BES1 and BZR1 Redundantly Promote Phloem and Xylem Differentiation

BES1 and BZR1 Redundantly Promote Phloem and Xylem Differentiation Abstract Vascular development is a good model for studying cell differentiation in plants. Two conductive tissues, the xylem and phloem, are derived from common stem cells known as procambial/cambial cells. Glycogen synthase kinase 3 proteins (GSK3s) play crucial roles in maintaining procambial/cambial cells by suppressing their differentiation into xylem or phloem cells. We previously designed an in vitro culture system for analyzing vascular cell differentiation named VISUAL (Vascular cell Induction culture System Using Arabidopsis Leaves). Using this system, we found that the transcription factor BRI1-EMS-SUPPRESSOR 1 (BES1) functions as a downstream target of GSK3s during xylem differentiation. However, the function of BES1 in vascular development remains largely unknown. Here, we found that, in addition to xylem differentiation, BES1 positively regulates phloem differentiation downstream of GSK3s. Transcriptome analysis using VISUAL confirmed that BES1 promotes bi-directional differentiation of procambial cells into xylem and phloem cells. Genetic analysis of loss-of-function mutants newly generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system revealed that BRASSINAZOLE RESISTANT 1 (BZR1), the closest homolog of BES1, functions in vascular development redundantly with BES1. Notably, BZR1 has a weaker impact on vascular cell differentiation than BES1, suggesting that they contribute differentially to this process. In conclusion, our findings indicate that BES1 and BZR1 are key regulators of both xylem and phloem cell differentiation from vascular stem cells. Introduction Vascular tissues, comprising xylem and phloem cells, are required for long-distance transport of various materials in plants. The xylem transports water and minerals from roots to shoots, whereas, in most cases, the phloem transports photoassimilates from shoots to roots. Although xylem and phloem cells have different structures and functions, these cells originate from common stem cells known as procambial/cambial cells. Previous studies identified master regulators of xylem and phloem cell fate. VASCULAR-RELATED NAC-DOMAINs (VNDs) facilitate xylem differentiation (Kubo et al. 2005), whereas ALTERED PHLOEM DEVELOPMENT (APL) is required for phloem development (Bonke et al. 2003). Molecular analyses of these master regulators have revealed the downstream components controlling various developmental events associated with xylem and phloem differentiation (Ohashi-Ito et al. 2010, Yamaguchi et al. 2011, Furuta et al. 2014b). Prior to the final determination of cell fate, glycogen synthase kinase 3 proteins (GSK3s) regulate vascular cell differentiation as earlier regulators (Kondo et al. 2014). The SKI and SKII subgroups of GSK3s redundantly maintain procambial cell populations by suppressing xylem differentiation. Indeed, the application of the GSK3 inhibitor bikinin (De Rybel et al. 2009) induces ectopic xylem cell differentiation in the presence of auxin and cytokinin (Kondo et al. 2014, Kondo et al. 2015). This feature allowed us to establish a powerful tissue culture system for vascular cell differentiation named VISUAL (Vascular cell Induction culture System Using Arabidopsis Leaves). Using VISUAL, we found that ectopic xylem differentiation induced by the inhibition of GSK3s is suppressed in mutants for BRI1-EMS-SUPPRESSOR 1 (BES1) (Kondo et al. 2014, Kondo et al. 2015), a direct substrate of GSK3s (Yin et al. 2002). Bikinin also has a promotive effect on phloem differentiation in VISUAL (Kondo et al. 2016). However, it remains to be elucidated whether BES1 regulates phloem differentiation downstream of GSK3s and, if so, how BES1 regulates both phloem and xylem differentiation. The GSK3s–BES1 signaling module is also involved in the brassinosteroid (BR) signaling pathway. The perception of BR by BRASSINOSTEROID-INSENSITIVE 1 (BRI1) leads to the inactivation of GSK3s (Li and Nam 2002). BES1 family transcription factors are then activated to control downstream gene expression (Yin et al. 2005). Arabidopsis contains six members of the BES1 family. Among these, BES1 and its closest homolog, BRASSINAZOLE RESISTANT 1 (BZR1), are considered to be major mediators of BR signaling. However, our understanding of the BR signaling pathway has primarily been obtained from studies using the gain-of-function mutants bes1-D and bzr1-D (Yin et al. 2002, Wang et al. 2002). In contrast, few loss-of-function studies investigating BES1 family members have been performed (Yin et al. 2005). In particular, loss-of-function mutants for BZR1 have not yet been reported. Therefore, our understanding of the contribution of BES1 and BZR1 to BR signaling remains incomplete. As for vascular development, we also analyzed the role of BES1 and BZR1 using gain-of-function mutants (Kondo et al. 2014). However, recent advances in genome editing techniques, including the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system, should allow the actual functions of genes of interest to be analyzed by generating knock-out mutants. In this study, we investigated the functions of BES1 and BZR1 in vascular cell differentiation using loss-of-function mutants generated via the CRISPR/Cas9 system. Comprehensive gene expression analysis of the bes1 mutants revealed that BES1 positively regulates both xylem and phloem differentiation, but not procambial cell differentiation from mesophyll cells, in VISUAL. Further genetic analysis with bes1 bzr1 mutants indicated that BES1 and BZR1 act redundantly in promoting both xylem and phloem differentiation. Interestingly, the phenotypes when cultured in VISUAL were weaker in bzr1 than in bes1, indicating that BES1 and BZR1 contribute differently to vascular development. Consistently, bes1 bzr1 plants displayed lower sensitivity to exogenous BR treatment than the bes1 single mutants but were still sensitive to BR, suggesting the further functional redundancy of BES1 family members. Results BES1 positively regulates xylem cell differentiation in VISUAL We previously found that bes1 mutants have defects in xylem differentiation when cultured in VISUAL (Kondo et al. 2014, Kondo et al. 2015). To investigate further the role of BES1 in vascular development, we obtained three T-DNA insertion lines for BES1: bes1-1 (He et al. 2005), bes1-2 (Lachowiec et al. 2013) and bes1-3 (previously named bes1-2 in Kang et al. 2015, and bes1 in Kondo et al. 2014) (Fig. 1A). None of the mutants showed visible phenotypes in terms of plant growth and xylem development in vivo. However, the cotyledons of these mutants commonly exhibited reduced xylem differentiation at 96 h after VISUAL induction (Fig. 1B–E). Differentiation rates in bes1-1 and bes1-2 mutants were lower than that in bes1-3, indicating that bes1-1 and bes1-2 are strong alleles and that bes1-3 is a weak allele. We performed detailed phenotypic analysis of bes1-3 (weak allele) and bes1-1 (strong allele) by monitoring the expression of vascular marker genes. RNA samples were collected at 72 h after induction, when xylem-related genes were expressed at maximum levels. Our quantitative reverse transcription PCR (qRT-PCR) analysis indicated that the expression levels of xylem marker genes such as IRREGULAR XYLEM 3 (IRX3) and XYLEM CYSTEINE PEPTIDASE 1 (XCP1) were lower in the bes1 mutants than in the wild type (WT) and were lower in bes1-1 than in bes1-3 (Fig. 2A, B). Next, we introduced a genomic fragment of BES1 (gBES1) into bes1-1. The introduction completely restored the reduced ectopic xylem differentiation rate and xylem-related gene expression in bes1-1 (Fig. 3). These results further support the notion that BES1 positively regulates xylem differentiation. Fig. 1 View largeDownload slide Phenotypes of bes1 T-DNA insertion mutants in VISUAL. (A) Schematic diagram of the exon–intron structures of BES1. Two splicing variants (Jiang et al. 2015) are shown. Exons, introns and untranslated regions are illustrated by boxes, black solid lines and dotted lines, respectively. Red lines represent putative insertion sites of T-DNA in each bes1 mutant. (B–E) Ectopic xylem cell formation in VISUAL. WT (B), bes1-1 (SALK_098634) (C), bes1-2 (WiscDsLox246D02) (D) and bes1-3 (SALK_091133) (E) cotyledons were cultured in medium containing 2,4-D, kinetin and bikinin for 4 d. Scale bars indicate 1 mm. (F) Quantification of xylem differentiation rates in (B–E). Error bars indicate the SD (n = 8–10). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). Fig. 1 View largeDownload slide Phenotypes of bes1 T-DNA insertion mutants in VISUAL. (A) Schematic diagram of the exon–intron structures of BES1. Two splicing variants (Jiang et al. 2015) are shown. Exons, introns and untranslated regions are illustrated by boxes, black solid lines and dotted lines, respectively. Red lines represent putative insertion sites of T-DNA in each bes1 mutant. (B–E) Ectopic xylem cell formation in VISUAL. WT (B), bes1-1 (SALK_098634) (C), bes1-2 (WiscDsLox246D02) (D) and bes1-3 (SALK_091133) (E) cotyledons were cultured in medium containing 2,4-D, kinetin and bikinin for 4 d. Scale bars indicate 1 mm. (F) Quantification of xylem differentiation rates in (B–E). Error bars indicate the SD (n = 8–10). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). Fig. 2 View largeDownload slide Gene expression analysis of bes1 mutants in VISUAL. (A–E) Expression levels of vascular-related marker genes in VISUAL. IRX3 (A) and XCP1 (B) are xylem-specific markers, TDR (C) is a procambium marker and APL (D) and SEOR1 (E) are phloem markers. Relative expression levels were calculated by comparison with gene expression levels at 72 h in the WT. Error bars indicate the SD (n = 3). Significant differences among samples after induction are indicated by different letters (Tamhane T2 test, α = 0.05). (F) Hierarchical clustering of vascular-related genes. Each gene expression level was normalized by setting the median value of the WT, bes1-3 and bes1-1 as 0 (see the Materials and Methods). Two major clades were defined as the bes1-down and bes1-not-down group based on their expression pattern. Relative gene expression levels are visualized in a heatmap image according to the color scale shown in the right-hand panel. (G) Comparison of the time course of expression patterns between the bes1-down and bes1-not-down group. Each gene expression level was normalized by dividing the level by its maximum. (H) Box plot showing the distribution of the half-time point for each gene. The half-time point is defined as the first time point when the relative expression level reached 50% of its maximum. (I) Comparison of the expression patterns of VISUAL phloem-specific genes (VPs; Kondo et al. 2016). Expression levels are visualized in a heatmap image according to the color scale shown in the right-hand panel. (J) Comparison of the expression patterns of VPs in the WT vs. bes1-1 and the WT vs. apl (Kondo et al. 2016) in VISUAL. The horizontal axis and vertical axis represent fold change in bes1-1 and apl against the WT at the log2 scale, respectively. (K) Schematic illustration of the bes1 mutant phenotype in VISUAL. The bes1 mutations repress xylem and phloem differentiation from procambial cells. Fig. 2 View largeDownload slide Gene expression analysis of bes1 mutants in VISUAL. (A–E) Expression levels of vascular-related marker genes in VISUAL. IRX3 (A) and XCP1 (B) are xylem-specific markers, TDR (C) is a procambium marker and APL (D) and SEOR1 (E) are phloem markers. Relative expression levels were calculated by comparison with gene expression levels at 72 h in the WT. Error bars indicate the SD (n = 3). Significant differences among samples after induction are indicated by different letters (Tamhane T2 test, α = 0.05). (F) Hierarchical clustering of vascular-related genes. Each gene expression level was normalized by setting the median value of the WT, bes1-3 and bes1-1 as 0 (see the Materials and Methods). Two major clades were defined as the bes1-down and bes1-not-down group based on their expression pattern. Relative gene expression levels are visualized in a heatmap image according to the color scale shown in the right-hand panel. (G) Comparison of the time course of expression patterns between the bes1-down and bes1-not-down group. Each gene expression level was normalized by dividing the level by its maximum. (H) Box plot showing the distribution of the half-time point for each gene. The half-time point is defined as the first time point when the relative expression level reached 50% of its maximum. (I) Comparison of the expression patterns of VISUAL phloem-specific genes (VPs; Kondo et al. 2016). Expression levels are visualized in a heatmap image according to the color scale shown in the right-hand panel. (J) Comparison of the expression patterns of VPs in the WT vs. bes1-1 and the WT vs. apl (Kondo et al. 2016) in VISUAL. The horizontal axis and vertical axis represent fold change in bes1-1 and apl against the WT at the log2 