TY - JOUR
AU - Xia, Gui-Xian
AB - Abstract The BLADE-ON-PETIOLE (BOP) genes of Arabidopsis (Arabidopsis thaliana) have been shown to play an essential role in floral abscission by specializing the abscission zone (AZ) anatomy. However, the molecular and cellular mechanisms that underlie differentiation of the AZ are largely unknown. In this study, we identified a tobacco (Nicotiana tabacum) homolog of BOP (designated NtBOP2) and characterized its cellular function. In tobacco plants, the NtBOP2 gene is predominantly expressed at the base of the corolla in an ethylene-independent manner. Both antisense suppression of NtBOP genes and overexpression of NtBOP2 in tobacco plants caused a failure in corolla shedding. Histological analysis revealed that the differentiation of the corolla AZ was blocked in the transgenic flowers. This blockage was due to uncontrolled cell elongation at the region corresponding to wild-type AZ. The role of NtBOP2 in regulating cell elongation was further demonstrated in Bright Yellow 2 single cells: perturbation of NtBOP2 function by a dominant negative strategy led to the formation of abnormally elongated cells. Subcellular localization analysis showed that NtBOP2-green fluorescent protein fusion proteins were targeted to both the nucleus and cytoplasm. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that NtBOP2 proteins interacted with TGA transcription factors. Taken together, these results indicated that NtBOP2 mediated the differentiation of AZ architecture by controlling longitudinal cell growth. Furthermore, NtBOP2 may achieve this outcome through interaction with the TGA transcription factors and via an ethylene-independent signaling pathway. It is common for a plant organ (leaf, flower, or fruit) to fall from the main body of the plant during its life span. This shedding takes place at the site termed the abscission zone (AZ; Sexton and Roberts, 1982; Roberts et al., 2002). Typically, the AZs consist of several layers of cytoplasmically condensed cells, which are anatomically divergent from the adjacent cells (Bleecker and Patterson, 1997; Roberts et al., 2000). Abscission can be triggered by both developmental signals and external stimuli. Ethylene signaling hastens organ abscission, whereas auxin signaling restrains the process. Environmental stresses, including drought, high temperature, and pathogen attack, can lead to premature organ abscission (Gonzalez-Carranza et al., 1998; Taylor and Whitelaw, 2001; Roberts et al., 2002). In many species of flowering plants, abscission of the entire flowers or floral parts (including petals, sepals, and stamens) from the flower base occurs (vanDoorn and Stead, 1997). The abscission process consists of two principal phases: differentiation of the AZ, and subsequent separation of the flower or floral parts from the main body through cell wall dissolution (Roberts et al., 2002). In Arabidopsis (Arabidopsis thaliana), a number of genes have been shown to be specifically and/or noticeably expressed in the floral AZs and to modulate floral abscission. These genes include the HAESA (HAE) and HAESA-LIKE2 (HSL2) genes, which encode receptor-like kinases (Jinn et al., 2000; Cho et al., 2008; Stenvik et al., 2008); the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) gene, which encodes a small protein that is considered to be the putative ligand of HAE (Butenko et al., 2003; Stenvik et al., 2006); and the genes encoding the components of the mitogen-activated protein kinase pathway (Cho et al., 2008). It has been proposed that the sequential action of IDA, HAE, and HSL2 and a mitogen-activated protein kinase cascade regulate floral organ abscission (Cho et al., 2008). More recently, three receptor-like cytoplasmic kinases were found to be the negative regulators of IDA/HAE signaling in AZ cells (Leslie et al., 2010; Lewis et al., 2010; Burr et al., 2011), and the KNOTTED-LIKE HOMEOBOX genes were shown to be the downstream components of the IDA signaling pathway (Shi et al., 2011). Apart from the genes associated with IDA signaling, a number of other genes were also shown to have important roles in floral abscission in Arabidopsis, including regulatory genes (Fernandez et al., 2000; Adamczyk et al., 2007; Cai and Lashbrook, 2008; Wei et al., 2010; Chen et al., 2011), cytoskeletal genes (Kandasamy et al., 2005a, 2005b), and genes encoding the cell wall hydrolytic enzymes (González-Carranza et al., 2007; Ogawa et al., 2009). Besides Arabidopsis, genes that take part in floral abscission have been identified from other plants. For example, the MADS box genes JOINTLESS and MACROCALYX were found to control pedicel AZ development in tomato (Solanum lycopersicum; Mao et al., 2000; Nakano et al., 2012), and several cellulase genes were shown to contribute to wall loosening at floral AZs in various plants (Lieberman et al., 1982; Kemmerer and Tucker, 1994; Lashbrook et al., 1994; del Campillo and Bennett, 1996). During the process of floral abscission, the above-mentioned genes act mainly in the later events, such as abscission initiation and the final separation of AZ cells. To date, far less is known about the genes functioning in the early stages of AZ development, especially the differentiation of AZ anatomy. The BLADE-ON-PETIOLE (BOP) genes in Arabidopsis belong to the NPR1 family (for nonexpressor of PR1), whose members play essential roles in systemic acquired resistance. It has been shown that AtBOP1 and AtBOP2 function redundantly to regulate leaf and flower patterning (Ha et al., 2003, 2004; Hepworth et al., 2005; Norberg et al., 2005; Xu et al., 2010b; Khan et al., 2012). Further studies demonstrated that BOPs functioned as the proximal-distal and adaxial-abaxial patterning determinants in Arabidopsis (Ha et al., 2007; Jun et al., 2010). Additionally, several studies demonstrated that BOP1 and BOP2 genes were essential for floral organ abscission. In floral organs, BOP genes were specifically expressed in the AZ regions of developing sepals, petals, and stamens, and these floral organs failed to shed in the bop1 bop2 double mutant (Hepworth et al., 2005; McKim et al., 2008). Based on their genetic and anatomical analyses, McKim et al. (2008) proposed that BOP1 and BOP2 proteins play a role in the differentiation of the AZ by promoting the formation of the specialized AZ anatomy that is necessary for abscission. In addition to Arabidopsis, BOP genes have been recently identified from the lower plant Physcomitrella patens, and two of these genes (PpBOP1 and PpBOP2) were shown to act as positive regulators of protonema differentiation during juvenile-to-adult gametophyte transition under the control of Pp-miR534a (Saleh et al., 2011). In this study, we identified a homolog BOP protein (NtBOP2) from tobacco (Nicotiana tabacum). Functional analyses of the protein in transgenic plants indicated that NtBOP2 plays a critical role in the differentiation of the corolla AZ. More importantly, our results revealed that NtBOP2 functions by controlling longitudinal cell expansion in the AZ region. Our results provide deeper insight into the cellular mechanisms involved in the development of the floral AZ in higher plants. RESULTS Isolation of NtBOP2 cDNA and Structural Analysis of the Protein A functional approach was taken to identify genes that are involved in the cytokinesis cascade in tobacco Bright Yellow 2 (BY-2) cells (Yu et al., 2007). Briefly, the cDNAs of BY-2 cells were inserted into the yeast expression vector pREP1 under the control of the thiamine-repressible nmt1 promoter and transformed into fission yeast cells. A number of tobacco genes that perturbed the terminal stage of cell division when ectopically expressed in yeast cells were isolated. Of these, a cDNA fragment that causes a defect in cell separation (Fig. 1, A and B) was chosen for further analysis in this study. The tobacco cDNA was isolated from the yeast transformant, and the sequence was determined. The cDNA is 1,701 bp in length with an open reading frame (ORF) of 1,428 bp and encodes a homolog (475 amino acids) of Arabidopsis BOP proteins. Sequence homology between the tobacco BOP and Arabidopsis BOP proteins was assessed. Figure 1, C and D, shows that the tobacco BOP shares the highest homology to AtBOP2, with a protein sequence identity of 77%. Accordingly, tobacco BOP was designated as NtBOP2 (GenBank accession no. EF051131). Domain analysis (Fig. 1E) indicated that NtBOP2 contains two domains typically found in BOP proteins, including a BTB/POZ (for Broad-Complex, Tramtrack, and Bric-a-Brac/POX virus and Zinc finger) domain and four ankyrin repeats involved in protein-protein interaction. Additionally, NtBOP2 contains a PEST sequence (enriched with Pro, Glu, Ser, and Thr) implicated in protein degradation. Figure 1. Open in new tabDownload slide Isolation of NtBOP2 and protein structure analysis. A and B, Phenotypes of fission yeast cells harboring the empty pREP1 vector (A) or pREP1-NtBOP2 (B). Transformed cells were stained with DAPI and calcofluor