A Novel System for Xylem Cell Differentiation in Arabidopsis thaliana Yuki Kondo, Takashi Fujita, Munetaka Sugiyama, Hiroo Fukuda  Molecular Plant  Volume 8, Issue 4, Pages 612-621 (April 2015) DOI: 10.1016/j.molp.2014.10.008 Copyright © 2015 The Author Terms and Conditions

Figure 1 A Leaf-Disk Culture System for the Study of Xylem Cell Differentiation. (A) The procedure for isolation and culture of Arabidopsis leaf disks. Leaf disks of 1 mm were isolated from 3- to 4-week-old Arabidopsis plants and cultured on MS liquid medium containing 1.25 mg l−1 2,4-D, 0.25 mg l−1 kinetin, and 10 μM bikinin for 3 days with shaking at 120 rpm. (B) Tracheary element differentiation in leaf disks cultured with 0, 5, and 10 μM bikinin in the presence or absence of plant hormones auxin and cytokinin. (C) A high-magnification image of induced leaf disks. (D) High-magnification images of tracheary elements isolated from bikinin-treated leaf disks. (E) An increase in ectopic tracheary element differentiation depending on concentrations of bikinin (n ≥ 12). Ectopic tracheary element differentiation is quantified by the areas occupied by differentiated tracheary elements. Error bars indicate SD. Asterisks indicate significant differences by Student's t-test (P < 0.01). (F) Four-week-old N. benthamiana plants. (G) Ectopic tracheary element differentiation in N. benthamiana leaf disks cultured for 6 days with bikinin. Both differential interference contrast (DIC) and UV images visualized secondary cell wall thickening. Scale bars indicate 5 mm for (A), 500 μm for (B) and (G), 100 μm for (C), and 50 μm for (D). Molecular Plant 2015 8, 612-621DOI: (10.1016/j.molp.2014.10.008) Copyright © 2015 The Author Terms and Conditions

Figure 2 Leaf Size Suitable for Tracheary Element Differentiation. (A) Classification of Arabidopsis true leaves according to the length of the leaf blade. (B) Effect of the leaf size on ectopic tracheary element differentiation. The average ectopic xylem area and its coefficient of variation were calculated for each category of true leaves to quantify differentiation potential and stability, respectively (n = 12–15). Molecular Plant 2015 8, 612-621DOI: (10.1016/j.molp.2014.10.008) Copyright © 2015 The Author Terms and Conditions

Figure 3 Progress of Differentiation Stages during Leaf-Disk Culture. (A–D) Spatiotemporal changes in TDRpro:GUS expression during culture of leaf disks with bikinin. Patterns of GUS expression and tracheary element differentiation allowed us to divide the process of vascular development into four stages (stages I–IV; see also the main text). Scale bars indicate 500 μm. (E–G) Progression of vascular development in leaf disks from apical and basal halves of leaves harboring TDRpro:GUS when cultured with bikinin. The proportion of individuals at each stage of development was calculated at 0, 24, 48, and 72 h after induction (hai) (n ≥ 10). Molecular Plant 2015 8, 612-621DOI: (10.1016/j.molp.2014.10.008) Copyright © 2015 The Author Terms and Conditions

Figure 4 Expression Analysis of Vascular-Related Genes in Cultured Leaf Disks. (A and B) Time-course expression profiles of procambium-related (A) and xylem-related (B) genes based on microarray data. Relative expression levels of genes in cultured leaf disks were calculated at each time point by comparing with those of genes before culture. Error bars indicate SD (n = 3). (C) Expression levels of 14 procambium-related genes in cultured leaf disks. Fourteen procambium-related genes were selected referring to previous reports. Fold changes of the 14 genes were calculated by comparing expression levels at 24 hai with those before induction. Error bars indicate SD. (D) Schematic illustration of the timetable of differentiation for procambial cells (PC) and xylem cells (XY) from mesophyll cells (MS) in leaf-disk culture. Molecular Plant 2015 8, 612-621DOI: (10.1016/j.molp.2014.10.008) Copyright © 2015 The Author Terms and Conditions

