root tips of control (C) and INTAM>>RFP-HUB1 (D) induced with 4-hydroxytamoxifen (24 h). Arrows (g) indicate gravity vector. (E) and (F) DR5rev:GFP expression pattern in auxin-induced lateral roots of control (E) and INTAM>>RFP-HUB1 (F). (G) Induction of HUB expression increases acropetal auxin transport in root meristems (26 h treatment with 2 μM 4-hydroxytamoxifen [Tam] or DMSO). Asterisk indicates P < 0.05; n = 9. Error bars are sd.

Figure 4.

Characterization of chc Mutant Alleles. (A) Schematic representation of the intron (bar)-exon (black box) structure in CHC1 and CHC2. Arrowheads indicate the T-DNA insertion sites. (B) RT-PCR from RNA extracts of the chc1 or chc2 single mutants and wild-type Columbia-0 (Col-0) plantlets. The positions of primers are shown in (A). (C) to (F) Delay of FM4-64 uptake in chc2 mutants. Intracellular accumulation of FM4-64 is apparent within 6 min after incubation in 2 μM FM4-64 in the wild type (C) and chc1-2 mutants (D). By contrast, the intracellular signal is much weaker in chc2 alleles ([E] and [F]). (G) Quantification of relative FM4-64 uptake. Error bars indicate sd. Asterisks indicate P < 0.0001 (Student’s t test); n = number of cells analyzed.

Figure 5.

Patterning Defects of Seedlings and Embryos in the chc2 Mutants. (A) to (D) Ten-day-old seedlings of the wild type (WT) (A) and chc2 mutants with trumpet-shaped cotyledons (B), collar-shaped cotyledons (C), and stick-shaped cotyledons (D). (E) and (F) Vascular pattern in cotyledons of 10-d-old seedlings of the wild type (E) and monocotyledonous chc2 mutants (F). (G) and (H) Lugol’s staining of primary root meristems of 10-d-old wild-type (G) and chc2 seedlings with stick-shaped cotyledons (H). Columella cells of these chc2 seedlings are highly disorganized. (I) to (N) Embryonic development in the wild type ([I] to [K]) and chc2 mutants ([L] to [N]). (O) to (Q) DR5rev:GFP expression pattern in wild-type ([O] and [P]) and chc2 (Q) embryos.

Figure 6.

PIN Localization Defects Caused by Impaired CHC Function. (A) to (F) Immunolocalization of PIN1 in globular stage embryos ([A], [C], and [E]) and in heart-stage embryos ([B], [D], and [F]) from wild-type Columbia-0 (Col-0) plants ([A] and [B]), chc2-1 plants ([C] and [D]), and chc2-2 plants ([E] and [F]). The left panels show medial view of embryos, and the right ones surface views of the same embryos in each panel. (G) to (H) Immunolocalization of PIN1 (stele and endodermis) and PIN2 (cortex and epidermis) in roots from a INTAM driver line seedling (G) and a INTAM>>RFP-HUB1 seedling (H). Arrowheads indicate individual cellular PIN polarities. En, endodermis; C, cortex; Ep, epidermis.