to visualize nuclei and septa, respectively. Bars = 10 µm. C, An unrooted phylogenetic tree of proteins predicted to contain BTB/POZ domains in Arabidopsis, tobacco, and moss. The scale indicates branch lengths. The accession numbers of the proteins are as follows: AtBOP1 (At3g57130), AtBOP2 (At2g41370), PpBOP1 (EDQ77565), PpBOP2 (EDQ75924), PpBOP3 (EDQ51035), NtNPR1 (ABH04326.1), AtNPR1 (At1g64280), AtNPR2 (At4g26120), AtNPR3 (At5g45110), and AtNPR4 (At4g19660). D, Alignment of the predicted amino acid sequence of NtBOP2 with AtBOP1 and AtBOP2 proteins. The BTB/POZ domain and ankyrin repeats are marked. Amino acids in black shading indicate identical amino acids among the proteins. E, Conserved domains in the NtBOP2 protein. The light gray box represents the BTB/POZ domain. Dark gray boxes show ankyrin-repeats (ANK). The black box indicates the PEST sequence enriched in Pro (P), Glu (E), Ser (S), and Thr (T). The PEST-rich region was identified using the program ePESTfind (http://expasy.org/tools/). [See online article for color version of this figure.] Figure 1. Open in new tabDownload slide Isolation of NtBOP2 and protein structure analysis. A and B, Phenotypes of fission yeast cells harboring the empty pREP1 vector (A) or pREP1-NtBOP2 (B). Transformed cells were stained with DAPI and calcofluor to visualize nuclei and septa, respectively. Bars = 10 µm. C, An unrooted phylogenetic tree of proteins predicted to contain BTB/POZ domains in Arabidopsis, tobacco, and moss. The scale indicates branch lengths. The accession numbers of the proteins are as follows: AtBOP1 (At3g57130), AtBOP2 (At2g41370), PpBOP1 (EDQ77565), PpBOP2 (EDQ75924), PpBOP3 (EDQ51035), NtNPR1 (ABH04326.1), AtNPR1 (At1g64280), AtNPR2 (At4g26120), AtNPR3 (At5g45110), and AtNPR4 (At4g19660). D, Alignment of the predicted amino acid sequence of NtBOP2 with AtBOP1 and AtBOP2 proteins. The BTB/POZ domain and ankyrin repeats are marked. Amino acids in black shading indicate identical amino acids among the proteins. E, Conserved domains in the NtBOP2 protein. The light gray box represents the BTB/POZ domain. Dark gray boxes show ankyrin-repeats (ANK). The black box indicates the PEST sequence enriched in Pro (P), Glu (E), Ser (S), and Thr (T). The PEST-rich region was identified using the program ePESTfind (http://expasy.org/tools/). [See online article for color version of this figure.] Alteration in NtBOP2 Expression Caused a Failure in Corolla Abscission To characterize the cellular function of NtBOP2 in plants, four types of transgenic tobacco plants were generated. These transgenics included plants overexpressing the intact or a truncated form of NtBOP2 as well as plants with NtBOP2 expression inhibited by either RNA interference (RNAi) or antisense approaches. The increased or inhibited expression of the NtBOP2 gene was verified by reverse transcription (RT)-PCR analysis (Fig. 2, A and B), and the phenotypes of the transgenic plants were examined. The RNAi plants (NtBOP2-RNAi) did not exhibit significant alteration in growth and development compared with the wild-type control. This lack of change is probably due to functional redundancy with other members of the BOP family. The antisense plants (NtBOP2-AS), however, showed a striking defect in corolla abscission. Whereas the wild-type corolla was shed about 4 d after fully opening, the transgenic plants retained the corolla that did not detach from the flower base eventually (Fig. 2C). Apart from the defect in corolla abscission, no significant aberration was observed in the transgenic plants during the course of growth and development (Fig. 2D). Interestingly, overexpression of either the intact or truncated NtBOP2 in the transgenic plants (named NtBOP2-OE and NtBOP2t-OE, respectively) also caused a defect in corolla shedding (data not shown) similar to that seen in the antisense transgenic plants. This defect was probably due to a dominant negative effect (see “Discussion”). Figure 2. Open in new tabDownload slide Phenotypic analysis of NtBOP2 transgenic plants. A and B, RT-PCR analysis of NtBOP2/NtBOP2t expression in transgenic plants. Total RNAs were extracted from the corollas of wild-type (WT) and transgenic plants, and the NtBOP2/NtBOP2t transcripts were PCR amplified. Transcripts of the intact and truncated NtBOP2 gene (NtBOP2/NtBOP2t) were amplified using primers BRT-F2 and BRT-R for 28 cycles (A); NtBOP2 transcripts in RNAi and antisense transgenic plants were amplified using primers BRT-F1 and BRT-R for 32 cycles (B). The 18S rRNA gene was used as an internal control for 16 cycles. C, Defective corolla abscission in an NtBOP2-AS plant. Infructescences of 3-month-old plants are shown. Left, the wild type; right, NtBOP2-AS. Bar = 1 cm. D, Similar phytomorphology of the wild-type and NtBOP2-AS plants grown in soil for 10 weeks. Bar = 5 cm. [See online article for color version of this figure.] Figure 2. Open in new tabDownload slide Phenotypic analysis of NtBOP2 transgenic plants. A and B, RT-PCR analysis of NtBOP2/NtBOP2t expression in transgenic plants. Total RNAs were extracted from the corollas of wild-type (WT) and transgenic plants, and the NtBOP2/NtBOP2t transcripts were PCR amplified. Transcripts of the intact and truncated NtBOP2 gene (NtBOP2/NtBOP2t) were amplified using primers BRT-F2 and BRT-R for 28 cycles (A); NtBOP2 transcripts in RNAi and antisense transgenic plants were amplified using primers BRT-F1 and BRT-R for 32 cycles (B). The 18S rRNA gene was used as an internal control for 16 cycles. C, Defective corolla abscission in an NtBOP2-AS plant. Infructescences of 3-month-old plants are shown. Left, the wild type; right, NtBOP2-AS. Bar = 1 cm. D, Similar phytomorphology of the wild-type and NtBOP2-AS plants grown in soil for 10 weeks. Bar = 5 cm. [See online article for color version of this figure.] Defective Corolla Abscission in the NtBOP2-AS Transgenic Flower Is Caused by the Lack of AZ In a previous study on the roles of the transglutaminase in developmental cell death, Della Mea et al. (2007) divided the life span of tobacco corolla into 10 stages. A brown ring corresponding to the AZ appeared at stage 7. The corolla abscised but remained in situ on the flower (supported by the calyx and the style) from stage 8 to 9. Finally, at stage 10, the entire corolla died. Based on this definition of corolla developmental stages, we compared the process of corolla abscission between the wild-type and NtBOP2-AS flowers. Under our experimental conditions, the development of the transgenic corollas appeared similar to that of the wild-type corollas during stages 1 to 6 (Fig. 3A). However, the aberration emerged in the later stages of corolla development. In the wild-type flower, the base of the corolla started to lose turgidity and structural integrity and a light brown AZ ring appeared at stage 7 (Fig. 3B, arrow), while the corolla was splitting open and the stigma was pushed out from the corolla tube by the growing ovary; the corolla detached but stayed on the flower due to the adhesion of the calyx and the style during stages 8 to 10. In contrast, the corolla of the transgenic flowers remained fully turgid from stage 7 to 10, as no AZ ring was visible from stage 7 (Fig. 3C) to flower senescence, and the corolla did not ultimately abscise from the flower (Fig. 3A). As a result of failed shedding, transgenic flowers showed a delay in corolla collapse and corolla necrosis compared with the wild-type flowers (Fig. 3D). Figure 3. Open in new tabDownload slide Corolla development in wild-type and NtBOP2-AS flowers. A, Difference in corolla development between wild-type (WT) and NtBOP2-AS flowers. Sepals in the inflorescence were forcibly removed. Corolla abscised at stage 8 in the wild-type flower (top), whereas corolla of the transgenic flowers remained on the receptacle without shedding eventually (bottom). The abscised wild-type corollas at stages 9 and 10 were manually removed from the ovaries. Numbers indicate the corolla developmental stages. Bar = 1 cm. B and C, Magnified views of the boxed regions in A. A light brown AZ ring appeared on the wild-type corolla (B) but not on the NtBOP2-AS corolla (C). The arrow indicates the AZ, and the arrowhead indicates the region from where sepals were removed. Bars = 2 mm. D, Measurement of the senescence time of corollas. The NtBOP2-AS corollas were delayed in petal collapse and necrosis compared with the wild-type control. Corolla collapse is present as the number of days after opening until the corolla loses turgidity, and corolla necrosis is present as the number of days after opening until necrosis of the corolla tube is complete (Yang et al., 2008). Values are means ± sd. [See online article for color version of this figure.] Figure 3. Open in new tabDownload slide Corolla development in wild-type and NtBOP2-AS flowers. A, Difference in corolla development between wild-type (WT) and NtBOP2-AS flowers. Sepals in the inflorescence were forcibly removed. Corolla abscised at stage 8 in the wild-type flower (top), whereas corolla of the transgenic flowers remained on the receptacle without shedding eventually (bottom). The abscised wild-type corollas at stages 9 and 10 were manually removed from the ovaries. Numbers indicate the corolla developmental stages. Bar = 1 cm. B and C, Magnified views of the boxed regions in A. A light brown AZ ring appeared on the wild-type corolla (B) but not on the NtBOP2-AS corolla (C). The arrow indicates the AZ, and the arrowhead indicates the region from where sepals were removed. Bars = 2 mm. D, Measurement of the senescence time of corollas. The NtBOP2-AS corollas were delayed in petal collapse and necrosis compared with the wild-type control. Corolla collapse is present as the number of days after opening until the corolla loses turgidity, and corolla necrosis is present as the number of days after opening until necrosis of the corolla tube is complete (Yang et al., 2008). Values are means ± sd. [See online article for color version of this figure.] Scanning electron microscopy analysis was conducted to examine the structural characteristics of the AZ site in the wild-type corolla or the region where the corolla AZ should form in the NtBOP2-AS. Corollas were either naturally shed or removed by external force. In the wild-type corolla, cells were ruptured at the split plane from stages 1 to 3 (Fig. 4, A and B). At stage 7, fewer cells were broken after the application of external force because some cells were rounded up (Fig. 4C, arrow). At stage 9, the corolla was naturally abscised and the surface of the abscission plane appeared smooth (Fig. 4D). By contrast, the corolla of the NtBOP2-AS flowers did not undergo abscission and had to be forcibly torn at all stages (Fig. 4, E–H). At stage 9, the cells were still ruptured in the split plane after pulling off the corolla (Fig. 4H). To quantify this abscission defect in the transgenic corollas, we conducted a force test to measure the strength required to tear the corolla from the receptacle. As shown in Figure 4I, the force required to tear the corolla from the receptacle of the wild-type flowers declined as corolla development progressed. The required force finally dropped to about 0 g, due to the degradation of the middle lamella of the AZ cells (Lease et al., 2006). However, this decrease in breakstrength did not occur in the corollas of the NtBOP2-AS flowers, even on the final day of testing (Fig. 4I), indicating that the cells in the region corresponding to wild-type AZ were more adhesive to one another compared with the wild-type control. Transmission electron microscopy examination of the ultrastructures of the AZ cells in the wild-type corolla and the cells at the equivalent position in the NtBOP2-AS corolla supported this interpretation. As Figure 4, J and K, shows, some wild-type AZ cells were well separated at stage 7, due to the dissolution of the middle lamella (Fig. 4J); in cells at the region corresponding to wild-type AZ in the NtBOP2-AS corolla, large amounts of osmiophilic material (undegraded middle lamella) appeared in the middle lamella (Fig. 4K), which indicates that the degradation of the middle lamella did not take place and neighboring cells remained adhered to one another. Collectively, these results suggested that the failure of corolla shedding in the NtBOP2-AS flowers was due to the lack of an AZ structure. Figure 4. Open in new tabDownload slide Scanning and transmission electron micrographs of wild-type corolla AZ and corresponding regions in NtBOP2-AS corollas. A to H, Scanning electron microscopy images. The wild-type corollas (A–D) naturally abscised at stage 9, whereas corollas of NtBOP2-AS (E–H) were forcibly removed from flowers and broken cells could be seen at all stages. Numbers indicate the corolla developmental stages. The arrow in C indicates the round cells. Bars = 100 µm. I, Measurement of the corolla breakstrength. Samples were taken from the day when flowers were fully open. The force required to remove wild-type corollas (black bars) decreased, whereas the breakstrength for the corollas (gray bars) in the transgenic flowers was almost unchanged. Ten corollas were used for each day measurement. Values are means ± sd. J and K, Transmission electron microscopy analysis of the middle lamella between wild-type (WT) AZ cells at stage 7 and corresponding cells of NtBOP2-AS. J, AZ cells in wild-type corolla. K, Cells in the region corresponding to wild-type AZ in the NtBOP2-AS corolla. cw, Cell wall; ml, middle lamella. Bars = 200 nm. Figure 4. Open in new tabDownload slide Scanning and transmission electron micrographs of wild-type corolla AZ and corresponding regions in NtBOP2-AS corollas. A to H, Scanning electron microscopy images. The wild-type corollas (A–D) naturally abscised at stage 9, whereas corollas of NtBOP2-AS (E–H) were forcibly removed from flowers and broken cells could be seen at all stages. Numbers indicate the corolla developmental stages. The arrow in C indicates the round cells. Bars = 100 µm. I, Measurement of the corolla breakstrength. Samples were taken from the day when flowers were fully open. The force required to remove wild-type corollas (black bars) decreased, whereas the breakstrength for the corollas (gray bars) in the transgenic flowers was almost unchanged. Ten corollas were used for each day measurement. Values are means ± sd. J and K, Transmission electron microscopy analysis of the middle lamella between wild-type (WT) AZ cells at stage 7 and corresponding cells of NtBOP2-AS. J, AZ cells in wild-type corolla. K, Cells in the region corresponding to wild-type AZ in the NtBOP2-AS corolla. cw, Cell wall; ml, middle lamella. Bars = 200 nm. Apart from NtBOP2-AS corollas, we also carried out the above analyses in NtBOP2-OE and NtBOP2t-OE plants, and similar results were obtained. Thus, we focused on the NtBOP2-AS plants in subsequent experiments. Analysis of the Expression Features of Some Abscission-Associated Genes in Wild-Type and NtBOP2-AS Corollas Several genes encoding for abscission-related hydrolytic enzymes such as cellulase, expansin, and polygalacturonase have been shown to be up-regulated in the differentiated AZ during organ abscission in plants (Lashbrook et al., 1994; Patterson, 2001; González-Carranza et al., 2007; Sane et al., 2007). If the corolla AZ structure was lacking in the NtBOP2-AS flowers, then expression of these genes should not be up-regulated in the region corresponding to wild-type AZ. Hence, we analyzed the expression features of several candidate abscission-related genes of tobacco, including five cellulase genes (named NtCel2, NtCel4, NtCel5, NtCel7, and NtCel8; Goellner et al., 2001), six expansin genes (NtEXPA1–NtEXPA6; Link and Cosgrove, 1998), one pectate lyase gene (Nt59; Kulikauskas and McCormick, 1997), and one polygalacturonase gene (Npg1; Tebbutt and Lonsdale, 1993). Real-time PCR was conducted to compare the expression status of these genes during AZ development in wild-type corolla. It turned out that among these genes, only NtCel5 and NtEXPA5 showed higher expression in corolla base (AZ region) as compared with the corolla tube (non-AZ region; Fig. 5, A and B) and exhibited highly different expression levels between differentiated AZ and undifferentiated tissues (Fig. 5, C and D). Expression features of these two genes in wild-type corolla prior to and after AZ formation and in the corresponding developmental stages of NtBOP2-AS corolla were then compared. Total RNAs were extracted from segments of corolla base (approximately 2 mm) at stage 4 (prior to AZ formation), stage 5 (AZ formed), stage 6 (abscission initiated), and stage 7 (abscission in process). Real-time PCR analysis revealed that expression levels of these two genes from stage 5 to stage 7 were significantly higher than those at stage 4 in the wild-type corollas, indicating that the expression of these genes was activated in differentiated AZ (Fig. 5, C and D). On the contrary, no significant changes in the transcript amounts of these genes were observed during the corresponding stages in the base cells of the transgenic corollas (Fig. 5, C and D). Such uninduced expression profiles of NtCel5 and NtEXPA5 are consistent with the lack of an AZ anatomy in the transgenic corollas. Figure 5. Open in new tabDownload slide Expression levels of two abscission-related genes in wild-type and NtBOP2-AS corollas. A and B, Expression patterns of NtCel5 and NtEXPA5 genes in wild-type corollas. Total RNAs were isolated from base (AZ; approximately 2 mm) and tube (non-AZ) segments of corollas. The expression levels were evaluated by real-time PCR, and the values from the corolla tube (non-AZ) were set as 1. C and D, Expression levels of NtCel5 (C) and NtEXPA5 (D) genes during stages 4 to 7 of corolla development. Total RNAs were extracted from differentiated AZ and undifferentiated tissues of wild-type (WT) corollas and the corresponding parts of the NtBOP2-AS corollas. The expression levels were evaluated by real-time PCR. The numbers indicate the