scale, respectively. (K) Schematic illustration of the bes1 mutant phenotype in VISUAL. The bes1 mutations repress xylem and phloem differentiation from procambial cells. Fig. 3 View largeDownload slide Complementation test. (A–C) Ectopic xylem cell formation in VISUAL. Cotyledons of the WT (A), bes1-1 (B) and bes1-1 harboring genomic BES1 (gBES1/bes1-1) (C) were cultured for 4 d. Scale bars indicate 1 mm. (D) Quantification of xylem differentiation rates in (A–C). Error bars indicate the SD (n = 8). Significant differences compared with the WT were examined by Tamhane T2 test (α = 0.05). (E) Expression levels of xylem and phloem marker genes in VISUAL. RNA was extracted from cotyledons at 72 h after VISUAL induction. Two independent complement lines were examined. Relative expression levels were calculated by comparison with the WT. Error bars indicate the SD (n = 3). Significant differences compared with the WT were examined by Tamhane T2 test (α = 0.05). Fig. 3 View largeDownload slide Complementation test. (A–C) Ectopic xylem cell formation in VISUAL. Cotyledons of the WT (A), bes1-1 (B) and bes1-1 harboring genomic BES1 (gBES1/bes1-1) (C) were cultured for 4 d. Scale bars indicate 1 mm. (D) Quantification of xylem differentiation rates in (A–C). Error bars indicate the SD (n = 8). Significant differences compared with the WT were examined by Tamhane T2 test (α = 0.05). (E) Expression levels of xylem and phloem marker genes in VISUAL. RNA was extracted from cotyledons at 72 h after VISUAL induction. Two independent complement lines were examined. Relative expression levels were calculated by comparison with the WT. Error bars indicate the SD (n = 3). Significant differences compared with the WT were examined by Tamhane T2 test (α = 0.05). BES1 promotes xylem cell differentiation from procambial cells in VISUAL In VISUAL, mesophyll cells first differentiate into procambial cells, followed by xylem cells (Kondo et al. 2015). We therefore examined whether BES1 is involved in the induction of procambial cells, xylem differentiation from procambial cells or both processes. First, we used the procambium-specific gene TDIF RECEPTOR (TDR) as a marker and found that this gene was not down-regulated in bes1 mutants in VISUAL. Next, to dissect downstream and upstream factors for BES1 comprehensively, we performed microarray analysis of cotyledons at 72 h after VISUAL induction in bes1-1, bes1-3 and the WT. We initially selected genes that were highly up-regulated at 72 h after VISUAL induction in the WT (>4-fold up-regulation, 1,534 genes; Kondo et al. 2016) as vascular-related genes. Hierarchical clustering classified the vascular-related genes into two major groups. Approximately 60% of the vascular-related genes were down-regulated in bes1 mutants. Then we designated these genes as a ‘bes1-down’ group (Fig. 2F). On the other hand, nearly 40% of vascular-related genes were expressed at the same or a higher level in the mutants. Therefore, we categorized these genes as a ‘bes1-not-down’ group (Fig. 2F). To characterize these two groups of genes, we analyzed the expression profiles of the bes1-down group and the bes1-not-down group using our previously reported time course microarray data from leaf disks cultured in VISUAL (Kondo et al. 2015). Most bes1-down genes were highly expressed 60 h after induction, whereas many bes1-not-down genes were induced 36 h after induction (Fig. 2G). This analysis indicated that the bes1-not-down and the bes1-down genes begin to be expressed at early and late stages in VISUAL, respectively. Further half-time point analysis, which evaluates the timing of gene expression in VISUAL, indicated that the expression of the bes1-not-down genes increased from 12 to 26 h and that of the bes1-down genes increased from 30 to 48 h in VISUAL (Fig. 2H). These results indicate that, in the bes1 mutants, the late stage of VISUAL is suppressed, whereas the early stage is not suppressed but is instead promoted. We then conducted Gene Ontology (GO) enrichment analysis to characterize the molecular functions of the bes1-down and bes1-not-down genes. In the bes1-down group, secondary cell wall-related genes, including xylan and lignin biosynthesis genes, were markedly enriched (Table 1). Additionally, genes related to xylem vessel cell differentiation were also enriched in this group (Table 1). These results coincide with the finding that bes1 mutants show defects in xylem differentiation in VISUAL. On the other hand, procambial cell-related genes were enriched in the bes1-not-down group (Table 1). Moreover, the bes1-not-down group includes many cell cycle and cell division genes (Table 1), including seven out of eight procambial-specific genes that were up-regulated in VISUAL only in the presence of bikinin (Kondo et al. 2015). Additionally, some bes1-not-down genes were annotated as auxin-related genes, which seem to be implicated in procambium formation in leaves (Furuta et al. 2014a). These results suggest that, in cultured bes1 cotyledons, procambial cells accumulate due to the inhibition of differentiation of procambial cells into xylem cells. Altogether, these data support our hypothesis that BES1 promotes xylem cell differentiation from procambial cells in VISUAL. Table 1 GO enrichment analysis of vascular-related genes Term  P-value  Fold enrichment  bes1-down genes  Regulation of secondary cell wall biogenesis  2.07E-15  24.88  Plant-type secondary cell wall biogenesis  1.29E-14  12.53  Xylan biosynthetic process  5.44E-13  15.88  Carbohydrate metabolic process  2.06E-07  2.75  Glucuronoxylan biosynthetic process  9.15E-07  17.87  Xylem development  1.56E-06  10.21  Xylan metabolic process  4.09E-06  20.42  Cell wall organization  4.79E-06  2.731  Lignin catabolic process  1.55E-05  11.91  Lignin biosynthetic process  6.65E-05  4.954  Xylem vessel member cell differentiation  7.05E-05  19.14  Cell wall thickening  1.35E-04  30.63  Phloem development  6.40E-04  20.42  Positive regulation of secondary cell wall biogenesis  6.40E-04  20.42  bes1-not-down genes  Microtubule-based movement  2.19E-19  15.32  Cell division  1.16E-14  6.134  Response to auxin  3.22E-12  4.77  Auxin-activated signaling pathway  3.10E-10  5.40  Cell cycle  1.40E-08  6.211  Mitotic nuclear division  4.34E-08  7.429  Regulation of cell cycle  1.15E-07  7.625  Multicellular organism development  6.54E-06  2.902  Procambium histogenesis  2.85E-05  24.76  Auxin polar transport  2.41E-04  6.368  Cotyledon vascular tissue pattern formation  5.90E-04  12.38  Term  P-value  Fold enrichment  bes1-down genes  Regulation of secondary cell wall biogenesis  2.07E-15  24.88  Plant-type secondary cell wall biogenesis  1.29E-14  12.53  Xylan biosynthetic process  5.44E-13  15.88  Carbohydrate metabolic process  2.06E-07  2.75  Glucuronoxylan biosynthetic process  9.15E-07  17.87  Xylem development  1.56E-06  10.21  Xylan metabolic process  4.09E-06  20.42  Cell wall organization  4.79E-06  2.731  Lignin catabolic process  1.55E-05  11.91  Lignin biosynthetic process  6.65E-05  4.954  Xylem vessel member cell differentiation  7.05E-05  19.14  Cell wall thickening  1.35E-04  30.63  Phloem development  6.40E-04  20.42  Positive regulation of secondary cell wall biogenesis  6.40E-04  20.42  bes1-not-down genes  Microtubule-based movement  2.19E-19  15.32  Cell division  1.16E-14  6.134  Response to auxin  3.22E-12  4.77  Auxin-activated signaling pathway  3.10E-10  5.40  Cell cycle  1.40E-08  6.211  Mitotic nuclear division  4.34E-08  7.429  Regulation of cell cycle  1.15E-07  7.625  Multicellular organism development  6.54E-06  2.902  Procambium histogenesis  2.85E-05  24.76  Auxin polar transport  2.41E-04  6.368  Cotyledon vascular tissue pattern formation  5.90E-04  12.38  GO enrichment analysis of genes up-regulated in VISUAL (>4-fold, 1,534 genes). The bes1-down and bes1-not-down groups were examined separately. Significantly enriched terms (P < 0.001) are shown. BES1 also promotes phloem cell differentiation in VISUAL In the WT, the differentiation of phloem sieve element-like cells also occurs in VISUAL (Kondo et al. 2016). Interestingly, several bes1-down genes were annotated as being related to phloem development (Table 1). Consistent with this notion, qRT-PCR analysis indicated that the expression levels of phloem marker genes APL and SIEVE-ELEMENT-OCCLUSION-RELATED 1 (SEOR1) were significantly reduced in the bes1 mutants compared with the WT, similar to xylem marker genes (Fig. 2D, E). These results suggest that phloem differentiation is also inhibited in bes1. To understand the role of BES1 in phloem development, we investigated the expression of VISUAL phloem-specific genes (VPs), which we previously identified by transcriptome analysis (Kondo et al. 2016), using the bes1 microarray data. Many VPs were down-regulated in the bes1 mutants (Fig. 2I). These down-regulated genes were also down-regulated in the apl mutants (Fig. 2J), which have strong defects in phloem development in VISUAL (Kondo et al. 2016). Considering the significant reduction in APL expression in bes1-1 (Fig. 2D), these data suggest that BES1 up-regulates a wide range of phloem sieve element-related genes, probably upstream of APL. In addition to the recovery of xylem differentiation, the decrease in expression of phloem genes in bes1-1 was fully complemented by the introduction of gBES1 (Fig. 3). These results strongly support the idea that BES1 promotes phloem differentiation as well as xylem differentiation (Fig. 2K). BZR1 acts redundantly with BES1 in vascular development In bes1 mutants, the induction of both xylem and phloem marker genes in VISUAL was not completely repressed, suggesting that other factors may contribute to the promotion of the differentiation process. BZR1 is the closest homolog of BES1, with 88% identity (Wang et al. 2002). We therefore employed genome editing techniques (Fauser et al. 2014) in Col-0 and bes1-1 to generate bzr1-1 and bes1-1 bzr1-2 plants, respectively. We succeeded in obtaining these homozygous lines, in which the Cas9 gene had been removed. The bzr1-1 mutant possesses a one base insertion in the second exon of BZR1, resulting in the appearance of a premature stop codon due to a frameshift (Fig. 4A, C). On the other hand, the bes1-1 bzr1-2 double mutant has lost 29 bases of BZR1 spanning from the 3' end of the intron to the start of the second exon (Fig. 4A). Sequencing of BZR1 transcripts revealed that the splicing site between the intron and the second exon is altered in bes1-1 bzr1-2 (Fig. 4B), leading to a premature stop codon due to a frameshift (Fig. 4C). Fig. 4 View largeDownload slide Generation of bzr1 mutants. (A) Schematic overview of the gene structure of BZR1 and the sequence around the selected site targeted by CRISPR/Cas9. Exons, the intron and untranslated regions are illustrated by boxes, a solid line and dotted lines, respectively. Exon and intron sequences in the WT are written in lower case and upper case letters, respectively. The protospacer adjacent motif (PAM) sequences of the target and the resulting mutations are highlighted in blue and red, respectively. A one base insertion was detected in bzr1-1, whereas 29 base deletions occurred in bes1-1 bzr1-2. (B) The mRNA sequences of BZR1 in the WT and bes1-1 bzr1-2. The splice site in each line is marked by an arrowhead, and spliced sequences are highlighted in green in (A). (C) Theoretical amino acid sequences of BZR1 around the mutated sites in the WT, bzr1-1 and bes1-1 bzr1-2. Asterisks represent premature stop codons. Fig. 4 View largeDownload slide Generation of bzr1 mutants. (A) Schematic overview of the gene structure of BZR1 and the sequence around the selected site targeted by CRISPR/Cas9. Exons, the intron and untranslated regions are illustrated by boxes, a solid line and dotted lines, respectively. Exon and intron sequences in the WT are written in lower case and upper case letters, respectively. The protospacer adjacent motif (PAM) sequences of the target and the resulting mutations are highlighted in blue and red, respectively. A one base insertion was detected in bzr1-1, whereas 29 base deletions occurred in bes1-1 bzr1-2. (B) The mRNA sequences of BZR1 in the WT and bes1-1 bzr1-2. The splice site in each line is marked by an arrowhead, and spliced sequences are highlighted in green in (A). (C) Theoretical amino acid sequences of BZR1 around the mutated sites in the WT, bzr1-1 and bes1-1 bzr1-2. Asterisks represent premature stop codons. We then investigated whether BZR1 functions in vascular development using VISUAL. The bzr1-1 single mutants showed slightly reduced xylem differentiation rates (Fig. 5A–D). Further gene expression analysis revealed that xylem marker genes were slightly repressed in bzr1-1 compared with the WT (Fig. 5E). The expression levels of phloem marker genes were not significantly different from those of the WT (P = 0.136 for APL and P = 0.036 for SEOR1), but they tended to be reduced in bzr1-1 (Fig. 5E). Thus, the mutant phenotypes of bzr1-1 were weaker than those of bes1-1 in terms of differentiation rate and gene expression level when cultured in VISUAL (Fig. 5). These results suggest that BZR1 is also involved in xylem and phloem differentiation, but to a lesser extent than BES1. We then investigated the relationship between the roles of BES1 and BZR1 in vascular cell differentiation using bes1-1 bzr1-2 double mutants. However, the bes1-1 mutants produced relatively small amounts of xylem cells under the original VISUAL conditions, preventing us from verifying the functional redundancy of BES1 and BZR1. In an effort to solve this problem, we increased the bikinin concentration in the culture medium to 20 µM, because this treatment induced the production of more ectopic xylem cells in bes1-1 than the original medium (Fig. 6A, B). Under this condition, the xylem differentiation rate was much lower in the bes1-1 bzr1-2 mutants than in bes1-1 (Fig. 6C, D). The down-regulation of xylem and phloem marker genes in the bes1 single mutants was also enhanced by the addition of the bzr1-2 mutation (Fig. 6E). On the other hand, the procambium marker gene TDR was not repressed in these mutants, even in bes1-1 bzr1-2 (Fig. 6E). Based on these results, it is likely that BZR1 acts redundantly with BES1 in xylem and phloem differentiation from procambial cells in VISUAL. Fig. 5 View largeDownload slide Mutant phenotypes of bzr1-1 in VISUAL. (A–C) Ectopic xylem cell formation in VISUAL. WT (A), bes1-1 (B) and bzr1-1 (C) cotyledons were cultured for 4 d. Scale bars indicate 1 mm. (D) Quantification of xylem differentiation rates in (A–C). Error bars indicate the SD (n = 8). