Arabidopsis IPGA1 is a microtubule-associated protein essential for cell expansion during petal morphogenesis

Abstract

Unlike animal cells, plant cells do not possess centrosomes that serve as microtubule organizing centers; how microtubule arrays are organized throughout plant morphogenesis remains poorly understood. We report here that Arabidopsis INCREASED PETAL GROWTH ANISOTROPY 1 (IPGA1), a previously uncharacterized microtubule-associated protein, regulates petal growth and shape by affecting cortical microtubule organization. Through a genetic screen, we showed that IPGA1 loss-of-function mutants displayed a phenotype of longer and narrower petals, as well as increased anisotropic cell expansion of the petal epidermis in the late phases of flower development. Map-based cloning studies revealed that IPGA1 encodes a previously uncharacterized protein that colocalizes with and directly binds to microtubules. IPGA1 plays a negative role in the organization of cortical microtubules into parallel arrays oriented perpendicular to the axis of cell elongation, with the ipga1-1 mutant displaying increased microtubule ordering in petal abaxial epidermal cells. The IPGA1 family is conserved among land plants and its homologs may have evolved to regulate microtubule organization. Taken together, our findings identify IPGA1 as a novel microtubule-associated protein and provide significant insights into IPGA1-mediated microtubule organization and petal growth anisotropy.

Keywords: Arabidopsis; INCREASED PETAL GROWTH ANISOTROPY 1 (IPGA1); cortical microtubule; growth anisotropy; microtubule-associated protein; petal.

Fig. 1.

Phenotypic analysis of the ipga1-1 mutant. (A) Mature flowers and petals from WT (Col-0) and the ipga1-1 mutant. The ipga1-1 mutant had longer and narrower petals than the WT. Scale bars: 1 mm. (B–D) Quantification of petal parameters of Col-0 and ipga1-1. Values are mean ±SD of 80 petals of 20 flowers from six plants. Asterisks indicate a significant difference (*P<0.05, Student’s t-test). n.s. indicates no significant difference (P>0.05, Student’s t-test).

Fig. 2.

Identification and characterization of IPGA1. (A) Schematic representation of the IPGA1 (At4g18570) gene, showing the nature and position of the ipga1 mutant alleles. Map-based cloning demonstrated that the igpa1-1 mutation carries a single nucleotide G-to-A mutation in codon 467 (TGG/TGA) of At4g18570 generating a premature stop codon. Black rectangles represent coding regions, and the black lines represent introns. Arrows indicate nucleotide substitutions, and triangles indicate T-DNA insertions. (B) RT-PCR monitoring of IPGA1 mRNA levels in Col-0, ipga1-2, and ipga1-3. EF1a mRNA was used as a control. (C) Mature flowers and petals from plants of the indicated genotypes. The petals from ipga1-2 and ipga1-3 displayed a longer and narrower shape compared with Col-0. Scale bars: 1 mm. (D–F) Quantification of petal parameters of Col-0, ipga1-2, and ipga1-3. Values are mean ±SD of 80 petals from 20 flowers from more than six plants. Asterisk indicates significant difference (*P<0.05, Student’s t-test).

Fig. 3.

IPGA1 functions as a negative regulator in modulating anisotropic cell expansion at late stages. (A) Cells from the top regions of petal abaxial epidermis of Col-0 and ipga1-1 throughout developmental stages 8–14. Scale bar: 20 µm. (B–E) Quantification of cell parameters of Col-0 and ipga1-1 from the indicated developmental stages. (B) Cell length. (C) Cell width. (D) Cell index, calculated by length divided by width. (E) Cell area. Values are mean ±SD of more than 600 cells of six petals from six plants. Asterisks indicate a significant difference (**P<0.01, Student’s t-test). n.s. indicates no significant difference (P>0.05).

Fig. 4.

