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FPX is a Novel Chemical Inducer that Promotes Callus Formation and Shoot Regeneration in Plants

FPX is a Novel Chemical Inducer that Promotes Callus Formation and Shoot Regeneration in Plants Abstract Auxin and cytokinin control callus formation from developed plant organs as well as shoot regeneration from callus. Dedifferentiation and regeneration of plant cells by auxin and cytokinin stimulation are considered to be caused by the reprogramming of callus cells, but this hypothesis is still argued to this day. Although an elucidation of the regulatory mechanisms of callus formation and shoot regeneration has helped advance plant biotechnology research, many plant species are intractable to transformation because of difficulties with callus formation. In this study, we identified fipexide (FPX) as a useful regulatory compound through a chemical biology-based screening. FPX was shown to act as a chemical inducer in callus formation, shoot regeneration and Agrobacterium infection. With regards to morphology, the cellular organization of FPX-induced calli differed from those produced under auxin/cytokinin conditions. Microarray analysis revealed that the expression of approximately 971 genes was up-regulated 2-fold after a 2 d FPX treatment compared with non-treated plants. Among these 971 genes, 598 genes were also induced by auxin/cytokinin, whereas 373 genes were specifically expressed upon FPX treatment only. FPX can promote callus formations in rice, poplar, soybean, tomato and cucumber, and thus can be considered a useful tool for revealing the mechanisms of plant development and for use in plant transformation technologies. Introduction The discovery of novel bioactive compounds has proven useful in biological research and applied bioscience. Within the plant science field, several types of phytohormones are well-known examples of useful bioactive compounds. Auxin was the first phytohormone to be identified by plant physiologists. In 1882, Charles Darwin predicted the existence of such phytohormone action by proposing that a compound transported from the shoot meristem to the hypocotyl after the perception of light was responsible for phototropism in plants (Darwin and Darwin 1880). The first auxin to be identified—IAA—was initially detected as a metabolite of human urine (Kögl et al. 1934), and then later identified in immature seeds of Zea mays when IAA was confirmed as a natural bioactive compound in higher plants (Haagen-Smit et al. 1946). Following the discovery of auxin, Skoog and Miller (1957) identified a compound that induced plant calli in the presence of IAA. Plant calli are unorganized, amorphous cell masses that are naturally present in plant tissues subjected to stresses such as wounding and pathogen infection. A compound inducing callus in the presence of IAA was identified as a product of DNA degradation and designated the synthetic cytokinin, kinetin (Miller et al. 1955). Naturally occurring cytokinins were subsequently discovered in higher plants, namely zeatin, which was identified from immature seeds of Z. mays in 1963 (Letham 1963), and dihydrozeatin, which was detected in immature seeds of lupin in 1967 (Koshimizu et al. 1968). Since its discovery, auxin has been revealed to function in the regulation of processes such as embryo development, germination, organ growth, flowering, phototropism and gravitropism. Cytokinin is a known regulator of organ growth, lateral bud formation, senescence, nutrition response and other activities (Davies 2010). In a study by Skoog and Miller (1957), the combination of auxin and cytokinin triggered callus production, from many kinds of plant organs. These callus cells divided and elongated on auxin/cytokinin medium, suggesting that auxin and cytokinin regulate and generally maintain plant cell division and elongation. Furthermore, the callus cells induced from ‘developed’ leaf or root cells had been reprogrammed to plant stem cell-like tissue possessing totipotency. The totipotent callus cells could newly generate shoot organs, including plant stems and leaves, in response to cytokinin treatment; they were also able to generate root organs when exposed to auxin. The combination of auxin and cytokinin could freely regulate differentiation, dedifferentiation and the regeneration of plant cells (Krikorian 1995). The use of auxin and cytokinin in plant biotechnology research and related applied sciences has been long established and continues to advance. The first application was with the growth of cultured plant cells in liquid media or on solid agarose medium. The improvement of regulatory techniques and the search for new plant species producing compounds beneficial to humans have also taken place over a long period. Some of the useful compounds consequently discovered include medicines, such as the anti-cancer drug paclitaxel, the anti-microbial berberine and the neural inhibitor scopolamine, and dyes such as shikonin (Evans et al. 1984, Sato 2013, Yazaki et al. 2017). Another important application of auxin/cytokinin-induced plant callus formation is in the stable genomic transformation of plants (Horsch et al. 1984) (De Block et al. 1984). Since their development, Agrobacterium infection or particle gun bombardment of plant callus have been the most popular transformation techniques. In the first step of Agrobacterium-mediated plant transformation, callus from plant leaves or embryos of immature seeds is induced by auxin and cytokinin and then infected by Agrobacterium harboring a Ti-plasmid-based transformation vector. Active cell division in the callus induced by auxin and cytokinin is important for the adequate infection activity of Agrobacterium. A transfected callus cell was induced to shoot and root by suitable concentrations of auxin and cytokinin. In this way, transformation of the whole plant is accomplished (Dunwell and Wetten 2012). As detailed in this present study, our phytochemical research laboratory recently discovered that the compound fipexide (FPX) can induce callus formation from plant seedlings, leaf and root tissues, and showed activities in several callus formation steps. Finally, using cell and molecular biological analyses, we investigated the molecular mechanism(s) underlying the activity of FPX as a chemical inducer. Results FPX induces calli on apical regions of Arabidopsis seedling shoots and roots To identify novel compounds useful for the regulation of plant development, we screened the RIKEN NPDepo chemical library (Kato et al. 2012) for compounds promoting or inhibiting hypocotyl elongation of Arabidopsis seedlings grown in the dark for 7 d. In these trials, FPX was first identified for its ability to inhibit hypocotyl elongation (Fig. 1A). To observe the effect of FPX on hypocotyls under long-term light treatment, we continued growing Arabidopsis seedlings on 15 or 45 μM FPX medium. After 2 weeks, we noted an unusual expansion of shoot and root apex regions. After 3 weeks, we observed callus formation around the shoot apical meristem region of Arabidopsis seedlings grown on 15 μM FPX medium and around the root apical meristem region of seedlings on 45 μM FPX medium (Fig. 1B). After 6 weeks, the calli were still enlarging. Fig. 1 View largeDownload slide FPX induces callus formation in Arabidopsis seedlings. (A) Chemical structure of FPX. (B) Phenotypes of light-grown Arabidopsis wild-type plants 8, 15 and 22 d, and 6 weeks after germination on medium containing 0, 15 or 45 µM FPX. Arrowheads indicate callus formation. Scale bars = 2 mm. Fig. 1 View largeDownload slide FPX induces callus formation in Arabidopsis seedlings. (A) Chemical structure of FPX. (B) Phenotypes of light-grown Arabidopsis wild-type plants 8, 15 and 22 d, and 6 weeks after germination on medium containing 0, 15 or 45 µM FPX. Arrowheads indicate callus formation. Scale bars = 2 mm. FPX exhibits callus formation activities We next compared the callus formation activity of FPX in Arabidopsis with that of previously established auxin/cytokinin conditions (Skoog and Miller 1957). Specifically, we analyzed the effects of FPX activity on callus formation from 10-day-old Arabidopsis roots that were divided into 1 cm sections corresponding to the root tip, middle and basal regions. Basal rosette leaf sections of 2–3 mm were also cut; all four sections were incubated and analyzed (Supplementary Fig. S1A). The callus induction conditions with auxin and cytokinin had been previously defined as 28.5 μM IAA, 2.3 μM 2,4-D and 1.3 μM kinetin with Murashige and Skoog (MS) medium (Gelvin 2006). Root tip section cells were first induced to callus after 50 d treatment in callus induction medium. Using the conditions for auxin/cytokinin-based callus induction (Gelvin 2006), callus could not be induced from approximately 30% of root tips with fresh weights <5 mg, and only a few calli grew to >50 mg (Fig. 2A, C). Callus formation from root tip sections was induced with very high efficiency with 15 μM FPX. Under this FPX treatment, approximately 70% of callus from root tips weighed >50 mg after 50 d treatment (Fig. 2B, C). This pattern of high callus formation activity of FPX was also observed in root middle sections and basal rosette leaf sections (Supplementary Fig. S1). With all three root sections, 15 μM FPX was more effective for inducing callus formation than the higher concentration of 45 μM. In contrast, in basal rosette leaf sections, 45 μM FPX induced callus formation with a higher efficiency than 15 μM FPX. Fig. 2 View largeDownload slide Callus formation activity of FPX and auxin/cytokinin for Arabidopsis root explants on MS medium. Root tip explants from 10-day-old light-grown seedlings were incubated for 50 d in the light on medium containing IAA, 2,4-D and kinetin (A) or 15 µM FPX (B). (C) The fresh weight of each callus was divided into six groups (0–5, 5–25, 25–50, 50–75, 75–100 and 100–150 mg) and callus number was counted in each group. The abundance ratio of each group is indicated in the graph (n ≥ 75 root tissues). Fig. 2 View largeDownload slide Callus formation activity of FPX and auxin/cytokinin for Arabidopsis root explants on MS medium. Root tip explants from 10-day-old light-grown seedlings were incubated for 50 d in the light on medium containing IAA, 2,4-D and kinetin (A) or 15 µM FPX (B). (C) The fresh weight of each callus was divided into six groups (0–5, 5–25, 25–50, 50–75, 75–100 and 100–150 mg) and callus number was counted in each group. The abundance ratio of each group is indicated in the graph (n ≥ 75 root tissues). Valvekens et al. (1988) identified another callus induction condition using 2.3 μM (0.5 mg l–1) 2,4-D and 0.23 μM (0.05 mg l–1) kinetin in Gamborg’s B5 medium, which has been used in several studies for understanding callus formation mechanisms (Atta et al. 2009, Sugimoto et al. 2010). In Gamborg’s B5 medium, calli could also be induced from root tip sections using 15 μM FPX for 34 d; despite being higher than when using 45 μM FPX, the other phytohormones showed a higher efficiency at inducing calli (Supplementary Fig. S2). FPX-induced calli display shoot regeneration activity Plant callus cells possess the ability to regenerate shoots—a phenomenon that is controlled by the phytohormones auxin and cytokinin. We compared the shoot regeneration activity of FPX-induced callus with that of already established auxin/cytokinin-induced callus in Arabidopsis, using root explants. In plant totipotency, the genetic formatting of plant cells—termed callus cell embryonalization—is very important (Sugimoto et al. 2011). Totipotency has been observed in phytohormone-induced calli, with subsequent shoot regeneration being triggered by a high concentration of cytokinin and a low concentration of auxin (Evans et al. 1984, Sato 2013). When we excised Arabidopsis root explants cultured on MS medium without phytohormones for 2 weeks, they never underwent shoot regeneration, not even when grown in shoot induction medium containing 0.86 μM (0.15 mg l–1) IAA and 12.3 μM (2.5 mg l–1) N6-Δ2-isopentenyladenine (2iPA), which we had identified as an optimized shoot induction condition for Arabidopsis (Fig. 3A;Supplementary Fig. S3). In contrast, when excised Arabidopsis root explants cultured on MS medium containing 15 μM FPX for 2 weeks were transferred to the same shoot-inducing medium, we observed shoot formation on almost all calli after 1 month (Fig. 3B). Fig. 3 View largeDownload slide Shoot regeneration efficiency from FPX-induced and auxin/cytokinin-induced callus on MS medium. (A) Shoot regeneration by FPX-induced callus from root explants. Root tip explants from light-grown seedlings were pre-incubated in the light on medium containing DMSO or 15 µM FPX and then transferred onto shoot-inducing medium. Photographs were taken 4 weeks after the transfer. (B, C) Root basal explants (B) and root tip explants (C) from 10-day-old light-grown seedlings were pre-incubated for 3 weeks in the light on medium containing auxin and cytokinin, or 15 µM FPX and then transferred onto shoot-inducing medium. Photographs were taken 4 weeks after the transfer. (D) Frequency of callus formation and shoot regeneration after pre-incubation on medium containing either IAA, 2,4-D and kinetin, or 15 µM FPX. Each value represents the average of four independent experiments, and bars indicate the SD (n = 25 root tissues). Fig. 3 View largeDownload slide Shoot regeneration efficiency from FPX-induced and auxin/cytokinin-induced callus on MS medium. (A) Shoot regeneration by FPX-induced callus from root explants. Root tip explants from light-grown seedlings were pre-incubated in the light on medium containing DMSO or 15 µM FPX and then transferred onto shoot-inducing medium. Photographs were taken 4 weeks after the transfer. (B, C) Root basal explants (B) and root tip explants (C) from 10-day-old light-grown seedlings were pre-incubated for 3 weeks in the light on medium containing auxin and cytokinin, or 15 µM FPX and then transferred onto