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). (E) Expression levels of xylem and phloem marker genes in the WT, bes1-1 and bzr1-1 in VISUAL. RNA was extracted from cotyledons at 72 h after VISUAL induction. Relative expression levels were calculated by comparison with the WT. Error bars indicate the SD (n = 3). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). Fig. 5 View largeDownload slide Mutant phenotypes of bzr1-1 in VISUAL. (A–C) Ectopic xylem cell formation in VISUAL. WT (A), bes1-1 (B) and bzr1-1 (C) cotyledons were cultured for 4 d. Scale bars indicate 1 mm. (D) Quantification of xylem differentiation rates in (A–C). Error bars indicate the SD (n = 8). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). (E) Expression levels of xylem and phloem marker genes in the WT, bes1-1 and bzr1-1 in VISUAL. RNA was extracted from cotyledons at 72 h after VISUAL induction. Relative expression levels were calculated by comparison with the WT. Error bars indicate the SD (n = 3). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). Fig. 6 View largeDownload slide Mutant phenotypes of bes1-1 bzr1-2 in VISUAL. (A–C) Ectopic xylem cell formation in VISUAL. WT (A), bes1-1 (B) and bes1-1 bzr1-2 (C) cotyledons were cultured with bikinin at twice the normal concentration (20 µM) for 4 d. Scale bars indicate 1 mm. (D) Quantification of xylem differentiation rates in (A–C). Error bars indicate the SD (n = 8–12). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). (E) Expression levels of xylem, phloem and procambium marker genes in the WT, bes1-1 and bes1-1 bzr1-2 in VISUAL in the presence of 20 µM bikinin. RNA was extracted from cotyledons at 72 h after induction. Relative expression levels were calculated by comparison with the WT. Error bars indicate the SD (n = 3). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). Fig. 6 View largeDownload slide Mutant phenotypes of bes1-1 bzr1-2 in VISUAL. (A–C) Ectopic xylem cell formation in VISUAL. WT (A), bes1-1 (B) and bes1-1 bzr1-2 (C) cotyledons were cultured with bikinin at twice the normal concentration (20 µM) for 4 d. Scale bars indicate 1 mm. (D) Quantification of xylem differentiation rates in (A–C). Error bars indicate the SD (n = 8–12). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). (E) Expression levels of xylem, phloem and procambium marker genes in the WT, bes1-1 and bes1-1 bzr1-2 in VISUAL in the presence of 20 µM bikinin. RNA was extracted from cotyledons at 72 h after induction. Relative expression levels were calculated by comparison with the WT. Error bars indicate the SD (n = 3). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). Growth defects are not observed in bes1-1 bzr1-2 mutants To investigate the functions of BES1 and BZR1 in in vivo vascular development, we compared cross-sections from WT vs. bes1-1 bzr1-2 plants. Although bes1-1 bzr1-2 mutants showed severe defects in VISUAL, no obvious defects were observed in the central xylem tissues of hypocotyls in 16-day-old or 6-week-old plants (Fig. 7A–D). Phloem tissues (visualized by toluidine blue and aniline blue staining) were also indistinguishable between the WT and bes1-1 bzr1-2 (Fig. 7A–D). Similarly, no defects in vascular development were observed in 6-week-old stems of bes1-1 bzr1-2 (Fig. 7E, F). These results suggest that the vascular phenotype of bes1-1 bzr1-2 is enhanced in the culture system VISUAL, in which exogenous bikinin induces rapid vascular cell differentiation apart from other developmental processes. Fig. 7 View largeDownload slide In vivo phenotype of bes1-1 bzr1-2. (A–F) Cross-sections of hypocotyls (A–D) and stems (E, F) of WT (A, C, E) and bes1-1 bzr1-2 (B, D, F) plants grown for 16 d (A, B) or 6 weeks (C–F). Toluidine blue (A, B) or aniline blue staining (C–F) was conducted. Blue and green fluorescence in (C–F) is autofluorescence from xylem cells and stained phloem cells after UV radiation, respectively. Scale bars indicate 200 µm for (C) and (D) and 50 µm for the others. (G, H) BL sensitivity of WT, bes1-1, bzr1-1 and bes1-1 bzr1-2 seedlings. Relative lengths of hypocotyls (G) and roots (H) were calculated by comparison with samples of each genotype grown without BL. Error bars indicate the SD (n = 30–32 except for n = 25 for bzr1-1 treated with 10 nM BL). (I) Hypocotyl elongation in plants grown in the dark. Seedlings were grown for 7 d. The scale bar indicates 0.5 cm. (J) Quantification of hypocotyl elongation in (I). Error bars indicate the SD (n = 3–6). Significant differences compared with the WT were examined by Tamhane T2 test (α = 0.05). Fig. 7 View largeDownload slide In vivo phenotype of bes1-1 bzr1-2. (A–F) Cross-sections of hypocotyls (A–D) and stems (E, F) of WT (A, C, E) and bes1-1 bzr1-2 (B, D, F) plants grown for 16 d (A, B) or 6 weeks (C–F). Toluidine blue (A, B) or aniline blue staining (C–F) was conducted. Blue and green fluorescence in (C–F) is autofluorescence from xylem cells and stained phloem cells after UV radiation, respectively. Scale bars indicate 200 µm for (C) and (D) and 50 µm for the others. (G, H) BL sensitivity of WT, bes1-1, bzr1-1 and bes1-1 bzr1-2 seedlings. Relative lengths of hypocotyls (G) and roots (H) were calculated by comparison with samples of each genotype grown without BL. Error bars indicate the SD (n = 30–32 except for n = 25 for bzr1-1 treated with 10 nM BL). (I) Hypocotyl elongation in plants grown in the dark. Seedlings were grown for 7 d. The scale bar indicates 0.5 cm. (J) Quantification of hypocotyl elongation in (I). Error bars indicate the SD (n = 3–6). Significant differences compared with the WT were examined by Tamhane T2 test (α = 0.05). BES1 and BZR1 are involved in various developmental processes. In particular, they function as major transcription factors that mediate the BR signaling pathway (Guo et al. 2013). To explore the impact of BES1 and BZR1 on BR signaling, we treated WT and bes1-1 bzr1-2 plants with brassinolide (BL). BL treatment promotes hypocotyl elongation and represses root growth in WT plants in the light. As previously reported (He et al. 2005, Kang et al. 2015), bes1-1 was slightly resistant to exogenous BL (Fig. 7G, H). Although bzr1-1 single mutants were partially resistant to exogenous BL only in root elongation, the bes1-1 bzr1-2 mutants were more insensitive to BL than the bes1 single mutants in terms of both hypocotyl and root elongation (Fig. 7G, H). Consistent with this observation, the changes in expression of a BR-responsive gene in response to BL treatment were also partially reduced in bes1-1 bzr1-2 (Supplementary Fig. S1). Next, we investigated the defects related to BR signaling in bes1-1 bzr1-2 in the absence of exogenous BL treatment. The bri1 mutant, a BR receptor mutant, shows severe defects in hypocotyl elongation under dark conditions (Li and Chory 1997). However, bes1-1 and bes1-1 bzr1-2 did not exhibit this phenotype, instead appearing like WT plants (Fig. 7I, J). Furthermore, stem growth in bes1-1 bzr1-2 was comparable with that in the WT under normal growth conditions, unlike the dwarf mutant bri1 (Supplementary Fig. S2). These results indicate that the BR-related phenotype in bes1-1 bzr1-2 appears only in the presence of exogenously applied BL. BES1 and BZR1 gain-of-function mutations promote phloem differentiation in vivo In contrast to bes1-1 bzr1-2 mutants, it is known that gain-of-function mutants bes1-D and bzr1-D exhibit pleiotropic phenotypes. For instance, hypocotyl elongation is promoted in these mutants under darkness (Li and Nam 2002, Yin et al. 2002). Moreover, it was reported that the gain-of-function mutant bes1-D sometimes exhibits reduced procambial cell layers between xylem and phloem cells due to the excess vascular differentiation from procambial cells (Kondo et al. 2014). Then we generated bes1-D bzr1-D double gain-of-function mutants and observed their vascular phenotype in detail (Fig. 8). The overall morphology of bes1-D bzr1-D was similar to that of bes1-D, in which the hypocotyl is elongated (Yin et al. 2002), but the thickness of the hypocotyl is comparable with that of the WT. In WT hypocotyls, usually two or three procambial cell layers were formed between phloem and xylem cells (Fig. 8A). In bes1-D bzr1-D, however, procambial cell layers were reduced, occasionally resulting in adjacency of xylem and phloem cells (Fig. 8B; red arrowhead), which is consistent with the result in bes1-D (Kondo et al. 2014). In addition, ectopic phloem cells, which are non-clustered phloem cells surrounded by undifferentiated cells, were sometimes formed only in bes1-D bzr1-D but not in the WT (Fig. 8A, C; yellow arrowhead). These results suggest that BES1 and BZR1 promote phloem differentiation from procambial cells in vivo. Fig. 8 View largeDownload slide Comparison of hypocotyl vasculature between WT and bes1-D bzr1-D. Cross-sections of hypocotyls of WT (A) and bes1-D bzr1-D (B, C) plants grown for 11 d. Orange and blue dotted lines indicate phloem (Ph) and xylem (Xy) cells, respectively. White solid lines indicate procambial cell (Pc) layers. Red and yellow arrowheads indicate the adjacency of xylem and phloem cells and non-clustered phloem cells, respectively. The fraction in the top and bottom of the panels represents the number of plants exhibiting adjacency of xylem and phloem cells and non-clustered phloem cells per total number of plants observed, respectively. Scale bars indicate 20 µm. Fig. 8 View largeDownload slide Comparison of hypocotyl vasculature between WT and bes1-D bzr1-D. Cross-sections of hypocotyls of WT (A) and bes1-D bzr1-D (B, C) plants grown for 11 d. Orange and blue dotted lines indicate phloem (Ph) and xylem (Xy) cells, respectively. White solid lines indicate procambial cell (Pc) layers. Red and yellow arrowheads indicate the adjacency of xylem and phloem cells and non-clustered phloem cells, respectively. The fraction in the top and bottom of the panels represents the number of plants exhibiting adjacency of xylem and phloem cells and non-clustered phloem cells per total number of plants observed, respectively. Scale bars indicate 20 µm. Discussion BES1 promotes xylem and phloem differentiation from procambial cells but not procambial cell differentiation from mesophyll cells In a previous paper, we reported that BES1 promotes xylem differentiation from mesophyll cells in VISUAL (Kondo et al. 2014). In the current study, microarray analysis of bes1 showed that BES1 promotes xylem differentiation from procambial cells but not procambial cell differentiation from mesophyll cells. We also found that the bes1 mutations suppressed phloem differentiation in VISUAL, suggesting that BES1 plays a positive role in phloem differentiation. Microarray analysis also indicated that BES1 and APL share downstream genes involved in phloem differentiation. The expression of APL was repressed in bes1. These results indicate that BES1 positively regulates phloem differentiation, probably upstream of APL. On the other hand, the transcription factor genes VND6 and VND7, encoding master regulators of xylem differentiation (Kubo et al. 2005), were also down-regulated in bes1 (Supplementary Table S1). Therefore, it is likely that BES1 functions early in vascular development before the induction of final differentiation by master regulators of xylem and phloem differentiation. These findings, together with the observation that a large number of procambial cells remained in the bes1 mutants after the induction of VISUAL, suggest that BES1 triggers the transition of stem cells to specialized cells into xylem or phloem