Logo of plntphysLink to Publisher's site
Plant Physiol. Oct 2006; 142(2): 651–661.
PMCID: PMC1586039

Down-Regulation of the 26S Proteasome Subunit RPN9 Inhibits Viral Systemic Transport and Alters Plant Vascular Development


Plant viruses utilize the vascular system for systemic movement. The plant vascular network also transports water, photosynthates, and signaling molecules and is essential for plant growth. However, the molecular mechanisms governing vascular development and patterning are still largely unknown. From viral transport suppressor screening using virus-induced gene silencing, we identified a 26S proteasome subunit, RPN9, which is required for broad-spectrum viral systemic transport. Silencing of RPN9 in Nicotiana benthamiana inhibits systemic spread of two taxonomically distinct viruses, Tobacco mosaic virus and Turnip mosaic virus. The 26S proteasome is a highly conserved eukaryotic protease complex controlling many fundamental biochemical processes, but the functions of many 26S proteasome regulatory subunits, especially in plants, are still poorly understood. We demonstrate that the inhibition of viral systemic transport after RPN9 silencing is largely due to alterations in the vascular tissue. RPN9-silenced plants display extra leaf vein formation with increased xylem and decreased phloem. We further illustrate that RPN9 functions at least in part through regulation of auxin transport and brassinosteroid signaling, two processes that are crucial for vascular formation. We propose that RPN9 regulates vascular formation by targeting a subset of regulatory proteins for degradation. The brassinosteroid-signaling protein BZR1 is one of the targets.

Virus movement within the plant occurs via local cell-to-cell movement through plasmodesmata and long-distance transport through the vasculature, mainly the phloem (Crawford and Zambryski, 1999; Scholthof, 2005). Compared with work on virus cell-to-cell movement (Zambryski and Crawford, 2000; Zambryski, 2004), there have been relatively few studies on virus long-distance movement in the vascular system (Santa Cruz, 1999). Using a modified Tobacco mosaic virus (TMV) expressing green fluorescent protein (GFP), Cheng and colleagues studied the systemic vascular invasion routes of TMV in Nicotiana benthamiana and determined that TMV could enter minor, major, or transport veins directly from nonvascular cells to produce a systemic infection (Cheng et al., 2000). Only a few host genes have been identified to be important for virus systemic spread (Scholthof, 2005). A vascular-specific Gly-rich protein inhibits the long-distance movement of Turnip vein-clearing virus, probably by inducing callose accumulation in the phloem cell walls (Ueki and Citovsky, 2002). RTM1 and RTM2, which function in phloem, were found to restrict long-distance movement of Tobacco etch virus (Chisholm et al., 2000, 2001; Whitham et al., 2000). However, the proteins identified so far only affect the systemic transport of specific viruses. Host components required for broad-spectrum virus systemic transport have yet to be identified.

An integrated vascular network is not only required for plant viral systemic transport, but is also essential for transporting water, nutrients, and signaling molecules during plant growth. The vascular system consists of phloem, xylem, and meristematic cells—procambium (Ye, 2002). The phloem forms a macromolecular trafficking network for transport of nutrients and signaling molecules that regulate physiological and developmental events at the whole-plant level (Ruiz-Medrano et al., 2001). The xylem consists of vessels and tracheary elements (TEs), which are essential for plant mechanical support and water transport. However, the molecular regulatory mechanisms of plant vascular development are still poorly understood.

A major research impediment in this field has been the small number of vascular development mutants because knockout mutations in central regulatory components of vascular development are likely to be lethal. Notably, most of the genetic mutants studied so far, including monopteros (arf5; Hardtke and Berleth, 1998), axr6 (Hobbie et al., 2000), bodenlos (iaa12; Hamann et al., 1999), vascular network defective 1 to 6 (van1-6; Koizumi et al., 2000, 2005), scarface (Deyholos et al., 2000; Sieburth and Deyholos, 2006), lop1/tornado1 (Carland and McHale, 1996), cotyledon vein pattern 1 and 2 (cvp1, cvp2; Carland et al., 2002; Carland and Nelson, 2004), orc (Willemsen et al., 2003), fackel (Jang et al., 2000), and ifl1 (Zhong and Ye, 1999), show either reduced vascular formation or discontinuous vascular patterning. Furthermore, all these mutants also display defects in other aspects of plant growth and development. Very few mutants exhibit increased vascular formation. Therefore, the in vitro zinnia (Zinnia elegans) TE induction system has long been used for vascular differentiation studies (Ye, 2002). Virus-induced gene silencing (VIGS) circumvents lethality by suppressing the expression of essential genes in developed plants and thus serves as an optimal technique for such research. Here we describe the identification of RPN9, which affects broad-spectrum viral systemic transport and regulates vascular development, from a viral transport suppressor screening using VIGS.