shoot-inducing medium. Photographs were taken 4 weeks after the transfer. (D) Frequency of callus formation and shoot regeneration after pre-incubation on medium containing either IAA, 2,4-D and kinetin, or 15 µM FPX. Each value represents the average of four independent experiments, and bars indicate the SD (n = 25 root tissues). To compare the shoot regeneration activity of FPX- and auxin/cytokinin-induced calli, three different root sections—root tip, middle and basal—were excised from 10-day-old Arabidopsis roots. The basal root explants were cultured and induced to form calli in callus-inducing medium containing 28.5 μM IAA, 2.3 μM 2,4-D and 1.3 μM kinetin (Gelvin 2006) with MS medium for 3 weeks and then transferred to shoot-inducing medium containing 0.86 μM IAA and 12.3 μM 2iPA. Under these conditions, approximately 50% of root sections formed callus, but only 4% of calli had regenerated shoots 4 weeks after the transfer. For the FPX treatment, similar root explants were cultured and induced to form calli in the presence of 15 μM FPX for 3 weeks, which resulted in approximately 100% callus induction (Fig. 3C, E). When calli were incubated for 3 weeks before transferring to a shoot induction medium, approximately 50% of the FPX-induced calli generated shoots. The effects of FPX were even more pronounced in root tip explants: approximately 5% of root tip explants were induced to form callus in the callus induction medium containing auxin and cytokinin, and only a few shoots were regenerated from auxin/cytokinin-induced callus in shoot induction medium. In contrast, approximately 100% of calli were induced in the presence of 15 μM FPX, with approximately 95% shoot regeneration observed from FPX-induced callus in the same shoot-inducing medium (Fig. 3D, E). These results suggest that FPX can awaken the shoot formation activity of plant cells. FPX and auxin/cytokinin differentially affect the cellular organization of calli from root explants To analyze the characteristics of FPX activity in detail, cellular organization of FPX- and auxin/cytokinin-induced calli was compared under a microscope. Arabidopsis was germinated for 10 d in the light. Root middle sections were then excised and cultured on callus-inducing medium containing 28.5 μM IAA, 2.3 μM 2,4-D and 1.3 μM kinetin or 50 μM FPX for 15 d (Gelvin 2006). Auxin/cytokinin-induced root primary calli were sliced vertically into segments (Fig. 4) and revealed multiple protuberances within the surfaces of the calli. Actively dividing cell regions, i.e. regions of small and/or cytosol-rich cells, were found beneath the outer epidermal region of calli containing non-uniform cells (Fig. 4A, square brackets). In the inner regions of auxin/cytokinin-induced calli, the differentiation of vessel elements was observed (Fig. 4a, black arrow). These results suggest that cellular differentiation was also promoted within auxin/cytokinin-induced calli. In contrast, FPX-induced calli showed simple structures, which contained epidermal regions with well-expanded cells and actively dividing regions (Fig. 4B, square brackets). The actively dividing cell regions in FPX-induced calli seemed to be larger than those of auxin/cytokinin-induced calli; however, vessel element formation was not observed in FPX-induced root cells. These observations suggest that cellular organization of FPX-induced calli differs from that of auxin/cytokinin-induced calli. Fig. 4 View largeDownload slide Cellular organization of FPX- and auxin/cytokinin-induced callus. Cross-sections of callus generated from root explants of 10-day-old light-grown Arabidopsis wild-type seedlings incubated for 15 d in the light on medium containing IAA, 2,4-D and kinetin (A) or 15 µM FPX (B). Scale bars = 100 µm. Right panels show magnifications of selected regions. Arrows indicate vessel element formation. Square brackets indicate actively dividing cell zones under each condition. Fig. 4 View largeDownload slide Cellular organization of FPX- and auxin/cytokinin-induced callus. Cross-sections of callus generated from root explants of 10-day-old light-grown Arabidopsis wild-type seedlings incubated for 15 d in the light on medium containing IAA, 2,4-D and kinetin (A) or 15 µM FPX (B). Scale bars = 100 µm. Right panels show magnifications of selected regions. Arrows indicate vessel element formation. Square brackets indicate actively dividing cell zones under each condition. Expressed genes induced by FPX and auxin/cytokinin show commonality and specificity To analyze the molecular mechanism of FPX functional expression, we examined the transcriptional regulation of genes after FPX stimulation in Arabidopsis by microarray analysis. In this analysis, the expression of induced and repressed genes was compared between FPX- and auxin/cytokinin-induced calli. Four days after germination in the light, Arabidopsis seedlings were treated with 45 μM FPX for 0, 2 or 8 d. Given that genes induced early by phytohormone treatments are usually expressed several hours after treatment (Goda et al. 2008), we considered treatment for 2 or 8 d as middle or long period treatments, respectively. Four-day-old Arabidopsis seedlings under the same conditions were treated with 2.26 μM 2,4-D and 0.46 μM kinetin. Total RNA was extracted from these plant materials, purified and then subjected to GeneChip analysis using an Arabidopsis Genome ATH1 array (Supplementary Fig. S4). According to the analysis results, 11,127 and 10,248 genes were differentially expressed during culture on FPX medium and auxin/cytokinin medium for 2 or 8 d, respectively, compared with Arabidopsis seedlings before either treatment (one-way analysis of variance (ANOVA), false discovery rate (FDR) <0.05] (Supplementary Fig. S4; Supplementary Tables S1–S10). A comparison of the gene sets indicated 9,259 commonly changed genes with similar expression patterns during culture (Supplementary Fig. S6), which suggests overlapping effects of the FPX treatment and the auxin/cytokinin treatment on the transcriptome. Genes with a fold change (FC) ratio between days 0 and 2 of treatment with FPX or auxin/cytokinin >2 were identified and compared with one another. In a comparison of the 2-fold up-regulated genes (FC > 2; FDR < 0.05), 598 genes overlapped between FPX and auxin/cytokinin conditions. In contrast, the expression of 368 genes was 2-fold induced only by FPX, while 162 genes were specifically up-regulated by auxin/cytokinin (Fig. 5A). Among the 2-fold down-regulated genes, 358 genes overlapped between FPX and auxin/cytokinin conditions. The expression of 239 genes was specifically suppressed 2-fold by FPX, whereas 165 genes were only inhibited by auxin/cytokinin (Fig. 5B). Fig. 5 View largeDownload slide Transcriptome analysis of FPX-treated seedlings. (A, B) Venn diagrams of up- (A) and down-regulated (B) genes after a 2 d treatment with FPX or auxin/cytokinin. Characteristic enriched Gene Ontology (GO) terms for each group (i.e. auxin/cytokinin- or FPX-specific- and commonly regulated genes) are indicated. The letter P indicates GO terms for biological process categories. For the full list of each group, see Supplementary Tables S1–S3. FC, fold change; FDR, false discovery rate. (C) GO term enrichment of genes specifically regulated by FPX. Green, blue and pink circles represent GO terms for cellular component, biological process and molecular function categories, respectively. The horizontal axis shows −log10 FDR. The size of each circle reflects fold enrichment, defined as the ratio of the number of proteins annotated with the GO term in the test set to the number of proteins annotated with the same term in the background set, i.e. the Arabidopsis genome. GO terms shown at the vertical axis are; organelle (GO:0043226), cell part (GO:0044464), intracellular (GO:0005622), cytoplasm (GO:0005737), Golgi apparatus (GO:0005794), endoplasmic reticulum (GO:0005783), nuclear outer membrane–endoplasmic reticulum membrane network (GO:0042175), DNA repair (GO:0006281), DNA metabolic process (GO:0006259), cellular process (GO:0009987), nitrogen compound metabolic process (GO:0006807), primary metabolic process (GO:0044238), metabolic process (GO:0008152), protein metabolic process (GO:0019538), biosynthetic process (GO:0009058), localization (GO:0051179), catabolic process (GO:0009056), transport (GO:0006810), intracellular protein transport (GO:0006886), intracellular signal transduction (GO:0035556), protein transport (GO:0015031), cellular amino acid metabolic process (GO:0006520), phosphate-containing compound metabolic process (GO:0006796), response to endogenous stimulus (GO:0009719), carbohydrate metabolic process (GO:0005975), protein targeting (GO:0006605), hydrolase activity (GO:0016787), catalytic activity (GO:0003824), oxidoreductase activity (GO:0016491), transferase activity (GO:0016740), transporter activity (GO:0005215), transmembrane transporter activity (GO:0022857), pyrophosphatase activity (GO:0016462), kinase activity (GO:0016301), protein kinase activity (GO:0004672), transferase activity, transferring glycosyl groups (GO:0016757), ATPase activity, coupled to transmembrane movement of substances (GO:0042626), transaminase activity (GO:0008483). For the full list of GO terms significantly enriched for each group, see Supplementary Tables S4–S9. (D) Hierarchical clustering of 348 genes actively regulated by FPX treatment (FC > 2 after 2 or 8 d, FDR < 0.05). Expression levels are shown as log2 FC. C, GO cellular component term. Fig. 5 View largeDownload slide Transcriptome analysis of FPX-treated seedlings. (A, B) Venn diagrams of up- (A) and down-regulated (B) genes after a 2 d treatment with FPX or auxin/cytokinin. Characteristic enriched Gene Ontology (GO) terms for each group (i.e. auxin/cytokinin- or FPX-specific- and commonly regulated genes) are indicated. The letter P indicates GO terms for biological process categories. For the full list of each group, see Supplementary Tables S1–S3. FC, fold change; FDR, false discovery rate. (C) GO term enrichment of genes specifically regulated by FPX. Green, blue and pink circles represent GO terms for cellular component, biological process and molecular function categories, respectively. The horizontal axis shows −log10 FDR. The size of each circle reflects fold enrichment, defined as the ratio of the number of proteins annotated with the GO term in the test set to the number of proteins annotated with the same term in the background set, i.e. the Arabidopsis genome. GO terms shown at the vertical axis are; organelle (GO:0043226), cell part (GO:0044464), intracellular (GO:0005622), cytoplasm (GO:0005737), Golgi apparatus (GO:0005794), endoplasmic reticulum (GO:0005783), nuclear outer membrane–endoplasmic reticulum membrane network (GO:0042175), DNA repair (GO:0006281), DNA metabolic process (GO:0006259), cellular process (GO:0009987), nitrogen compound metabolic process (GO:0006807), primary metabolic process (GO:0044238), metabolic process (GO:0008152), protein metabolic process (GO:0019538), biosynthetic process (GO:0009058), localization (GO:0051179), catabolic process (GO:0009056), transport (GO:0006810), intracellular protein transport (GO:0006886), intracellular signal transduction (GO:0035556), protein transport (GO:0015031), cellular amino acid metabolic process (GO:0006520), phosphate-containing compound metabolic process (GO:0006796), response to endogenous stimulus (GO:0009719), carbohydrate metabolic process (GO:0005975), protein targeting (GO:0006605), hydrolase activity (GO:0016787), catalytic activity (GO:0003824), oxidoreductase activity (GO:0016491), transferase activity (GO:0016740), transporter activity (GO:0005215), transmembrane transporter activity (GO:0022857), pyrophosphatase activity (GO:0016462), kinase activity (GO:0016301), protein kinase activity (GO:0004672), transferase activity, transferring glycosyl groups (GO:0016757), ATPase activity, coupled to transmembrane movement of substances (GO:0042626), transaminase activity (GO:0008483). For the full list of GO terms significantly enriched for each group, see Supplementary Tables S4–S9. (D) Hierarchical clustering of 348 genes actively regulated by FPX treatment (FC > 2 after 2 or 8 d, FDR < 0.05). Expression levels are shown as log2 FC. C, GO cellular component term. These results suggest that 598 induced genes and approximately 358 suppressed genes have common functions in FPX- and auxin/cytokinin-induced callus formation. Gene Ontology (GO) term analysis revealed that the genes commonly up-regulated at early stages of treatment (FC > 2; FDR < 0.05) were enriched in categories related to ‘cell cycle’, ‘root development’ and ‘response to auxin stimulus’ (Fig. 5A; Supplementary Tables S1, S4, S7). This finding is in accordance with previous reports of callus induction by auxin treatment (Atta et al. 2009, Sugimoto et al. 2010). These results also suggest that FPX can induce auxin response, at least in part, to initiate callus formation. The finding of the FPX specifically induced 368 genes and 239 suppressed genes implies that FPX treatment may have additional effects to auxin/cytokinin treatment. Gene expression profiles obtained from the microarray data were used to identify genes potentially important for callus induction that were specifically stimulated by FPX (Fig. 5A–D; Supplementary Tables S1–S10). Gene sets specifically affected by FPX or auxin/cytokinin with significant FCs were also detected (Fig. 5A, B; Supplementary Tables S2, S3, S5, S6, S8, S9). In the FPX-specific gene set (1,868 genes; Supplementary Tables S3, S6, S9), genes annotated as related to metabolism were dominant, with the GO terms ‘catalytic activity’, ‘transferase activity’ and ‘phosphate-containing compound metabolic process’ particularly statistically significantly enriched (Fig. 5C). Metabolism-related genes were also over-represented among genes up- or down-regulated specifically by FPX after a 2 d treatment (FC > 2; FDR < 0.05; Fig. 5A, B); thus, FPX may influence cellular metabolic