Expression patterns of IPGA1. (A, B) IPGA1 mRNA expression levels through plant development. Data were collected from publicly available databases at (A) eFP browser (microarray) and (B) TRAVAdb (RNA-seq). (C) qRT-PCR monitoring of IPGA1 mRNA levels throughout petal development stages 8–14. Petals from various developmental stages were used for reverse transcription (*P<0.05, **P<0.01, Student’s t-test). (D, E) IPGA1 was expressed in developing petals. Histochemical GUS staining of inflorescence (D) and petals (E) from a representative proIPGA1:GUS transgenic line. Scale bars: 1 mm.

Fig. 5.

IPGA1 localizes to and directly binds to microtubules. (A) Subcellular localization of GFP–IPGA1. Confocal images of the 35S::GFP-IPGA1 constructs transiently expressing in Col-0 pavement cells. GFP–IPGA1 exhibited filamentous structures in cotyledon pavement cells, and these filamentous structures visualized in this cell were disrupted by oryzalin treatment. Notably, these structures remained intact in the presence of LatA. Scale bars: 10 µm. (B) IPGA1 colocalized with cortical microtubules in Col-0 pavement cells. pIPGA1::GFP-IPGA1 was transiently expressed in cotyledon pavement cells from the microtubule marker line mCherry-TUA5. Scale bar: 10 µm. (C–E) FRAP analysis of GFP–IPGA1. (C) 35S::GFP-IPGA1 signal in the cotyledon pavement cell. The yellow rectangle outlines the photobleached area. Scale bar: 5 µm. (D) Time series of GFP–IPGA1 signal recovery during a FRAP experiment within the area indicated by the rectangle in (C). The numbers show the time in seconds when each of the frames were collected with 0 corresponding to the image before the photobleaching onset and 6.440 just after the photobleaching. (E) Quantification of fluorescence signal. The first FRAP measurement was performed just before the photobleaching and corresponds to point 0. The grey sector indicates the duration of photobleaching, after which 20 images were collected and quantified at approximately 3 s intervals. The fluorescence signals were measured in 20 cells and expressed as the mean percentage of the signal before photobleaching. The error bars represent the SD.

Fig. 6.

IPGA1 affects microtubule organization and stability. (A) Analysis of microtubule organization at indicated petal developmental stages using the microtubule maker line GFP-TUA6. The ipga1-1 mutant displayed well-ordered transverse microtubule arrays in abaxial epidermal cells of the top part of petal blades at stage 10 and beyond. The red dots depict cell outlines. Scale bar: 5 µm. (B) Quantification of microtubule alignment. The microtubule alignment measurement was carried out with OrientationJ, an ImageJ plug-in, to calculate the directional coherency coefficient of the fibers (Fonck et al., 2009). A coherency coefficient close to 1 represents a strongly coherent orientation of the microtubules. Values are mean ±SD (n=70 cells from 15 petals). Asterisks indicate a significant difference (*P<0.05, **P<0.01, Student’s t-test). (C) Cortical microtubules were observed in epidermal cells of the basal region of GFP-TUA6 and ipga1-1 GFP-TUA6 etiolated hypocotyls after treatment with 20 μM oryzalin for 0, 2, 5, and 10 min. Oryzalin was then washed off and cortical microtubules were imaged after 2 h. Scale bar: 10 μm. (D) Quantification of number of microtubules in hypocotyl epidermal cells using ImageJ software. Values are mean ±SD (n>50 cells from each sample). Asterisks indicate a significant difference (*P<0.05, **P<0.01, Student’s t-test). (E) Cortical microtubules were observed in abaxial epidermal cells of the middle region of GFP-TUA6 and GFP-TUA6 ipga1-1 petals after treatment with water or 20 μM oryzalin for 10 min. Scale bar: 10 μm. (F) Quantification of the numbers of microtubules using ImageJ software. Values are mean ±SD (n>30 cells from each sample). Asterisks indicate a significant difference (**P<0.01, Student’s t-test).