SEARCH

SEARCH BY CITATION

Keywords:

  • NAC domain protein;
  • VND7;
  • xylem vessel differentiation;
  • transcriptional activation;
  • proteasome-mediated degradation;
  • tobacco BY-2 cell

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The Arabidopsis thaliana NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), plays a pivotal role in regulating the differentiation of root protoxylem vessels. In order to understand the mechanisms underscoring the function of VND7 in vessel differentiation in more detail, we conducted extensive molecular analyses in yeast (Saccharomyces cerevisiae), Arabidopsis, and Nicotiana tabacum L. cv. Bright Yellow 2 (tobacco BY-2) cells. The transcriptional activation activity of VND7 was confirmed in yeast and Arabidopsis, and the C-terminal region was shown to be required for VND7 transcriptional activation. Expression of the C-terminus-truncated VND7 protein under the control of the native VND7 promoter resulted in inhibition of the normal development of metaxylem vessels in roots and vessels in aerial organs, as well as protoxylem vessels in roots. The expression pattern of VND7 overlapped that of VND2 to VND5 in most of the differentiating vessels. Furthermore, a yeast two-hybrid assay revealed the ability of VND7 to form homodimers and heterodimers with other VND proteins via their N-termini, which include the NAC domain. The heterologous expression of VND7 in tobacco BY-2 cells demonstrated that the stability of VND7 could be regulated by proteasome-mediated degradation. Together these data suggest that VND7 regulates the differentiation of all types of vessels in roots and shoots, possibly in cooperation with VND2 to VND5 and other regulatory proteins.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The vascular system of plants consists of two types of elongated cell files: xylem, through which water and dissolved minerals are passed, and phloem, in which amino acids and sucrose are transmitted. In Arabidopsis roots, the primary vascular xylem exhibits a diarch pattern, while two phloem poles are oriented perpendicularly to the xylem. In the primary xylem, protoxylem vessels with helical secondary wall thickenings are positioned at the end of the diarch xylem adjacent to two pericycle cells, between which three or four metaxylem vessels with reticulated secondary wall thickenings are located (Baum et al., 2002; Demura and Fukuda, 2007; Kobayashi et al., 2002; Ye et al., 2002). The vessels developing in the shoots of young seedlings are protoxylem vessels with helical secondary wall thickenings (Kobayashi et al., 2002).

The Zinnia elegans experimental system, in which single isolated mesophyll cells transdifferentiate synchronously into vessel elements, is an excellent model for studying the program of xylem vessel development (reviewed in Fukuda, 1996, 2004; Turner et al., 2007). A comprehensive transcriptome analysis has identified a number of genes whose expression is closely associated with the in vitro transdifferentiation (Demura et al., 2002; Milioni et al., 2002; Pesquet et al., 2005). One of these genes is Ze567, which encodes a NAC (for NAM, ATAF1/2 and CUC2) domain protein, and is transiently expressed prior to the formation of the characteristic secondary wall structure (Demura et al., 2002). Recently, we established a vessel element transdifferentiation system using Arabidopsis suspension cells (Kubo et al., 2005). Microarray analysis revealed that the expression levels of seven Arabidopsis NAC genes, which were closely related to Ze567, were significantly elevated during transdifferentiation. Moreover, analysis of promoter activity confirmed that all seven genes were preferentially expressed in vascular cells. Therefore, we designated these seven genes as VASCULAR-RELATED NAC-DOMAIN1 (VND1) to VND7. Interestingly, overexpression of VND6 and VND7 driven by the CaMV 35S (CaMV35S) promoter induced transdifferentiation into metaxylem and protoxylem vessel cells, respectively, in various tissues, while transgenic plants overexpressing VND6 and VND7 fused to the SRDX strong repression domain driven by the CaMV35S promoter inhibited the normal differentiation of inner-metaxylem and protoxylem vessel cells, respectively. Together these data indicate that vessel differentiation at the inner metaxylem and protoxylem in roots is regulated by VND6 and VND7, respectively (Kubo et al., 2005). However, little is known about the mechanisms underlying the differentiation of other vessels, including a vessel at the outermost metaxylem of roots and vessels in shoots.

Within the NAC domain proteins, VND1 to VND7, are classified in the IIb/OsNAC7 subgroup, together with NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1), NST2, and SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1 (SND1)/NST3/ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN012 (ANAC012) (Ko et al., 2007; Mitsuda et al., 2005, 2007; Ooka et al., 2003; Zhong et al., 2006, 2007). Overexpression of NST1, NST2 and SND1/NST3/ANAC012 induced ectopic lignified secondary cell wall thickenings in various tissues (Ko et al., 2007; Mitsuda et al., 2005, 2007; Zhong et al., 2006). Promoter analysis showed that NST1 and SND1/NST3/ANAC012 were expressed in fibers in the inflorescence stem (Ko et al., 2007; Mitsuda et al., 2005, 2007; Zhong et al., 2006). Double knockouts of the nst1 and snd1/nst3/anac012 loci showed defects in secondary cell wall thickenings of fiber cells (Mitsuda et al., 2007; Zhong et al., 2007). These results indicated that NST1 and SND1/NST3/ANAC012 also function in secondary wall formation of vascular tissues.

Members of the NAC domain protein family function as plant-specific transcriptional factors (reviewed in Olsen et al., 2005), and comprise approximately 100 genes in the Arabidopsis genome (Ooka et al., 2003). Although only a small number of NAC domain genes have been characterized thus far, several studies implicate the NAC domain proteins in diverse processes, such as the establishment of the shoot apical meristem (Aida et al., 1997; Souer et al., 1996), lateral root formation (Xie et al., 2000), the signaling pathway involved in abiotic stress (Fujita et al., 2004; Tran et al., 2004), and defense responses (Delessert et al., 2005; Ren et al., 2000; Xie et al., 1999).