RPN9 encodes a subunit of the 26S proteasome, a highly conserved eukaryotic protease responsible for intracellular protein degradation. The 26S proteasome consists of a 20S catalytic core particle (CP) and a 19S regulatory particle (RP), and controls many fundamental biochemical processes by programmed degradation of regulatory protein targets besides its role in removing damaged or misfolded proteins. The ubiquitin/26S proteasome pathway is implicated in numerous diseases, including cancer and neurodegenerative diseases (Mani and Gelmann, 2005). In plants, the 26S proteasome degrades various regulators of diverse cellular processes, including cell division, stress responses, and hormone-signaling pathways (Chinnusamy et al., 2003; Moon et al., 2004; Smalle and Vierstra, 2004).

The capacity of protein selection for degradation by the proteasome is gained through the 19S RP, which recognizes, binds, and unfolds the target proteins, cleaves ubiquitin chains, and directs the target proteins into the lumen of the CP for degradation (Wolf and Hilt, 2004). However, our knowledge of these functions is still limited. The 19S RP is divided further into two subcomplexes, known as the base and the lid. The base contains six ATPase subunits, RPT1 to 6, and three non-ATPase subunits, RPN1, 2, and 10. The RPT subunits use ATP hydrolysis to facilitate channel opening, polyubiquitin chain binding, and protein unfolding (Lam et al., 2002; Hartmann-Petersen et al., 2003). RPN10 binds polyubiquitin chains and contributes to the turnover of multiple proteasome targets (Mayor et al., 2005). In plants, RPN10 regulates abscisic acid (ABA) signaling by targeting ABA-signaling proteins for degradation (Smalle et al., 2003). RPT2a is involved in the maintenance of Arabidopsis (Arabidopsis thaliana) meristems (Ueda et al., 2004). RPN1 is essential for Arabidopsis embryogenesis (Brukhin et al., 2005). The lid contains eight subunits, RPN3, RPN5 to 9, and RPN11 to 12. The functions of lid subunits are poorly understood. Some of them may function in a target-specific manner, presumably by recruiting specific E3 ligases or carrier proteins to deliver ubiquitinated targets to the 26S proteasome (Smalle and Vierstra, 2004). Arabidopsis RPN12 is required for normal cytokinin responses, likely by identifying specific proteins controlling these processes for degradation (Smalle et al., 2002; Smalle and Vierstra, 2004). However, the roles of RPN9 and other subunits of the 19S RP in higher plants, particularly during morphogenesis, are still unknown.

Here we demonstrate that down-regulation of RPN9, but not two other 26S proteasome subunits, inhibits the systemic spread of two taxonomically distinct viruses. Inhibition of virus spread may be largely due to the alteration of vascular formation with reduced phloem and induced xylem. Our further analysis shows that RPN9 functions at least in part through regulation of auxin transport and brassinosteroid (BR) signaling. Given the established role of the 26S proteasome in programmed degradation of regulatory proteins, we propose that RPN9 regulates plant vascular development by targeting a subset of regulatory proteins for degradation. Our data suggest that the BR-signaling protein BZR1 is one such target.


RPN9-Silenced Plants Inhibit Systemic Transport of Two Taxonomically Distinct Viruses

The RPN9 gene was identified in a functional high-throughput VIGS screen to identify genes necessary for viral infection and systemic spread in N. benthamiana, one of the best hosts for plant virus studies. A cDNA library was enriched for TMV-induced genes by suppression subtractive hybridization between cDNAs prepared from TMV-challenged and untreated N. benthamiana plants. The cDNA library was cloned into Potato virus X (PVX)-derived silencing vector pGr106 and introduced into Agrobacterium for high-throughput silencing in wild-type N. benthamiana. GFP-labeled TMV was inoculated on the silenced leaves (start from the third or fourth leaf above the VIGS agroinfiltration sites) 2 weeks after Agrobacterium inoculation for visualization of the viral infection and systemic spread in the silenced plants (Jin et al., 2002). One of the genes that attenuated viral systemic spread after silencing was RPN9 (DQ226994), which encodes a subunit of the 26S proteasome regulatory complex.