regulation in addition to inducing auxin response. The GO term ‘hormone stimulus’ was significantly enriched in the auxin/cytokinin-specific gene set (993 genes; Supplementary Tables S2, S5, S8) but not in the FPX-specific gene set (Supplementary Tables S3, S6). The results of hierarchical clustering analysis of 349 genes actively regulated by FPX treatment (FC > 2 after 2 or 8 d; FDR < 0.05) are shown in Fig. 5D. Two-thirds of these genes were up-regulated by FPX after 2 or 8 d of culture, and genes with the GO terms ‘extracellular region’ and ‘plasma membrane’ were over-represented (Fig. 5D; Supplementary Table S10). The list of genes is further described in detail in the Discussion. FPX promotes callus formation and shoot regeneration, and facilitates Agrobacterium transformation of monocots and tree species Prior to this study, almost no analysis of FPX activity had been performed in plants other than the model plant Arabidopsis. To determine the universality of FPX activity in different plant families, we investigated whether FPX could exert biological activity in monocot crops and tree species from several other plant families. Embryos of immature seeds have generally been used for callus formation in rice. When dry seeds of Oryza sativa ‘Nipponbare’ were sown on medium containing 45 μM FPX, callus formation was observed in the germinated seedlings after 30 d under light conditions (Fig. 6A). Callus formation in rice is induced in the embryonic region of the seed; this contrasts with Arabidopsis, where FPX-induced callus formation occurs in hypocotyl or root apical meristem regions. Fig. 6 View largeDownload slide Callus formation in rice, poplar and Brachypodium distachyon by FPX. (A) Phenotypes of 30-day-old light-grown rice on medium containing 1% DMSO, 15 µM FPX or 45 µM FPX. Right panels show magnifications of selected regions. (B) FPX-induced callus formation in poplar. Stem explants from poplar were incubated for 4 weeks on medium containing 0.4 µM NAA, 30 µM FPX or 45 µM FPX. (C) FPX-induced shoot regeneration. Stem and root explants from poplar were pre-incubated on medium containing 30 µM FPX and then incubated on shoot induction medium for 30 d. (D) Transformation of FPX-induced callus from poplar and GUS analysis of poplar callus tissue harboring 35S:GUS from Agrobacterium-mediated transformation. (E) FPX-induced callus formation on mature Brachypodium seeds. Phenotypes of Brachypodium plants 4 and 14 d after germination on medium containing DMSO, 45 µM FPX, 100 µM FPX or 2.5 mg l−1 2,4-D are shown. Arrowheads indicate callus formation. (F) FPX-induced callus formation on Brachypodium roots. Phenotypes of Brachypodium plants 21 d after germination on medium containing DMSO, 45 µM FPX or 100 µM FPX are shown. Arrowheads indicate callus formation. (G) Shoot regeneration from FPX-induced callus produced from immature seeds. Immature seeds of Brachypodium were pre-incubated on medium containing 75 µM FPX and then transferred onto regular shoot induction medium. Photographs were taken 14 d after the transfer. (H) Transformation of FPX-induced callus from Brachypodium. GUS analysis of Brachypodium callus tissue harboring BdUbi::GUS from Agrobacterium-mediated transformation. (I) Effect of FPX-induced callus from Brachypodium on transformation efficiency. Fig. 6 View largeDownload slide Callus formation in rice, poplar and Brachypodium distachyon by FPX. (A) Phenotypes of 30-day-old light-grown rice on medium containing 1% DMSO, 15 µM FPX or 45 µM FPX. Right panels show magnifications of selected regions. (B) FPX-induced callus formation in poplar. Stem explants from poplar were incubated for 4 weeks on medium containing 0.4 µM NAA, 30 µM FPX or 45 µM FPX. (C) FPX-induced shoot regeneration. Stem and root explants from poplar were pre-incubated on medium containing 30 µM FPX and then incubated on shoot induction medium for 30 d. (D) Transformation of FPX-induced callus from poplar and GUS analysis of poplar callus tissue harboring 35S:GUS from Agrobacterium-mediated transformation. (E) FPX-induced callus formation on mature Brachypodium seeds. Phenotypes of Brachypodium plants 4 and 14 d after germination on medium containing DMSO, 45 µM FPX, 100 µM FPX or 2.5 mg l−1 2,4-D are shown. Arrowheads indicate callus formation. (F) FPX-induced callus formation on Brachypodium roots. Phenotypes of Brachypodium plants 21 d after germination on medium containing DMSO, 45 µM FPX or 100 µM FPX are shown. Arrowheads indicate callus formation. (G) Shoot regeneration from FPX-induced callus produced from immature seeds. Immature seeds of Brachypodium were pre-incubated on medium containing 75 µM FPX and then transferred onto regular shoot induction medium. Photographs were taken 14 d after the transfer. (H) Transformation of FPX-induced callus from Brachypodium. GUS analysis of Brachypodium callus tissue harboring BdUbi::GUS from Agrobacterium-mediated transformation. (I) Effect of FPX-induced callus from Brachypodium on transformation efficiency. Next, we tried to analyze FPX activity in trees. Incubation of Populus tremula×tremuloides ‘T89’ stem organs on medium with 30 or 45 μM FPX resulted in callus formation after 4 weeks, whereas no visible callus was detected in the callus induction medium containing 0.4 μM naphthaleneacetic acid (NAA) (Fig. 6B) (Eriksson et al. 2000, Ohtani et al. 2011). After ≥4 weeks of incubation, FPX-induced callus formation in poplar was observed. In a subsequent step, the FPX-induced callus was transferred to shoot-inducing medium containing 0.5 μM indole-3-butyric acid and 0.8 μM benzyl adenine. Four weeks after transfer, shoot regeneration was observed from stem- and root-induced callus. In addition, poplar stem explants were infected by Agrobacterium harboring a 35S:GUS (β-glucuronidase) vector and then cultured on medium containing 30 μM FPX and antibiotics for 4 weeks. All of the obtained calli tested positive for GUS staining. FPX is thus able to induce tree callus that has shoot regeneration ability and the capacity for Agrobacterium-mediated transformation. We also analyzed the effect of FPX on the model grass plant species Brachypodium distachyon (L.) (hereafter Brachypodium). For initial analysis of Brachypodium in the presence of FPX, mature seeds were germinated, and roots explants were cultured in medium containing FPX. Four days after germination, 45 and 100 μM FPX induced callus from mature Brachypodium seeds (Fig. 6E). Callus formation on Brachypodium root explants was also observed upon culture with 45 and 100 μM FPX for 21 d (Fig. 6F). Embryos of immature seeds are frequently used for callus formation and shoot regeneration in monocots. We found that 75 μM FPX could induce callus formation on embryos of immature Brachypodium seeds after 14 d of culture, while Brachypodium shoots could be regenerated from the FPX-induced embryo callus after 14 d induction (Fig. 6G). Because the floral dip method is generally used for Agrobacterium-mediated transformation of Arabidopsis, we analyzed the transformation efficiency of FPX-induced callus by Agrobacterium using Brachypodium callus. Embryogenic callus induced by 75 or 100 μM FPX and 11.3 μM 2,4-D was co-cultured with Agrobacterium harboring a 35S:GUS vector for 2 d. The Agrobacterium-infected callus was cultured for 9 d and then stained with X-gluc. Analysis of 50 calli on each of three plates indicated that FPX-induced embryogenic callus tended to have a high transformation efficiency (Fig. 6H). FPX induces callus formation in vegetable plants To analyze the diversity of FPX-induced activity in callus formation, we applied FPX to several plant species generally consumed as vegetables and belonging to different families. Glycine max L. ‘Tsurunoko’ (soybean), a member of the family Fabaceae, was bred in the Hokkaido area of Japan. When mature seeds of this soybean cultivar were germinated on medium containing FPX for 22 d, callus induction was observed over a wide range of FPX concentrations: from 15 to 105 μM (Fig. 7). Solanum lycopersicum L. ‘Micro-Tom’ is a dwarf tomato variety that has been extensively used as a model experimental plant in the family Solanaceae (Saito et al. 2011). Callus was induced from mature germinated Micro-Tom seeds using 15 μM FPX for 32 d. In the case of this tomato variety, higher concentrations of FPX may have inhibited seedling growth, as callus formation was not observed at concentrations of 45–105 μM (Fig. 7). Cucumis sativus L. ‘Natsusuzumi’ (cucumber) belongs to Cucurbitaceae, a major vegetable family along with Fabaceae, Solanaceae and Brassicaceae. When mature seeds of cucumber were germinated in MS medium containing FPX for 35 d, callus induction was observed over a wide range of FPX concentrations, from 15 to 105 μM (Fig. 7). These results suggest that FPX has callus-inducing activity in many different plant families. Fig. 7 View largeDownload slide Callus formation in soybean, tomato and cucumber by FPX. Phenotypes of light-grown soybean, Micro-Tom tomato and cucumber on medium containing the indicated concentration of FPX on the indicated number of days after germination are shown. Right panels show magnifications of selected regions. Fig. 7 View largeDownload slide Callus formation in soybean, tomato and cucumber by FPX. Phenotypes of light-grown soybean, Micro-Tom tomato and cucumber on medium containing the indicated concentration of FPX on the indicated number of days after germination are shown. Right panels show magnifications of selected regions. Discussion FPX is a psychoactive drug used to treat senile dementia (Bompani and Scali 1986) and memory impairment (Genkova-Papasova and Lazarova-Bakurova 1988) in mammals, though the detailed mode of action and the target protein(s) of FPX have not yet been identified. FPX was initially identified in plants in a screen for compounds promoting or inhibiting hypocotyl elongation of Arabidopsis seedlings, but the effect of FPX on plants has not been fully investigated. Here, we showed that FPX is able to induce callus formation and shoot regeneration in plant seedlings and leaf and root tissues. These activities suggest that FPX is a chemical inducer for callus and shoot formation in plants. Extensive previous research has revealed that plant callus cells are induced by auxin and cytokinin from already developed leaf, stem and root cells. Callus formation has an important role in the plant life cycle, as plant callus cells regain the totipotency to develop multiple plant organs that differ from the original cells of the callus. Callus formation has been observed on insect-infected, bacterium-infected and wounded plant organs (Ikeuchi et al. 2013). Although exogenous application of auxin and cytokinin does not occur naturally, research on plant cell totipotency using this approach has helped reveal previously unknown and interesting molecular mechanisms of plant development. Recent mammalian research has suggested that induced pluripotent stem cells (iPS cells) can be developed by the transformation of four mammalian genes. This finding has had a huge impact both in the field of basic animal biology as well as in applied medical science (Takahashi and Yamanaka 2016). The scientific concept of mammalian iPS cells is similar to that of plant totipotent callus cells. The discovery of the chemical inducer FPX may add new knowledge to plant developmental research and applied plant science. Treatment with FPX resulted in high callus formation efficiency in Arabidopsis leaf and root sections. In shoot-inducing medium, FPX-induced callus exhibited a high regeneration efficiency. To analyze the activity of FPX in callus formation, we examined the organization of callus cells induced from root explants by FPX and auxin/cytokinin under an optical microscope. Recent studies have attempted to reveal the molecular mechanism of callus formation induced by combined auxin and cytokinin using Arabidopsis developmental gene markers and mutants (Atta et al. 2009, Sugimoto et al. 2010). In intact plants, lateral roots were induced from the main roots in response to the auxin-induced signaling system. Similar to lateral root formation, callus formation from roots was initiated in the initial stages of pericycle cell division. These lateral root apical meristem (LRM)-like structures induced by auxin and cytokinin were termed LRM-like protuberances. Several marker genes expressed in lateral root formation were also induced in the LRM-like protuberances and observed in the resulting callus cells. After 5 d culture on auxin/cytokinin medium, the protuberances increased in size. During auxin/cytokinin treatment, auxin induced the reactivation of pericycle cells and the initiation of LRM-like structures, whereas cytokinin induced re-entry of pericycle cells into the cell cycle. This combination caused high cell division activity in the outer epidermal region (Atta et al. 2009). Similarly active cell division was observed in the outer epidermal region of roots treated with auxin and cytokinin in our study (Fig. 4). We observed that actively dividing cell regions in FPX-induced callus from root explants seemed to be larger than those of auxin/cytokinin-induced callus. The vessel element formation that was observed in auxin/cytokinin-induced callus was not observed in FPX-induced root cells. These results suggest that the cellular organization of the actively dividing cell region in FPX-induced callus and auxin/cytokinin-induced callus was different. Although the initiation cell of FPX-stimulated callus induction has not yet been identified, root development-related genes were highly expressed by both auxin/cytokinin and FPX induction (Fig. 5; Supplementary Figs. S1–S6). We additionally analyzed the expression of lateral root formation genes (Orman-Ligeza et al. 2013). ARF7 and LBD29 expression was induced by FPX and auxin/cytokinin at the same levels. ARF19, LBD16, LBD17 and LBD18 expression was induced by FPX and auxin/cytokinin, but the expression strengths of these genes in the presence of