The NAC domain proteins contain a conserved NAC domain at the N-terminus, which is implicated in nuclear localization, DNA binding, and the formation of homodimers or heterodimers with other NAC domain proteins (Duval et al., 2002; Ernst et al., 2004; Hegedus et al., 2003; Olsen et al., 2004; Xie et al., 2000). On the other hand, the C-terminal regions of the proteins are highly diverse, with the exception of several small motifs shared among the subgroups (Ko et al., 2007; Ooka et al., 2003), and confer regulation of transcriptional activation activity (Duval et al., 2002; Hegedus et al., 2003; Ko et al., 2007; Xie et al., 2000).

The NAC domain proteins have also been demonstrated to interact with a wide variety of cellular factors involved in multiple cellular processes. The RING domain protein SINA of Arabidopsis thaliana 5 (SINAT5) interacts with NAC1 and promotes its degradation through the ubiquitination pathway (Xie et al., 2002). Likewise, ABSCISIC ACID-RESPONSIVE NAC (ANAC)/ANAC019 interacts with other RING domain proteins (Greve et al., 2003). Wheat GEMINIVIRUS RepA-BINDING1 (GRAB1) and GRAB2, Arabidopsis TCV-INTERACTING PROTEIN, and barley HvSPY-INTERACTING NAC were isolated as binding partners with viral proteins (Ren et al., 2000; Robertson, 2004; Xie et al., 1999). Antirrhinum CUPULIFORMIS interacts with a TCP (for TEOSINTE BRANCHED1, CYCLOIDEA and PCF) domain protein, a member of another family of transcription factors (Weir et al., 2004). Recently, Tran et al. (2007) reported that ANAC/ANAC019, ANAC055 and ANAC072 interacted with a zinc finger homeodomain protein. Together these data suggest that the transcriptional activity of NAC domain proteins could be modulated by their interactions with other cellular factors.

In this study, we conducted a comprehensive and detailed analysis of the multiple functions of VND7, including a study of VND7 transcriptional activation activity in yeast and Arabidopsis, identification of the functional domain required for transdifferentiation of vessels in Arabidopsis, analysis of VND7 dimerization with other VND proteins in yeast, and determination of the stability of VND7 protein in tobacco (Nicotiana tabacum) BY-2 cells. Our results show that VND7 plays a critical role in regulating vessel formation not only in the root protoxylem but also in the root metaxylem and in shoots.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The C-terminal region of VND7 confers transcriptional activation

Most NAC domain proteins have been previously characterized as transcriptional activators, and the C-terminal regions have been demonstrated to confer the transcriptional activation (reviewed in Olsen et al., 2005). To determine whether VND7 has transcriptional activation activity, full-length VND7 was fused to the GAL4 DNA-binding domain (GAL4-BD) and introduced into the AH109 yeast strain. GAL4-BD alone was used as a negative control. The cells transformed with full-length VND7 fused to GAL4-BD grew on the selective media without histidine or without histidine and adenine, indicating that VND7 exhibits transcriptional activation activity (Figure 1a). To investigate in detail the regions required for the transcriptional activation, truncated VND7 proteins deleted from the C-terminus were fused to GAL4-BD and expressed in AH109. The NAC domain proteins in the IIb/OsNAC7 subgroup contain two highly conserved domains, the LP-box and the WQ-box, within the C-terminal regions (Ko et al., 2007). VND71–270 and VND71–242, which contain the LP-box but lack almost all or all of the WQ-box, promoted growth on selective medium lacking histidine but not on medium lacking histidine and adenine (Figure 1a), indicating that VND71–270 or VND71–242 are weaker transcriptional activators than full-length VND7. Little or no growth was observed in the cells transformed with VND71–216 and VND71–188, which lack both the LP- and WQ-boxes (Figure 1a).

image

Figure 1.  Transcriptional activation activity of VND7. (a) Yeast assay. The indicated VND7 truncation mutants or the multicloning site (MCS) fused to the GAL4 DNA-binding domain (GAL4-BD) were introduced into the AH109 yeast strain. Transformants were incubated on the indicated medium at 30°C for 5 days. I–V represent subdomains I–V of the NAC domain. LP and WQ represent the LP- and WQ-boxes conserved in the IIb/OsNAC7 subgroup (Ko et al., 2007). (b) Schematic diagrams of the reporter and effector constructs used for the dual luciferase assay. (c) Dual luciferase transient transfection assay. The effector, reporter and reference plasmids were delivered to Arabidopsis leaves by particle bombardment and LUC activity was assayed. Error bars represent the standard deviation of three biological replicates.

Download figure to PowerPoint

The transcriptional activation activity of VND7 in plants was further assessed using dual luciferase assays (Figure 1b,c). The reporter plasmid containing the firefly (Photinus pyralis) luciferase (LUC) linked to GAL4-binding sites and the effector plasmid containing full-length or VND7 deletion mutants fused to GAL4-BD under the control of the CaMV35S promoter were delivered to Arabidopsis leaves by particle bombardment. Luciferase activity increased at least four-fold when GAL4-BD-VND7full was expressed, while no such increase was detected in LUC activity when only the NAC domain of VND7 (VND71–161) was expressed (Figure 1b,c). We next tested whether the C-terminal region of VND7 functions as a portable transcriptional activation domain. Expression of the entire C-terminal region (VND7162–324) fused to GAL4-BD exhibited stronger LUC activity than that of the truncated C-terminal region (VND7217–324). These results suggested that VND7 has transcriptional activation activity, and that the entire C-terminal region containing the LP- and WQ-boxes is required for full activity.