corolla developmental stages. The values in undifferentiated AZ (stage 4) of wild-type corollas were set as 1. The expression levels were normalized to those of Histone3. Error bars indicate the sd of three technical replicates of one biological experiment. At least three biological repeats were performed. Figure 5. Open in new tabDownload slide Expression levels of two abscission-related genes in wild-type and NtBOP2-AS corollas. A and B, Expression patterns of NtCel5 and NtEXPA5 genes in wild-type corollas. Total RNAs were isolated from base (AZ; approximately 2 mm) and tube (non-AZ) segments of corollas. The expression levels were evaluated by real-time PCR, and the values from the corolla tube (non-AZ) were set as 1. C and D, Expression levels of NtCel5 (C) and NtEXPA5 (D) genes during stages 4 to 7 of corolla development. Total RNAs were extracted from differentiated AZ and undifferentiated tissues of wild-type (WT) corollas and the corresponding parts of the NtBOP2-AS corollas. The expression levels were evaluated by real-time PCR. The numbers indicate the corolla developmental stages. The values in undifferentiated AZ (stage 4) of wild-type corollas were set as 1. The expression levels were normalized to those of Histone3. Error bars indicate the sd of three technical replicates of one biological experiment. At least three biological repeats were performed. Expression Pattern of the NtBOP2 Gene To determine whether the function of NtBOP2 in corolla abscission is correlated to its expression pattern, RT-PCR analysis was conducted with the total RNA extracted from various organs of the tobacco plants with gene-specific primers. To analyze the expression pattern of NtBOP2 in corolla, segments (approximately 2 mm) of the corolla bases (AZ) were collected. The corolla tube region (non-AZ) was used as a control tissue. The results showed that NtBOP2 was expressed at a low level in the roots, and the transcripts were hardly detected in the leaves and stems. In corollas, NtBOP2 was primarily expressed in the AZ region, whereas the transcript level was rather low in the adjacent tissues (Fig. 6A). This expression pattern of NtBOP2 was further confirmed by GUS expression. A 1.5-kb fragment upstream of the NtBOP2 ORF was cloned by genomic walking, and the GUS cDNA was cloned under the control of this fragment in a plant expression vector. The NtBOP2pro:GUS construct was transformed into tobacco plants, and the expression pattern was detected. As seen in Figure 6B, GUS expression was predominantly exhibited at the base of the corolla during stages 1 to 5 (before corolla shedding). No GUS staining was detected in the immature tobacco seedlings. Figure 6. Open in new tabDownload slide Expression pattern of NtBOP2 in tobacco plants. A, RT-PCR analysis of NtBOP2 expression in various organs of wild-type tobacco plants. The organs were harvested from 10-week-old soil-grown plants. The transcript of NtBOP2 was amplified using primers BRT-F2 and BRT-R. The 18S rRNA gene was used as an internal control. NtBOP2, 30 cycles; 18S, 16 cycles. B, Histochemical GUS staining of flowers from plants transformed with the NtBOP2pro:GUS construct. GUS activity was predominantly exhibited at the base of corollas from budding to fully open stages during floral development. The corolla development stages are indicated at the bottom. [See online article for color version of this figure.] Figure 6. Open in new tabDownload slide Expression pattern of NtBOP2 in tobacco plants. A, RT-PCR analysis of NtBOP2 expression in various organs of wild-type tobacco plants. The organs were harvested from 10-week-old soil-grown plants. The transcript of NtBOP2 was amplified using primers BRT-F2 and BRT-R. The 18S rRNA gene was used as an internal control. NtBOP2, 30 cycles; 18S, 16 cycles. B, Histochemical GUS staining of flowers from plants transformed with the NtBOP2pro:GUS construct. GUS activity was predominantly exhibited at the base of corollas from budding to fully open stages during floral development. The corolla development stages are indicated at the bottom. [See online article for color version of this figure.] NtBOP2 Plays a Role in Controlling Longitudinal Cell Expansion Required for AZ Differentiation The phenotype exhibited by the NtBOP2-AS plants and the corolla-specific expression in wild-type plants demonstrate the important role of the gene in corolla abscission. Moreover, the lack of an AZ in NtBOP2-AS plants indicated that NtBOP2 acted in the process of AZ differentiation. To assess this function of the NtBOP2 gene at the cellular level, the anatomical structures of the AZ in wild-type corolla and the region corresponding to wild-type AZ in the NtBOP2-AS corolla were examined and compared. At early stages of corolla development, the wild-type and transgenic corollas had similar cellular structures, with short cells from the proximal to the distal region (Fig. 7, A, B, F, and G). Later, AZ appeared in the wild-type corolla by the formation of several layers of shorter cells that were established by inhibited expansion in the longitudinal direction. Although these AZ cells did not show the typically dense cytoplasm, they were morphologically different from adjacent cells, particularly being much shorter than the distal cells (Fig. 7, C and K). Subsequently, the middle lamella of the AZ cells dissolved (Fig. 7, D and L) and the corolla detached from the receptacle (Fig. 7E). Unlike the wild-type control, the cells in the region corresponding to wild-type AZ in the NtBOP2-AS corolla were unable to assume a shorter cell morphology, but they continued to grow longitudinally in all stages and formed elongated cells (Fig. 7, H–J). As a result, the distinct cell layers at the junction of the corolla and receptacle observed in the wild-type flowers were absent in the NtBOP2-AS flowers (Fig. 7, H–J, M, and N). These histological data revealed that NtBOP2 functioned in corolla abscission by controlling longitudinal cell expansion, which is required for differentiation of the AZ. Figure 7. Open in new tabDownload slide Anatomical comparisons between AZ of wild-type corolla and the corresponding region in NtBOP2-AS. A to J, Light microscopic analysis of longitudinal sections of the flower bases in wild-type (A–E) and NtBOP2-AS (F–J) plants. The boxed regions show AZ in wild-type corolla and the corresponding part in transgenic corolla. The numbers show the developmental stages of the corollas. C, Corolla; cw, carpel wall. Bars = 250 µm. K to N, Magnified views of AZ cells in wild-type corollas (K and L) and corresponding cells in the NtBOP2-AS corollas (M and N). K and M, Stage 5. L and N, Stage 7. Bars = 200 µm. O, Schematic illustration of the AZ differentiation and abscission process in wild-type (WT) corolla and the alterations in the NtBOP2-AS corolla. Dark gray rounded rectangles represent AZ cells, and light gray rounded rectangles represent non-AZ cells. Figure 7. Open in new tabDownload slide Anatomical comparisons between AZ of wild-type corolla and the corresponding region in NtBOP2-AS. A to J, Light microscopic analysis of longitudinal sections of the flower bases in wild-type (A–E) and NtBOP2-AS (F–J) plants. The boxed regions show AZ in wild-type corolla and the corresponding part in transgenic corolla. The numbers show the developmental stages of the corollas. C, Corolla; cw, carpel wall. Bars = 250 µm. K to N, Magnified views of AZ cells in wild-type corollas (K and L) and corresponding cells in the NtBOP2-AS corollas (M and N). K and M, Stage 5. L and N, Stage 7. Bars = 200 µm. O, Schematic illustration of the AZ differentiation and abscission process in wild-type (WT) corolla and the alterations in the NtBOP2-AS corolla. Dark gray rounded rectangles represent AZ cells, and light gray rounded rectangles represent non-AZ cells. A schematic diagram was drawn to show the process of corolla AZ development in the wild-type flower and the elongated cells at the region corresponding to wild-type AZ in the NtBOP2-AS flower (Fig. 7O). Perturbed Expression of NtBOP2 in BY-2 Cells Caused the Formation of Highly Elongated Cells To better characterize the function of NtBOP2 in controlling longitudinal cell expansion, we tested its role in the BY-2 single cell system. A truncated form (NtBOP2t) of NtBOP2, which caused a phenotype similar to that seen in NtBOP2-AS when overexpressed in tobacco, was overexpressed in BY-2 cells (Fig. 8A). The phenotype of the transgenic cells was examined and compared with the wild-type control. As seen in Figure 8, B and C, most of the BY-2 cells were spherical under the experimental condition, but the majority of NtBOP2t-OE transgenic cells (approximately three-fourths) were elongated. To determine whether cell division was affected in these cells, fluorescent staining was performed using the nuclear indicator 4′,6-diamino-phenylindole (DAPI) and the cell wall indicator calcofluor. The nuclear division and cell plate formation proceeded normally in the NtBOP2t-OE transgenic cells (Fig. 8, D and