auxin/cytokinin were higher than those under FPX induction (Supplementary Fig. S5). These results suggest that the initiation of callus formation under FPX treatment may begin with LRM formation, while the activity of FPX in the induction of lateral root formation genes may be weaker than that of auxin. Auxin strongly induced LRM-like protuberances in root epidermal regions of calli, and differentiation to form vessel elements may have started in the inner region of calli. In FPX-induced callus, continuous cell division may continue to the inner region such that differentiation to vessel element occurred late. Future observations of FPX-induced callus initiation cells and an expression analysis of marker genes related to root and shoot meristem regulation should reveal the molecular mechanism underlying FPX function. Microarray analysis allowed us to examine the similarity and specificity of genes regulated in FPX- and auxin/cytokinin-induced calli. Most of the 598 genes that were commonly and significantly induced by FPX and auxin/cytokinin (Fig. 5A; Supplementary Table S4) were related to the cell cycle, root development and auxin response. These 598 genes suggested commonality between FPX activity and auxin/cytokinin activity. Other auxin response genes were highly induced only in auxin/cytokinin-stimulated callus (Supplementary Fig. S5). These results suggest that FPX-induced callus formation is less dependent on auxin signaling than is auxin/cytokinin-induced callus formation. In the FPX-specific gene set (1,868 genes; Supplementary Tables S6, S10), annotated genes related to metabolism were highly abundant. The GO terms ‘metabolic process’ in the biological process category and ‘catalytic activity’, ‘transferase activity’ and ‘phosphate-containing compound metabolic process’ in the molecular function category were particularly statistically significantly enriched (Fig. 5C; Supplementary Table S6). Among the 1,868 genes specifically induced by FPX, 359 genes that were 2-fold up-regulated (FC > 2, FDR < 0.05) by FPX were selected and subjected to GO annotation. Five types of metabolism-related genes (highlighted in orange in Supplementary Table S10) were identified, namely genes related to sugar metabolism, lipid metabolism, amino acid metabolism, glutathione transfer and nicotiamine metabolism. FPX function might suggest unknown metabolic signaling that may be involved in plant cell division activity through direct or auxiliary effects. According to the results of the hierarchical clustering analysis of 368 genes actively regulated by FPX treatment (FC > 2 after 2 or 8 d; FDR < 0.05), two-thirds of these genes were up-regulated by FPX after 2 or 8 d of culture, among which genes associated with the GO terms ‘extracellular region’ and ‘plasma membrane’ were over-represented (Fig. 5D; highlighted in green in Supplementary Table S10). In particular, groups 1–3 contained several plasma membrane-associated protein kinases related to the regulation of cell division and expansion, such as NIMA-RELATED KINASE 5 (Motose et al. 2011); membrane-bound proteins such as the transporter YELLOW STRIPE LIKE 7 (YSL7) (Hofstetter et al. 2013) and phosphate transporter PHO1 (Hamburger et al. 2002); and AMINOPHOSPHOLIPID ATPASE10 (ALA10), an ATPase flippase that internalizes exogenous phospholipids across the plasma membrane (Poulsen et al. 2015). FPX may therefore also activate plasma membrane-associated cellular signaling. FPX exhibited high activity in callus formation, and high shoot regeneration activity was observed from FPX-induced callus. Finally, we also determined that FPX-induced callus can be transformed by Agrobacterium. Callus formation, shoot regeneration and Agrobacterium infection are three important steps in the plant transformation technique in plant biotechnology. Our results suggest that FPX can be used in these three steps of plant transformation as an additional and complementary chemical inducer with the already developed auxin/cytokinin system. In 1996, cultivated transgenic crops and vegetables occupied 1.7 Mha of land. Twenty years later, in 2016, the area under transgenic cultivation had expanded approximately 110-fold, to 1,800 Mha worldwide (ISAAA 2016). Despite the popularity of genetically modified organisms, many useful plant species have not been subjected to genetic transformation because of overwhelming difficulties due to the insufficient activity of available phytohormones to induce plant callus formation and regenerate shoots or roots from callus. Genome editing has recently been applied in the field of plant science. This technique can be exploited for the transformation of plant callus by using Agrobacterium harboring genome-editing enzyme-encoded vectors. The use of FPX would increase the number of plant species amenable to genetic transformation and genome editing. Materials and Methods Plant materials and growth conditions Arabidopsis thaliana ecotype Columbia (Col-0) seeds were germinated on half-strength MS (1/2 MS) medium (Duchefa) containing 0.8% phytoagar (Duchefa) and 1.5% sucrose. Arabidopsis plants were grown at 22°C under white light using a 16 h light/8 h dark cycle to simulate long-day conditions. Nipponbare rice, Tsurunoko soybean, Micro-Tom tomato and Natsusuzumi cucumber plants were grown under a 16 h light/8 h dark cycle at 25°C. Chemical screening and growth conditions A total of 24,275 compounds from a chemical library in the RIKEN Natural Products Depository were screened for hypocotyl phenotypes at a final concentration of 50 µM in 2% dimethylsulfoxide (DMSO). Five Arabidopsis seeds were sown in individual wells of 96-well plates containing 1/2 MS medium supplemented with 0.5% phytoagar, 1.5% sucrose and one of the tested compounds. Following treatment for 2 d at 4°C to induce germination, seeds were incubated in a growth chamber in the dark at 22°C. Arabidopsis callus formation analyses on MS medium Ten days after germination, Arabidopsis rosette leaves were cut into explants having mid-section widths of 2–3 mm, and roots (excluding 1 cm at each end) were subdivided into three explants (Supplementary Fig. S1). These explants were placed onto 1/2 MS medium containing either 28.5 µM IAA, 2.3 µM 2,4-D and 1.3 µM kinetin, or 15 or 45 µM FPX solidified with 0.7% agarose and sucrose. After 50 d of incubation, the explants were weighed. Arabidopsis callus formation analyses on Gamborg’s B5 medium Seven days after germination, Arabidopsis root tips (Supplementary Fig. S1A) were placed onto Gamborg’s B5 medium (Wakon) with 20 g l−1 glucose, 0.5 g l−1 MES and 1 ml l−1 Gamborg’s vitamin solution (Sigma-Aldrich) solidified with 0.8% phytoagar containing the indicated chemicals. After 34 d of incubation, the explants were weighed. Arabidopsis shoot regeneration assays Root explants were excised from seedlings 10 d after germination. The roots were cut into three pieces as shown in Supplementary Fig. S1. Explants were pre-cultured on 1/2 MS medium containing either 28.5 µM IAA, 2.3 µM 2,4-D and 1.3 µM kinetin, or 15 µM FPX (agarose and sucrose) for 3 weeks and then transferred to shoot induction medium (1/2 MS medium containing 0.85 µM IAA and 7.4 µM 2iPA) to induce shoots. Histological analysis Sterilized wild-type Arabidopsis seeds were sown on plates containing solid MS medium supplemented with 1% sucrose, 5 mg l−1 nicotinic acid, 0.5 mg l−1 pyridoxine, 3 mg l−1 thiamine-HCl and 7.5 g Bacto Agar. Ten days after germination, roots were subdivided into three explants. These explants were placed onto MS medium containing 2% glucose, 0.5 mg l−1 nicotinic acid, 0.5 mg l−1 pyridoxine, 0.5 mg l−1 thiamine-HCl, 0.05 M MES, 100 mg l−1 myoinositol and 7.5 g l−1 Bacto Agar supplemented with either 5 mg l−1 IAA, 0.5 mg/ml−1 2,4-D and 0.3 mg ml−1 kinetin or with 50 µM FPX for 15 d. The induced calli were processed for histological analysis according to the method of Wang et al. (2015) with minor modifications. Cross-sections were made from calli after fixation for a few days at 4°C in 45% ethanol/5% formaldehyde/5% acetic acid in 50 mM sodium phosphate buffer (pH 7.2), dehydration through graded ethanol and t-butanol series, and embedding in Paraplast Plus (McCormick Scientific). Microtome sections (10 µm thick) were mounted on MAS-coated glass slides (Matsunami Glass). The sections were deparaffinized in xylene, hydrated through a graded ethanol to distilled water series, and stained with toluidine blue O. GeneChip analysis of FPX-treated seedlings Four-day-old seedlings grown on the MS medium were transferred to MS medium containing 45 µM FPX (FPX plate), or 0.5 mg l−1 2,4-D and 0.1 mg l−1 kinetin (auxin/cytokinin plate). Twenty seedlings cultured for 0, 2 and 8 d were collected and frozen with liquid nitrogen immediately, and stored at −80°C. Total RNAs were isolated from the stored samples using the RNeasy Plant Mini Kit (Qiagen). Microarray analysis was performed using the GeneChip® Arabidopsis Genome ATH1 Array (Affymetrix) on three independent biological replicates, according to Ohtani et al. (2011) and Song et al. (2016). Subsequent procedures of quality control, statistical analysis and filtering were carried out using GeneSpring GX software v13.0.1 (Agilent Technologies). P-values were calculated for each probe by one-way ANOVA (n = 3), for differences among 0, 2 and 8 d treatments with FPX or auxin/cytokinin. The Benjamini–Hochberg FDR method was applied for controlling false positives, and a corrected P-value cut-off of 0.05 was used to select the regulated genes with the lowest FDR. FC values were also computed by GeneSpring GX to select the probes up- or down-regulated by >2-fold after 2 d treatment (Fig. 5A, B and D). For hierarchical clustering analysis, the Cluster 3.0 software was used with Euclidean distance measurement method (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm). GO term analysis was performed via the website of the Gene Ontology Consortium (http://geneontology.org/page/download-ontology). Microarray data presented in this study were submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) and can be retrieved via accession number GSE116939. Analysis of callus formation, shoot regeneration and transformation in poplar T89 poplar plants were used for this analysis. To induce callus, stem segments from poplar were pre-incubated with Agrobacterium containing a 35S:GUS vector and then incubated for 4 weeks on 1/2 MS medium supplemented with hygromycin and containing 0.4 µM NAA, 30 µM FPX or 45 µM FPX. To induce shoot regeneration, stem or root explants were first cultured on 1/2 MS medium containing 30 µM FPX to induce callus and then transferred to shoot induction medium (0.5 µM indole-3-butyric acid and 0.8 µM benzyl adenine) to induce shoots. Histochemical GUS staining was performed on callus using 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid at 37°C after pre-treatment in 90% (v/v) acetone at 4°C. GUS-stained tissue was cleared by incubation in 70% (v/v) ethanol. Analysis of callus formation, shoot regeneration and transformation in Brachypodium Plants of B. distachyon line Bd21 (USDA National Plant Germplasm System) were used as the wild type. The method used for seed sterilization has been previously described (Himuro et al. 2014). To induce callus, mature seeds and roots of Brachypodium were incubated on MS medium containing DMSO, FPX or 2,4-D. To induce shoot regeneration, immature seeds were pre-cultured on MS medium containing 75 µM FPX to induce callus and then transferred to shoot induction medium containing 0.2 mg l−1 kinetin to induce shoots. Histochemical GUS staining was performed on callus using 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid at 37°C after pre-treatment in 90% (v/v) acetone at 4°C. GUS-stained tissue was cleared by incubation in 70% (v/v) ethanol. Quantitative real-time PCR The methods for total RNA isolation, cDNA synthesis and real-time PCR have been previously described (Yamagami et al. 2017). The sequences of the gene-specific primers for real-time PCR were as follows: for ARF7, 5'-GGAGTTCGTCGGTATATGGG-3' and 5'-GAAACTCGACTGGGCCTATC-3'; for ARF19, 5'-GGCTCACAATGGCGTAATC-3' and 5'-GGAGTTATGACGGGTTCGAT-3'; for LBD16, 5'-AGCTCGGAAAGTACCAACCA-3' and 5'-CGAGACCGGATTGTTAGGG-3'; for LBD17, 5'-CATCATGACGTCGTGCTACC-3' and 5'-TTCGCTGCAGCCACTAGAG-3'; for LBD18, 5'-AGTGTGTGCCGGGATG-3' and 5'-CTCCGAACACTTTATGCACC-3'; for LBD29, 5'-GCAGCCATTCACAAGGTC-3' and 5'-AGCCATAGATGGGATCTTGA-3'; and for ACT2, 5'-CGCCATCCAAGCTGTTCTC-3' and 5'-TCACGTCCAGCAAGGTCAAG-3'. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Japan Science and Technology Agency [grants from CREST to T.N. and T.A.]; NARO Bio-oriented Technology Research Advancement [BRAIN; to T.N. and T.A.]; and the Cabinet Office, Government of Japan [Cross-ministerial Strategic Innovation Promotion Program (SIP; to S.H.)]. Acknowledgments We thank K. Saeki, Y. Suzuki and K. Maekawa for their technical assistance. Tomato (Micro-Tom) seed was provided by University of Tsukuba, gene research center, through the National Bio-Resource Project of the AMED, Japan. Disclosures The authors have no conflicts of interest to declare. References Atta R. , Laurens L. , Boucheron-Dubuisson E. , Guivarc'h A. , Carnero E. , Giraudat-Pautot V. ( 2009 ) Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro . Plant J . 57 : 626 – 644 . Google Scholar CrossRef Search ADS PubMed Bompani R. , Scali G. 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Genetics 199 : 95 – 104 . Google Scholar CrossRef Search ADS PubMed Yamagami A. , Saito C. , Nakazawa M. , Fujioka S. , Uemura T. , Matsui M. , et al. ( 2017 ) Evolutionarily conserved BIL4 suppresses the degradation of brassinosteroid receptor BRI1 and regulates cell elongation . Sci. Rep. 7 : 5739 . Google Scholar CrossRef Search ADS PubMed Yazaki K. , Arimura G.I. , Ohnishi T. ( 2017 ) ‘Hidden’ terpenoids in plants: their biosynthesis, localization and ecological roles . Plant Cell Physiol. 58 : 1615 – 1621 . Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations FC fold change FDR false discovery rate FPX fipexide GO Gene Ontology GUS β-glucuronidase 2iPA N6-Δ2-isopentenyladenine LRM lateral root apical meristem MS Murashige and Skoog NAA naphthaleneacetic acid © 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 This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