The onset of viral infection foci was clearly visible on the inoculated leaves of both RPN9-silenced plants (RPN9) and pGr106 empty vector-treated controls at 4 days postinoculation (4 DPI) of TMV-GFP (Fig. 1A). The infection foci enlarged to a similar size in both RPN9 plants and control leaves, which indicates that cell-to-cell movement of TMV-GFP was not affected. However, the virus failed to achieve systemic spread in RPN9-silenced plants because no GFP fluorescence was detected on the upper uninoculated leaves at 10 DPI (Fig. 1A). In contrast, the virus invaded the vascular tissue of control plants and spread systemically to upper uninoculated leaves (Fig. 1A, 10 DPI). Real-time reverse transcription (RT)-PCR analysis showed that RPN9 mRNA was reduced by 80% to 95% in the silenced leaves beginning at 8 days postsilencing induction (DPS) relative to the empty vector-treated control (Fig. 1B). VIGS induces RNA degradation based on nucleotide sequence identity. Southern analysis under stringent conditions showed at most two copies of RPN9, which might share greater than 80% identity in the N. benthamiana genome (data not shown). Database searches revealed that two RPN9 genes sharing more than 87% identity are present in Arabidopsis and tomato (Lycopersicon esculentum). VIGS of RPN9 in N. benthamiana very likely silences both homologs because the silencing insertion is about 1 kb and covers the conserved region.

Figure 1.
RPN9 is required for TMV systemic spread. A, Systemic spread of TMV-GFP is inhibited in RPN9 plants. TMV-GFP infection foci were green under UV light and the noninfected area showed red due to chlorophyll. Arrows indicate inoculated leaves. B, ...

Previous studies have identified host components that only affect the systemic transport of specific viruses. To determine whether RPN9-silenced plants inhibit systemic transport of other viruses, we tested Turnip mosaic virus (TuMV), a potyvirus that is taxonomically very different from TMV, a tobamovirus. The systemic transport of GFP-tagged TuMV was also attenuated after RPN9 silencing (Fig. 1C); no virus was visualized in the upper uninoculated leaves. Therefore, RPN9 is required for systemic transport of both TMV and TuMV.

RPN9 Plants Exhibit Greatly Increased Leaf Vein Formation

The inhibition of systemic spread of two taxonomically distinct viruses suggested that a common structure required for viral systemic transport, such as the vascular system, might be altered in RPN9 plants. Leaf-clearing experiments did not reveal obvious differences in vein patterning between RPN9 and control leaves. However, veins were slightly denser and clearly thicker in RPN9 leaves than the same order of veins in control leaves (Fig. 2A). Scanning electron microscopy (SEM) revealed that, at 14 DPS, double- and even triple-twisted veins were present on the abaxial surface of RPN9 leaves (Fig. 2B). Interestingly, the additional veins emerged adjacent to the existing veins (Fig. 2B, arrows), including both midveins and high-order veins. In wild-type plants, vascular bundles with normal auxin transport suppress the formation of new vascular strands in their immediate vicinity (Sachs, 1991). This inhibition is suppressed in RPN9 plants.

Figure 2.
Increased vein formation on RPN9 leaves observed by leaf clearing (A) and SEM (B). Leaf tissue was collected at 14 DPS. Leaf clearing was done as described in Sieburth (1999). The abaxial side of leaves was examined under SEM. Arrows indicate ...

Increased Xylem and Reduced Phloem Were Observed in RPN9 Plants

Increased vein formation might be expected to aid viral systemic spread. However, long-distance movement of viruses was inhibited in RPN9 plants. To further explore this unexpected finding, we characterized the fine structures of the veins and stems of RPN9 plants. Compared to control plants that formed veins with a single vascular bundle, two or three vascular bundles were observed in RPN9 plants as early as 12 DPS (Fig. 3A, black circles). These vascular bundles contained increased xylem and decreased phloem in both veins (Fig. 3A) and stems (Fig. 3B). The phenotype became more severe at later stages of silencing (16 DPS or later) and the highly organized bicollateral vascular bundle patterning was disrupted (Fig. 3, A and B). Lignin deposition was greatly increased in RPN9 plants, which confirms that RPN9 silencing promotes xylem formation (Fig. 3C). These data indicate that RPN9 silencing suppresses phloem and promotes xylem, which may be the major cause of viral transport inhibition.

Figure 3.
RPN9 plants exhibit multiple vascular bundles in the veins and increased xylem and reduced phloem in both veins and stems. X, Xylem (red); P, phloem (black). A, Cross section of leaf veins stained with toluidine blue. Vascular bundles are circled. ...