E). These results indicated that cell elongation was not coordinately controlled in the transgenic cells, consistent with the data from the studies of transgenic tobacco plants. Figure 8. Open in new tabDownload slide Morphological changes of the transgenic BY-2 cells overexpressing NtBOP2t. A, RT-PCR analysis of NtBOP2t/NtBOP2 expression in wild-type (WT) and transgenic BY-2 cells. Total RNA was extracted from exponentially growing cells. The 18S rRNA gene was used as an internal control. NtBOP2t/NtBOP2 was amplified using primers BRT-F2 and BRT-R. NtBOP2t/NtBOP2, 30 cycles; 18S, 16 cycles. B to E, Morphological differences of wild-type and NtBOP2t-OE BY-2 cells after a 7-d subculture. Bright-field (B and C) and fluorescence (D and E) analyses are shown. Bars = 100 µm. Figure 8. Open in new tabDownload slide Morphological changes of the transgenic BY-2 cells overexpressing NtBOP2t. A, RT-PCR analysis of NtBOP2t/NtBOP2 expression in wild-type (WT) and transgenic BY-2 cells. Total RNA was extracted from exponentially growing cells. The 18S rRNA gene was used as an internal control. NtBOP2t/NtBOP2 was amplified using primers BRT-F2 and BRT-R. NtBOP2t/NtBOP2, 30 cycles; 18S, 16 cycles. B to E, Morphological differences of wild-type and NtBOP2t-OE BY-2 cells after a 7-d subculture. Bright-field (B and C) and fluorescence (D and E) analyses are shown. Bars = 100 µm. Subcellular Localization of NtBOP2 Proteins In order to understand the molecular mechanism by which NtBOP2 exerts its function, the subcellular distribution of the NtBOP2 proteins was visualized using both transient expression and stable expression systems. Plasmid vectors encoding the N-terminal or the C-terminal GFP fusion of NtBOP2 were introduced into the protoplasts of BY-2 cells and tobacco leaf epidermal cells, respectively. GFP fluorescence was examined by confocal laser scanning. As can be seen in Figure 9, A to F, transiently expressed NtBOP2-GFP fusion proteins were distributed in both the nucleus and cytoplasm of BY-2 protoplasts and leaf epidermal cells. This subcellular distribution pattern was further confirmed by expressing the two types of NtBOP2-GFP fusion proteins in stable transgenics of BY-2 cells (Fig. 9, G–I). Figure 9. Open in new tabDownload slide Subcellular localization of NtBOP2-GFP fusion proteins. A to I, Confocal images of GFP fluorescence in BY-2 protoplasts (A–C), infiltrated tobacco leaf epidermal cells (D–F), and BY-2 cells (G–I). GFP and NtBOP2-GFP fusion proteins were transiently expressed in A to C and D to F and stably expressed in G to I. n, Nucleus. Bars = 25 µm. J, Western-blot analysis of GFP and GFP fusion proteins in 35S:GFP, 35S:GFP-NtBOP2, and 35S:NtBOP2-GFP transgenic BY-2 cells. Proteins were extracted from cells subcultured for 7 d, and western-blot analysis was conducted with the GFP antibody. The bottom panel shows the SDS-polyacrylamide gel that indicates the equal loading of protein samples. [See online article for color version of this figure.] Figure 9. Open in new tabDownload slide Subcellular localization of NtBOP2-GFP fusion proteins. A to I, Confocal images of GFP fluorescence in BY-2 protoplasts (A–C), infiltrated tobacco leaf epidermal cells (D–F), and BY-2 cells (G–I). GFP and NtBOP2-GFP fusion proteins were transiently expressed in A to C and D to F and stably expressed in G to I. n, Nucleus. Bars = 25 µm. J, Western-blot analysis of GFP and GFP fusion proteins in 35S:GFP, 35S:GFP-NtBOP2, and 35S:NtBOP2-GFP transgenic BY-2 cells. Proteins were extracted from cells subcultured for 7 d, and western-blot analysis was conducted with the GFP antibody. The bottom panel shows the SDS-polyacrylamide gel that indicates the equal loading of protein samples. [See online article for color version of this figure.] As we noticed that the fluorescent signal pattern of cells containing GFP alone appeared similar to that of the cells expressing the two types of GFP fusion proteins, a western-blot experiment was carried out to check the size of the GFP fusion proteins in BY-2 cells using GFP antibody. An approximately 80-kD band corresponding to the predicted size of NtBOP2-GFP fusion proteins was detected in the cells transformed with the plasmid constructs encoding NtBOP2-GFP fusion proteins, whereas in the cells transformed with the plasmid encoding GFP, the protein band corresponded to the size of the GFP (approximately 26 kD) alone (Fig. 9J). This result indicated that the fluorescence in the NtBOP2-GFP transgenic cells was emitted by the fusion proteins. NtBOP2 Interacts with TGA Transcription Factors and Forms Dimers The nuclear localization of the NtBOP2 proteins suggested that it may act in the nucleus by interacting with some other nuclear factors. In a previous study, Arabidopsis BOP proteins were shown to physically interact with the TGA transcription factor PERIANTHIA in yeast (Hepworth et al., 2005). As NtBOP2 shows high homology to the two BOPs in Arabidopsis and contains the conserved protein domains, we predicted that NtBOP2 would also possess this activity. To verify this speculation, five tobacco TGAs were identified from an available database, and the ORF of each gene was cloned into the yeast two-hybrid (Y2H) pGADT7 vector containing the transcriptional activation domain (AD) of GAL4. The NtBOP2 ORF was then cloned into the pGBKT7 vector that contains the sequence encoding the DNA-binding domain (BD) of GAL4. The yeast cotransformed with vectors harboring NtBOP2-BD and the pGADT7 empty vector could not grow on the plate with synthetic dextrose/-Ade/-His/-Leu/-Trp medium. However, the yeast cells transformed with NtBOP2-BD and TGA-AD vectors grew normally. This interaction of the proteins was tested using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) filter assay and β-galactosidase quantitative activity measurement. The results indicated that NtBOP2 was able to interact with these five TGA transcription factors in yeast (Fig. 10A). In addition to Y2H, the luciferase (Luc) complementation imaging assay was also performed to see the interaction between NtBOP2 and the TGAs. For each test, NtBOP2 was fused N terminal to the NLuc fragment, whereas individuals of the TGA transcription factors were fused C terminal to the CLuc fragment. As expected, coinjection of the vector constructs into the tobacco leaves resulted in the same interaction features of the proteins (Fig. 10B) as observed in Y2H. To further verify the interaction of NtBOP2 with TGAs, an in vitro pull-down assay was carried out. The TGA proteins fused to the glutathione S-transferase (GST) tag and NtBOP2 protein fused to the maltose-binding protein (MBP) tag were expressed in the bacterial system and purified. GST fusion proteins binding to glutathione beads were used as baits and incubated with MBP fusion proteins. As shown in Figure 10C, GST-TGA fusion proteins pulled down the MBP-NtBOP2 proteins, but the GST proteins alone did not, indicating that TGAs interacted with NtBOP2 in vitro. Figure 10. Open in new tabDownload slide NtBOP2 proteins interact with TGA transcription factors. A, Y2H analyses of interactions between NtBOP2 and TGA transcription factors and homodimerization of NtBOP2 proteins. NtBOP2 was used as the bait protein. Prey proteins are shown on the left. β-Galactosidase activity was tested by X-gal filter assay and quantitatively measured using o-nitrophenyl-β-galactopranoside as the substrate. Error bars represent se of three independent transformants. E, Empty vector. B, Firefly Luc complementation imaging assay showing the interaction of NtBOP2 with TGAs. Vectors expressing NtBOP2-NLuc and CLuc fused with TGA transcription factors were cotransformed into expanding tobacco leaves by infiltration. E, Empty vector. C, Pull-down assay showing the interactions between NtBOP2 and TGA transcription factors and the dimerization of NtBOP2 proteins. GST-TGA or GST-NtBOP2 fusion proteins were used as bait, and NtBOP2 fused with MBP was used as prey. The anti-GST and anti-MBP antibodies were used to detect bait and prey proteins, respectively. Asterisks indicate the bait proteins. GST protein was used as a negative control. [See online article for color version of this figure.] Figure 10. Open in new tabDownload slide NtBOP2 proteins interact with TGA transcription factors. A, Y2H analyses of interactions between NtBOP2 and TGA transcription factors and homodimerization of NtBOP2 proteins. NtBOP2 was used as the bait protein. Prey proteins are shown on the left. β-Galactosidase activity was tested by X-gal filter assay and quantitatively measured using o-nitrophenyl-β-galactopranoside as the substrate. Error bars represent se of three independent transformants. E, Empty vector. B, Firefly Luc complementation imaging assay showing the interaction of NtBOP2 with TGAs. Vectors expressing NtBOP2-NLuc and CLuc fused with TGA transcription factors were cotransformed into expanding tobacco leaves by infiltration. E, Empty vector. C, Pull-down assay showing the interactions between NtBOP2 and TGA