FPX is a Novel Chemical Inducer that Promotes Callus Formation and Shoot Regeneration in Plants

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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
DOI
10.1093/pcp/pcy139
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30053249
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Abstract

Abstract Auxin and cytokinin control callus formation from developed plant organs as well as shoot regeneration from callus. Dedifferentiation and regeneration of plant cells by auxin and cytokinin stimulation are considered to be caused by the reprogramming of callus cells, but this hypothesis is still argued to this day. Although an elucidation of the regulatory mechanisms of callus formation and shoot regeneration has helped advance plant biotechnology research, many plant species are intractable to transformation because of difficulties with callus formation. In this study, we identified fipexide (FPX) as a useful regulatory compound through a chemical biology-based screening. FPX was shown to act as a chemical inducer in callus formation, shoot regeneration and Agrobacterium infection. With regards to morphology, the cellular organization of FPX-induced calli differed from those produced under auxin/cytokinin conditions. Microarray analysis revealed that the expression of approximately 971 genes was up-regulated 2-fold after a 2 d FPX treatment compared with non-treated plants. Among these 971 genes, 598 genes were also induced by auxin/cytokinin, whereas 373 genes were specifically expressed upon FPX treatment only. FPX can promote callus formations in rice, poplar, soybean, tomato and cucumber, and thus can be considered a useful tool for revealing the mechanisms of plant development and for use in plant transformation technologies. Introduction The discovery of novel bioactive compounds has proven useful in biological research and applied bioscience. Within the plant science field, several types of phytohormones are well-known examples of useful bioactive compounds. Auxin was the first phytohormone to be identified by plant physiologists. In 1882, Charles Darwin predicted the existence of such phytohormone action by proposing that a compound transported from the shoot meristem to the hypocotyl after the perception of light was responsible for phototropism in plants (Darwin and Darwin 1880). The first auxin to be identified—IAA—was initially detected as a metabolite of human urine (Kögl et al. 1934), and then later identified in immature seeds of Zea mays when IAA was confirmed as a natural bioactive compound in higher plants (Haagen-Smit et al. 1946). Following the discovery of auxin, Skoog and Miller (1957) identified a compound that induced plant calli in the presence of IAA. Plant calli are unorganized, amorphous cell masses that are naturally present in plant tissues subjected to stresses such as wounding and pathogen infection. A compound inducing callus in the presence of IAA was identified as a product of DNA degradation and designated the synthetic cytokinin, kinetin (Miller et al. 1955). Naturally occurring cytokinins were subsequently discovered in higher plants, namely zeatin, which was identified from immature seeds of Z. mays in 1963 (Letham 1963), and dihydrozeatin, which was detected in immature seeds of lupin in 1967 (Koshimizu et al. 1968). Since its discovery, auxin has been revealed to function in the regulation of processes such as embryo development, germination, organ growth, flowering, phototropism and gravitropism. Cytokinin is a known regulator of organ growth, lateral bud formation, senescence, nutrition response and other activities (Davies 2010). In a study by Skoog and Miller (1957), the combination of auxin and cytokinin triggered callus production, from many kinds of plant organs. These callus cells divided and elongated on auxin/cytokinin medium, suggesting that auxin and cytokinin regulate and generally maintain plant cell division and elongation. Furthermore, the callus cells induced from ‘developed’ leaf or root cells had been reprogrammed to plant stem cell-like tissue possessing totipotency. The totipotent callus cells could newly generate shoot organs, including plant stems and leaves, in response to cytokinin treatment; they were also able to generate root organs when exposed to auxin. The combination of auxin and cytokinin could freely regulate differentiation, dedifferentiation and the regeneration of plant cells (Krikorian 1995). The use of auxin and cytokinin in plant biotechnology research and related applied sciences has been long established and continues to advance. The first application was with the growth of cultured plant cells in liquid media or on solid agarose medium. The improvement of regulatory techniques and the search for new plant species producing compounds beneficial to humans have also taken place over a long period. Some of the useful compounds consequently discovered include medicines, such as the anti-cancer drug paclitaxel, the anti-microbial berberine and the neural inhibitor scopolamine, and dyes such as shikonin (Evans et al. 1984, Sato 2013, Yazaki et al. 2017). Another important application of auxin/cytokinin-induced plant callus formation is in the stable genomic transformation of plants (Horsch et al. 1984) (De Block et al. 1984). Since their development, Agrobacterium infection or particle gun bombardment of plant callus have been the most popular transformation techniques. In the first step of Agrobacterium-mediated plant transformation, callus from plant leaves or embryos of immature seeds is induced by auxin and cytokinin and then infected by Agrobacterium harboring a Ti-plasmid-based transformation vector. Active cell division in the callus induced by auxin and cytokinin is important for the adequate infection activity of Agrobacterium. A transfected callus cell was induced to shoot and root by suitable concentrations of auxin and cytokinin. In this way, transformation of the whole plant is accomplished (Dunwell and Wetten 2012). As detailed in this present study, our phytochemical research laboratory recently discovered that the compound fipexide (FPX) can induce callus formation from plant seedlings, leaf and root tissues, and showed activities in several callus formation steps. Finally, using cell and molecular biological analyses, we investigated the molecular mechanism(s) underlying the activity of FPX as a chemical inducer. Results FPX induces calli on apical regions of Arabidopsis seedling shoots and roots To identify novel compounds useful for the regulation of plant development, we screened the RIKEN NPDepo chemical library (Kato et al. 2012) for compounds promoting or inhibiting hypocotyl elongation of Arabidopsis seedlings grown in the dark for 7 d. In these trials, FPX was first identified for its ability to inhibit hypocotyl elongation (Fig. 1A). To observe the effect of FPX on hypocotyls under long-term light treatment, we continued growing Arabidopsis seedlings on 15 or 45 μM FPX medium. After 2 weeks, we noted an unusual expansion of shoot and root apex regions. After 3 weeks, we observed callus formation around the shoot apical meristem region of Arabidopsis seedlings grown on 15 μM FPX medium and around the root apical meristem region of seedlings on 45 μM FPX medium (Fig. 1B). After 6 weeks, the calli were still enlarging. Fig. 1 View largeDownload slide FPX induces callus formation in Arabidopsis seedlings. (A) Chemical structure of FPX. (B) Phenotypes of light-grown Arabidopsis wild-type plants 8, 15 and 22 d, and 6 weeks after germination on medium containing 0, 15 or 45 µM FPX. Arrowheads indicate callus formation. Scale bars = 2 mm. Fig. 1 View largeDownload slide FPX induces callus formation in Arabidopsis seedlings. (A) Chemical structure of FPX. (B) Phenotypes of light-grown Arabidopsis wild-type plants 8, 15 and 22 d, and 6 weeks after germination on medium containing 0, 15 or 45 µM FPX. Arrowheads indicate callus formation. Scale bars = 2 mm. FPX exhibits callus formation activities We next compared the callus formation activity of FPX in Arabidopsis with that of previously established auxin/cytokinin conditions (Skoog and Miller 1957). Specifically, we analyzed the effects of FPX activity on callus formation from 10-day-old Arabidopsis roots that were divided into 1 cm sections corresponding to the root tip, middle and basal regions. Basal rosette leaf sections of 2–3 mm were also cut; all four sections were incubated and analyzed (Supplementary Fig. S1A). The callus induction conditions with auxin and cytokinin had been previously defined as 28.5 μM IAA, 2.3 μM 2,4-D and 1.3 μM kinetin with Murashige and Skoog (MS) medium (Gelvin 2006). Root tip section cells were first induced to callus after 50 d treatment in callus induction medium. Using the conditions for auxin/cytokinin-based callus induction (Gelvin 2006), callus could not be induced from approximately 30% of root tips with fresh weights <5 mg, and only a few calli grew to >50 mg (Fig. 2A, C). Callus formation from root tip sections was induced with very high efficiency with 15 μM FPX. Under this FPX treatment, approximately 70% of callus from root tips weighed >50 mg after 50 d treatment (Fig. 2B, C). This pattern of high callus formation activity of FPX was also observed in root middle sections and basal rosette leaf sections (Supplementary Fig. S1). With all three root sections, 15 μM FPX was more effective for inducing callus formation than the higher concentration of 45 μM. In contrast, in basal rosette leaf sections, 45 μM FPX induced callus formation with a higher efficiency than 15 μM FPX. Fig. 2 View largeDownload slide Callus formation activity of FPX and auxin/cytokinin for Arabidopsis root explants on MS medium. Root tip explants from 10-day-old light-grown seedlings were incubated for 50 d in the light on medium containing IAA, 2,4-D and kinetin (A) or 15 µM FPX (B). (C) The fresh weight of each callus was divided into six groups (0–5, 5–25, 25–50, 50–75, 75–100 and 100–150 mg) and callus number was counted in each group. The abundance ratio of each group is indicated in the graph (n ≥ 75 root tissues). Fig. 2 View largeDownload slide Callus formation activity of FPX and auxin/cytokinin for Arabidopsis root explants on MS medium. Root tip explants from 10-day-old light-grown seedlings were incubated for 50 d in the light on medium containing IAA, 2,4-D and kinetin (A) or 15 µM FPX (B). (C) The fresh weight of each callus was divided into six groups (0–5, 5–25, 25–50, 50–75, 75–100 and 100–150 mg) and callus number was counted in each group. The abundance ratio of each group is indicated in the graph (n ≥ 75 root tissues). Valvekens et al. (1988) identified another callus induction condition using 2.3 μM (0.5 mg l–1) 2,4-D and 0.23 μM (0.05 mg l–1) kinetin in Gamborg’s B5 medium, which has been used in several studies for understanding callus formation mechanisms (Atta et al. 2009, Sugimoto et al. 2010). In Gamborg’s B5 medium, calli could also be induced from root tip sections using 15 μM FPX for 34 d; despite being higher than when using 45 μM FPX, the other phytohormones showed a higher efficiency at inducing calli (Supplementary Fig. S2). FPX-induced calli display shoot regeneration activity Plant callus cells possess the ability to regenerate shoots—a phenomenon that is controlled by the phytohormones auxin and cytokinin. We compared the shoot regeneration activity of FPX-induced callus with that of already established auxin/cytokinin-induced callus in Arabidopsis, using root explants. In plant totipotency, the genetic formatting of plant cells—termed callus cell embryonalization—is very important (Sugimoto et al. 2011). Totipotency has been observed in phytohormone-induced calli, with subsequent shoot regeneration being triggered by a high concentration of cytokinin and a low concentration of auxin (Evans et al. 1984, Sato 2013). When we excised Arabidopsis root explants cultured on MS medium without phytohormones for 2 weeks, they never underwent shoot regeneration, not even when grown in shoot induction medium containing 0.86 μM (0.15 mg l–1) IAA and 12.3 μM (2.5 mg l–1) N6-Δ2-isopentenyladenine (2iPA), which we had identified as an optimized shoot induction condition for Arabidopsis (Fig. 3A;Supplementary Fig. S3). In contrast, when excised Arabidopsis root explants cultured on MS medium containing 15 μM FPX for 2 weeks were transferred to the same shoot-inducing medium, we observed shoot formation on almost all calli after 1 month (Fig. 3B). Fig. 3 View largeDownload slide Shoot regeneration efficiency from FPX-induced and auxin/cytokinin-induced callus on MS medium. (A) Shoot regeneration by FPX-induced callus from root explants. Root tip explants from light-grown seedlings were pre-incubated in the light on medium containing DMSO or 15 µM FPX and then transferred onto shoot-inducing medium. Photographs were taken 4 weeks after the transfer. (B, C) Root basal explants (B) and root tip explants (C) from 10-day-old light-grown seedlings were pre-incubated for 3 weeks in the light on medium containing auxin and cytokinin, or 15 µM FPX and then transferred onto shoot-inducing medium. Photographs were taken 4 weeks after the transfer. (D) Frequency of callus formation and shoot regeneration after pre-incubation on medium containing either IAA, 2,4-D and kinetin, or 15 µM FPX. Each value represents the average of four independent experiments, and bars indicate the SD (n = 25 root tissues). Fig. 3 View largeDownload slide Shoot regeneration efficiency from FPX-induced and auxin/cytokinin-induced callus on MS medium. (A) Shoot regeneration by FPX-induced callus from root explants. Root