The C-terminal region of VND7 is required for formation of protoxylem vessels

Overexpression of VND7 in Arabidopsis induces the transdifferentiation of non-vascular cells into protoxylem-like vessel elements (Kubo et al., 2005). To elucidate the functional domain in VND7 required for transdifferentiation, we generated transgenic Arabidopsis plants overexpressing full-length or C-terminal truncated VND7 (see Figure 1a) fused to the yellow fluorescent protein (YFP) and under the control of the CaMV35S promoter. While fluorescence signals in transgenic control plants overexpressing YFP alone were detected in both the nucleus and cytosol (Figure 2a), the VND7full protein fused to YFP localized only to the nucleus, as described previously (Kubo et al., 2005) (Figure 2b). Moreover, all the C-terminal truncated VND7 proteins, including VND71–161 containing only the NAC domain, were localized specifically to the nucleus (Figure 2c), indicating that the NAC domain of VND7 is sufficient for nuclear localization. In control plants overexpressing YFP alone, vascular development in roots was normal – two protoxylem vessels were continuously formed and metaxylem vessels were located between these protoxylem vessels at the central region of the primary roots (Figure 2d,e). Overexpression of VND7full-YFP induced transdifferentiation of the various types of non-vascular cells into the xylem vessel elements in roots (Figure 2f,g; Kubo et al., 2005). However, we found that the cell fate of the original metaxylem vessel elements was unaffected by VND7full-YFP overexpression (Figure 2g). This result suggested that overexpression of VND7full-YFP driven by the CaMV35S promoter is sufficient for many cells to transdifferentiate but insufficient to change cell fate at the metaxylem position. Corresponding with the reduced transcriptional activation activities observed in yeast, the VND71–270 and VND71–242 proteins induced transdifferentiation of vessel elements with fainter secondary cell walls compared with those induced by overexpression of VND7full, and at a lower frequency (Table 1); in contrast, overexpression of VND71–216, VND71–188 and VND71–161 failed to induce any transdifferentiated cells (Table 1). These results suggest that the entire C-terminal region containing LP- and WQ-boxes is required to induce vessel transdifferentiation.

image

Figure 2.  Root phenotypes of transgenic plants overexpressing full-length or truncated VND7. (a–c) Subcellular localization of yellow fluorescent protein (YFP)-tagged proteins. Transgenic plants harboring (a) CaMV35Spro:YFP, (b) CaMV35Spro:VND7-YFP and (c) CaMV35Spro:VND71–161-YFP. Differential interference contrast (DIC) and YFP fluorescence images were merged. Bar = 200 μm. (d,e) Xylem vessel formation in young roots of 9-day-old control plants expressing CaMV35S-driven YFP. Panel (e) shows a magnified view of the region indicated by the black frame in (d). At the central region of the primary roots of control plants, two protoxylem vessels (px, black arrowheads) were continuously formed between which two outermost-metaxylem vessels (mx, white arrowheads) developed. Bar = 50 μm. (f, g) Various non-vascular cells transdifferentiated into the xylem vessel elements in primary roots of 9-day-old transgenic seedlings expressing CaMV35S-driven VND7full-YFP. Panel (g) shows a magnified view of the region indicated by the black frame in (f). The formation of original xylem vessel elements was not affected in the roots. Bar = 50 μm. Black arrowheads, protoxylem vessels; white arrowheads, outermost metaxylem vessels; arrows, transdifferentiated vessels. (h, i) Dominant negative phenotypes, showing discontinuous formation of (h) one or (i) both protoxylem vessel columns in primary roots of 9-day-old seedlings harboring CaMV35Spro:VND71–161-YFP. Bar = 50 μm.

Download figure to PowerPoint

Table 1.   Frequency of transdifferentiated vessel elements in primary roots of transgenic plants (T1 generation)
ConstructTotalNumber of transdifferentiated vessel elements per root
01–1011–2021–5051–100101–200200+
  1. Nine-day-old roots with indicated number of transdifferentiated vessel elements were counted under a differential interference contrast (DIC) microscope.

  2. Values in parentheses are expressed as percentage (%) of the total.

YFP1010 (100)000000
VND7full-YFP2411 (45.8)1 (4.2)01 (4.2)02 (8.3)9 (37.5)
VND71–270-YFP16 8 (50)4 (25.0)2 (12.5)1 (6.3)1 (6.3)00
VND71–242-YFP12 3 (25.0)1 (8.3)2 (16.7)4 (33.3)1 (8.3)1 (8.3)0
VND71–216-YFP1717 (100)000000
VND71–188-YFP2727 (100)000000
VND71–161-YFP1313 (100)000000

VND7 regulates the formation of metaxylem vessels in roots and vessels in shoots

We found that overexpression of VND71–216, VND71–188 and VND71–161 failed to induce transdifferentiation of vessels and instead resulted in discontinuous formation of one or both protoxylem vessel columns (Figure 2h,i), a phenotype similar to transgenic plants overexpressing VND7 fused to the SRDX strong repression domain (Kubo et al., 2005). These data suggest that the C-terminus-truncated VND7 proteins exert a dominant negative effect on the formation of protoxylem vessels.