cells. Functional redundancy among BES1 family members There are six BES1-like genes in the Arabidopsis genome: BES1, BZR1 and BES1/BZR1 HOMOLOG 1–4 (BEH1–BEH4) (Yin et al. 2002, Wang et al. 2002, Yin et al. 2005). BES1 and BZR1 are thought to act as central players that regulate various developmental processes in plants, playing prominent roles in BR signaling (Guo et al. 2013). However, in many cases, functional analyses of these proteins have been performed using gain-of-function mutants (bes1-D and bzr1-D), and few analyses have been performed using loss-of-function mutants. Here, we produced bzr1-1 (a loss-of-function mutant of BZR1) and the bes1-1 bzr1-2 double mutant to analyze the roles of BES1 and BZR1. The bes1, bzr1-1 and bes1-1 bzr1-2 mutants did not show any visible phenotypes in planta, including a dwarf phenotype and photomorphogenesis in the dark, which are characteristic phenotypes of BR-deficient mutants such as bri1. This result strongly suggests that BES1 family members function redundantly. We then examined the phenotypes of these mutants in physiological aspects. The only mutant phenotype observed in bes1-1 bzr1-2 was reduced sensitivity to exogenous BL application. To account for this phenomenon, the following findings about other multiple mutants should be considered. The arabidopsis response regulator 10 (arr10) arr12 double mutants are resistant to exogenous cytokinin but grow well on Murashige and Skoog (MS) agar plates and in soil (Yokoyama et al. 2007). The addition of the arr1 mutation to arr10 arr12 results in plants with a dwarf phenotype in the absence of cytokinin, which was also observed in another cytokinin dominant-negative mutant, wooden leg (Yokoyama et al. 2007). Consistent with this idea, bes1-RNAi (RNA interference) lines, which exhibit repressed expression not only of BES1 and BZR1 (Yin et al. 2005), but also of several BEH genes (Wang et al. 2013), show defects in stem growth and shoot branching. Our results obtained using loss-of-function mutants raise the possibility that BEHs function redundantly with BES and BZR1. Further genetic analysis is needed to understand fully the functions of BES1 family proteins. BZR1 is also a positive regulator of xylem and phloem differentiation from procambial cells Although we did not detect mutant vascular phenotypes in bzr1 in planta, the VISUAL system could visualize them. VISUAL using bzr1-1 and bes1-1 bzr1-2 revealed that BZR1 as well as BES1 positively regulates both xylem and phloem differentiation from procambial cells. This finding is consistent with the previous finding that both bes1-D and bzr1-D can partially rescue the phenotype of the octopus mutant, in which root protophloem differentiation is prone to be repressed (Anne et al. 2015). Our observation that phloem cells are ectopically produced in hypocotyls of bes1-D bzr1-D also supports the molecular functions of BES1 and BZR1 found in VISUAL. In the current study, bes1 exhibited a stronger vascular phenotype than bzr1-1, and the phenotype induced by these mutations was additive, suggesting that BES1 and BZR1 redundantly function in xylem and phloem differentiation to a greater and lesser extent, respectively. This result is also in accordance with the finding that bes1-D, but not bzr1-D, shows defects in the hypocotyl vasculature (Kondo et al. 2014). In VISUAL, mesophyll cells initially differentiate into procambial cells, followed by xylem or phloem cells (Kondo et al. 2015, Kondo et al. 2016). Bikinin promotes procambial cell differentiation (Kondo et al. 2015) as well as the differentiation of procambial cells into xylem and phloem cells (Kondo et al. 2016). However, BES1 and BZR1 only regulate differentiation into xylem and phloem cells. Therefore, it is likely that GSK3s also control procambial cell differentiation, but factors other than BES1 and BZR1 are involved in this process downstream of the GSK3s. Indeed, GSK3s phosphorylate various developmental regulators such as ENHANCER OF GLABRA 3 (EGL3) and TRANSPARENT TESTA GLABRA 1 (TTG1) (Cheng et al. 2014) for root hair formation and AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 for lateral root formation (Cho et al. 2014). Therefore, it will be interesting to identify a new target(s) of GSK3s that regulates procambial cell differentiation. Materials and Methods Plant materials Seeds of the Arabidopsis thaliana bes1-1 (SALK_098634), bes1-2 (WiscDsLox246D02) and bes1-3 (SALK_091133) mutants were obtained from the ABRC stock center. The progeny of heterozygous bri1-48 (SALK_041648) were gifts from Dr. Takeshi Nakano, and homozygous mutants were identified based on the presence of severely shortened hypocotyls. bes1-D and bzr1-D mutants were also gifts from Dr. Takeshi Nakano, and bes1-D bzr1-D mutants were generated by crossing. All mutants in this study were in the Col-0 background. To produce the complementation lines, DNA fragments of gBES1 containing the 2,000 bp promoter sequence and the 1,000 bp sequence downstream of the stop codon were cloned and introduced into the pGWB1 vector (Nakagawa et al. 2007), followed by transformation into bes1-1 by the floral dip method (Clough and Bent 1998). Plants were grown on conventional 1/2 MS agar plates (pH 5.7) at 22°C under continuous light unless otherwise mentioned. For later harvesting, seedlings were transferred to pots containing a mixture of vermiculite (VS Kakou) and PRO-MIX BX (Premier Horticulture). CRISPR The target sequence was selected using CRISPRdirect software (Naito et al. 2015). DNA fragments containing 20 bp of the target sequence neighboring the PAM (protospacer adjacent motif) sequence and overhang sequences were generated by hybridizing two primers, followed by ligation into the pEn-Chimera vector (Fauser et al. 2014) that had been digested with BbsI. The single guide RNA (sgRNA)-containing sequence was then transferred into the pDe-CAS9 vector (Fauser et al. 2014) via LR reaction (Thermo Fisher Scientific). The resulting construct was transformed into WT and bes1-1 plants. Transformants were obtained by bialaphos selection. T2 seeds from single insertion lines were incubated on MS agar plates without antibiotics. Cas9-free mutants were selected from the T2 population by PCR and sequencing using the primers listed in Supplementary Table S2. To check the sequence of the BZR1 transcript in bes1-1 bzr1-2, total RNA was extracted from cotyledons of 6-day-old WT and bes1-1 bzr1-2 plants using an RNeasy Plant Mini kit (Qiagen). Reverse transcription was performed using SuperScript III (Thermo Fisher Scientific). PCR was performed using synthesized cDNA and the primers listed in Supplementary Table S2, followed by sequencing using the same primer. Data were visualized using Chromas 2.6 (http://technelysium.com.au/wp/). VISUAL The detailed protocol for VISUAL was described previously (Kondo et al. 2016). In brief, cotyledons of 6-day-old seedlings grown in liquid 1/2 MS medium were cultured in MS-based medium containing 0.25 mg l−1 2,4-D, 1.25 mg l−1 kinetin and 10 µM bikinin under continuous light. To compare the phenotypes of bes1-1 vs. bes1-1 bzr1-2, the bikinin concentration was changed to 20 µM. To observe ectopic xylem differentiation, cotyledons cultured for 4 d were fixed in ethanol/acetic acid (3 : 1) and mounted with clearing solution (chloral hydrate/glycerol/water; 8 : 1 : 2). The differentiation rates were determined based on the area of ectopic xylem cells in a cotyledon per total area of the cotyledon blade. These areas were calculated using ImageJ. qRT-PCR Total RNA was extracted from cotyledons before or after culture for 72 h (in the VISUAL experiments) or from whole seedlings treated with dimethylsulfoxide (DMSO) or 100 µM BL for 3 h (in the BL sensitivity tests) using an RNeasy Plant Mini kit (Qiagen). Quantitative PCR was performed using a LightCycler (Roche Diagnostics). UBIQUITIN 14 (UBQ14) was used for the VISUAL experiments, and ACTIN 2 (ACT2) was used for the BL sensitivity tests as an internal control. The gene-specific primer sets are listed in Supplementary Table S2. Microarray experiment Microarray analyses were performed as previously described (Ohashi-Ito et al. 2010). For clustering, normalization of gene expression levels was performed as follows: first, the average fold change between values before and after induction in each experiment was calculated. These values were then normalized by dividing them by the median value of the WT, bes1-3 and bes1-1. Finally, these values were converted to the log2 scale, producing a value defined as the Processed signal. Hierarchical clustering via the Pearson correlation method and heatmap generation were performed using Subio Platform software (Subio). The half-time point was defined as the first time point when the relative expression level reached 50% of its maximum value in a set of time course microarray data (Kondo et al. 2015) in which the expression levels between observed values were postulated to change linearly. GO enrichment analysis was performed using DAVID Bioinformatics Resources 6.8 (Huang et al. 2009; https://david.ncifcrf.gov/). Histological analysis Cross-sections of hypocotyls of 11- or 16-day-old plants grown in liquid 1/2 MS medium were produced as previously described (Kondo et al. 2014). Sliced samples were stained with 0.1% toluidine blue for 1 min. To produce cross-sections of 6-week-old plants, hypocotyls or the basal parts of stems were fixed in 70% ethanol. Sections of hypocotyls and stems 2 cm above the rosette at 80 µm thickness were produced using a LEICA VT1200S vibratome. Sliced samples were stained with 0.05% aniline blue in 100 mM phosphate buffer (pH 7.2) for 1 h and observed under UV radiation. BR treatment To observe hypocotyl or root elongation, plants were grown on 1/2 MS agar plates containing DMSO or BL. After 7 d, the plates were scanned with a CanoScan 9000F (Canon). Hypocotyl and root lengths were measured using ImageJ. For gene expression analysis, plants were grown on 1/2 MS agar plates. After 10 d, the seedlings were incubated in a solution of DMSO or 100 µM BL for 3 h and collected for RNA extraction. Phenotypic observation of dark-grown plants Plants were grown on 1/2 MS agar plates in the dark after white light irradiation to promote germination. After 7 d, the plates were scanned using a CanoScan 9000F (Canon). Hypocotyl length was measured using ImageJ. Statistical analysis For multiple comparisons, Tamhane T2 tests were employed in this study due to inhomogeneous variances obtained by the Levene tests (Wallner et al. 2017). The significance thresholds were set to α = 0.05. The P-values for each comparison were calculated by Welch’s t-test. The significance threshold of each comparison was set to α’ = 1 – (1 – α)1/k, where k is the total number of comparisons. For example, if k = 3, the significance threshold of the P-value of each comparison (α’) is approximately 0.0167. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grants-in-Aid No. 17H06476 to Y.K. and No. 15H05958 to H.F.] and the Japan Society for the Promotion of Science [grant No. 16J06222 to M.S., No. 17H05008 to Y.K. and No. 16H06377 to H.F.]. Acknowledgments We thank Yukiko Sugisawa, Yasuko Ozawa and Satoshi Endo for technical support. We also thank Holger Puchta and Takeshi Nakano for providing materials. Disclosures The authors have no conflicts of interest to declare. References Anne P., Azzopardi M., Gissot L., Beaubiat S., Hématy K., Palauqui J.C. ( 2015) OCTOPUS negatively regulates BIN2 to control phloem differentiation in Arabidopsis thaliana. Curr. Biol . 25: 2584– 2590. Google Scholar CrossRef Search ADS PubMed  Bonke M., Thitamadee S., Mähönen A.P., Hauser M.T., Helariutta Y. ( 2003) APL regulates vascular tissue identity in Arabidopsis. 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Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations APL ALTERED PHLOEM DEVELOPMENT ARF AUXIN RESPONSE FACTOR ARR ARABIDOPSIS RESPONSE REGULATOR BEH BES1/BZR1 HOMOLOG BES1 BRI1-EMS-SUPPRESSOR 1 BL brassinolide BR brassinosteroid BRI1 BRASSINOSTEROID-INSENSITIVE 1 BZR1 BRASSINAZOLE RESISTANT 1 CRISPR clustered regularly interspaced short palindromic repeats Cas9 CRISPR-associated protein 9 DMSO dimethylsulfoxide GO Gene Ontology GSK3 glycogen synthase kinase 3 protein IRX3 IRREGULAR XYLEM 3 MS Murashige and Skoog PAM, protospacer adjacent motif; qRT-PCR quantitative reverse transcription PCR SEOR1 SIEVE-ELEMENT-OCCLUSION-RELATED 1 sgRNA, single guide RNA; TDR TDIF RECEPTOR UBQ14 UBIQUITIN 14 VISUAL Vascular cell Induction culture System Using Arabidopsis Leaves VND VASCULAR-RELATED NAC-DOMAIN VP VISUAL phloem-specific gene WT wild type XCP1 XYLEM CYSTEINE PEPTIDASE 1 Footnote Footnote Microarray data have been deposited in Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). Accession number is GSE110199 © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

BES1 and BZR1 Redundantly Promote Phloem and Xylem Differentiation

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0032-0781
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1471-9053