tip explants from light-grown seedlings were pre-incubated in the light on medium containing DMSO or 15 µM FPX and then transferred onto shoot-inducing medium. Photographs were taken 4 weeks after the transfer. (B, C) Root basal explants (B) and root tip explants (C) from 10-day-old light-grown seedlings were pre-incubated for 3 weeks in the light on medium containing auxin and cytokinin, or 15 µM FPX and then transferred onto shoot-inducing medium. Photographs were taken 4 weeks after the transfer. (D) Frequency of callus formation and shoot regeneration after pre-incubation on medium containing either IAA, 2,4-D and kinetin, or 15 µM FPX. Each value represents the average of four independent experiments, and bars indicate the SD (n = 25 root tissues). To compare the shoot regeneration activity of FPX- and auxin/cytokinin-induced calli, three different root sections—root tip, middle and basal—were excised from 10-day-old Arabidopsis roots. The basal root explants were cultured and induced to form calli in callus-inducing medium containing 28.5 μM IAA, 2.3 μM 2,4-D and 1.3 μM kinetin (Gelvin 2006) with MS medium for 3 weeks and then transferred to shoot-inducing medium containing 0.86 μM IAA and 12.3 μM 2iPA. Under these conditions, approximately 50% of root sections formed callus, but only 4% of calli had regenerated shoots 4 weeks after the transfer. For the FPX treatment, similar root explants were cultured and induced to form calli in the presence of 15 μM FPX for 3 weeks, which resulted in approximately 100% callus induction (Fig. 3C, E). When calli were incubated for 3 weeks before transferring to a shoot induction medium, approximately 50% of the FPX-induced calli generated shoots. The effects of FPX were even more pronounced in root tip explants: approximately 5% of root tip explants were induced to form callus in the callus induction medium containing auxin and cytokinin, and only a few shoots were regenerated from auxin/cytokinin-induced callus in shoot induction medium. In contrast, approximately 100% of calli were induced in the presence of 15 μM FPX, with approximately 95% shoot regeneration observed from FPX-induced callus in the same shoot-inducing medium (Fig. 3D, E). These results suggest that FPX can awaken the shoot formation activity of plant cells. FPX and auxin/cytokinin differentially affect the cellular organization of calli from root explants To analyze the characteristics of FPX activity in detail, cellular organization of FPX- and auxin/cytokinin-induced calli was compared under a microscope. Arabidopsis was germinated for 10 d in the light. Root middle sections were then excised and cultured on callus-inducing medium containing 28.5 μM IAA, 2.3 μM 2,4-D and 1.3 μM kinetin or 50 μM FPX for 15 d (Gelvin 2006). Auxin/cytokinin-induced root primary calli were sliced vertically into segments (Fig. 4) and revealed multiple protuberances within the surfaces of the calli. Actively dividing cell regions, i.e. regions of small and/or cytosol-rich cells, were found beneath the outer epidermal region of calli containing non-uniform cells (Fig. 4A, square brackets). In the inner regions of auxin/cytokinin-induced calli, the differentiation of vessel elements was observed (Fig. 4a, black arrow). These results suggest that cellular differentiation was also promoted within auxin/cytokinin-induced calli. In contrast, FPX-induced calli showed simple structures, which contained epidermal regions with well-expanded cells and actively dividing regions (Fig. 4B, square brackets). The actively dividing cell regions in FPX-induced calli seemed to be larger than those of auxin/cytokinin-induced calli; however, vessel element formation was not observed in FPX-induced root cells. These observations suggest that cellular organization of FPX-induced calli differs from that of auxin/cytokinin-induced calli. Fig. 4 View largeDownload slide Cellular organization of FPX- and auxin/cytokinin-induced callus. Cross-sections of callus generated from root explants of 10-day-old light-grown Arabidopsis wild-type seedlings incubated for 15 d in the light on medium containing IAA, 2,4-D and kinetin (A) or 15 µM FPX (B). Scale bars = 100 µm. Right panels show magnifications of selected regions. Arrows indicate vessel element formation. Square brackets indicate actively dividing cell zones under each condition. Fig. 4 View largeDownload slide Cellular organization of FPX- and auxin/cytokinin-induced callus. Cross-sections of callus generated from root explants of 10-day-old light-grown Arabidopsis wild-type seedlings incubated for 15 d in the light on medium containing IAA, 2,4-D and kinetin (A) or 15 µM FPX (B). Scale bars = 100 µm. Right panels show magnifications of selected regions. Arrows indicate vessel element formation. Square brackets indicate actively dividing cell zones under each condition. Expressed genes induced by FPX and auxin/cytokinin show commonality and specificity To analyze the molecular mechanism of FPX functional expression, we examined the transcriptional regulation of genes after FPX stimulation in Arabidopsis by microarray analysis. In this analysis, the expression of induced and repressed genes was compared between FPX- and auxin/cytokinin-induced calli. Four days after germination in the light, Arabidopsis seedlings were treated with 45 μM FPX for 0, 2 or 8 d. Given that genes induced early by phytohormone treatments are usually expressed several hours after treatment (Goda et al. 2008), we considered treatment for 2 or 8 d as middle or long period treatments, respectively. Four-day-old Arabidopsis seedlings under the same conditions were treated with 2.26 μM 2,4-D and 0.46 μM kinetin. Total RNA was extracted from these plant materials, purified and then subjected to GeneChip analysis using an Arabidopsis Genome ATH1 array (Supplementary Fig. S4). According to the analysis results, 11,127 and 10,248 genes were differentially expressed during culture on FPX medium and auxin/cytokinin medium for 2 or 8 d, respectively, compared with Arabidopsis seedlings before either treatment (one-way analysis of variance (ANOVA), false discovery rate (FDR) <0.05] (Supplementary Fig. S4; Supplementary Tables S1–S10). A comparison of the gene sets indicated 9,259 commonly changed genes with similar expression patterns during culture (Supplementary Fig. S6), which suggests overlapping effects of the FPX treatment and the auxin/cytokinin treatment on the transcriptome. Genes with a fold change (FC) ratio between days 0 and 2 of treatment with FPX or auxin/cytokinin >2 were identified and compared with one another. In a comparison of the 2-fold up-regulated genes (FC > 2; FDR < 0.05), 598 genes overlapped between FPX and auxin/cytokinin conditions. In contrast, the expression of 368 genes was 2-fold induced only by FPX, while 162 genes were specifically up-regulated by auxin/cytokinin (Fig. 5A). Among the 2-fold down-regulated genes, 358 genes overlapped between FPX and auxin/cytokinin conditions. The expression of 239 genes was specifically suppressed 2-fold by FPX, whereas 165 genes were only inhibited by auxin/cytokinin (Fig. 5B). Fig. 5 View largeDownload slide Transcriptome analysis of FPX-treated seedlings. (A, B) Venn diagrams of up- (A) and down-regulated (B) genes after a 2 d treatment with FPX or auxin/cytokinin. Characteristic enriched Gene Ontology (GO) terms for each group (i.e. auxin/cytokinin- or FPX-specific- and commonly regulated genes) are indicated. The letter P indicates GO terms for biological process categories. For the full list of each group, see Supplementary Tables S1–S3. FC, fold change; FDR, false discovery rate. (C) GO term enrichment of genes specifically regulated by FPX. Green, blue and pink circles represent GO terms for cellular component, biological process and molecular function categories, respectively. The horizontal axis shows −log10 FDR. The size of each circle reflects fold enrichment, defined as the ratio of the number of proteins annotated with the GO term in the test set to the number of proteins annotated with the same term in the background set, i.e. the Arabidopsis genome. GO terms shown at the vertical axis are; organelle (GO:0043226), cell part (GO:0044464), intracellular (GO:0005622), cytoplasm (GO:0005737), Golgi apparatus (GO:0005794), endoplasmic reticulum (GO:0005783), nuclear outer membrane–endoplasmic reticulum membrane network (GO:0042175), DNA repair (GO:0006281), DNA metabolic process (GO:0006259), cellular process (GO:0009987), nitrogen compound metabolic process (GO:0006807), primary metabolic process (GO:0044238), metabolic process (GO:0008152), protein metabolic process (GO:0019538), biosynthetic process (GO:0009058), localization (GO:0051179), catabolic process (GO:0009056), transport (GO:0006810), intracellular protein transport (GO:0006886), intracellular signal transduction (GO:0035556), protein transport (GO:0015031), cellular amino acid metabolic process (GO:0006520), phosphate-containing compound metabolic process (GO:0006796), response to endogenous stimulus (GO:0009719), carbohydrate metabolic process (GO:0005975), protein targeting (GO:0006605), hydrolase activity (GO:0016787), catalytic activity (GO:0003824), oxidoreductase activity (GO:0016491), transferase activity (GO:0016740), transporter activity (GO:0005215), transmembrane transporter activity (GO:0022857), pyrophosphatase activity (GO:0016462), kinase activity (GO:0016301), protein kinase activity (GO:0004672), transferase activity, transferring glycosyl groups (GO:0016757), ATPase activity, coupled to transmembrane movement of substances (GO:0042626), transaminase activity (GO:0008483). For the full list of GO terms significantly enriched for each group, see Supplementary Tables S4–S9. (D) Hierarchical clustering of 348 genes actively regulated by FPX treatment (FC > 2 after 2 or 8 d, FDR < 0.05). Expression levels are shown as log2 FC. C, GO cellular component term. Fig. 5 View largeDownload slide Transcriptome analysis of FPX-treated seedlings. (A, B) Venn diagrams of up- (A) and down-regulated (B) genes after a 2 d treatment with FPX or auxin/cytokinin. Characteristic enriched Gene Ontology (GO) terms for each group (i.e. auxin/cytokinin- or FPX-specific- and commonly regulated genes) are indicated. The letter P indicates GO terms for biological process categories. For the full list of each group, see Supplementary Tables S1–S3. FC, fold change; FDR, false discovery rate. (C) GO term enrichment of genes specifically regulated by FPX. Green, blue and pink circles represent GO terms for cellular component, biological process and molecular function categories, respectively. The horizontal axis shows −log10 FDR. The size of each circle reflects fold enrichment, defined as the ratio of the number of proteins annotated with the GO term in the test set to the number of proteins annotated with the same term in the background set, i.e. the Arabidopsis genome. GO terms shown at the vertical axis are; organelle (GO:0043226), cell part (GO:0044464), intracellular (GO:0005622), cytoplasm (GO:0005737), Golgi apparatus (GO:0005794), endoplasmic reticulum (GO:0005783), nuclear outer membrane–endoplasmic reticulum membrane network (GO:0042175), DNA repair (GO:0006281), DNA metabolic process (GO:0006259), cellular process (GO:0009987), nitrogen compound metabolic process (GO:0006807), primary metabolic process (GO:0044238), metabolic process (GO:0008152), protein metabolic process (GO:0019538), biosynthetic process (GO:0009058), localization (GO:0051179), catabolic process (GO:0009056), transport (GO:0006810), intracellular protein transport (GO:0006886), intracellular signal transduction (GO:0035556), protein transport (GO:0015031), cellular amino acid metabolic process (GO:0006520), phosphate-containing compound metabolic process (GO:0006796), response to endogenous stimulus (GO:0009719), carbohydrate metabolic process (GO:0005975), protein targeting (GO:0006605), hydrolase activity (GO:0016787), catalytic activity (GO:0003824), oxidoreductase activity (GO:0016491), transferase activity (GO:0016740), transporter activity (GO:0005215), transmembrane transporter activity (GO:0022857), pyrophosphatase activity (GO:0016462), kinase activity (GO:0016301), protein kinase activity (GO:0004672), transferase activity, transferring glycosyl groups (GO:0016757), ATPase activity, coupled to transmembrane movement of substances (GO:0042626), transaminase activity (GO:0008483). For the full list of GO terms significantly enriched for each group, see Supplementary Tables S4–S9. (D) Hierarchical clustering of 348 genes actively regulated by FPX treatment (FC > 2 after 2 or 8 d, FDR < 0.05). Expression levels are shown as log2 FC. C, GO cellular component term. These results suggest that 598 induced genes and approximately 358 suppressed genes have common functions in FPX- and auxin/cytokinin-induced callus formation. Gene Ontology (GO) term analysis revealed that the genes commonly up-regulated at early stages of treatment (FC > 2; FDR < 0.05) were enriched in categories related to ‘cell cycle’, ‘root development’ and ‘response to auxin stimulus’ (Fig. 5A; Supplementary Tables S1, S4, S7). This finding is in accordance with previous reports of callus induction by auxin treatment (Atta et al. 2009, Sugimoto et al. 2010). These results also suggest that FPX can induce auxin response, at least in part, to initiate callus formation. The finding of the FPX specifically induced 368 genes and 239 suppressed genes implies that FPX treatment may have additional effects to auxin/cytokinin treatment. Gene expression profiles obtained from the microarray data were used to identify genes potentially important for callus induction that were specifically stimulated by FPX (Fig. 5A–D; Supplementary Tables S1–S10). Gene sets specifically affected by FPX or auxin/cytokinin with significant FCs were also detected (Fig. 5A, B; Supplementary Tables S2, S3, S5, S6, S8, S9). In the FPX-specific gene set (1,868 genes; Supplementary Tables S3, S6, S9), genes