Figure 5 Bikinin-Dependent Activation of TDR Expression. (A and B) TDRpro:GUS expression patterns in leaf disks cultured without (A) and with (B) bikinin for 24 h. Scale bars indicate 500 μm. (C) TDR expression in leaf disks cultured without or with bikinin for 24 h quantified by real-time RT–PCR. Fold changes in expression levels were calculated by comparing with those before culture (n = 3). Error bars indicate SD. An asterisk indicates a significant difference by the paired t-test (P < 0.05). (D and E) TDRpro:YFPnls fluorescent signals in young true leaves cultured without or with bikinin for 24 h. Green dotted lines indicate the margin of leaves. Similar results were obtained for at least five independent experimental sets. Culture media contain auxin and cytokinin. Scale bars indicate 500 μm. Molecular Plant 2015 8, 612-621DOI: (10.1016/j.molp.2014.10.008) Copyright © 2015 The Author Terms and Conditions

Figure 6 Effect of Bikinin and TDIF on Tracheary Element Differentiation in Cultured Leaf Disks. (A) Outline of the experiment to evaluate the effects of TDIF and bikinin on the step during the differentiation of procambial cells into xylem cells in leaf-disk culture. Leaf disks were precultured with bikinin for the first 24 h to produce procambial cells. These leaf disks were transferred to media containing no bikinin or TDIF (control), 5 μM TDIF, or both 5 μM TDIF and 10 μM bikinin, then cultured for the next 48 h. Auxin and cytokinin are included in all media. (B–E) Effects of TDIF and bikinin on tracheary element differentiation. Leaf disks from wild-type (WT) and TDRpro:GUS plants were precultured with bikinin and then were transferred to media without TDIF or bikinin (B), with TDIF (C and D), or with TDIF and bikinin (E). Scale bars indicate 500 μm. (F) Quantitative analysis of tracheary element differentiation when cultured under the same conditions as (B), (C), and (E). Error bars indicate SD (n ≥ 10). Asterisks indicate significant differences by Student’s t-test (P < 0.01). (G) VND6 expression in leaf disks from WT and tdr-1 plants, when cultured without TDIF or bikinin, with TDIF, or with TDIF and bikinin for next 48 h. Error bars indicate SD (n = 3). An asterisk indicates a significant difference by the paired t-test (P < 0.05). Culture media contain auxin and cytokinin. Molecular Plant 2015 8, 612-621DOI: (10.1016/j.molp.2014.10.008) Copyright © 2015 The Author Terms and Conditions

Figure 7 Transcriptome Analysis of Cultured Leaf Disks. Clustering analysis of 117 genes up-regulated during culture. As highly (more than eight-fold) and significantly (P < 0.001) up-regulated genes, 117 genes were selected (see also main text). These genes were classified into three groups (clusters A–C) by K-means clustering based on gene expression profiles (log2 scale). Expression profiles of genes belonging to each cluster are shown in the top panels. Panels in the middle row show fold changes in gene expression levels between at 12 and 24 hai, and between at 24 and 36 hai. In the bottom panels, genes in each cluster were categorized according to their functions based on the information obtained from TAIR (http://www.Arabidopsis.org/): cell cycle, cell division, cell wall, cell death, others, and unknown. Molecular Plant 2015 8, 612-621DOI: (10.1016/j.molp.2014.10.008) Copyright © 2015 The Author Terms and Conditions

Figure 8 Expression Profiles of Cluster Genes in Two Other Arabidopsis Xylogenic Cultures. (A) Expression profiles of cluster A, B, and C genes in boron-treated xylogenic culture (Kubo et al., 2005). Expression levels for genes of each cluster at 0, 2, 4, 6, 8, and 10 days after induction were visualized by tree clustering and heat maps. (B) Expression profiles of cluster A, B, and C genes in the culture of WT and inducible VND6-harboring suspension cells (Ohashi-Ito et al., 2010). Expression levels for genes of each cluster at 0 and 12 h after estradiol induction were visualized by tree clustering and heat maps. Processed signal was calculated by median-centering normalization for each gene. Color-coded heat maps were generated by the Subio Platform (Subio) according to the scales shown on the right. Molecular Plant 2015 8, 612-621DOI: (10.1016/j.molp.2014.10.008) Copyright © 2015 The Author Terms and Conditions

Figure 9 Ectopic Tracheary Element Differentiation in Leaf Disks from bes1 and vnd6 Mutants. Leaf disks of WT, bes1, and vnd6 mutants were cultured with bikinin for 72 h. (A–C) DIC images of cultured leaf disks of WT, bes1, and vnd6. Scale bars indicate 500 μm. (D–F) Histograms of ectopic tracheary element differentiation areas in leaf disks from WT, bes1, and vnd6 mutants (n = 24–30). Molecular Plant 2015 8, 612-621DOI: (10.1016/j.molp.2014.10.008) Copyright © 2015 The Author Terms and Conditions