transcription factors and the dimerization of NtBOP2 proteins. GST-TGA or GST-NtBOP2 fusion proteins were used as bait, and NtBOP2 fused with MBP was used as prey. The anti-GST and anti-MBP antibodies were used to detect bait and prey proteins, respectively. Asterisks indicate the bait proteins. GST protein was used as a negative control. [See online article for color version of this figure.] It has been recently reported that AtBOP proteins form homodimers in vivo (Jun et al., 2010). The presence of several Cys residues in NtBOP2 also implied that the protein may form a dimer. Y2H analysis was performed to test this possibility. The NtBOP2 ORF was cloned into BD and AD vectors, and homodimer formation was tested by examining the interaction of two proteins expressed from each vector. The results showed that NtBOP2 proteins interacted in the Y2H assay (Fig. 10A). This self-interaction of NtBOP2 proteins was confirmed by the in vitro pull-down assay (Fig. 10C). Thus, the activity of NtBOP2 may rely upon homodimerization or the formation of heterodimers with other BOP homologs. NtBOP2 Mediates Corolla Abscission Probably via an Ethylene-Independent Pathway In Arabidopsis, both ethylene-dependent and -independent pathways have been reported to be involved in floral abscission (Patterson and Bleecker, 2004). In tobacco, it has been reported that transgenic plants ectopically expressing the Arabidopsis ETR1-1 gene exhibited a silenced ethylene response and corolla abscission deficiency (Yang et al., 2008). To determine in which pathway NtBOP2 participates to mediate corolla abscission, wild-type and NtBOP2-AS plants grown in soil for 10 weeks were sprayed with 0.1 g L−1 ethephon (the precursor of ethylene). The corolla abscission in NtBOP2-AS plants was still blocked, which indicated that external application of ethylene was unable to restore the defective corolla abscission in the NtBOP2-AS plants (data not shown). To verify this result, expanded flowers were cultured in vitro in medium containing 0.5 g L−1 ethephon. After 24 h, a brown ring formed in the base of the wild-type corolla, yet this ring was not observed with the explants incubated in the medium without ethephon. Conversely, the NtBOP2-AS corollas did not display the ring structure regardless of whether the ethephon was present or absent in the medium (Fig. 11, A and B). These results indicated that although ethylene promoted the process of corolla abscission in the wild-type plant, it did not restore the corolla abscission deficiency in the NtBOP2-AS plant, indicating that the function of NtBOP2 was not associated with ethylene signaling. Subsequent real-time PCR analysis of NtBOP2 expression in the wild-type plant confirmed this result. As shown in Figure 11C, the expression levels of NtBOP2 did not change after treatment with 0.5 g L−1 ethephon for 6 and 18 h in wild-type corollas. As it has been reported that the expression of two tomato cellulase genes (Cel1 and Cel2) was induced by the external application of ethylene (Lashbrook et al., 1994), we tested the expression profile of NtCel5 (a homolog of tomato cellulase genes) as a reference experiment. Like the tomato cellulase genes, the expression of NtCel5 was clearly induced by ethylene (Fig. 11D). Together, these results suggested that NtBOP2 mediates corolla abscission in an ethylene-independent manner. Figure 11. Open in new tabDownload slide NtBOP2-mediated corolla AZ differentiation in an ethylene-independent manner. A and B, The bases of wild-type (WT) and NtBOP2-AS flowers without or with ethephon treatment. Explants were cultured in the absence (A) or presence of ethephon (B) for 24 h. C and D, Real-time PCR analysis of NtBOP2 (C) and NtCel5 (D) expression after ethephon treatment in wild-type corollas. Results were normalized to the expression of Histone3, then to the value of an untreated explant that was set to 1. Error bars indicate sd of three technical repeats. Two independent biological replicates were performed. [See online article for color version of this figure.] Figure 11. Open in new tabDownload slide NtBOP2-mediated corolla AZ differentiation in an ethylene-independent manner. A and B, The bases of wild-type (WT) and NtBOP2-AS flowers without or with ethephon treatment. Explants were cultured in the absence (A) or presence of ethephon (B) for 24 h. C and D, Real-time PCR analysis of NtBOP2 (C) and NtCel5 (D) expression after ethephon treatment in wild-type corollas. Results were normalized to the expression of Histone3, then to the value of an untreated explant that was set to 1. Error bars indicate sd of three technical repeats. Two independent biological replicates were performed. [See online article for color version of this figure.] DISCUSSION The morphology of the cells in AZs is usually distinct from the surrounding tissues. According to the cell anatomy features, the following three features of floral AZs have been characterized: (1) cells in the AZ are relatively smaller and more isodiametric than the neighboring cells; (2) AZ cells are small and oblong compared with the adjacent cells; (3) cells in the AZ are similar in size to the surrounding cells (vanDoorn and Stead, 1997). In Arabidopsis, the floral AZ consists of a few layers of small and densely cytoplasmic cells that, unlike the surrounding tissues, are unable to enlarge (Bleecker and Patterson, 1997; Patterson, 2001). Here, we observed that the tobacco corolla AZ cells were distinct. They appeared shorter than the neighboring cells but shared similar cell width with them. In addition, the cytoplasm of these cells was not typically dense. These variations in AZ cell morphology indicate that their formation follows active cell-shaping programs in different plant species or floral parts. NtBOP2 Contributes to the Differentiation of the Corolla AZ by Controlling Longitudinal Cell Expansion In Arabidopsis, the loss of floral organ abscission in bop1 bop2 is correlated with an absence of the typical cellular anatomy of the AZ (McKim et al., 2008). Based on these results, the authors proposed that the AZ anatomy is necessary for abscission and that BOP may initiate AZ differentiation. In this study, we also found a deficiency in the formation of the typical corolla AZ anatomy in NtBOP2 transgenic plants (NtBOP2-AS, NtBOP2-OE, and NtBOP2t-OE). Thus, data from both Arabidopsis and tobacco demonstrated that BOP proteins were required for the differentiation of floral AZs. To understand the process of AZ differentiation in tobacco corolla, we examined the cell morphology of tobacco corollas at different developmental stages. The results showed that, in the corolla AZ of wild-type flowers, cells were unable to elongate longitudinally, as compared with the distally adjacent cells. This phenomenon resulted in the formation of shorter cells that constituted the AZ. In the NtBOP2-AS plants, however, such a cell shape variation in the region corresponding to wild-type AZ did not occur. Instead of forming shorter cells, the majority of the cells appeared fairly elongated and the specialized wild-type AZ anatomy was absent. These results demonstrated that NtBOP2 is involved in the regulation of longitudinal cell growth during AZ differentiation. In support of this notion, we found that overexpression of a dominant negative-type NtBOP2 resulted in the formation of elongated BY-2 cells, indicating that longitudinal elongation of these cells was uncontrolled. Again, this result from the study of the single-cell system indicated that NtBOP2 had a critical role in regulating cell length. NtBOP2 May Act as a Coregulator of TGA Transcription Factors BOP proteins belong to the NPR1 family, whose members play key roles in systemic acquired resistance in plants. NPR1 specifically interacts with five members of the Arabidopsis TGA family (TGA2, TGA3, TGA5, TGA6, and TGA7) to regulate pathogenesis-related genes (Zhang et al., 1999; Després et al., 2000; Boyle et al., 2009). It has been reported that Arabidopsis BOP proteins interacted with TGA transcription factors; therefore, these proteins are considered to be transcription regulators (Hepworth et al., 2005). Our experimental results from the Y2H, Luc complementation imaging, and in vitro pull-down assays demonstrated that NtBOP2 interacted with five TGA transcription factors. Because our study indicates a role for NtBOP2 in the regulation of cell elongation, this raised the question of whether such a function of NtBOP2 was associated with the TGA transcription factors. Interestingly, Fukazawa et al. (2000) showed that a TGA transcription factor, RSG (for repression of shoot growth), participated in regulating plant cell elongation through the transcriptional control of a GA biosynthetic enzyme. In the RSG dominant negative mutant, the plant appeared dwarf and the cell elongation of stems was severely inhibited. Based on this information, we assume that the NtBOP2 protein may work as a transcription coregulator by interacting with TGA to regulate the cell elongation process