To elucidate the dominant negative effects of expression of truncated VND7 on vessel formation, we generated transgenic plants expressing VND71–161 and VND7full fused to YFP under the control of the native VND7 promoter. The YFP signal in these transgenic seedlings was localized specifically in the nuclei in both immature protoxylem and metaxylem vessels, whereas the YFP signal of VND7pro:YFP-SV40 nuclear localizing signal (NLS) seedlings was detected not only in immature vessels but also in various vascular cells of older part of the roots, near the location of emergence of the lateral root (Figure 3a, Figure S1). Among 49 first-generation transgenic seedlings carrying VND7pro:VND71–161-YFP, 34 exhibited a dwarf phenotype in the aerial part (Figure 3b,f). In roots of these seedlings, discontinuous formation of protoxylem vessel was observed (Figure S2), as seen in transgenic seedlings expressing CaMV35Spro:VND71–161-YFP (Figure 2i) or CaMV35Spro:VND7full-SRDX (Kubo et al., 2005). Moreover, the VND7pro:VND71–161-YFP seedlings often showed inhibition of metaxylem vessel formation in roots as well as discontinuous vessel formation in the aerial parts, including the petioles and leaves (Figure 3c,e, Figure S2). In addition, more than half of the VND7pro:VND7full-SRDX lines (15 out of 24 lines) exhibited a dwarf phenotype with defects in vessel formation (data not shown), similar to the phenotype of VND7pro:VND71–161-YFP seedlings (Figure 3, Figure S2). These results strongly suggest that VND7 has a critical role not only in the formation of protoxylem vessels in roots, as shown previously (Kubo et al., 2005), but also in the formation of other types of vessels. Recently, it was reported that mis-expression of XYLEM NAC DOMAIN 1 (XND1), encoding another NAC domain protein, resulted in extreme dwarfism associated with the absence of xylem vessels (Zhao et al., 2008), suggesting a possible functional relationship between VND7 and XND1.

image

Figure 3.  Expression of C-terminal truncated VND7 driven by the VND7 promoter caused a dwarf phenotype and defects in formation of various types of vessels. (a) Vessel-specific localization of VND7full-YFP driven by the VND7 promoter. The yellow fluorescent protein (YFP) signal was detected in nuclei of protoxylem (black arrowheads) and metaxylem (white arrowheads) vessels. Bar = 50 μm. (b) Fourteen-day-old T1 seedlings harboring VND7pro:VND7full-YFP (left-most two plants) and VND7pro:VND71–161-YFP (right-most three plants). Bar = 1 cm. Among 49 seedlings carrying VND7pro:VND71–161-YFP, 34 exhibited a dwarf phenotype in the aerial part. (c) Root phenotype of the VND7pro:VND71–161-YFP seedling. The outermost metaxylem vessel columns were discontinuous (white arrowhead). Black arrowheads indicate the protoxylem vessel columns. Bar = 50 μm. (d, e) Dark field images of first true leaves of 8-day-old T1 seedlings harboring (d) VND7pro:VND7full-YFP and (e) VND7pro:VND71–161-YFP. Bar = 500 μm. (f) Forty-five-day-old T1 plants harboring VND7pro:VND7full-YFP (left-most two plants) and VND7pro:VND71–161-YFP (right-most two plants and inset). Bar = 5 cm.

Download figure to PowerPoint

VND7 forms homodimers and heterodimers with other VND members

Several NAC domain proteins form homodimers, and heterodimerize with other family members (Duval et al., 2002; Ernst et al., 2004; Hegedus et al., 2003; Olsen et al., 2004; Xie et al., 2000), suggesting that VND7 may associate with other VND proteins. To investigate this possibility, we conducted a yeast two-hybrid assay. As full-length VND7 functions as a transcriptional activator in yeast, as described above, only the N-terminal regions containing the NAC domains of VND family proteins (VNDNTERM) were used as bait and prey. Although the yeast cells carrying NAC11–199 fused to the GAL4-BD (GAL4-BD-NAC11–199) grew on the selective medium lacking histidine when co-transformed with NAC11–199 fused to the GAL4 transactivating domain (GAL4-AD-NAC11–199), as previously reported by Xie et al. (2000), GAL4-BD-NAC11–199 co-transformed with other VNDNTERM proteins fused to GAL4-AD or CUC21–228-GAL4-AD failed to grow on the selective medium (Figure 4). The transformants co-expressing GAL4-BD-VND71–188 along with any of the GAL4-AD-VNDNTERM constructs all grew on the selective medium, though co-expression with GAL4-AD-VND11–187, VND21–188 and VND31–192 exhibited better growth than other VNDNTERM proteins (Figure 4). However, transformants expressing GAL4-BD-VND71–188 with GAL4-AD-NAC11–199 or CUC21–228 failed to grow on the selective medium (Figure 4). These data suggest that VND7 might homodimerize and heterodimerize with other VND members. In contrast, the transformants carrying GAL4-BD-VND61–186 showed strong reporter expression only with co-transformed GAL4-AD-VND61–186, suggesting that VND6 preferentially formed homodimers (Figure 4). It is possible that the distinct dimerization patterns of VND6 and VND7 reflect their differential function in the regulation of vessel formation.

image

Figure 4.  Dimer formation among NAC domain proteins. The multi cloning site (MCS) or the N-terminal region of VND family proteins, NAC1 or CUC2 fused to the GAL4 DNA-binding domain (GAL4-BD) or GAL4 transactivating domain (GAL4-AD) were introduced into the yeast strain AH109 and grown on the indicated medium at 30°C for 7 days. P indicates the positive control.

Download figure to PowerPoint

Co-expression of VND members in differentiating xylem vessels

To identify the cells in which VND family proteins may associate with each other via homo- and heterodimerization, we analyzed the detailed expression patterns of the VND family genes using a promoter assay with GUS as a reporter driven by the promoters of the VND family genes (Kubo et al., 2005; Figure 5, Table 2, Figure S3). The vascular expression of VND7pro:GUS was detected in protoxylem poles of root procambium, differentiating protoxylem and metaxylem vessels of roots, differentiating vessels of hypocotyls and leaves (Figure 5, Table 2). The expression pattern of VND7pro:GUS in the root procambium partially overlapped with that of VND2pro:GUS and VND3pro:GUS (Table 2, Figure S3). GUS expression driven by the VND2 to VND5 promoters in differentiating vessels was observed in all organs examined (roots, hypocotyls, cotyledons and leaves), and was similar to the pattern of GUS expression driven by the VND7 promoter (Table 2, Figure S3). VND6pro:GUS expression was restricted in the inner metaxylem vessels in roots and several vessels in hypocotyls, where VND7pro:GUS was also expressed (Table 2, Figure S3). These data suggest that VND7 might collaboratively function with other VND members in differentiating xylem vessels of all organs.

