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. 2012 Jun 6;12:81.
doi: 10.1186/1471-2229-12-81.

Myosins XI-K, XI-1, and XI-2 Are Required for Development of Pavement Cells, Trichomes, and Stigmatic Papillae in Arabidopsis

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Myosins XI-K, XI-1, and XI-2 Are Required for Development of Pavement Cells, Trichomes, and Stigmatic Papillae in Arabidopsis

Eve-Ly Ojangu et al. BMC Plant Biol. .
Free PMC article

Abstract

Background: The positioning and dynamics of vesicles and organelles, and thus the growth of plant cells, is mediated by the acto-myosin system. In Arabidopsis there are 13 class XI myosins which mediate vesicle and organelle transport in different cell types. So far the involvement of five class XI myosins in cell expansion during the shoot and root development has been shown, three of which, XI-1, XI-2, and XI-K, are essential for organelle transport.

Results: Simultaneous depletion of Arabidopsis class XI myosins XI-K, XI-1, and XI-2 in double and triple mutant plants affected the growth of several types of epidermal cells. The size and shape of trichomes, leaf pavement cells and the elongation of the stigmatic papillae of double and triple mutant plants were affected to different extent. Reduced cell size led to significant size reduction of shoot organs in the case of triple mutant, affecting bolt formation, flowering time and fertility. Phenotype analysis revealed that the reduced fertility of triple mutant plants was caused by delayed or insufficient development of pistils.

Conclusions: We conclude that the class XI myosins XI-K, XI-1 and XI-2 have partially redundant roles in the growth of shoot epidermis. Myosin XI-K plays more important role whereas myosins XI-1 and XI-2 have minor roles in the determination of size and shape of epidermal cells, because the absence of these two myosins is compensated by XI-K. Co-operation between myosins XI-K and XI-2 appears to play an important role in these processes.