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10.1093/pcp/pcy012
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Abstract

Abstract Vascular development is a good model for studying cell differentiation in plants. Two conductive tissues, the xylem and phloem, are derived from common stem cells known as procambial/cambial cells. Glycogen synthase kinase 3 proteins (GSK3s) play crucial roles in maintaining procambial/cambial cells by suppressing their differentiation into xylem or phloem cells. We previously designed an in vitro culture system for analyzing vascular cell differentiation named VISUAL (Vascular cell Induction culture System Using Arabidopsis Leaves). Using this system, we found that the transcription factor BRI1-EMS-SUPPRESSOR 1 (BES1) functions as a downstream target of GSK3s during xylem differentiation. However, the function of BES1 in vascular development remains largely unknown. Here, we found that, in addition to xylem differentiation, BES1 positively regulates phloem differentiation downstream of GSK3s. Transcriptome analysis using VISUAL confirmed that BES1 promotes bi-directional differentiation of procambial cells into xylem and phloem cells. Genetic analysis of loss-of-function mutants newly generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system revealed that BRASSINAZOLE RESISTANT 1 (BZR1), the closest homolog of BES1, functions in vascular development redundantly with BES1. Notably, BZR1 has a weaker impact on vascular cell differentiation than BES1, suggesting that they contribute differentially to this process. In conclusion, our findings indicate that BES1 and BZR1 are key regulators of both xylem and phloem cell differentiation from vascular stem cells. Introduction Vascular tissues, comprising xylem and phloem cells, are required for long-distance transport of various materials in plants. The xylem transports water and minerals from roots to shoots, whereas, in most cases, the phloem transports photoassimilates from shoots to roots. Although xylem and phloem cells have different structures and functions, these cells originate from common stem cells known as procambial/cambial cells. Previous studies identified master regulators of xylem and phloem cell fate. VASCULAR-RELATED NAC-DOMAINs (VNDs) facilitate xylem differentiation (Kubo et al. 2005), whereas ALTERED PHLOEM DEVELOPMENT (APL) is required for phloem development (Bonke et al. 2003). Molecular analyses of these master regulators have revealed the downstream components controlling various developmental events associated with xylem and phloem differentiation (Ohashi-Ito et al. 2010, Yamaguchi et al. 2011, Furuta et al. 2014b). Prior to the final determination of cell fate, glycogen synthase kinase 3 proteins (GSK3s) regulate vascular cell differentiation as earlier regulators (Kondo et al. 2014). The SKI and SKII subgroups of GSK3s redundantly maintain procambial cell populations by suppressing xylem differentiation. Indeed, the application of the GSK3 inhibitor bikinin (De Rybel et al. 2009) induces ectopic xylem cell differentiation in the presence of auxin and cytokinin (Kondo et al. 2014, Kondo et al. 2015). This feature allowed us to establish a powerful tissue culture system for vascular cell differentiation named VISUAL (Vascular cell Induction culture System Using Arabidopsis Leaves). Using VISUAL, we found that ectopic xylem differentiation induced by the inhibition of GSK3s is suppressed in mutants for BRI1-EMS-SUPPRESSOR 1 (BES1) (Kondo et al. 2014, Kondo et al. 2015), a direct substrate of GSK3s (Yin et al. 2002). Bikinin also has a promotive effect on phloem differentiation in VISUAL (Kondo et al. 2016). However, it remains to be elucidated whether BES1 regulates phloem differentiation downstream of GSK3s and, if so, how BES1 regulates both phloem and xylem differentiation. The GSK3s–BES1 signaling module is also involved in the brassinosteroid (BR) signaling pathway. The perception of BR by BRASSINOSTEROID-INSENSITIVE 1 (BRI1) leads to the inactivation of GSK3s (Li and Nam 2002). BES1 family transcription factors are then activated to control downstream gene expression (Yin et al. 2005). Arabidopsis contains six members of the BES1 family. Among these, BES1 and its closest homolog, BRASSINAZOLE RESISTANT 1 (BZR1), are considered to be major mediators of BR signaling. However, our understanding of the BR signaling pathway has primarily been obtained from studies using the gain-of-function mutants bes1-D and bzr1-D (Yin et al. 2002, Wang et al. 2002). In contrast, few loss-of-function studies investigating BES1 family members have been performed (Yin et al. 2005). In particular, loss-of-function mutants for BZR1 have not yet been reported. Therefore, our understanding of the contribution of BES1 and BZR1 to BR signaling remains incomplete. As for vascular development, we also analyzed the role of BES1 and BZR1 using gain-of-function mutants (Kondo et al. 2014). However, recent advances in genome editing techniques, including the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system, should allow the actual functions of genes of interest to be analyzed by generating knock-out mutants. In this study, we investigated the functions of BES1 and BZR1 in vascular cell differentiation using loss-of-function mutants generated via the CRISPR/Cas9 system. Comprehensive gene expression analysis of the bes1 mutants revealed that BES1 positively regulates both xylem and phloem differentiation, but not procambial cell differentiation from mesophyll cells, in VISUAL. Further genetic analysis with bes1 bzr1 mutants indicated that BES1 and BZR1 act redundantly in promoting both xylem and phloem differentiation. Interestingly, the phenotypes when cultured in VISUAL were weaker in bzr1 than in bes1, indicating that BES1 and BZR1 contribute differently to vascular development. Consistently, bes1 bzr1 plants displayed lower sensitivity to exogenous BR treatment than the bes1 single mutants but were still sensitive to BR, suggesting the further functional redundancy of BES1 family members. Results BES1 positively regulates xylem cell differentiation in VISUAL We previously found that bes1 mutants have defects in xylem differentiation when cultured in VISUAL (Kondo et al. 2014, Kondo et al. 2015). To investigate further the role of BES1 in vascular development, we obtained three T-DNA insertion lines for BES1: bes1-1 (He et al. 2005), bes1-2 (Lachowiec et al. 2013) and bes1-3 (previously named bes1-2 in Kang et al. 2015, and bes1 in Kondo et al. 2014) (Fig. 1A). None of the mutants showed visible phenotypes in terms of plant growth and xylem development in vivo. However, the cotyledons of these mutants commonly exhibited reduced xylem differentiation at 96 h after VISUAL induction (Fig. 1B–E). Differentiation rates in bes1-1 and bes1-2 mutants were lower than that in bes1-3, indicating that bes1-1 and bes1-2 are strong alleles and that bes1-3 is a weak allele. We performed detailed phenotypic analysis of bes1-3 (weak allele) and bes1-1 (strong allele) by monitoring the expression of vascular marker genes. RNA samples were collected at 72 h after induction, when xylem-related genes were expressed at maximum levels. Our quantitative reverse transcription PCR (qRT-PCR) analysis indicated that the expression levels of xylem marker genes such as IRREGULAR XYLEM 3 (IRX3) and XYLEM CYSTEINE PEPTIDASE 1 (XCP1) were lower in the bes1 mutants than in the wild type (WT) and were lower in bes1-1 than in bes1-3 (Fig. 2A, B). Next, we introduced a genomic fragment of BES1 (gBES1) into bes1-1. The introduction completely restored the reduced ectopic xylem differentiation rate and xylem-related gene expression in bes1-1 (Fig. 3). These results further support the notion that BES1 positively regulates xylem differentiation. Fig. 1 View largeDownload slide Phenotypes of bes1 T-DNA insertion mutants in VISUAL. (A) Schematic diagram of the exon–intron structures of BES1. Two splicing variants (Jiang et al. 2015) are shown. Exons, introns and untranslated regions are illustrated by boxes, black solid lines and dotted lines, respectively. Red lines represent putative insertion sites of T-DNA in each bes1 mutant. (B–E) Ectopic xylem cell formation in VISUAL. WT (B), bes1-1 (SALK_098634) (C), bes1-2 (WiscDsLox246D02) (D) and bes1-3 (SALK_091133) (E) cotyledons were cultured in medium containing 2,4-D, kinetin and bikinin for 4 d. Scale bars indicate 1 mm. (F) Quantification of xylem differentiation rates in (B–E). Error bars indicate the SD (n = 8–10). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). Fig. 1 View largeDownload slide Phenotypes of bes1 T-DNA insertion mutants in VISUAL. (A) Schematic diagram of the exon–intron structures of BES1. Two splicing variants (Jiang et al. 2015) are shown. Exons, introns and untranslated regions are illustrated by boxes, black solid lines and dotted lines, respectively. Red lines represent putative insertion sites of T-DNA in each bes1 mutant. (B–E) Ectopic xylem cell formation in VISUAL. WT (B), bes1-1 (SALK_098634) (C), bes1-2 (WiscDsLox246D02) (D) and bes1-3 (SALK_091133) (E) cotyledons were cultured in medium containing 2,4-D, kinetin and bikinin for 4 d. Scale bars indicate 1 mm. (F) Quantification of xylem differentiation rates in (B–E). Error bars indicate the SD (n = 8–10). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). Fig. 2 View largeDownload slide Gene expression analysis of bes1 mutants in VISUAL. (A–E) Expression levels of vascular-related marker genes in VISUAL. IRX3 (A) and XCP1 (B) are xylem-specific markers, TDR (C) is a procambium marker and APL (D) and SEOR1 (E) are phloem markers. Relative expression levels were calculated by comparison with gene expression levels at 72 h in the WT. Error bars indicate the SD (n = 3). Significant differences among samples after induction are indicated by different letters (Tamhane T2 test, α = 0.05). (F) Hierarchical clustering of vascular-related genes. Each gene expression level was normalized by setting the median value of the WT, bes1-3 and bes1-1 as 0 (see the Materials and Methods). Two major clades were defined as the bes1-down and bes1-not-down group based on their expression pattern. Relative gene expression levels are visualized in a heatmap image according to the color scale shown in the right-hand panel. (G) Comparison of the time course of expression patterns between the bes1-down and bes1-not-down group. Each gene expression level was normalized by dividing the level by its maximum. (H) Box plot showing the distribution of the half-time point for each gene. The half-time point is defined as the first time point when the relative expression level reached 50% of its maximum. (I) Comparison of the expression patterns of VISUAL phloem-specific genes (VPs; Kondo et al. 2016). Expression levels are visualized in a heatmap image according to the color scale shown in the right-hand panel. (J) Comparison of the expression patterns of VPs in the WT vs. bes1-1 and the WT vs. apl (Kondo et al. 2016) in VISUAL. The horizontal axis and vertical axis represent fold change in bes1-1 and apl against the WT at the log2 scale, respectively. (K) Schematic illustration of the bes1 mutant phenotype in VISUAL. The bes1 mutations repress xylem and phloem differentiation from procambial cells. Fig. 2 View largeDownload slide Gene expression analysis of bes1 mutants in VISUAL. (A–E) Expression levels of vascular-related marker genes in VISUAL. IRX3 (A) and XCP1 (B) are xylem-specific markers, TDR (C) is a procambium marker and APL (D) and SEOR1 (E) are phloem markers. Relative expression levels were calculated by comparison with gene expression levels at 72 h in the WT. Error bars indicate the SD (n = 3). Significant differences among samples after induction are indicated by different letters (Tamhane T2 test, α = 0.05). (F) Hierarchical clustering of vascular-related genes. Each gene expression level was normalized by setting the median value of the WT, bes1-3 and bes1-1 as 0 (see the Materials and Methods). Two major clades were defined as the bes1-down and bes1-not-down group based on their expression pattern. Relative gene expression levels are visualized in a heatmap image according to the color scale shown in the right-hand panel. (G) Comparison of the time course of expression patterns between the bes1-down and bes1-not-down group. Each gene expression level was normalized by dividing the level by its maximum. (H) Box plot showing the distribution of the half-time point for each gene. The half-time point is defined as the first time point when the relative expression level reached 50% of its maximum. (I) Comparison of the expression patterns of VISUAL phloem-specific genes (VPs; Kondo et al. 2016). Expression levels are visualized in a heatmap image according to the color scale shown in the right-hand panel. (J) Comparison of the expression patterns of VPs in the WT vs. bes1-1 and the WT vs. apl (Kondo et al. 2016) in VISUAL. The horizontal axis and vertical axis represent fold change in bes1-1 and apl against the WT at the log2 scale, respectively. (K) Schematic