image

Figure 5.  Expression of VND7pro:GUS in 7-day-old seedlings. (a) GUS staining in the root tip. The black frame indicates the procambium region. The black arrowhead indicates where secondary cell walls begin to thicken in differentiating protoxylem vessels. Bar = 200 μm. (b–e) GUS staining of (b) protoxylem poles of root procambium, (c) differentiating protoxylem vessels, (d) differentiating outermost metaxylem vessels and (e) differentiating inner metaxylem vessels. The black arrowhead in (c) indicates where secondary cell walls begin to thicken in differentiating protoxylem vessels. White arrowheads in (d) and in (e) indicate outermost metaxylem and inner metaxylem vessels, respectively. Bar = 20 μm. (f) GUS staining in the aerial part. Bar = 200 μm. (g, h) GUS staining of differentiating vessels without (g) and with (h) visible secondary wall thickenings in true leaves. Bar = 20 μm.

Download figure to PowerPoint

Table 2.   Summary of localization of promoter:GUS expression of VND family genes in 7-day-old seedlings
 VND1VND2VND3VND4VND5VND6VND7
  1. Expression levels: +++, strong; ++, medium; +, weak.

  2. aLateral root primordia.

  3. bA part of cells in columella root caps. Epidermal and cortex cells in the older part of roots and the root–hypocotyl junctions.

  4. cVascular cells other than vessels and pericycle cells in the older part of roots.

  5. dThe terms, pre-procambium and procambium, were used based on Scarpella and Meijer (2004).

  6. eHydathodes of leaves.

  7. fEpidermal cells surrounding trichomes. Distal parts of true leaves 3 and 4. Marginal regions of true leaves 1 and 2.

  8. gBasal cells of stipules.

  9. hShoot apical regions.

Root
 Procambial zone
  Protoxylem pole ++++++   ++
  Metaxylem pole+++++++    
 Elongation/differentiation zone
  Protoxylem vessel +++++++++ +++
  Outermost metaxylem vessel +++++++++ +++
  Inner metaxylem vessel +++++++++++++++
  Other cells +++a ++b  ++c
Hypocotyl       
 Vessel +++++++++++
 Other cells  ++b    
Shoot
 Pre-procambiumd++++     
 Procambiumd+++++++    
 Vessel ++++++++++ +++
 Other cells+e+++e,f +e,g  +++h

Post-transcriptional modifications of VND7

While YFP-tagged VND7 expression driven by the VND7 promoter was only observed in differentiating vessels in the roots of transgenic VND7pro:VND7full-YFP Arabidopsis seedlings, the YFP signal in VND7pro:YFP-NLS seedlings and GUS staining in VND7pro:GUS seedlings were observed not only in differentiating vessels but also in other vascular cells and pericycle cells in the older areas of roots (Figure 5, Figure S1). These data suggest that VND7 gene expression may be regulated post-transcriptionally. The expression of several NAC genes, such as CUC1, CUC2 and NAC1, was shown to be regulated via mRNA cleavage mediated by miR164 (Guo et al., 2005; Laufs et al., 2004). However, sequence analysis did not reveal any potential cleavage sites for currently characterized miRNAs in the VND7 mRNA. We therefore postulated that VND7 expression levels could be controlled by protein stability, and thus investigated the stability of the VND7 protein in plant cells.

Transformed tobacco BY-2 cells expressing VND7-YFP and YFP were prepared using an estrogen receptor-based chemical-inducible system (Zuo et al., 2000). Two days after subculture, the transformed cells were treated with or without 10 μm 17-β-estradiol for 24 h. In the absence of 17-β-estradiol, none of the cell lines had detectable YFP signals (data not shown). In the presence of 17-β-estradiol, the cells expressing YFP alone exhibited fluorescence signals in both the cytosol and nucleus, while those expressing VND7-YFP showed only nuclear signals (Figure 6a). Cells were cultured in the presence or absence of 17-β-estradiol for 16 h and were subsequently treated with or without 100 μm MG-132, a proteasome inhibitor (Genschik et al., 1998), for an additional 8 h. Immunoblot analysis with anti-GFP, which recognizes GFP derivatives including YFP and cyan fluorescent protein, and anti-tubulin antibodies showed that the VND7-YFP protein accumulated upon treatment with MG-132, while YFP alone and tubulin were unaffected by MG-132 (Figure 6b). This result suggested that the VND7 protein is regulated by proteasome-mediated degradation. VND7-YFP was observed to migrate as two bands (Figure 6b), which could be the result of post-transcriptional modifications, such as alternative mRNA splicing, partial protein degradation, protein glycosylation or protein phosphorylation.

image

Figure 6.  Heterologous expression of VND7 protein in tobacco BY-2 cells. (a) Subcellular localization of VND7 in BY-2 cells. BY-2 cells were transformed with pER8 vectors expressing yellow fluorescent protein (YFP) alone (left) or YFP-tagged VND7 (right), and treated with 10 μm 17-β-estradiol for 24 h. Differential interference contrast (DIC) and YFP fluorescence images were merged. Bar = 100 μm. (b) Enhanced accumulation of VND7 protein upon treatment with the MG-132 proteasome inhibitor. Protein extracts from transgenic BY-2 cells treated with (+) or without (−) 10 μm 17-β-estradiol and/or 100 μm MG-132 were immunoblotted with YFP (top) and tubulin antibodies (bottom). (c) Transdifferentiation of BY-2 cells into vessel elements. The transgenic BY-2 cells expressing VND7-YFP were treated with 10 μm 17-β-estradiol for 4 days. The DIC (left), 4′-6-diamidino-2-phenylinodole (DAPI) fluorescence signal (center), and merged images (right) of transdifferentiated vessel elements are shown. Arrowheads indicate transdifferentiated vessel elements in which nuclei were not detected. Bar = 50 μm.