in tobacco. In this regard, it will be interesting to investigate the functional link between these two families of proteins in the regulation of cell length in our future work. Multiple BOPs Are Involved in Regulating Corolla Abscission in Tobacco In our study, we generated the following four types of transgenic tobacco plants: overexpressing intact NtBOP2 (NtBOP2-OE), overexpressing truncated NtBOP2 (NtBOP2t-OE), underexpressing NtBOP2 by RNAi (NtBOP2-RNAi), and underexpressing NtBOP2 by antisense approach (NtBOP2-AS). We first examined the NtBOP2-RNAi transgenic plants, which did not exhibit significant phenotypic change as compared with the wild-type control. This implied a functional redundancy of NtBOP2 with other members of the BOP family in tobacco. A similar phenomenon has been reported in Arabidopsis (Ha et al., 2004; Hepworth et al., 2005). However, the antisense NtBOP2 transgenic plants exhibited a complete loss of corolla shedding, suggesting that functional redundancy of the BOP proteins was overcome due to the inhibition of BOP genes by antisense NtBOP2. To test this hypothesis, three BOP homolog genes in tobacco were amplified by 3′ RACE and designated as NtBOP1, NtBOP3, and NtBOP4, and their sequence homology with NtBOP2 was analyzed (Supplemental Fig. S1). The expression levels of these three BOP genes in wild-type, NtBOP2-OE, NtBOP2t-OE, NtBOP2-RNAi, and NtBOP2-AS plants were detected by real-time PCR (Supplemental Fig. S2). In NtBOP2-AS plants, the expression of three BOP genes was inhibited compared with the wild-type control. In NtBOP2-RNAi plants, the expression of NtBOP1, which shares 92% sequence identity with NtBOP2, was down-regulated, but the expression levels of NtBOP3 and NtBOP4 genes were unaffected, supporting the hypothesis that the absence of phenotypic alteration of NtBOP2-RNAi plants was probably due to the function redundancy of these BOPs with NtBOP2. Although this result indicated that more than one tobacco BOP gene was involved in corolla abscission, as was seen in Arabidopsis, the role of NtBOP2 was made further apparent by the phenotypes of the transgenic plants overexpressing either the intact or truncated NtBOP2 genes. In these two types of transgenic plants, the expression levels of NtBOP1, NtBOP3, and NtBOP4 genes remained almost unchanged as compared with the wild-type control, but the plants showed similar defects in corolla shedding to the antisense transgenics, supporting the role of NtBOP2 in corolla AZ development. The Corolla Abscission in Tobacco May Be Modulated by a Protein Complex The defects in corolla abscission caused by overexpression of either an intact or a truncated NtBOP2 gene suggested that excess production of the proteins had a dominant negative effect on the function of endogenous NtBOP2 or other interacting proteins. It is known that a dominant negative effect typically occurs in association with proteins that form dimers or macromolecular complexes, such as the transcriptional complex (Herskowitz, 1987; Veitia, 2007; Hirano et al., 2011). A recent study by Xu et al. (2010b) demonstrated that AtBOP1/2 proteins were recruited to the promoter of AP1 through direct interaction with TGA transcription factors during the floral transition in Arabidopsis. Accordingly, we found that NtBOP2 formed homodimers and that it interacts with the TGA transcription factors. These data suggest that NtBOP2 may exert its function in corolla AZ differentiation through a protein complex that contains TGAs and other BOPs. It is possible that overproduced NtBOP2t or NtBOP2 proteins competed with the endogenous BOP proteins or caused stoichiometric imbalance among the protein components and hence disturbed the formation of a functional protein complex and resulted in the mutant phenotypes. The dominant negative effect of BOP proteins was also reported for the Arabidopsis BOPs. The Arabidopsis bop1-1 mutant showed an enhanced expression of BOP1-1 mutant protein (containing four additional amino acids at the C terminus) but exhibited a phenotype similar to that of the bop1 bop2 double knockdown mutant. The authors speculated that this phenotype was caused by a dominant negative effect, possibly by interference with the activity of an interacting partner, such as AtBOP2 (Ha et al., 2004; Jun et al., 2010). Because the Arabidopsis bop1 bop2 double mutant and the NtBOP2 transgenic plants showed similar defects in floral abscission, we tested the functional complementation activity of NtBOP2 to the Arabidopsis mutant. It turned out that ectopic overexpression of NtBOP2 did not complement the Arabidopsis mutant (Supplemental Fig. S3). This result suggested that NtBOP2 may have alternative interacting proteins in the tobacco AZ. Alternatively, the large amount of NtBOP2 proteins may have had a dominant negative effect on Arabidopsis floral abscission, as was seen in the NtBOP2-OE tobacco plants. MATERIALS AND METHODS Plant Material and Cell Culture Tobacco (Nicotiana tabacum ‘Petit Havana SR1’) and Arabidopsis (Arabidopsis thaliana ecotype Columbia-1 and bop1 bop2 mutant) plants were grown in the growth chamber under 16-h-light/8-h-dark conditions at 23°C and watered weekly with Murashige and Skoog nutrient solution. Tobacco BY-2 cells were maintained as described previously (Nagata and Kumagai, 1999). Sequence Alignment and Phylogenetic Analysis The protein sequences were aligned using ClustalW, and the neighbor-joining method was used to produce the phylogenetic tree using the MEGA program version 4 (Tamura et al., 2007). Vector Construction and Plant Transformation The cDNA of NtBOP2 (nucleotides 1–1,684) was inserted into the binary expression vector pPZP111 (Hajdukiewicz et al., 1994) in both sense and antisense orientations under the control of the cauliflower mosaic virus (CaMV) 35S promoter to generate overexpression (35S:NtBOP2) and antisense (35S:antiNtBOP2) expression vectors. The cDNA fragment encoding the truncated form of NtBOP2 lacking 75 amino acids in the C-terminal end was amplified using the primers BN-F and BN-R (Supplemental Table S1) and cloned into pPZP111. The resulting construct was named 35S:NtBOP2t. To construct the RNAi vector, an approximately 0.6-kb cDNA of NtBOP2 (nucleotides 1,031–1,622) was amplified using the primers RNAi-F and RNAi-R (Supplemental Table S1) and cloned into the vector pHANNIBAL in the sense and antisense orientations to create a hairpin structure. The NotI-digested fragment was subcloned into the binary expression vector pART27. All vectors were introduced into the Agrobacterium tumefaciens strain EHA105. Transgenic tobacco plants and BY-2 cells were generated by A. tumefaciens-mediated leaf disc (Horsch et al., 1985) and cell (Nagata et al., 1992) transformation, respectively. Assessment of the functional complementation of the Arabidopsis bop1 bop2 mutant was performed by the floral dip method as described previously (Clough and Bent, 1998). RT-PCR and Quantitative Real-Time PCR Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. First-strand cDNAs were synthesized from 1 µg of DNase I (Takara)-treated RNA using random primer p(N)6 and ReverTra Ace reverse transcriptase (TOYOBO). One microliter of the first-strand cDNA reaction was used as a template for RT-PCR using LA Taq polymerase (Takara). The 18S rRNA gene was used as an internal control. Quantitative real-time PCR assays were performed by using the SYBR Green Real-Time PCR Master Mix (Toyobo) and the DNA Engine Opticon 2 Real-Time PCR Detection System (MJ Research). The tobacco Histone3 gene was used as an internal control. All reactions were performed in triplicate. Primer sequences are listed in Supplemental Table S1. Western-Blot Analysis Total proteins were extracted from BY-2 suspension cells 7 d after subculture using a buffer containing 50 mm NaPO4 (pH 7.4), 0.5 m NaCl, 1 mm EDTA, 1% (v/v) β-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, and 10% (w/v) polyvinylpolypyrrolidone. Twenty micrograms of protein was separated on a 15% SDS-PAGE gel and transferred to a nitrocellulose membrane (Amersham Biosciences). The filter was blocked overnight with 5% (w/v) milk powder at 4°C and subsequently incubated with GFP antibody (1:2,000; Abmart) for 1 h at 37°C. After three washes, the filter was incubated for 1 h with goat anti-mouse IgG conjugated to horseradish peroxidase (1:3,000; Sungene Biotechnology) and received three postincubation washes. Signals were visualized using the enhanced chemiluminescence detection system according to the manufacturer’s instructions (GE Healthcare UK). Y2H Assay The coding region of NtBOP2 was cloned into the yeast expression vector pGBKT7 (Clontech) to generate the construct NtBOP2-GAL4-BD as bait. The ORFs of TGA1a (GI:19679), PG13 (GI:170284), TGA2.2 (GI:6288681), TGA2.1 (GI:2281448), and RSG (GI:8777511) were PCR amplified from the cDNAs that were prepared from tobacco corollas using gene-specific primers and then cloned into the AD vector pGADT7 (Clontech) as prey constructs. To detect dimerization