illustration of the bes1 mutant phenotype in VISUAL. The bes1 mutations repress xylem and phloem differentiation from procambial cells. Fig. 3 View largeDownload slide Complementation test. (A–C) Ectopic xylem cell formation in VISUAL. Cotyledons of the WT (A), bes1-1 (B) and bes1-1 harboring genomic BES1 (gBES1/bes1-1) (C) were cultured for 4 d. Scale bars indicate 1 mm. (D) Quantification of xylem differentiation rates in (A–C). Error bars indicate the SD (n = 8). Significant differences compared with the WT were examined by Tamhane T2 test (α = 0.05). (E) Expression levels of xylem and phloem marker genes in VISUAL. RNA was extracted from cotyledons at 72 h after VISUAL induction. Two independent complement lines were examined. Relative expression levels were calculated by comparison with the WT. Error bars indicate the SD (n = 3). Significant differences compared with the WT were examined by Tamhane T2 test (α = 0.05). Fig. 3 View largeDownload slide Complementation test. (A–C) Ectopic xylem cell formation in VISUAL. Cotyledons of the WT (A), bes1-1 (B) and bes1-1 harboring genomic BES1 (gBES1/bes1-1) (C) were cultured for 4 d. Scale bars indicate 1 mm. (D) Quantification of xylem differentiation rates in (A–C). Error bars indicate the SD (n = 8). Significant differences compared with the WT were examined by Tamhane T2 test (α = 0.05). (E) Expression levels of xylem and phloem marker genes in VISUAL. RNA was extracted from cotyledons at 72 h after VISUAL induction. Two independent complement lines were examined. Relative expression levels were calculated by comparison with the WT. Error bars indicate the SD (n = 3). Significant differences compared with the WT were examined by Tamhane T2 test (α = 0.05). BES1 promotes xylem cell differentiation from procambial cells in VISUAL In VISUAL, mesophyll cells first differentiate into procambial cells, followed by xylem cells (Kondo et al. 2015). We therefore examined whether BES1 is involved in the induction of procambial cells, xylem differentiation from procambial cells or both processes. First, we used the procambium-specific gene TDIF RECEPTOR (TDR) as a marker and found that this gene was not down-regulated in bes1 mutants in VISUAL. Next, to dissect downstream and upstream factors for BES1 comprehensively, we performed microarray analysis of cotyledons at 72 h after VISUAL induction in bes1-1, bes1-3 and the WT. We initially selected genes that were highly up-regulated at 72 h after VISUAL induction in the WT (>4-fold up-regulation, 1,534 genes; Kondo et al. 2016) as vascular-related genes. Hierarchical clustering classified the vascular-related genes into two major groups. Approximately 60% of the vascular-related genes were down-regulated in bes1 mutants. Then we designated these genes as a ‘bes1-down’ group (Fig. 2F). On the other hand, nearly 40% of vascular-related genes were expressed at the same or a higher level in the mutants. Therefore, we categorized these genes as a ‘bes1-not-down’ group (Fig. 2F). To characterize these two groups of genes, we analyzed the expression profiles of the bes1-down group and the bes1-not-down group using our previously reported time course microarray data from leaf disks cultured in VISUAL (Kondo et al. 2015). Most bes1-down genes were highly expressed 60 h after induction, whereas many bes1-not-down genes were induced 36 h after induction (Fig. 2G). This analysis indicated that the bes1-not-down and the bes1-down genes begin to be expressed at early and late stages in VISUAL, respectively. Further half-time point analysis, which evaluates the timing of gene expression in VISUAL, indicated that the expression of the bes1-not-down genes increased from 12 to 26 h and that of the bes1-down genes increased from 30 to 48 h in VISUAL (Fig. 2H). These results indicate that, in the bes1 mutants, the late stage of VISUAL is suppressed, whereas the early stage is not suppressed but is instead promoted. We then conducted Gene Ontology (GO) enrichment analysis to characterize the molecular functions of the bes1-down and bes1-not-down genes. In the bes1-down group, secondary cell wall-related genes, including xylan and lignin biosynthesis genes, were markedly enriched (Table 1). Additionally, genes related to xylem vessel cell differentiation were also enriched in this group (Table 1). These results coincide with the finding that bes1 mutants show defects in xylem differentiation in VISUAL. On the other hand, procambial cell-related genes were enriched in the bes1-not-down group (Table 1). Moreover, the bes1-not-down group includes many cell cycle and cell division genes (Table 1), including seven out of eight procambial-specific genes that were up-regulated in VISUAL only in the presence of bikinin (Kondo et al. 2015). Additionally, some bes1-not-down genes were annotated as auxin-related genes, which seem to be implicated in procambium formation in leaves (Furuta et al. 2014a). These results suggest that, in cultured bes1 cotyledons, procambial cells accumulate due to the inhibition of differentiation of procambial cells into xylem cells. Altogether, these data support our hypothesis that BES1 promotes xylem cell differentiation from procambial cells in VISUAL. Table 1 GO enrichment analysis of vascular-related genes Term  P-value  Fold enrichment  bes1-down genes  Regulation of secondary cell wall biogenesis  2.07E-15  24.88  Plant-type secondary cell wall biogenesis  1.29E-14  12.53  Xylan biosynthetic process  5.44E-13  15.88  Carbohydrate metabolic process  2.06E-07  2.75  Glucuronoxylan biosynthetic process  9.15E-07  17.87  Xylem development  1.56E-06  10.21  Xylan metabolic process  4.09E-06  20.42  Cell wall organization  4.79E-06  2.731  Lignin catabolic process  1.55E-05  11.91  Lignin biosynthetic process  6.65E-05  4.954  Xylem vessel member cell differentiation  7.05E-05  19.14  Cell wall thickening  1.35E-04  30.63  Phloem development  6.40E-04  20.42  Positive regulation of secondary cell wall biogenesis  6.40E-04  20.42  bes1-not-down genes  Microtubule-based movement  2.19E-19  15.32  Cell division  1.16E-14  6.134  Response to auxin  3.22E-12  4.77  Auxin-activated signaling pathway  3.10E-10  5.40  Cell cycle  1.40E-08  6.211  Mitotic nuclear division  4.34E-08  7.429  Regulation of cell cycle  1.15E-07  7.625  Multicellular organism development  6.54E-06  2.902  Procambium histogenesis  2.85E-05  24.76  Auxin polar transport  2.41E-04  6.368  Cotyledon vascular tissue pattern formation  5.90E-04  12.38  Term  P-value  Fold enrichment  bes1-down genes  Regulation of secondary cell wall biogenesis  2.07E-15  24.88  Plant-type secondary cell wall biogenesis  1.29E-14  12.53  Xylan biosynthetic process  5.44E-13  15.88  Carbohydrate metabolic process  2.06E-07  2.75  Glucuronoxylan biosynthetic process  9.15E-07  17.87  Xylem development  1.56E-06  10.21  Xylan metabolic process  4.09E-06  20.42  Cell wall organization  4.79E-06  2.731  Lignin catabolic process  1.55E-05  11.91  Lignin biosynthetic process  6.65E-05  4.954  Xylem vessel member cell differentiation  7.05E-05  19.14  Cell wall thickening  1.35E-04  30.63  Phloem development  6.40E-04  20.42  Positive regulation of secondary cell wall biogenesis  6.40E-04  20.42  bes1-not-down genes  Microtubule-based movement  2.19E-19  15.32  Cell division  1.16E-14  6.134  Response to auxin  3.22E-12  4.77  Auxin-activated signaling pathway  3.10E-10  5.40  Cell cycle  1.40E-08  6.211  Mitotic nuclear division  4.34E-08  7.429  Regulation of cell cycle  1.15E-07  7.625  Multicellular organism development  6.54E-06  2.902  Procambium histogenesis  2.85E-05  24.76  Auxin polar transport  2.41E-04  6.368  Cotyledon vascular tissue pattern formation  5.90E-04  12.38  GO enrichment analysis of genes up-regulated in VISUAL (>4-fold, 1,534 genes). The bes1-down and bes1-not-down groups were examined separately. Significantly enriched terms (P < 0.001) are shown. BES1 also promotes phloem cell differentiation in VISUAL In the WT, the differentiation of phloem sieve element-like cells also occurs in VISUAL (Kondo et al. 2016). Interestingly, several bes1-down genes were annotated as being related to phloem development (Table 1). Consistent with this notion, qRT-PCR analysis indicated that the expression levels of phloem marker genes APL and SIEVE-ELEMENT-OCCLUSION-RELATED 1 (SEOR1) were significantly reduced in the bes1 mutants compared with the WT, similar to xylem marker genes (Fig. 2D, E). These results suggest that phloem differentiation is also inhibited in bes1. To understand the role of BES1 in phloem development, we investigated the expression of VISUAL phloem-specific genes (VPs), which we previously identified by transcriptome analysis (Kondo et al. 2016), using the bes1 microarray data. Many VPs were down-regulated in the bes1 mutants (Fig. 2I). These down-regulated genes were also down-regulated in the apl mutants (Fig. 2J), which have strong defects in phloem development in VISUAL (Kondo et al. 2016). Considering the significant reduction in APL expression in bes1-1 (Fig. 2D), these data suggest that BES1 up-regulates a wide range of phloem sieve element-related genes, probably upstream of APL. In addition to the recovery of xylem differentiation, the decrease in expression of phloem genes in bes1-1 was fully complemented by the introduction of gBES1 (Fig. 3). These results strongly support the idea that BES1 promotes phloem differentiation as well as xylem differentiation (Fig. 2K). BZR1 acts redundantly with BES1 in vascular development In bes1 mutants, the induction of both xylem and phloem marker genes in VISUAL was not completely repressed, suggesting that other factors may contribute to the promotion of the differentiation process. BZR1 is the closest homolog of BES1, with 88% identity (Wang et al. 2002). We therefore employed genome editing techniques (Fauser et al. 2014) in Col-0 and bes1-1 to generate bzr1-1 and bes1-1 bzr1-2 plants, respectively. We succeeded in obtaining these homozygous lines, in which the Cas9 gene had been removed. The bzr1-1 mutant possesses a one base insertion in the second exon of BZR1, resulting in the appearance of a premature stop codon due to a frameshift (Fig. 4A, C). On the other hand, the bes1-1 bzr1-2 double mutant has lost 29 bases of BZR1 spanning from the 3' end of the intron to the start of the second exon (Fig. 4A). Sequencing of BZR1 transcripts revealed that the splicing site between the intron and the second exon is altered in bes1-1 bzr1-2 (Fig. 4B), leading to a premature stop codon due to a frameshift (Fig. 4C). Fig. 4 View largeDownload slide Generation of bzr1 mutants. (A) Schematic overview of the gene structure of BZR1 and the sequence around the selected site targeted by CRISPR/Cas9. Exons, the intron and untranslated regions are illustrated by boxes, a solid line and dotted lines, respectively. Exon and intron sequences in the WT are written in lower case and upper case letters, respectively. The protospacer adjacent motif (PAM) sequences of the target and the resulting mutations are highlighted in blue and red, respectively. A one base insertion was detected in bzr1-1, whereas 29 base deletions occurred in bes1-1 bzr1-2. (B) The mRNA sequences of BZR1 in the WT and bes1-1 bzr1-2. The splice site in each line is marked by an arrowhead, and spliced sequences are highlighted in green in (A). (C) Theoretical amino acid sequences of BZR1 around the mutated sites in the WT, bzr1-1 and bes1-1 bzr1-2. Asterisks represent premature stop codons. Fig. 4 View largeDownload slide Generation of bzr1 mutants. (A) Schematic overview of the gene structure of BZR1 and the sequence around the selected site targeted by CRISPR/Cas9. Exons, the intron and untranslated regions are illustrated by boxes, a solid line and dotted lines, respectively. Exon and intron sequences in the WT are written in lower case and upper case letters, respectively. The protospacer adjacent motif (PAM) sequences of the target and the resulting mutations are highlighted in blue and red, respectively. A one base insertion was detected in bzr1-1, whereas 29 base deletions occurred in bes1-1 bzr1-2. (B) The mRNA sequences of BZR1 in the WT and bes1-1 bzr1-2. The splice site in each line is marked by an arrowhead, and spliced sequences are highlighted in green in (A). (C) Theoretical amino acid sequences of BZR1 around the mutated sites in the WT, bzr1-1 and bes1-1 bzr1-2. Asterisks represent premature stop codons. We then investigated whether BZR1 functions in vascular development using VISUAL. The bzr1-1 single mutants showed slightly reduced xylem differentiation rates (Fig. 5A–D). Further gene expression analysis revealed that xylem marker genes were slightly repressed in bzr1-1 compared with the WT (Fig. 5E). The expression levels of phloem marker genes were not significantly different from those of the WT (P = 0.136 for APL and P = 0.036 for SEOR1), but they tended to be reduced in bzr1-1 (Fig. 5E). Thus, the mutant phenotypes of bzr1-1 were weaker than those of bes1-1 in terms of differentiation rate and gene expression level when cultured in VISUAL (Fig. 5). These results suggest that BZR1 is also involved in xylem and phloem differentiation, but to a lesser extent than BES1. We then investigated the relationship between the roles of BES1 and BZR1 in vascular cell differentiation using bes1-1 bzr1-2 double mutants. However, the bes1-1 mutants produced relatively small amounts of xylem cells under the original VISUAL conditions, preventing us from verifying the functional redundancy of BES1 and BZR1. In an effort to solve this problem, we increased the bikinin concentration in the culture medium to 20 µM, because this treatment induced the production of more ectopic xylem cells in bes1-1 than the original medium (Fig. 6A, B). Under this condition, the xylem differentiation rate was much lower in the bes1-1 bzr1-2 mutants than in bes1-1 (Fig. 6C, D). The down-regulation of xylem and phloem marker genes in the bes1 single mutants was also enhanced by the addition of the bzr1-2 mutation (Fig. 6E). On the other hand, the procambium marker gene TDR was not repressed in these mutants, even in bes1-1 bzr1-2 (Fig. 6E). Based on these results, it is likely that BZR1 acts redundantly with BES1 in xylem and phloem differentiation from procambial cells in VISUAL. Fig. 5 View largeDownload slide Mutant phenotypes of bzr1-1 in VISUAL. (A–C) Ectopic xylem cell formation in VISUAL. WT (A), bes1-1 (B) and bzr1-1 (C) cotyledons were cultured for 4 d. Scale bars indicate 1 mm. (D) Quantification of xylem differentiation rates in (A–C). Error bars indicate the SD (n = 8). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). (E) Expression levels of xylem and phloem marker genes in the WT, bes1-1 and bzr1-1 in VISUAL. RNA was extracted from cotyledons at 72 h after VISUAL induction. Relative expression levels were calculated by comparison with the WT. Error bars indicate the SD (n = 3). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). Fig. 5 View largeDownload slide Mutant phenotypes of bzr1-1 in VISUAL. (A–C) Ectopic xylem cell formation in VISUAL. WT (A), bes1-1 (B) and bzr1-1 (C) cotyledons were cultured for 4 d. Scale bars indicate 1 mm. (D) Quantification of xylem differentiation rates in (A–C). Error bars indicate the SD (n = 8). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). (E) Expression levels of xylem and phloem marker genes in the WT, bes1-1 and bzr1-1 in VISUAL. RNA was extracted from cotyledons at 72 h after VISUAL induction. Relative expression levels were calculated by comparison with the WT. Error bars indicate the SD (n = 3). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). Fig. 6 View largeDownload slide Mutant phenotypes of bes1-1 bzr1-2 in VISUAL. (A–C) Ectopic xylem cell formation in VISUAL. WT (A), bes1-1 (B) and bes1-1 bzr1-2 (C) cotyledons were cultured with bikinin at twice the normal concentration (20 µM) for 4 d. Scale bars indicate 1 mm. (D) Quantification of xylem differentiation rates in (A–C). Error bars indicate the SD (n = 8–12). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). (E) Expression levels of xylem, phloem and procambium marker genes in the WT, bes1-1 and bes1-1 bzr1-2 in VISUAL in the presence of 20 µM bikinin. RNA was extracted from cotyledons at 72 h after induction. Relative expression levels were calculated by comparison with the WT. Error bars indicate the SD (n = 3). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). Fig. 6 View largeDownload slide Mutant phenotypes of bes1-1 bzr1-2 in VISUAL. (A–C) Ectopic xylem cell formation in VISUAL. WT (A), bes1-1 (B) and bes1-1 bzr1-2 (C) cotyledons were cultured with bikinin at twice the normal concentration (20 µM) for 4 d. Scale bars indicate 1 mm. (D) Quantification of xylem differentiation rates in (A–C). Error bars indicate the SD (n = 8–12). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). (E) Expression levels of xylem, phloem and procambium marker genes in the WT, bes1-1 and bes1-1 bzr1-2 in VISUAL in the presence of 20 µM bikinin. RNA was extracted from cotyledons at 72 h after induction. Relative expression levels were calculated by comparison with the WT. Error bars indicate the SD (n = 3). Significant differences are indicated by different letters (Tamhane T2 test, α = 0.05). Growth defects are not observed in bes1-1 bzr1-2 mutants To investigate the functions of BES1 and BZR1 in in vivo vascular development, we compared cross-sections from WT vs. bes1-1 bzr1-2 plants. Although bes1-1 bzr1-2 mutants showed severe defects in VISUAL, no obvious defects were observed in the central xylem tissues of hypocotyls in 16-day-old or 6-week-old plants (Fig. 7A–D). Phloem tissues (visualized by toluidine blue and aniline blue staining) were also indistinguishable between the WT and bes1-1 bzr1-2 (Fig. 7A–D). Similarly, no defects in vascular development were observed in 6-week-old stems of bes1-1 bzr1-2 (Fig. 7E, F). These results suggest that the vascular phenotype of bes1-1 bzr1-2 is enhanced in the culture system VISUAL, in which exogenous bikinin induces rapid vascular cell differentiation apart from other developmental processes. Fig. 7 View largeDownload slide In vivo phenotype of bes1-1 bzr1-2. (A–F) Cross-sections of hypocotyls (A–D) and stems (E, F) of WT (A, C, E) and bes1-1 bzr1-2 (B, D, F) plants grown for 16 d (A, B) or 6 weeks (C–F). Toluidine blue (A, B) or aniline blue staining (C–F) was conducted. Blue and green fluorescence in (C–F) is autofluorescence from xylem cells and stained phloem cells after UV radiation, respectively. Scale bars indicate 200 µm for (C) and (D) and 50 µm for the others. (G, H) BL sensitivity of WT, bes1-1, bzr1-1 and bes1-1 bzr1-2 seedlings. Relative lengths of hypocotyls (G) and roots (H) were calculated by comparison with samples of each genotype grown without BL. Error bars indicate the SD (n = 30–32 except for n = 25 for bzr1-1 treated with 10 nM BL). (I) Hypocotyl elongation in plants grown in the dark. Seedlings were grown for 7 d. The scale bar indicates 0.5 cm. (J) Quantification of hypocotyl elongation in (I). Error bars indicate the SD (n = 3–6). Significant differences compared with the WT were examined by Tamhane T2 test (α = 0.05). Fig. 7 View largeDownload slide In vivo phenotype of bes1-1 bzr1-2. (A–F) Cross-sections of hypocotyls (A–D) and stems (E, F) of WT (A, C, E) and bes1-1 bzr1-2 (B, D, F) plants grown for 16 d (A, B) or 6 weeks (C–F). Toluidine blue (A, B) or aniline blue staining (C–F) was conducted. Blue and green fluorescence in (C–F) is autofluorescence from xylem cells and stained phloem cells after UV radiation, respectively. Scale bars indicate 200 µm for (C) and (D) and 50 µm for the others. (G, H) BL sensitivity of WT, bes1-1, bzr1-1 and bes1-1 bzr1-2 seedlings. Relative lengths of hypocotyls (G) and roots (H) were calculated by comparison with samples of each genotype grown without BL. Error bars indicate the SD (n = 30–32 except for n = 25 for bzr1-1 treated with 10 nM BL). (I) Hypocotyl elongation in plants grown in the dark. Seedlings were grown for 7 d. The scale bar indicates 0.5 cm. (J) Quantification of hypocotyl elongation in (I). Error bars indicate the SD (n = 3–6). Significant differences compared with the WT were examined by Tamhane T2 test (α = 0.05). BES1 and BZR1 are involved in various developmental processes. In particular, they function as major transcription factors that mediate the BR signaling pathway (Guo et al. 2013). To explore the impact of BES1 and BZR1 on BR signaling, we treated WT and bes1-1 bzr1-2 plants with brassinolide (BL). BL treatment promotes hypocotyl elongation and represses root growth in WT plants in the light. As previously reported (He et al. 2005, Kang et al. 2015), bes1-1 was slightly resistant to exogenous BL (Fig. 7G, H). Although bzr1-1 single mutants were partially resistant to exogenous BL only in root elongation, the bes1-1 bzr1-2 mutants were more insensitive to BL than the bes1 single mutants in terms of both hypocotyl and root elongation (Fig. 7G, H). Consistent with this observation, the changes in expression of a BR-responsive gene in response to BL treatment were also partially reduced in bes1-1 bzr1-2 (Supplementary Fig. S1). Next, we investigated the defects related to BR signaling in bes1-1 bzr1-2 in the absence of exogenous BL treatment. The bri1 mutant, a BR receptor mutant, shows severe defects in hypocotyl elongation under dark conditions (Li and Chory 1997). However, bes1-1 and bes1-1 bzr1-2 did not exhibit this phenotype, instead appearing like WT plants (Fig. 7I, J). Furthermore, stem growth in bes1-1 bzr1-2 was comparable with that in the WT under normal growth conditions, unlike the dwarf mutant bri1 (Supplementary Fig. S2). These results indicate that the BR-related phenotype in bes1-1 bzr1-2 appears only in the presence of exogenously applied BL. BES1 and BZR1 gain-of-function mutations promote phloem differentiation in vivo In contrast to bes1-1 bzr1-2 mutants, it is known that gain-of-function mutants bes1-D and bzr1-D exhibit pleiotropic phenotypes. For instance, hypocotyl elongation is promoted in these mutants under darkness (Li and Nam 2002, Yin et al. 2002). Moreover, it was reported that the gain-of-function mutant bes1-D sometimes exhibits reduced procambial cell layers between xylem and phloem cells due to the excess vascular differentiation from procambial cells (Kondo et al. 2014). Then we generated bes1-D bzr1-D double gain-of-function mutants and observed their vascular phenotype in detail (Fig. 8). The overall morphology of bes1-D bzr1-D was similar to that of bes1-D, in which the hypocotyl is elongated (Yin et al. 2002), but the thickness of the hypocotyl is comparable with that of the WT. In WT hypocotyls, usually two or three procambial cell layers were formed between phloem and xylem cells (Fig. 8A). In bes1-D bzr1-D, however, procambial cell layers were reduced, occasionally resulting in adjacency of xylem and phloem cells (Fig. 8B; red arrowhead), which is consistent with the result in bes1-D (Kondo et al. 2014). In addition, ectopic phloem cells, which are non-clustered phloem cells surrounded by undifferentiated cells, were sometimes formed only in bes1-D bzr1-D but not in the WT (Fig. 8A, C; yellow arrowhead). These results suggest that BES1 and BZR1 promote phloem differentiation from procambial cells in vivo. Fig. 8 View largeDownload slide Comparison of hypocotyl vasculature between WT and bes1-D bzr1-D. Cross-sections of hypocotyls of WT (A) and bes1-D bzr1-D (B, C) plants grown for 11 d. Orange and blue dotted lines indicate phloem (Ph) and xylem (Xy) cells, respectively. White solid lines indicate procambial cell (Pc) layers. Red and yellow arrowheads indicate the adjacency of xylem and phloem cells and non-clustered phloem cells, respectively. The fraction in the top and bottom of the panels represents the number of plants exhibiting adjacency of xylem and phloem cells and non-clustered phloem cells per total number of plants observed, respectively. Scale bars indicate 20 µm. Fig. 8 View largeDownload slide Comparison of hypocotyl vasculature between WT and bes1-D bzr1-D. Cross-sections of hypocotyls of WT (A) and bes1-D bzr1-D (B, C) plants grown for 11 d. Orange and blue dotted lines indicate phloem (Ph) and xylem (Xy) cells, respectively. White solid lines indicate procambial cell (Pc) layers. Red and yellow arrowheads indicate the adjacency of xylem and phloem cells and non-clustered phloem cells, respectively. The fraction in the top and bottom of the panels represents the number of plants exhibiting adjacency of xylem and phloem cells and non-clustered phloem cells per total number of plants observed, respectively. Scale bars indicate 20 µm. Discussion BES1 promotes xylem and phloem differentiation from procambial cells but not procambial cell differentiation from mesophyll cells In a previous paper, we reported that BES1 promotes xylem differentiation from mesophyll cells in VISUAL (Kondo et al. 2014). In the current study, microarray analysis of bes1 showed that BES1 promotes xylem differentiation from procambial cells but not procambial cell differentiation from mesophyll cells. We also found that the bes1 mutations suppressed phloem differentiation in VISUAL, suggesting that BES1 plays a positive role in phloem differentiation. Microarray analysis also indicated that BES1 and APL share downstream genes involved in phloem differentiation. The expression of APL was repressed in bes1. These results indicate that BES1 positively regulates phloem differentiation, probably upstream of APL. On the other hand, the transcription factor genes VND6 and VND7, encoding master regulators of xylem differentiation (Kubo et al. 2005), were also down-regulated in bes1 (Supplementary Table S1). Therefore, it is likely that BES1 functions early in vascular development before the induction of final differentiation by master regulators of xylem and phloem differentiation. These findings, together with the observation that a large number of procambial cells remained in the bes1 mutants after the induction of VISUAL, suggest that BES1 triggers the transition of stem cells to specialized