Figures

Figure 1
Figure 1
RT-PCR analysis of theXI-1T-DNA insertional line Salk_022140. Two different primer pairs were used to amplify two regions within the XI-1 mRNA: a – primers spanning the T-DNA insertion site; b - primers for downstream region of the T-DNA insertion site; c - control amplified with GAPB primers. gDNA – genomic DNA; WT - wild type.
Figure 2
Figure 2
Leaf and cell size. A) Size of the rosette leaves of five-week-old plants. Rosette leaves of xi-2/xi-k and xi-1/xi-2/xi-k in comparison with wild type (WT). Leaves on the lower panel originate from the rosettes on upper panel. Bar = 10 mm. B) Mean size of leaf blades and petioles (±SD). The 5th to 10th rosette leaves were used for the size measurements. Note shorter petioles and smaller leaf blades of the xi-2/xi-k and xi-1/xi-2/xi-k plants in comparison with WT. Asterisks indicate statistical significance (** p < 0.01, Repeated Measures ANOVA with Dunn’s Multiple Comparisons Test, n = 18). C) Mean cell areas of pavement and mesophyll cells of five-week-old rosette leaves (±SD). In the case of xi-2/xi-k and xi-1/xi-2/xi-k the cell area is smaller compared to WT (* p < 0.05 and ** p < 0.01, One-Way ANOVA with Dunnett’s Multiple Comparisons Test). D) Shape of pavement cells of leaf abaxial epidermis. Note less extended lobes of the xi-1/xi-2/xi-k pavement cells in comparison with WT (black cells on the image). Bar = 50 μm. E) Circularity values of pavement cells on leaf abaxial epidermis (mean ± SD). Circularity reflects the ratio of pavement cell area to the perimeter. Cells with values near one are more circular, meaning that xi-1/xi-2/xi-k cells have more round morphology than WT cells (*** p < 0.001, unpaired t-test with Welch correction, n = 13-16).
Figure 3
Figure 3
Size and morphology of leaf trichomes.A) Stalk and branch length of leaf trichomes (mean ± SD): BR1 – first branch; BR2 – second branch; BR3 – third branch; WT – wild type. B) Ratios of mean lengths of trichome branches and stalk (±SD). In WT, xi-1, xi-2, xi-k and xi-1/xi-2 the stalk length constitutes approximately half of the branch length. In xi-1/xi-k and xi-2/xi-k the stalk is abnormally increased and in xi-1/xi-2/xi-k the branches are equal or slightly longer than stalk. Asterisks indicate statistical significance (* p < 0.05 and *** p < 0.001, Kruskal-Wallis with Dunn’s Multiple Comparisons Test, n = 73-109). C) Morphology of leaf trichomes. Numbers and arrows show: (1) trichomes with elongated interbranch zone, (2) unproportionally elongated individual branches, (3) unproportionally elongated stalks, (4) sword-shaped trichomes. Note also that modest bending of trichomes is visible in the case of all mutants. The number of trichomes with irregular shape was quantified by counting the presence of at least one of the phenotypes. Bar = 300 μm.
Figure 4
Figure 4
Morphology of leaf trichomes at different developmental stages. A-D) Overall view of trichomes of the two-week-old leaves. Bar = 250 μm. E-H) Close-up view of trichomes. Numbers indicate the different developmental stages of trichomes. Arrows indicate the irregular shape or elongation of stage 5 and 6 trichome branches of xi-1/xi-k, xi-2/xi-k and xi-1/xi-2/xi-k plants. Bar = 100 μm.
Figure 5
Figure 5
Shape of leaf trichome nuclei. A) Hoechst stained nuclei of two-week-old leaf trichomes. Abnormally elongated nuclei of xi-2/xi-k and xi-1/xi-2/xi-k trichomes are shown. Bar = 15 μm. B) Sphericity of trichome nuclei (mean ± SD). Sphericity is the ratio of the volume of a nucleus to the surface area of the nucleus, which is the smallest in xi-2/xi-k mutants. Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, and *** p < 0.001, Kruskal-Wallis with Dunn’s Multiple Comparisons Test, n = 55-157).
Figure 6
Figure 6
Length of siliques of the primary shoot. In xi-1/xi-2/xi-k, siliques are significantly shorter, containing much less seeds than those in wild type (WT). Bar = 5 mm.
Figure 7
Figure 7
Silique size and seed development. A) Shoots of the eight-week-old plants. Arrows indicate the unpollinated pistils on the primary stem of xi-1/xi-2/xi-k. B) Representative wild type (WT) and xi-1/xi-2/xi-k siliques are shown. Note the heterogenous development of triple mutant siliques on the main stem. Bar = 2 mm. C) Scanning electron micrographs of developing seeds in manually opened siliques. Arrows point to the unfertilized ovules in xi-1/xi-2/xi-k siliques. Left panel, bar = 500 μm; right panel, bar = 150 μm.
Figure 8
Figure 8
Development of pollen tubes. Pollen tube growth was followed in cross-pollinated pistils: WT/triple – wild type (WT) pistil was pollinated with xi-1/xi-2/xi-k; triple/WT – xi-1/xi-2/xi-k pistil was pollinated with WT. Note the number of pollen grains attached to the stigmas and the number of pollen tubes formed. In the case of triple/WT, WT pollen grains were not able to attach effectively on the surface of xi-1/xi-2/xi-k stigma and only few pollen tubes were formed. Aniline blue staining of pollen tubes was performed 12 hours after hand-pollination. Pg – pollen grains, St – stigma, Pt – pollen tubes. Bar = 50 μm.
Figure 9
Figure 9
Size of floral organs. A) Developmental stages of flowers from inflorescence of the primary shoot are shown. Note the smaller size of xi-1/xi-2/xi-k flowers and flower buds. Bar = 1 mm. B) Mean length of peduncles, sepals, petals and flower buds (±SD). Opened flowers and flower buds of the inflorescences (from main stems) were selected for measurements. The average size of floral organs of xi-1/xi-2/xi-k is smaller than in wild type (WT). Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, and *** p < 0.001, unpaired t-test with Welch correction, n = 6-12).
Figure 10
Figure 10
Flower development ofxi-1/xi-2/xi-k. A) Scanning electron micrographs of stage 12 flower buds of wild type (WT) and xi-1/xi-2/xi-k. Epidermal surface of the stigma has papillar structure. Note that stigmatic papillae (Sp) of xi-1/xi-2/xi-k are shorter than those of WT. B) Scanning electron micrographs of stage 14 flowers of xi-1/xi-2/xi-k. As xi-1/xi-2/xi-k flowers develop heterogeneously, both well developed (pollinated) as well as underdeveloped flower is shown. In the case of underdeveloped flower the development of whole pistil is retarded. Arrows indicate the retarded growth of the pistil, arrested in developmental stage 10. On the left panel: overall view of the flower. Bar = 500 μm. On the right panel: close-up view of the pistil. Bar = 100 μm. Sp – stigmatic papillae.

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