of the NtBOP2 proteins, the coding region of NtBOP2 was also cloned into pGADT7 vector. The bait and each of the prey constructs were cotransformed into the yeast strain AH109 (Clontech). Interactions were visually detected using an X-gal filter assay, and β-galactosidase activity was quantitatively measured using o-nitrophenyl-β-galactopranoside (Sigma; N-1127) as the substrate according to the manufacturer’s protocol (Clontech Laboratories). Subcellular Localization Analysis The coding region of GFP was fused to that of NtBOP2 at either the 5′ or 3′ terminus under the control of the CaMV 35S promoter in expression vector pPZP111 to generate pPZP-GFP-NtBOP2 and pPZP-NtBOP2-GFP constructs. Recombinant plasmids were transformed into A. tumefaciens strain EHA105 and subsequently used to transform BY-2 cells. For transient expression of GFP fusion proteins in tobacco leaf epidermal cells, fully expanded Nicotiana benthamiana leaves were infiltrated with A. tumefaciens harboring pPZP-GFP, pPZP-GFP-NtBOP2, and pPZP-NtBOP2-GFP following the method described previously (Sparkes et al., 2006). The infiltrated plants were incubated for 48 h at 23°C, and the GFP signal was then visualized with a confocal microscope (Leica TCS SP5; Leica Microsystems) at 488 nm. For transient expression in BY-2 protoplasts, the cDNAs of GFP-NtBOP2 and NtBOP2-GFP were subcloned into pBI221 vector under the control of the CaMV 35S promoter. The derived constructs were named pBI221-GFP-NtBOP2 and pBI221-NtBOP2-GFP and used for protoplast transfection. BY-2 protoplasts were transfected as described previously (Miao and Jiang, 2007) with minor modifications. Briefly, 3-d subcultured BY-2 cells were incubated in enzyme solution (1% [w/v] cellulase Onozuka RS, 0.05% [w/v] pectinase, 0.2% [w/v] driselase, 0.4 m mannitol, 20 mm KCl, 10 mm CaCl2, and 200 mm MES, pH 5.6) for 3 h at 28°C. The polyethylene glycol-calcium transfection was conducted following Yoo et al. (2007). The transfected protoplasts were incubate at 28°C for 24 h. GFP signals were visualized with a confocal microscope (Leica TCS SP5; Leica Microsystems) at 488 nm. Firefly Luc Complementation Imaging Assay The method of firefly Luc complementation imaging and the vectors were described previously (Chen et al., 2008; Xu et al., 2010a). Briefly, the ORFs of TGA1a, PG13, TGA2.2, TGA2.1, and RSG were ligated to the cDNA encoding the C-terminal end of dissected Luc in a pCAMBIA-based plasmid, and the constructs were named CLuc-TGAs. The ORF of NtBOP2 was ligated to the cDNA coding for the N-terminal end of dissected Luc in pCAMBIA-based plasmid and named NtBOP2-NLuc. The vectors were transformed into the A. tumefaciens EHA105 strain. Equal volumes of A. tumefaciens cultures harboring each of the CLuc and NLuc constructs were mixed to a final optical density at 600 nm = 1.0 in the infiltration buffer (10 mm MES, pH 5.6, 10 mm MgCl2, and 200 µm acetosyringone) and infiltrated into fully expanded tobacco leaves with a 1-mL needleless syringe. The agroinfiltrated tobacco plants were grown in the dark for 24 h and then exposed to a 16-h-light/8-h-dark cycle for 48 h at 23°C. Luc activity was visualized with a CCD imaging apparatus (Andor iXon). In Vitro Pull-Down Assay The ORFs of TGA1a, PG13, TGA2.2, TGA2.1, RSG, and NtBOP2 were fused to the coding sequence of the GST tag in pGEX6P-1 vector. The ORF of NtBOP2 was fused to the coding sequence of the MBP tag in pMAL-p2X vector. The constructs were transformed into Escherichia coli BL21 strain. The in vitro pull-down assay was performed according to the protocol of the ProFound Pull-Down GST Protein Kit (Pierce Biotechnology). The eluted proteins were separated on a SDS-PAGE gel and detected by western blot using anti-MBP or anti-GST antibodies (1:5,000; Sungene Biotechnology). GUS Staining An approximately 1.5-kb DNA fragment upstream of the start codon of NtBOP2 was amplified by PCR and fused upstream of the cDNA encoding GUS reporter in the binary vector pBI121. The recombinant plasmid was introduced into tobacco by A. tumefaciens-mediated leaf disc transformation. The T1 transgenic plants were used for GUS staining. Histochemical staining for GUS activity in transgenic plants was performed as described by Jefferson et al. (1987). Histological Analysis Flowers for histological analysis were fixed overnight at 4°C in a solution containing 50% (v/v) ethanol, 3.7% (v/v) formaldehyde, and 5% (v/v) acetic acid. The samples were dehydrated in a series of ethanol solutions (50%–100%, v/v) and incubated in 100% ethanol for 1 h. The Leica Historesin Embedding Kit (Leica Microsystems) was used for sample embedding. Serial sections (3 μm thick) of the specimens were cut with a rotary microtome (Leica Microsystems), stained with 0.25% (w/v) toluidine blue, and viewed with a microscope. Microscopy Analyses The nucleus and cell wall of the yeast and BY-2 cells were stained with DAPI and calcofluor (fluorescent brightener), respectively. Photographs were taken with a fluorescence microscope (Olympus BX51). For scanning electron microscopy analysis, corollas were fixed in 2.5% (v/v) glutaraldehyde in 0.2 m sodium phosphate buffer (pH 7.4) at 4°C overnight. After dehydration in a graduated ethanol series (50%–100%, v/v), the samples were treated with 100% isopentyl acetate at 4°C overnight. The specimens were subsequently dried using a critical point dryer (model HCP-2; Hitachi), sputter coated with gold in an E-1010 ion sputter (Hitachi), and observed with a scanning electron microscope (Quanta200; FEI). For transmission electron microscopy analysis, segments were cut from the junction region of corolla and receptacle and fixed in 2.5% (v/v) glutaraldehyde in 0.2 M sodium phosphate buffer (pH 7.4) at 4°C overnight. After three washes with phosphate-buffered saline buffer, the materials were incubated in 1% (w/v) osmium tetroxide for 4 h at room temperature. The specimens were washed three times and dehydrated in a series of ethanol and epoxy ethane; next, they were embedded in Epon 812 resin for 48 h at 60°C. The specimens were cut into thin sections (50 nm) with an LKB III Ultratome, stained with uranyl acetate followed by lead citrate, then examined and photographed with a JEM-1400 transmission electron microscope (JEOL). Measurement of Breakstrength The force required to pull the corollas from their receptacles was measured using force measurement gauges (Aogu). The corollas were cut into four parts along the longitudinal axis using a razor blade. The breakstrength was tested for each part. Ten flowers were used for each analysis. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers EF051131 (NtBOP2), AF362951 (NtCel5), AF049354 (NtEXPA5), X16449 (NtTGA1a), AF031487 (NtTGA2.2), U90214 (NtTGA2.1), M62855 (NtPG13), and AB040471 (NtRSG). Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. Multiple sequence alignment and phylogenetic analysis of NtBOP1 (JN165230), NtBOP2 (EF051131), NtBOP3 (JN165231), and NtBOP4 (JN165232) proteins. Supplemental Figure S2. Real-time PCR analyses of the expression levels of NtBOP1, NtBOP3, and NtBOP4 genes in wild-type, NtBOP2-OE, NtBOP2t-OE, NtBOP2-RNAi, and NtBOP2-AS plants. Supplemental Figure S3. NtBOP2 failed to complement the Arabidopsis bop1 bop2 mutant phenotype. Supplemental Table S1. Primer sequences used in this study. ACKNOWLEDGMENTS We are grateful to Dr. George W. Haughn (University of British Columbia) for providing us with the Arabidopsis bop1 bop2 seeds, to Prof. Yi-Hua Zhou (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for her help in transmission electron microscopy analysis, and to Dr. Zhao-Sheng Kong (Institute of Microbiology, Chinese Academy of Sciences) for his critical reading of the manuscript. Glossary AZ abscission zone BY-2 Bright Yellow 2 ORF open reading frame RNAi RNA interference RT reverse transcription DAPI 4′,6-diamino-phenylindole Y2H yeast two-hybrid GST glutathione S-transferase MBP maltose-binding protein CaMV cauliflower mosaic virus LITERATURE CITED Adamczyk BJ Lehti-Shiu MD Fernandez DE ( 2007 ) The MADS domain factors AGL15 and AGL18 act redundantly as repressors of the floral transition in Arabidopsis . Plant J 50 : 1007 – 1019 Google Scholar Crossref Search ADS PubMed WorldCat Bleecker AB Patterson SE ( 1997 ) Last exit: senescence, abscission, and meristem arrest in Arabidopsis . 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The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Gui-Xian Xia (xiagx@im.ac.cn). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.112.193482 © 2012 American Society of Plant Biologists. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - The Tobacco BLADE-ON-PETIOLE2 Gene Mediates Differentiation of the Corolla Abscission Zone by Controlling Longitudinal Cell Expansion    
JF - Plant Physiology
DO - 10.1104/pp.112.193482
DA - 2012-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-tobacco-blade-on-petiole2-gene-mediates-differentiation-of-the-5COc53GYuQ
SP - 835
EP - 850
VL - 159
IS - 2
DP - DeepDyve
ER -