annotated as related to metabolism were dominant, with the GO terms ‘catalytic activity’, ‘transferase activity’ and ‘phosphate-containing compound metabolic process’ particularly statistically significantly enriched (Fig. 5C). Metabolism-related genes were also over-represented among genes up- or down-regulated specifically by FPX after a 2 d treatment (FC > 2; FDR < 0.05; Fig. 5A, B); thus, FPX may influence cellular metabolic regulation in addition to inducing auxin response. The GO term ‘hormone stimulus’ was significantly enriched in the auxin/cytokinin-specific gene set (993 genes; Supplementary Tables S2, S5, S8) but not in the FPX-specific gene set (Supplementary Tables S3, S6). The results of hierarchical clustering analysis of 349 genes actively regulated by FPX treatment (FC > 2 after 2 or 8 d; FDR < 0.05) are shown in Fig. 5D. Two-thirds of these genes were up-regulated by FPX after 2 or 8 d of culture, and genes with the GO terms ‘extracellular region’ and ‘plasma membrane’ were over-represented (Fig. 5D; Supplementary Table S10). The list of genes is further described in detail in the Discussion. FPX promotes callus formation and shoot regeneration, and facilitates Agrobacterium transformation of monocots and tree species Prior to this study, almost no analysis of FPX activity had been performed in plants other than the model plant Arabidopsis. To determine the universality of FPX activity in different plant families, we investigated whether FPX could exert biological activity in monocot crops and tree species from several other plant families. Embryos of immature seeds have generally been used for callus formation in rice. When dry seeds of Oryza sativa ‘Nipponbare’ were sown on medium containing 45 μM FPX, callus formation was observed in the germinated seedlings after 30 d under light conditions (Fig. 6A). Callus formation in rice is induced in the embryonic region of the seed; this contrasts with Arabidopsis, where FPX-induced callus formation occurs in hypocotyl or root apical meristem regions. Fig. 6 View largeDownload slide Callus formation in rice, poplar and Brachypodium distachyon by FPX. (A) Phenotypes of 30-day-old light-grown rice on medium containing 1% DMSO, 15 µM FPX or 45 µM FPX. Right panels show magnifications of selected regions. (B) FPX-induced callus formation in poplar. Stem explants from poplar were incubated for 4 weeks on medium containing 0.4 µM NAA, 30 µM FPX or 45 µM FPX. (C) FPX-induced shoot regeneration. Stem and root explants from poplar were pre-incubated on medium containing 30 µM FPX and then incubated on shoot induction medium for 30 d. (D) Transformation of FPX-induced callus from poplar and GUS analysis of poplar callus tissue harboring 35S:GUS from Agrobacterium-mediated transformation. (E) FPX-induced callus formation on mature Brachypodium seeds. Phenotypes of Brachypodium plants 4 and 14 d after germination on medium containing DMSO, 45 µM FPX, 100 µM FPX or 2.5 mg l−1 2,4-D are shown. Arrowheads indicate callus formation. (F) FPX-induced callus formation on Brachypodium roots. Phenotypes of Brachypodium plants 21 d after germination on medium containing DMSO, 45 µM FPX or 100 µM FPX are shown. Arrowheads indicate callus formation. (G) Shoot regeneration from FPX-induced callus produced from immature seeds. Immature seeds of Brachypodium were pre-incubated on medium containing 75 µM FPX and then transferred onto regular shoot induction medium. Photographs were taken 14 d after the transfer. (H) Transformation of FPX-induced callus from Brachypodium. GUS analysis of Brachypodium callus tissue harboring BdUbi::GUS from Agrobacterium-mediated transformation. (I) Effect of FPX-induced callus from Brachypodium on transformation efficiency. Fig. 6 View largeDownload slide Callus formation in rice, poplar and Brachypodium distachyon by FPX. (A) Phenotypes of 30-day-old light-grown rice on medium containing 1% DMSO, 15 µM FPX or 45 µM FPX. Right panels show magnifications of selected regions. (B) FPX-induced callus formation in poplar. Stem explants from poplar were incubated for 4 weeks on medium containing 0.4 µM NAA, 30 µM FPX or 45 µM FPX. (C) FPX-induced shoot regeneration. Stem and root explants from poplar were pre-incubated on medium containing 30 µM FPX and then incubated on shoot induction medium for 30 d. (D) Transformation of FPX-induced callus from poplar and GUS analysis of poplar callus tissue harboring 35S:GUS from Agrobacterium-mediated transformation. (E) FPX-induced callus formation on mature Brachypodium seeds. Phenotypes of Brachypodium plants 4 and 14 d after germination on medium containing DMSO, 45 µM FPX, 100 µM FPX or 2.5 mg l−1 2,4-D are shown. Arrowheads indicate callus formation. (F) FPX-induced callus formation on Brachypodium roots. Phenotypes of Brachypodium plants 21 d after germination on medium containing DMSO, 45 µM FPX or 100 µM FPX are shown. Arrowheads indicate callus formation. (G) Shoot regeneration from FPX-induced callus produced from immature seeds. Immature seeds of Brachypodium were pre-incubated on medium containing 75 µM FPX and then transferred onto regular shoot induction medium. Photographs were taken 14 d after the transfer. (H) Transformation of FPX-induced callus from Brachypodium. GUS analysis of Brachypodium callus tissue harboring BdUbi::GUS from Agrobacterium-mediated transformation. (I) Effect of FPX-induced callus from Brachypodium on transformation efficiency. Next, we tried to analyze FPX activity in trees. Incubation of Populus tremula×tremuloides ‘T89’ stem organs on medium with 30 or 45 μM FPX resulted in callus formation after 4 weeks, whereas no visible callus was detected in the callus induction medium containing 0.4 μM naphthaleneacetic acid (NAA) (Fig. 6B) (Eriksson et al. 2000, Ohtani et al. 2011). After ≥4 weeks of incubation, FPX-induced callus formation in poplar was observed. In a subsequent step, the FPX-induced callus was transferred to shoot-inducing medium containing 0.5 μM indole-3-butyric acid and 0.8 μM benzyl adenine. Four weeks after transfer, shoot regeneration was observed from stem- and root-induced callus. In addition, poplar stem explants were infected by Agrobacterium harboring a 35S:GUS (β-glucuronidase) vector and then cultured on medium containing 30 μM FPX and antibiotics for 4 weeks. All of the obtained calli tested positive for GUS staining. FPX is thus able to induce tree callus that has shoot regeneration ability and the capacity for Agrobacterium-mediated transformation. We also analyzed the effect of FPX on the model grass plant species Brachypodium distachyon (L.) (hereafter Brachypodium). For initial analysis of Brachypodium in the presence of FPX, mature seeds were germinated, and roots explants were cultured in medium containing FPX. Four days after germination, 45 and 100 μM FPX induced callus from mature Brachypodium seeds (Fig. 6E). Callus formation on Brachypodium root explants was also observed upon culture with 45 and 100 μM FPX for 21 d (Fig. 6F). Embryos of immature seeds are frequently used for callus formation and shoot regeneration in monocots. We found that 75 μM FPX could induce callus formation on embryos of immature Brachypodium seeds after 14 d of culture, while Brachypodium shoots could be regenerated from the FPX-induced embryo callus after 14 d induction (Fig. 6G). Because the floral dip method is generally used for Agrobacterium-mediated transformation of Arabidopsis, we analyzed the transformation efficiency of FPX-induced callus by Agrobacterium using Brachypodium callus. Embryogenic callus induced by 75 or 100 μM FPX and 11.3 μM 2,4-D was co-cultured with Agrobacterium harboring a 35S:GUS vector for 2 d. The Agrobacterium-infected callus was cultured for 9 d and then stained with X-gluc. Analysis of 50 calli on each of three plates indicated that FPX-induced embryogenic callus tended to have a high transformation efficiency (Fig. 6H). FPX induces callus formation in vegetable plants To analyze the diversity of FPX-induced activity in callus formation, we applied FPX to several plant species generally consumed as vegetables and belonging to different families. Glycine max L. ‘Tsurunoko’ (soybean), a member of the family Fabaceae, was bred in the Hokkaido area of Japan. When mature seeds of this soybean cultivar were germinated on medium containing FPX for 22 d, callus induction was observed over a wide range of FPX concentrations: from 15 to 105 μM (Fig. 7). Solanum lycopersicum L. ‘Micro-Tom’ is a dwarf tomato variety that has been extensively used as a model experimental plant in the family Solanaceae (Saito et al. 2011). Callus was induced from mature germinated Micro-Tom seeds using 15 μM FPX for 32 d. In the case of this tomato variety, higher concentrations of FPX may have inhibited seedling growth, as callus formation was not observed at concentrations of 45–105 μM (Fig. 7). Cucumis sativus L. ‘Natsusuzumi’ (cucumber) belongs to Cucurbitaceae, a major vegetable family along with Fabaceae, Solanaceae and Brassicaceae. When mature seeds of cucumber were germinated in MS medium containing FPX for 35 d, callus induction was observed over a wide range of FPX concentrations, from 15 to 105 μM (Fig. 7). These results suggest that FPX has callus-inducing activity in many different plant families. Fig. 7 View largeDownload slide Callus formation in soybean, tomato and cucumber by FPX. Phenotypes of light-grown soybean, Micro-Tom tomato and cucumber on medium containing the indicated concentration of FPX on the indicated number of days after germination are shown. Right panels show magnifications of selected regions. Fig. 7 View largeDownload slide Callus formation in soybean, tomato and cucumber by FPX. Phenotypes of light-grown soybean, Micro-Tom tomato and cucumber on medium containing the indicated concentration of FPX on the indicated number of days after germination are shown. Right panels show magnifications of selected regions. Discussion FPX is a psychoactive drug used to treat senile dementia (Bompani and Scali 1986) and memory impairment (Genkova-Papasova and Lazarova-Bakurova 1988) in mammals, though the detailed mode of action and the target protein(s) of FPX have not yet been identified. FPX was initially identified in plants in a screen for compounds promoting or inhibiting hypocotyl elongation of Arabidopsis seedlings, but the effect of FPX on plants has not been fully investigated. Here, we showed that FPX is able to induce callus formation and shoot regeneration in plant seedlings and leaf and root tissues. These activities suggest that FPX is a chemical inducer for callus and shoot formation in plants. Extensive previous research has revealed that plant callus cells are induced by auxin and cytokinin from already developed leaf, stem and root cells. Callus formation has an important role in the plant life cycle, as plant callus cells regain the totipotency to develop multiple plant organs that differ from the original cells of the callus. Callus formation has been observed on insect-infected, bacterium-infected and wounded plant organs (Ikeuchi et al. 2013). Although exogenous application of auxin and cytokinin does not occur naturally, research on plant cell totipotency using this approach has helped reveal previously unknown and interesting molecular mechanisms of plant development. Recent mammalian research has suggested that induced pluripotent stem cells (iPS cells) can be developed by the transformation of four mammalian genes. This finding has had a huge impact both in the field of basic animal biology as well as in applied medical science (Takahashi and Yamanaka 2016). The scientific concept of mammalian iPS cells is similar to that of plant totipotent callus cells. The discovery of the chemical inducer FPX may add new knowledge to plant developmental research and applied plant science. Treatment with FPX resulted in high callus formation efficiency in Arabidopsis leaf and root sections. In shoot-inducing medium, FPX-induced callus exhibited a high regeneration efficiency. To analyze the activity of FPX in callus formation, we examined the organization of callus cells induced from root explants by FPX and auxin/cytokinin under an optical microscope. Recent studies have attempted to reveal the molecular mechanism of callus formation induced by combined auxin and cytokinin using Arabidopsis developmental gene markers and mutants (Atta et al. 2009, Sugimoto et al. 2010). In intact plants, lateral roots were induced from the main roots in response to the auxin-induced signaling system. Similar to lateral root formation, callus formation from roots was initiated in the initial stages of pericycle cell division. These lateral root apical meristem (LRM)-like structures induced by auxin and cytokinin were termed LRM-like protuberances. Several marker genes expressed in lateral root formation were also induced in the LRM-like protuberances and observed in the resulting callus cells. After 5 d culture on auxin/cytokinin medium, the protuberances increased in size. During auxin/cytokinin treatment, auxin induced the reactivation of pericycle cells and the initiation of LRM-like structures, whereas cytokinin induced re-entry of pericycle cells into the cell cycle. This combination caused high cell division activity in the outer epidermal region (Atta et al. 2009). Similarly active cell division was observed in the outer epidermal region of roots treated with auxin and cytokinin in our study (Fig. 4). We observed that actively dividing cell regions in FPX-induced callus from root explants seemed to be larger than those of auxin/cytokinin-induced callus. The vessel element formation that was observed in auxin/cytokinin-induced callus was not observed in FPX-induced root cells. These results suggest that the cellular organization of the actively dividing cell region in FPX-induced callus and auxin/cytokinin-induced callus was different. Although the initiation cell of FPX-stimulated callus induction has not yet been identified, root development-related