Download figure to PowerPoint

Notably, vessel elements were induced in the BY-2 cells overexpressing VND7-YFP under the control of the estrogen receptor-based chemical-inducible system (Figure 6c). The rate of differentiation reached up to 2% of cells 4 days after treatment with 17-β-estradiol. No vessel elements were found with the transgenic cells cultured in the absence of 17-β-estradiol or from cells overexpressing YFP alone (data not shown). Furthermore, 4′-6-diamidino-2-phenylinodole (DAPI) staining showed that the nuclei of some differentiated vessel elements from the VND7-YFP cells disappeared (Figure 6c). These data indicate that VND7 could induce the complete program of vessel element formation including programmed cell death even in BY-2 cells, which have been sustained as proliferating cells for several decades.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We previously demonstrated that two closely related NAC domain proteins, VND6 and VND7, are master regulators in the development of two types of xylem cells, metaxylem and protoxylem vessel elements, respectively, based on three major observations (Kubo et al., 2005). First, expression of VND6 and VND7 was restricted to metaxylem and protoxylem vessels, respectively, in the root tip. Second, CaMV35S promoter-driven overexpression of VND6 and VND7 induced transdifferentiation of various cells into metaxylem and protoxylem vessel elements, respectively. Third, dominant repression of VND6 and VND7 specifically inhibited metaxylem and protoxylem formation, respectively, in roots. In this study, we showed that the VND7 promoter was also active in immature metaxylem vessels in roots and vessels in the aerial parts of seedlings, and that VND7 promoter-driven expression of truncated VND7 proteins lacking the C-terminal transcriptional activation domain caused defects in vessel formation in protoxylem and metaxylem of roots and in aerial parts. We therefore concluded that VND7 regulates not only the formation of protoxylem vessels in roots, but also the formation of metaxylem vessels in roots and vessels in the aerial parts of seedlings.

Our studies in yeast revealed that although the truncated VND7 protein lacking most of the C-terminal region but containing an intact NAC domain (VND71–188) cannot confer transcriptional activation, this mutant is still able to dimerize with other VND proteins. Overexpression of the truncated VND7 proteins (VND71–216, VND71–188 and VND71–161) fused to YFP and driven by the CaMV35S promoter caused defects in the development of protoxylem vessels, including thinner secondary cell walls, a loosely coiled spiral pattern and discontinuous vessel formation. Moreover, expression of YFP-tagged VND71–161 driven by the VND7 promoter often resulted in a dwarf phenotype with defects in vessel formation in protoxylem and metaxylem of roots and in veins of petioles and leaves. Thus, the truncated VND7 protein might form inactive heterodimers with native VND family proteins or inactive homodimers, which might compete with active native dimers. Because the expression profile of VND7 correlates well with that of VND2 to VND5, and as the VND7 protein interacts with these VND family proteins in yeast, VND2 to VND5 might be preferential partners for VND7 heterodimerization, and together with VND7 could regulate xylem vessel differentiation. However, since vessel transdifferentiation was not induced by the overexpression of VND2 to VND5 driven by the CaMV35S promoter, as was observed with VND7 (Kubo et al., 2005), VND7 probably functions as the principal regulator of vessel differentiation.

Each VND family gene showed a slightly different expression pattern in vascular cells. For instance, VND1, VND2 and VND3 are expressed in the pre-procambial and/or procambial cells of root and shoot, VND7 is expressed in the protoxylem pole of root procambial zone, and VND6 is specifically expressed in the inner-metaxylem vessels (Table 2, Figure S3). These distinct expression patterns would give rise to various combinations of dimers, depending on the state of cell differentiation. The mechanisms of the development of floral organs (petal, sepal, stamen and carpel) have been shown to be controlled by different combinations of MADS-box transcriptional factors (Robles and Pelaz, 2005). Similarly, different VND family protein homo- and heterodimers might mediate distinct activities in vascular development.

Several studies have reported the dominant negative effects of transcription factors lacking the activation domain (Fukazawa et al., 2000; Heinekamp et al., 2004). Similarly, expression of truncated YFP-tagged VND7 that lacks the activation domain exhibited a dominant negative effect on vessel formation (Figures 2 and 3). This effect was much more prominent when expression was driven by the native VND7 promoter compared with the CaMV35S promoter. One possibility is that we could not obtain transformants with high levels of ectopic expression of the truncated VND7 proteins driven by the CaMV35S promoter due to embryonic or seedling lethality. Alternatively, VND7 promoter-mediated expression could be stronger than that of the CaMV35S promoter in causing defects in vessel differentiation. Expression of NST1 and NST2 with the SRDX domain showed stronger effects on dominant repression with native NST promoters than with the CaMV35S promoter (Mitsuda et al., 2005). These observations suggest that truncated transcriptional activators lacking activation domains and expressed from native promoters may provide more physiological insights into the functions of these transcription factors.

The expression and activity of several NAC domain proteins are strictly regulated (Olsen et al., 2005). Although mRNAs for several Arabidopsis NAC genes, including CUC1, CUC2 and NAC1, were shown to be post-transcriptionally regulated via miR164-directed cleavage (Guo et al., 2005; Laufs et al., 2004), we have not yet identified any miRNAs that might direct the cleavage of VND7 mRNA. The accumulation of the NAC1 protein has been shown to be controlled post-translationally at the level of proteasome-mediated protein degradation; the level of NAC1 protein in roots increased considerably with MG-132 treatment, and the SINAT5 RING finger protein, identified as a NAC1-binding partner in a yeast two-hybrid screen, acts as an E3 ubiquitin-protein ligase that targets NAC1 for proteasome-mediated degradation (Xie et al., 2002). The stability of VND7 protein could also be regulated by proteasome-mediated degradation, as observed in tobacco BY-2 cells treated with MG-132 (Figure 6). The YFP signal from non-vessel cells in the basal part of roots of transgenic seedlings carrying VND7pro:VND7full-YFP was hardly detectable in spite of the strong VND7 promoter activity in vasculature as shown by VND7pro:YFP-NLS or VND7pro:GUS; this observation could be due to continuous proteasome-mediated degradation of VND7 in the vasculature of the basal part of roots (Figures 3a and 5, Figure S1). Therefore, it is important to elucidate the mechanisms underlying cell-specific degradation or stabilization of the VND7 protein to fully understand the pathways controlling vessel formation.