cells into xylem or phloem cells. Functional redundancy among BES1 family members There are six BES1-like genes in the Arabidopsis genome: BES1, BZR1 and BES1/BZR1 HOMOLOG 1–4 (BEH1–BEH4) (Yin et al. 2002, Wang et al. 2002, Yin et al. 2005). BES1 and BZR1 are thought to act as central players that regulate various developmental processes in plants, playing prominent roles in BR signaling (Guo et al. 2013). However, in many cases, functional analyses of these proteins have been performed using gain-of-function mutants (bes1-D and bzr1-D), and few analyses have been performed using loss-of-function mutants. Here, we produced bzr1-1 (a loss-of-function mutant of BZR1) and the bes1-1 bzr1-2 double mutant to analyze the roles of BES1 and BZR1. The bes1, bzr1-1 and bes1-1 bzr1-2 mutants did not show any visible phenotypes in planta, including a dwarf phenotype and photomorphogenesis in the dark, which are characteristic phenotypes of BR-deficient mutants such as bri1. This result strongly suggests that BES1 family members function redundantly. We then examined the phenotypes of these mutants in physiological aspects. The only mutant phenotype observed in bes1-1 bzr1-2 was reduced sensitivity to exogenous BL application. To account for this phenomenon, the following findings about other multiple mutants should be considered. The arabidopsis response regulator 10 (arr10) arr12 double mutants are resistant to exogenous cytokinin but grow well on Murashige and Skoog (MS) agar plates and in soil (Yokoyama et al. 2007). The addition of the arr1 mutation to arr10 arr12 results in plants with a dwarf phenotype in the absence of cytokinin, which was also observed in another cytokinin dominant-negative mutant, wooden leg (Yokoyama et al. 2007). Consistent with this idea, bes1-RNAi (RNA interference) lines, which exhibit repressed expression not only of BES1 and BZR1 (Yin et al. 2005), but also of several BEH genes (Wang et al. 2013), show defects in stem growth and shoot branching. Our results obtained using loss-of-function mutants raise the possibility that BEHs function redundantly with BES and BZR1. Further genetic analysis is needed to understand fully the functions of BES1 family proteins. BZR1 is also a positive regulator of xylem and phloem differentiation from procambial cells Although we did not detect mutant vascular phenotypes in bzr1 in planta, the VISUAL system could visualize them. VISUAL using bzr1-1 and bes1-1 bzr1-2 revealed that BZR1 as well as BES1 positively regulates both xylem and phloem differentiation from procambial cells. This finding is consistent with the previous finding that both bes1-D and bzr1-D can partially rescue the phenotype of the octopus mutant, in which root protophloem differentiation is prone to be repressed (Anne et al. 2015). Our observation that phloem cells are ectopically produced in hypocotyls of bes1-D bzr1-D also supports the molecular functions of BES1 and BZR1 found in VISUAL. In the current study, bes1 exhibited a stronger vascular phenotype than bzr1-1, and the phenotype induced by these mutations was additive, suggesting that BES1 and BZR1 redundantly function in xylem and phloem differentiation to a greater and lesser extent, respectively. This result is also in accordance with the finding that bes1-D, but not bzr1-D, shows defects in the hypocotyl vasculature (Kondo et al. 2014). In VISUAL, mesophyll cells initially differentiate into procambial cells, followed by xylem or phloem cells (Kondo et al. 2015, Kondo et al. 2016). Bikinin promotes procambial cell differentiation (Kondo et al. 2015) as well as the differentiation of procambial cells into xylem and phloem cells (Kondo et al. 2016). However, BES1 and BZR1 only regulate differentiation into xylem and phloem cells. Therefore, it is likely that GSK3s also control procambial cell differentiation, but factors other than BES1 and BZR1 are involved in this process downstream of the GSK3s. Indeed, GSK3s phosphorylate various developmental regulators such as ENHANCER OF GLABRA 3 (EGL3) and TRANSPARENT TESTA GLABRA 1 (TTG1) (Cheng et al. 2014) for root hair formation and AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 for lateral root formation (Cho et al. 2014). Therefore, it will be interesting to identify a new target(s) of GSK3s that regulates procambial cell differentiation. Materials and Methods Plant materials Seeds of the Arabidopsis thaliana bes1-1 (SALK_098634), bes1-2 (WiscDsLox246D02) and bes1-3 (SALK_091133) mutants were obtained from the ABRC stock center. The progeny of heterozygous bri1-48 (SALK_041648) were gifts from Dr. Takeshi Nakano, and homozygous mutants were identified based on the presence of severely shortened hypocotyls. bes1-D and bzr1-D mutants were also gifts from Dr. Takeshi Nakano, and bes1-D bzr1-D mutants were generated by crossing. All mutants in this study were in the Col-0 background. To produce the complementation lines, DNA fragments of gBES1 containing the 2,000 bp promoter sequence and the 1,000 bp sequence downstream of the stop codon were cloned and introduced into the pGWB1 vector (Nakagawa et al. 2007), followed by transformation into bes1-1 by the floral dip method (Clough and Bent 1998). Plants were grown on conventional 1/2 MS agar plates (pH 5.7) at 22°C under continuous light unless otherwise mentioned. For later harvesting, seedlings were transferred to pots containing a mixture of vermiculite (VS Kakou) and PRO-MIX BX (Premier Horticulture). CRISPR The target sequence was selected using CRISPRdirect software (Naito et al. 2015). DNA fragments containing 20 bp of the target sequence neighboring the PAM (protospacer adjacent motif) sequence and overhang sequences were generated by hybridizing two primers, followed by ligation into the pEn-Chimera vector (Fauser et al. 2014) that had been digested with BbsI. The single guide RNA (sgRNA)-containing sequence was then transferred into the pDe-CAS9 vector (Fauser et al. 2014) via LR reaction (Thermo Fisher Scientific). The resulting construct was transformed into WT and bes1-1 plants. Transformants were obtained by bialaphos selection. T2 seeds from single insertion lines were incubated on MS agar plates without antibiotics. Cas9-free mutants were selected from the T2 population by PCR and sequencing using the primers listed in Supplementary Table S2. To check the sequence of the BZR1 transcript in bes1-1 bzr1-2, total RNA was extracted from cotyledons of 6-day-old WT and bes1-1 bzr1-2 plants using an RNeasy Plant Mini kit (Qiagen). Reverse transcription was performed using SuperScript III (Thermo Fisher Scientific). PCR was performed using synthesized cDNA and the primers listed in Supplementary Table S2, followed by sequencing using the same primer. Data were visualized using Chromas 2.6 (http://technelysium.com.au/wp/). VISUAL The detailed protocol for VISUAL was described previously (Kondo et al. 2016). In brief, cotyledons of 6-day-old seedlings grown in liquid 1/2 MS medium were cultured in MS-based medium containing 0.25 mg l−1 2,4-D, 1.25 mg l−1 kinetin and 10 µM bikinin under continuous light. To compare the phenotypes of bes1-1 vs. bes1-1 bzr1-2, the bikinin concentration was changed to 20 µM. To observe ectopic xylem differentiation, cotyledons cultured for 4 d were fixed in ethanol/acetic acid (3 : 1) and mounted with clearing solution (chloral hydrate/glycerol/water; 8 : 1 : 2). The differentiation rates were determined based on the area of ectopic xylem cells in a cotyledon per total area of the cotyledon blade. These areas were calculated using ImageJ. qRT-PCR Total RNA was extracted from cotyledons before or after culture for 72 h (in the VISUAL experiments) or from whole seedlings treated with dimethylsulfoxide (DMSO) or 100 µM BL for 3 h (in the BL sensitivity tests) using an RNeasy Plant Mini kit (Qiagen). Quantitative PCR was performed using a LightCycler (Roche Diagnostics). UBIQUITIN 14 (UBQ14) was used for the VISUAL experiments, and ACTIN 2 (ACT2) was used for the BL sensitivity tests as an internal control. The gene-specific primer sets are listed in Supplementary Table S2. Microarray experiment Microarray analyses were performed as previously described (Ohashi-Ito et al. 2010). For clustering, normalization of gene expression levels was performed as follows: first, the average fold change between values before and after induction in each experiment was calculated. These values were then normalized by dividing them by the median value of the WT, bes1-3 and bes1-1. Finally, these values were converted to the log2 scale, producing a value defined as the Processed signal. Hierarchical clustering via the Pearson correlation method and heatmap generation were performed using Subio Platform software (Subio). The half-time point was defined as the first time point when the relative expression level reached 50% of its maximum value in a set of time course microarray data (Kondo et al. 2015) in which the expression levels between observed values were postulated to change linearly. GO enrichment analysis was performed using DAVID Bioinformatics Resources 6.8 (Huang et al. 2009; https://david.ncifcrf.gov/). Histological analysis Cross-sections of hypocotyls of 11- or 16-day-old plants grown in liquid 1/2 MS medium were produced as previously described (Kondo et al. 2014). Sliced samples were stained with 0.1% toluidine blue for 1 min. To produce cross-sections of 6-week-old plants, hypocotyls or the basal parts of stems were fixed in 70% ethanol. Sections of hypocotyls and stems 2 cm above the rosette at 80 µm thickness were produced using a LEICA VT1200S vibratome. Sliced samples were stained with 0.05% aniline blue in 100 mM phosphate buffer (pH 7.2) for 1 h and observed under UV radiation. BR treatment To observe hypocotyl or root elongation, plants were grown on 1/2 MS agar plates containing DMSO or BL. After 7 d, the plates were scanned with a CanoScan 9000F (Canon). Hypocotyl and root lengths were measured using ImageJ. For gene expression analysis, plants were grown on 1/2 MS agar plates. After 10 d, the seedlings were incubated in a solution of DMSO or 100 µM BL for 3 h and collected for RNA extraction. Phenotypic observation of dark-grown plants Plants were grown on 1/2 MS agar plates in the dark after white light irradiation to promote germination. After 7 d, the plates were scanned using a CanoScan 9000F (Canon). Hypocotyl length was measured using ImageJ. Statistical analysis For multiple comparisons, Tamhane T2 tests were employed in this study due to inhomogeneous variances obtained by the Levene tests (Wallner et al. 2017). The significance thresholds were set to α = 0.05. The P-values for each comparison were calculated by Welch’s t-test. The significance threshold of each comparison was set to α’ = 1 – (1 – α)1/k, where k is the total number of comparisons. For example, if k = 3, the significance threshold of the P-value of each comparison (α’) is approximately 0.0167. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grants-in-Aid No. 17H06476 to Y.K. and No. 15H05958 to H.F.] and the Japan Society for the Promotion of Science [grant No. 16J06222 to M.S., No. 17H05008 to Y.K. and No. 16H06377 to H.F.]. Acknowledgments We thank Yukiko Sugisawa, Yasuko Ozawa and Satoshi Endo for technical support. We also thank Holger Puchta and Takeshi Nakano for providing materials. Disclosures The authors have no conflicts of interest to declare. References Anne P., Azzopardi M., Gissot L., Beaubiat S., Hématy K., Palauqui J.C. ( 2015) OCTOPUS negatively regulates BIN2 to control phloem differentiation in Arabidopsis thaliana. Curr. Biol . 25: 2584– 2590. Google Scholar CrossRef Search ADS PubMed  Bonke M., Thitamadee S., Mähönen A.P., Hauser M.T., Helariutta Y. ( 2003) APL regulates vascular tissue identity in Arabidopsis. 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Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations APL ALTERED PHLOEM DEVELOPMENT ARF AUXIN RESPONSE FACTOR ARR ARABIDOPSIS RESPONSE REGULATOR BEH BES1/BZR1 HOMOLOG BES1 BRI1-EMS-SUPPRESSOR 1 BL brassinolide BR brassinosteroid BRI1 BRASSINOSTEROID-INSENSITIVE 1 BZR1 BRASSINAZOLE RESISTANT 1 CRISPR clustered regularly interspaced short palindromic repeats Cas9 CRISPR-associated protein 9 DMSO dimethylsulfoxide GO Gene Ontology GSK3 glycogen synthase kinase 3 protein IRX3 IRREGULAR XYLEM 3 MS Murashige and Skoog PAM, protospacer adjacent motif; qRT-PCR quantitative reverse transcription PCR SEOR1 SIEVE-ELEMENT-OCCLUSION-RELATED 1 sgRNA, single guide RNA; TDR TDIF RECEPTOR UBQ14 UBIQUITIN 14 VISUAL Vascular cell Induction culture System Using Arabidopsis Leaves VND VASCULAR-RELATED NAC-DOMAIN VP VISUAL phloem-specific gene WT wild type XCP1 XYLEM CYSTEINE PEPTIDASE 1 Footnote Footnote Microarray data have been deposited in Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). Accession number is GSE110199 © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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Plant and Cell PhysiologyOxford University Press

Published: Mar 1, 2018

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