genes were highly expressed by both auxin/cytokinin and FPX induction (Fig. 5; Supplementary Figs. S1–S6). We additionally analyzed the expression of lateral root formation genes (Orman-Ligeza et al. 2013). ARF7 and LBD29 expression was induced by FPX and auxin/cytokinin at the same levels. ARF19, LBD16, LBD17 and LBD18 expression was induced by FPX and auxin/cytokinin, but the expression strengths of these genes in the presence of auxin/cytokinin were higher than those under FPX induction (Supplementary Fig. S5). These results suggest that the initiation of callus formation under FPX treatment may begin with LRM formation, while the activity of FPX in the induction of lateral root formation genes may be weaker than that of auxin. Auxin strongly induced LRM-like protuberances in root epidermal regions of calli, and differentiation to form vessel elements may have started in the inner region of calli. In FPX-induced callus, continuous cell division may continue to the inner region such that differentiation to vessel element occurred late. Future observations of FPX-induced callus initiation cells and an expression analysis of marker genes related to root and shoot meristem regulation should reveal the molecular mechanism underlying FPX function. Microarray analysis allowed us to examine the similarity and specificity of genes regulated in FPX- and auxin/cytokinin-induced calli. Most of the 598 genes that were commonly and significantly induced by FPX and auxin/cytokinin (Fig. 5A; Supplementary Table S4) were related to the cell cycle, root development and auxin response. These 598 genes suggested commonality between FPX activity and auxin/cytokinin activity. Other auxin response genes were highly induced only in auxin/cytokinin-stimulated callus (Supplementary Fig. S5). These results suggest that FPX-induced callus formation is less dependent on auxin signaling than is auxin/cytokinin-induced callus formation. In the FPX-specific gene set (1,868 genes; Supplementary Tables S6, S10), annotated genes related to metabolism were highly abundant. The GO terms ‘metabolic process’ in the biological process category and ‘catalytic activity’, ‘transferase activity’ and ‘phosphate-containing compound metabolic process’ in the molecular function category were particularly statistically significantly enriched (Fig. 5C; Supplementary Table S6). Among the 1,868 genes specifically induced by FPX, 359 genes that were 2-fold up-regulated (FC > 2, FDR < 0.05) by FPX were selected and subjected to GO annotation. Five types of metabolism-related genes (highlighted in orange in Supplementary Table S10) were identified, namely genes related to sugar metabolism, lipid metabolism, amino acid metabolism, glutathione transfer and nicotiamine metabolism. FPX function might suggest unknown metabolic signaling that may be involved in plant cell division activity through direct or auxiliary effects. According to the results of the hierarchical clustering analysis of 368 genes actively regulated by FPX treatment (FC > 2 after 2 or 8 d; FDR < 0.05), two-thirds of these genes were up-regulated by FPX after 2 or 8 d of culture, among which genes associated with the GO terms ‘extracellular region’ and ‘plasma membrane’ were over-represented (Fig. 5D; highlighted in green in Supplementary Table S10). In particular, groups 1–3 contained several plasma membrane-associated protein kinases related to the regulation of cell division and expansion, such as NIMA-RELATED KINASE 5 (Motose et al. 2011); membrane-bound proteins such as the transporter YELLOW STRIPE LIKE 7 (YSL7) (Hofstetter et al. 2013) and phosphate transporter PHO1 (Hamburger et al. 2002); and AMINOPHOSPHOLIPID ATPASE10 (ALA10), an ATPase flippase that internalizes exogenous phospholipids across the plasma membrane (Poulsen et al. 2015). FPX may therefore also activate plasma membrane-associated cellular signaling. FPX exhibited high activity in callus formation, and high shoot regeneration activity was observed from FPX-induced callus. Finally, we also determined that FPX-induced callus can be transformed by Agrobacterium. Callus formation, shoot regeneration and Agrobacterium infection are three important steps in the plant transformation technique in plant biotechnology. Our results suggest that FPX can be used in these three steps of plant transformation as an additional and complementary chemical inducer with the already developed auxin/cytokinin system. In 1996, cultivated transgenic crops and vegetables occupied 1.7 Mha of land. Twenty years later, in 2016, the area under transgenic cultivation had expanded approximately 110-fold, to 1,800 Mha worldwide (ISAAA 2016). Despite the popularity of genetically modified organisms, many useful plant species have not been subjected to genetic transformation because of overwhelming difficulties due to the insufficient activity of available phytohormones to induce plant callus formation and regenerate shoots or roots from callus. Genome editing has recently been applied in the field of plant science. This technique can be exploited for the transformation of plant callus by using Agrobacterium harboring genome-editing enzyme-encoded vectors. The use of FPX would increase the number of plant species amenable to genetic transformation and genome editing. Materials and Methods Plant materials and growth conditions Arabidopsis thaliana ecotype Columbia (Col-0) seeds were germinated on half-strength MS (1/2 MS) medium (Duchefa) containing 0.8% phytoagar (Duchefa) and 1.5% sucrose. Arabidopsis plants were grown at 22°C under white light using a 16 h light/8 h dark cycle to simulate long-day conditions. Nipponbare rice, Tsurunoko soybean, Micro-Tom tomato and Natsusuzumi cucumber plants were grown under a 16 h light/8 h dark cycle at 25°C. Chemical screening and growth conditions A total of 24,275 compounds from a chemical library in the RIKEN Natural Products Depository were screened for hypocotyl phenotypes at a final concentration of 50 µM in 2% dimethylsulfoxide (DMSO). Five Arabidopsis seeds were sown in individual wells of 96-well plates containing 1/2 MS medium supplemented with 0.5% phytoagar, 1.5% sucrose and one of the tested compounds. Following treatment for 2 d at 4°C to induce germination, seeds were incubated in a growth chamber in the dark at 22°C. Arabidopsis callus formation analyses on MS medium Ten days after germination, Arabidopsis rosette leaves were cut into explants having mid-section widths of 2–3 mm, and roots (excluding 1 cm at each end) were subdivided into three explants (Supplementary Fig. S1). These explants were placed onto 1/2 MS medium containing either 28.5 µM IAA, 2.3 µM 2,4-D and 1.3 µM kinetin, or 15 or 45 µM FPX solidified with 0.7% agarose and sucrose. After 50 d of incubation, the explants were weighed. Arabidopsis callus formation analyses on Gamborg’s B5 medium Seven days after germination, Arabidopsis root tips (Supplementary Fig. S1A) were placed onto Gamborg’s B5 medium (Wakon) with 20 g l−1 glucose, 0.5 g l−1 MES and 1 ml l−1 Gamborg’s vitamin solution (Sigma-Aldrich) solidified with 0.8% phytoagar containing the indicated chemicals. After 34 d of incubation, the explants were weighed. Arabidopsis shoot regeneration assays Root explants were excised from seedlings 10 d after germination. The roots were cut into three pieces as shown in Supplementary Fig. S1. Explants were pre-cultured on 1/2 MS medium containing either 28.5 µM IAA, 2.3 µM 2,4-D and 1.3 µM kinetin, or 15 µM FPX (agarose and sucrose) for 3 weeks and then transferred to shoot induction medium (1/2 MS medium containing 0.85 µM IAA and 7.4 µM 2iPA) to induce shoots. Histological analysis Sterilized wild-type Arabidopsis seeds were sown on plates containing solid MS medium supplemented with 1% sucrose, 5 mg l−1 nicotinic acid, 0.5 mg l−1 pyridoxine, 3 mg l−1 thiamine-HCl and 7.5 g Bacto Agar. Ten days after germination, roots were subdivided into three explants. These explants were placed onto MS medium containing 2% glucose, 0.5 mg l−1 nicotinic acid, 0.5 mg l−1 pyridoxine, 0.5 mg l−1 thiamine-HCl, 0.05 M MES, 100 mg l−1 myoinositol and 7.5 g l−1 Bacto Agar supplemented with either 5 mg l−1 IAA, 0.5 mg/ml−1 2,4-D and 0.3 mg ml−1 kinetin or with 50 µM FPX for 15 d. The induced calli were processed for histological analysis according to the method of Wang et al. (2015) with minor modifications. Cross-sections were made from calli after fixation for a few days at 4°C in 45% ethanol/5% formaldehyde/5% acetic acid in 50 mM sodium phosphate buffer (pH 7.2), dehydration through graded ethanol and t-butanol series, and embedding in Paraplast Plus (McCormick Scientific). Microtome sections (10 µm thick) were mounted on MAS-coated glass slides (Matsunami Glass). The sections were deparaffinized in xylene, hydrated through a graded ethanol to distilled water series, and stained with toluidine blue O. GeneChip analysis of FPX-treated seedlings Four-day-old seedlings grown on the MS medium were transferred to MS medium containing 45 µM FPX (FPX plate), or 0.5 mg l−1 2,4-D and 0.1 mg l−1 kinetin (auxin/cytokinin plate). Twenty seedlings cultured for 0, 2 and 8 d were collected and frozen with liquid nitrogen immediately, and stored at −80°C. Total RNAs were isolated from the stored samples using the RNeasy Plant Mini Kit (Qiagen). Microarray analysis was performed using the GeneChip® Arabidopsis Genome ATH1 Array (Affymetrix) on three independent biological replicates, according to Ohtani et al. (2011) and Song et al. (2016). Subsequent procedures of quality control, statistical analysis and filtering were carried out using GeneSpring GX software v13.0.1 (Agilent Technologies). P-values were calculated for each probe by one-way ANOVA (n = 3), for differences among 0, 2 and 8 d treatments with FPX or auxin/cytokinin. The Benjamini–Hochberg FDR method was applied for controlling false positives, and a corrected P-value cut-off of 0.05 was used to select the regulated genes with the lowest FDR. FC values were also computed by GeneSpring GX to select the probes up- or down-regulated by >2-fold after 2 d treatment (Fig. 5A, B and D). For hierarchical clustering analysis, the Cluster 3.0 software was used with Euclidean distance measurement method (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm). GO term analysis was performed via the website of the Gene Ontology Consortium (http://geneontology.org/page/download-ontology). Microarray data presented in this study were submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) and can be retrieved via accession number GSE116939. Analysis of callus formation, shoot regeneration and transformation in poplar T89 poplar plants were used for this analysis. To induce callus, stem segments from poplar were pre-incubated with Agrobacterium containing a 35S:GUS vector and then incubated for 4 weeks on 1/2 MS medium supplemented with hygromycin and containing 0.4 µM NAA, 30 µM FPX or 45 µM FPX. To induce shoot regeneration, stem or root explants were first cultured on 1/2 MS medium containing 30 µM FPX to induce callus and then transferred to shoot induction medium (0.5 µM indole-3-butyric acid and 0.8 µM benzyl adenine) to induce shoots. Histochemical GUS staining was performed on callus using 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid at 37°C after pre-treatment in 90% (v/v) acetone at 4°C. GUS-stained tissue was cleared by incubation in 70% (v/v) ethanol. Analysis of callus formation, shoot regeneration and transformation in Brachypodium Plants of B. distachyon line Bd21 (USDA National Plant Germplasm System) were used as the wild type. The method used for seed sterilization has been previously described (Himuro et al. 2014). To induce callus, mature seeds and roots of Brachypodium were incubated on MS medium containing DMSO, FPX or 2,4-D. To induce shoot regeneration, immature seeds were pre-cultured on MS medium containing 75 µM FPX to induce callus and then transferred to shoot induction medium containing 0.2 mg l−1 kinetin to induce shoots. Histochemical GUS staining was performed on callus using 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid at 37°C after pre-treatment in 90% (v/v) acetone at 4°C. GUS-stained tissue was cleared by incubation in 70% (v/v) ethanol. Quantitative real-time PCR The methods for total RNA isolation, cDNA synthesis and real-time PCR have been previously described (Yamagami et al. 2017). The sequences of the gene-specific primers for real-time PCR were as follows: for ARF7, 5'-GGAGTTCGTCGGTATATGGG-3' and 5'-GAAACTCGACTGGGCCTATC-3'; for ARF19, 5'-GGCTCACAATGGCGTAATC-3' and 5'-GGAGTTATGACGGGTTCGAT-3'; for LBD16, 5'-AGCTCGGAAAGTACCAACCA-3' and 5'-CGAGACCGGATTGTTAGGG-3'; for LBD17, 5'-CATCATGACGTCGTGCTACC-3' and 5'-TTCGCTGCAGCCACTAGAG-3'; for LBD18, 5'-AGTGTGTGCCGGGATG-3' and 5'-CTCCGAACACTTTATGCACC-3'; for LBD29, 5'-GCAGCCATTCACAAGGTC-3' and 5'-AGCCATAGATGGGATCTTGA-3'; and for ACT2, 5'-CGCCATCCAAGCTGTTCTC-3' and 5'-TCACGTCCAGCAAGGTCAAG-3'. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Japan Science and Technology Agency [grants from CREST to T.N. and T.A.]; NARO Bio-oriented Technology Research Advancement [BRAIN; to T.N. and T.A.]; and the Cabinet Office, Government of Japan [Cross-ministerial Strategic Innovation Promotion Program (SIP; to S.H.)]. Acknowledgments We thank K. Saeki, Y. Suzuki and K. Maekawa for their technical assistance. Tomato (Micro-Tom) seed was provided by University of Tsukuba, gene research center, through the National Bio-Resource Project of the AMED, Japan. Disclosures The authors have no conflicts of interest to declare. References Atta R. , Laurens L. , Boucheron-Dubuisson E. , Guivarc'h A. , Carnero E. , Giraudat-Pautot V. ( 2009 ) Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro . Plant J . 57 : 626 – 644 . Google Scholar CrossRef Search ADS PubMed Bompani R. , Scali G. 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Plant and Cell PhysiologyOxford University Press

Published: Aug 1, 2018

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