Overexpressed VND7 protein in BY-2 cells migrated as two bands, suggesting post-translational modification of VND7 (Figure 6). Phosphorylation of a transcription factors is a well-known mechanism for regulating stability, activity and subcellular localization (reviewed in Whitmarsh and Davis, 2000), and several plant transcription factors such as BRI1-EMS-SUPPRESSOR1 (Yin et al., 2002, 2005), REPRESSION OF SHOOT GROWTH (Fukazawa et al., 2000; Igarashi et al., 2001) and ABRE-BINDING PROTEIN1 (Furihata et al., 2006) have been shown to be regulated by phosphorylation. Whether VND7 is phosphorylated and whether it is critical for its stability, activation or localization is currently unknown. Further analysis of the modification of VND7, including phosphorylation and degradation, will shed light upon the mechanisms underlying transcriptional regulation by NAC domain proteins during vessel formation.

Tobacco BY-2 cells are known to proliferate 100- to 125-fold in a week, and thus have been employed to analyze various cellular processes, such as the cell cycle and metabolite biosynthesis (Nagata et al., 1992). Expression of VND7 under the control of the estrogen receptor-based chemical-inducible system induced vessel element differentiation in BY-2 cells (Figure 6c), indicating that these cells are invaluable tools for use in analysis of secondary wall formation and programmed cell death. The efficiency of differentiation might be improved by the modification of growth conditions and inducible systems.

Based on the defects in vessel formation in the protoxylem and metaxylem of roots and in aerial parts by VND7 promoter-driven expression of truncated VND7 proteins lacking the C-terminal transcriptional activation, we concluded that VND7 plays a central role in regulating the differentiation of various types of vessels in roots and aerial parts. However, we should not exclude the possibility that the truncated VND7 proteins that may have a longer half-life – supported by the stronger signal intensity of VND71–161-YFP compared with that of VND7full-YFP (Figure S1) – stably dimerize with native VND proteins and consequently just perturb the normal vessel formation, regardless of the original function of VND7 proteins. Loss-of-function analysis of VND7 could be effective in verifying the possibility. Transfer-DNA (T-DNA) insertion mutants of VND7 and transgenic plants that expressed antisense RNA or RNA interference (RNAi) for VND7 did not show any detectable defects in morphology (Kubo et al., 2005). As the expression of VND7 overlaps with that of VND2 to VND5 (Table 2, Figure S3), this suggests that VND2 to VND5 might function collaboratively and/or redundantly with VND7 in xylem vessel differentiation. To further analyze the collaborative and/or redundant functions of VND family members, simultaneous loss of function of the VND family genes by T-DNA insertion or RNAi/antisense expression should be next employed. An important focus for elucidating the functions of VND family members will be the identification of downstream genes and direct targets of VND7 and other VND genes; this could be accomplished with inducible expression systems, such as the estrogen receptor-based chemical-inducible system used in this study.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Vector constructions

To generate the destination vectors for the two-hybrid assay under the GATEWAY system, pBD-GAL4 Cam and pAD-GAL4-2.1 (Stratagene, http://www.stratagene.com), which contain a GAL4 DNA-binding domain (GAL4-BD) and GAL4 transactivating domain (GAL4-AD), respectively, were digested with EcoRI/PstI or EcoRI/XhoI and blunted using the BKL kit (Takara Bio, http://www.takara-bio.com/). These linearized fragments were ligated to an EcoRV-digested GATEWAY Reading Frame Cassette (GWRFC) B (Invitrogen, http://www.invitrogen.com/). The resultant plasmids were named pBD-GAL4-GWRFC and pAD-GAL4-GWRFC, respectively. The pH35GY and pH35GEAR vectors (Kubo et al., 2005) were digested with HindIII and XbaI, blunted using the BKL kit, and self-ligated to generate pHGY and pHGEAR, which lack the CaMV35S promoter sequence and contain YFP and SRDX, respectively. Full-length or truncated cDNAs of VND family genes, NAC1 or CUC2 with or without the native promoters were subcloned into the pENTR/D-TOPO vector (Invitrogen) and then integrated into GATEWAY destination vectors, such as pH35GY (Kubo et al., 2005), pBD-GAL4-GWRFC, pAD-GAL4-GWRFC, pHGY or pHGEAR, using LR clonase (Invitrogen). The nucleotide sequence of the multicloning site (MCS) fragment is 5′-CACCTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGTGATG-3′, and includes a stop codon at the 5′ end and a start codon at the 3′ end. The GATEWAY destination vectors containing the MCS fragments were used as controls. Complementary DNA fragments of YFP and VND7 fused to the 3′ end of YFP were amplified by PCR from the pH35GY-VND7 plasmid, and then subcloned into the XhoI/SpeI sites of the pER8 vector, an estrogen receptor-based chemical-inducible system for use in transgenic plants (Zuo et al., 2000). For the dual luciferase transient transfection assay, VND7 cDNA fragments were inserted into the SmaI/SalI sites of the p35S-GAL4DB effector plasmid (Ohta et al., 2000); reporter and reference plasmids containing firefly LUC and Renilla reniformis LUC, respectively, were prepared as described previously (Ohta et al., 2000).

