Twin Embryos in Arabidopsis thaliana KATANIN 1 Mutants

Abstract

Regulation of microtubule dynamics is crucial during key developmental transitions such as gametogenesis, fertilization, embryogenesis, and seed formation, where cells undergo rapid changes in shape and function. In plants, katanin plays an essential role in microtubule dynamics. This study investigates two seed developmental mutants in Arabidopsis thaliana, named elk5-1D (erecta-like 5, ELK5) and loo1 (lollipop 1), which are characterized by round seeds, dwarfism, and fertility defects. Notably, elk5-1D exhibits a dominant inheritance pattern, whereas loo1 is recessive. Through positional cloning, we identified both mutants as new alleles of the KATANIN 1 (KTN1) gene, which encodes a microtubule-severing enzyme critical for cell division and morphology. Mutations in KTN1 disrupt embryo cell division and lead to the emergence of a twin embryo phenotype. Our findings underscore the essential role of KTN1 in fertility and early embryonic development, potentially influencing the fate of reproductive cells.

Keywords: Arabidopsis thaliana; KATANIN 1; embryogenesis; microtubule; twin embryo.

Figure 1

Abnormal seed development in elk5-1D (erecta-like 5, ELK5) and loo1 (lollipop 1) mutants. (A) Representative images of mature seeds in Col-0 and elk5-1D and loo1 mutants. Scale bars = 0.5 mm. (B,C) Quantitative comparisons of seed area and of seed length/width ratio among Col-0, elk5-1D, and loo1. Final calculations were based on data collection from 443 to 457 seeds. Asterisks indicate significant differences compared to Col-0. Asterisks indicate significant differences analyzed by one-way ANOVA tests (ns: not significant p > 0.05, ****: p < 0.0001).

Figure 2

Observation of elk5-1D and loo1 fertility. (A) Morphology of 8-week-old plants. The main inflorescence stem of elk5-1D and loo1 (middle and right) is much shorter than that of wild-type (left). Scale bars = 2 cm. (B) Representative images of flowers of Col-0, elk5-1D, and loo1. Scale bars = 0.5 mm. (C) Number of seeds per silique in Col-0, elk5-1D, loo1, and reciprocal crosses, with elk5-1D and loo1 subjected to artificial pollination. (D) Mature pollen grains were stained with Alexander solution; viable pollen appeared purple. Scale bars = 0.2 mm. (E) Wild-type siliques showing a complete seed set, while siliques of elk5-1D and loo1 plants subjected to artificial pollination show many unfertilized ovules (indicated by black arrows). Scale bars = 0.5 mm.

Figure 3

Morphologies of the elk5-1D and loo1 mutants. The phenotypes of rosette leaves (A), individual leaves (B), inflorescences (C), flowers (D), main stems (E), and root lengths (F) among Col-0, elk5-1D, and loo1. The scale bars measure 2 cm in (A,B) and 0.5 cm in (C–F).

Figure 4

Diagram of the position of the elk5-1D and loo1 gene. (A) Fine mapping of the elk5-1D and loo1. The numbers below the markers indicate the number of recombinants detected between the markers. (B) Gene structure of KTN1 and the location of SNPs in mutant elk5-1D and the site of deletions in mutant loo1. (C) ktn1 transcript in loo1 detected by RT-PCR. (D) A schematic diagram of the KTN1 protein, indicating the Kp60-NTD, P-loop_NTPase, AAA_lid_3, and Vps4_C (oligomerization domain) dimer domains, is indicated in the bottom panel. The mutation positions of the elk5-1D and loo1 mutants are shown.

Figure 5

Rosette leaves and fertility phenotypes of plants that harbor the elk5-1D allele. Representative images of rosette leaves (A) and fertility phenotypes (B) from wild-type Col-0, heterozygous elk5-1D mutants, homozygous elk5-1D mutants, and transgenic plants harboring the pKTN1::elk5-1D and p35S::elk5-1D constructs. The scale bars measure 1 cm in (A) and 2 cm in (B).

Figure 6

Aberrant embryo development in elk5-1D and loo1 mutants. Col-0 embryos at different developmental stages, including 2 to 4 cells (A), octant (B), globular (C), and heart-shaped (D). Embryos of the elk5-1D and loo1 mutant at the early globular stage (E,F,I,J), globular stage (G), and heart shape stage (H,L). Developmental arrest in the loo1 mutant embryo (K). Scale bar = 50 μm.

Figure 7

Abnormal cell division in the suspensor. Transverse cell division occurs in the hypophysis of elk5-1D (A–C) and loo1 (D,F) as indicated by the arrow. The suspensor of loo1 abnormally differentiates into a proembryo (E). Scale bar = 50 μm.

Figure 8

Twin embryos in elk5-1D and loo1. There are two embryos within elk5-1D (A–C) and loo1 (D–F) seeds. Scale bar = 50 μm.