Yeast transformations

The resultant plasmids constructed on pBD-GAL4-GWRFC and/or pAD-GAL4-GWRFC were introduced into S. cerevisiae strain AH109 (Clontech, http://www.clontech.com/) by the lithium acetate method (Gietz et al., 1992). The transformants were incubated at 30°C on minimal SD medium (Clontech) either lacking tryptophan and leucine, tryptophan, leucine and histidine, or tryptophan, leucine, histidine and adenine. pBD-wt and pAD-wt (Stratagene) were used as the positive controls.

Dual luciferase transient transfection assay

The effector, reporter and reference plasmids were delivered to rosette leaves of 4-week-old Arabidopsis by particle bombardment (GE Healthcare, http://www.gelifesciences.com/) and LUC activity was assayed with the Dual-Luciferase Reporter Assay System (Promega, http://www.promega.com/) using the Mithras LB940 (Berthold, http://www.bertholdtech.com/).

Plant transformations

The resultant plasmids were electroporated into Agrobacterium strain GV3101::pMP90, which was used to transform Arabidopsis ecotype Col-0 and BY-2. The T1 generation of transgenic Arabidopsis seedlings was selected by growth medium (GM) supplemented with 20 μg mL−1 hygromycin under conditions of continuous light. Six days after germination, the antibiotic-resistant plants were transferred to antibiotic-free medium. The T1 generation of transgenic Arabidopsis containing VND7pro:VND7 was selected by GM supplemented with hygromycin for 7 days, transferred to antibiotic-free medium for 7 days, and planted into Jiffy seven pots (Sakata Seed, http://www.sakata.com/) under 16-h light/8-h dark conditions. The observation of transgenic plants, including the promoter:reporter (YFP-NLS or GUS) lines, was carried out according to Kubo et al. (2005). Transformation of BY-2 cells was essentially as previously described by An (1987). Transgenic BY-2 cells from the single colonies were maintained in Linsmaier–Skoog (LS) medium (Linsmaier and Skoog, 1965). BY-2 cells were stained with DAPI to visualize nuclei and safranin to visualize the cell wall.

Protein expression assays

To block proteasome-mediated protein degradation, transgenic BY-2 cells were subcultured for 2 days and first treated with 10 μm of 17-β-estradiol (Wako Pure Chemical, http://www.wako-chem.co.jp/) for 16 h and then with 100 μm MG-132 (Wako Pure Chemical) for an additional 8 h. Protein was extracted using an extraction buffer [25 mm 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)-HCl pH 7.6, 75 mm NaCl, 15 mm MgCl2, 15 mm EGTA, 60 mmβ-glycerophosphate, 0.1% (v/v) NP-40, 0.1 mm Na3VO4, 1 mm NaF, 1 mm DTT] containing Protease Inhibitor cocktail (Sigma-Aldrich, http://www.sigmaaldrich.com/) and 100 μm MG-132. Protein samples were fractionated by SDS-PAGE, subjected to immunoblotting with the anti-GFP antibody (MBL, http://www.mbl.co.jp/) or anti-tubulin antibody (Igarashi et al., 2000), and signals were detected with ECL plus (GE Healthcare).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Dr Nam-Hai Chua (The Rockefeller University, USA) and Dr Takashi Aoyama (Kyoto University, Japan) for providing the pER8 vector, Dr Ken Matsuoka (Kyushu University, Japan) for providing tobacco BY-2 cells, Dr Masaru Ohme-Takagi and Dr Nobutaka Mitsuda (AIST, Japan) for providing plasmids and technical advice for the dual luciferase transient transfection assay, Dr Seiji Sonobe (University of Hyogo, Japan) and Dr Hisako Igarashi (RIKEN, Japan) for providing the anti-tubulin antibody, and Hiromi Ogawa, Ayumi Ihara, Mitsutaka Araki, Sachiko Oyama, Tomoko Matsumoto and Ryoko Hiroyama (RIKEN, Japan) for excellent technical assistance. This work was supported by a Grant-in-Aid for Scientific Research (grant no. 18770045, MY and no. 20061029, TD).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Localization of YFP-NLS, VND7full-YFP and VND71−161-YFP expressed by the VND7 promoter in transgenic roots. Regions shown are approximately 5 cm from the root tip.

Figure S2. Defects in vessel formation in fourteen-day old of seedlings. (a) and (b) DIC images of roots of the VND7pro:VND71−161-YFP seedlings. Discontinuous vessel formation in protoxylem (black arrowheads), outermost-metaxylem (white arrowheads) and central-metaxylem (white arrow) was observed in roots. Bar = 50 μm. (c) and (d) Dark field images of the fifth and first true leaves of seedlings harboring (d) VND7pro:VND7full-YFP and (e) VND7pro:VND71−161-YFP, respectively. Bar = 500 μm. (e) and (f) DIC images of veins of petiole (c) and leaf blade (d) of the VND7pro:VND71−161-YFP seedlings. Bar = 50 μm.

Figure S3. Expression Patterns of VNDpro:GUS in Seven-Day-Old Seedlings. (a) GUS staining in VNDpro:GUS transgenic plants in root tips (upper row), cotyledon (middle row) and true leaves (lower row). Bar = 100 μm (roots) and 500 μm (cotyledons and true leaves). (b) GUS staining in vessels or non-vessel cells. From left to right: inner-metaxylem, lateral root primordia, epidermal cells at root-hypocotyl junction, preprocambial cells (outlined in red) of leaves, procambial cells (outlined in yellow) of leaves, differentiating vessels of leaves, epidermal cells surrounding trichomes, and basal cells of stipules. Bar = 50 μm.

Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

FilenameFormatSizeDescription
TPJ_3533_sm_figs.pdf1587KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.