The Function of RPN9 in Vascular Development Is Specific

RPN9 is a subunit of the 19S RP lid, which is a part of the 26S proteasome. To determine whether the phenotype observed is specific to RPN9 silencing or is a common phenomenon of silencing any of the 26S proteasome subunits, genes encoding two other subunits, the α4-subunit of the 20S CP and RPN8 of the 19S lid complex, were silenced. Silencing the α4-subunit resulted in whole-plant death at 10 DPS (Fig. 4A), which indicates that the α4-subunit is absolutely required for plant growth. Silencing of RPN8 induced early senescence and plant death at 14 to 16 DPS, but did not alter leaf morphology (Fig. 4A). No difference in vasculature was observed between control and RPN8 plants by either leaf clearing or SEM (data not shown). In contrast, RPN9 plants survived for 30 to 40 DPS, developing distinctive curled and crinkled leaf morphology. RPN9 leaves were slightly darker green and wider than the control (Fig. 4B). Interestingly, leaf senescence of RPN9 plants initiated from the veins after 28 DPS, whereas the control leaves were still green and healthy (Fig. 4B). Because TE differentiation is tightly coupled with secondary wall formation and leads to autonomous programmed cell death (Jones, 2001; Obara and Fukuda, 2003), the increased formation of xylem in the veins after RPN9 silencing could explain the early senescence that initiated from veins. Real-time RT-PCR showed that the silencing efficiency was similar for all three subunits (Fig. 4C), indicating that the different VIGS-induced phenotypes were due to specific effects of each subunit rather than to a different degree of silencing. Thus, the phenotype of RPN9 silencing in vascular development is rather specific.

Auxin Transport Is Reduced in RPN9 Plants

Various phytohormones and steroids have been implicated in vascular development and vein patterning (Fukuda, 2004). Polar auxin flow is essential for continuous vascular pattern formation (Scarpella et al., 2006). Auxin transport inhibitors induce the formation of a strong continuous vasculature along leaf margins (Mattsson et al., 1999; Sieburth, 1999). The asymmetrically localized auxin-efflux carrier, AtPIN1, is responsible for polarized auxin flow, which may lead to the formation of continuous columns of procambial cells (Galweiler et al., 1998; Fukuda, 2004). The Ser-Thr kinase PINOID is preferentially expressed in young vascular tissues and acts as a binary switch controlling the polar localization of PIN proteins (Christensen et al., 2000; Friml et al., 2004). Vascular bundles with high rates of auxin transport were found to inhibit the development of new vascular strands in their immediate vicinity (Sachs, 1991). RPN9 plants show double- and even triple-twisted veins (Fig. 2) and the extra veins emerged adjacent to the existing veins (Fig. 2, arrows). These phenotypes suggested that auxin transport might be altered in RPN9 plants, although the multiple veins were not concentrated along the leaf margins.

To test this hypothesis, we first analyzed the gene expression level of PIN1 and PINOID, two major players in auxin transport. PIN1 mRNA levels were similar in control and RPN9 plants (data not shown). In contrast, PINOID mRNA levels were reduced in RPN9 plants compared to the control (Fig. 5A). PINOID is preferentially expressed in young vascular tissues and controls the polar localization of PIN proteins for auxin transport (Christensen et al., 2000; Friml et al., 2004). The down-regulation of PINOID provides another indication that auxin transport might be affected in RPN9 plants. Therefore, we directly measured auxin polar transport activity in midveins using 14C-labeled radioactive indole-3-acetic acid (IAA), a major form of endogenous auxin. IAA polar transport in RPN9 veins was reduced to approximately 57% of the control beginning at 10 DPS (Fig. 5, B and C). We detected IAA transport only in a basipetal direction, which was inhibited in the presence of 1-N-naphthylphtalamic acid (NPA), an auxin transport inhibitor (Fig. 5, B and C). The double and triple veins were not typically observed in RPN9 leaves until after 12 DPS and IAA transport did not show substantially more severe inhibition at later time points, which suggests that reduced IAA transport is likely the cause of the extra vein formation instead of a secondary effect of reduced phloem.

Figure 5.
Auxin transport is reduced in RPN9 plants. A, PINOID mRNA is reduced at 8 DPS in RPN9 plants. Three biological samples were tested and gave similar results. Error bars represent the sd of the mean. B, Distribution of [14C]IAA in the ...

To confirm that the inhibition of auxin transport really induces vein formation in N. benthamiana, we germinated seeds in Murashisge and Skoog medium containing the auxin transport inhibitor NPA. The presence of 25 μm NPA induced thick vein formation (Fig. 5D), mimicking the phenotype of extra vein formation in RPN9 plants (Fig. 3C). Plants germinated on NPA arrested and could not be used for the viral infection assay. To examine the effect of extra vein formation on viral systemic transport, we sprayed 3-week-old N. benthamiana with 150 μm of NPA as described in Nemhauser et al. (2000). Leaves became crinkly and extra veins were observed in these leaves 1 week after NPA treatment, especially along the leaf margins (Fig. 5E). Systemic transport of TMV-GFP was clearly inhibited in plants that had been sprayed with NPA, but was not completely blocked (Fig. 5E). Virus could enter the vascular system and could occasionally be transported to the upper uninfected leaves (data not shown). These results suggest that inhibition of auxin transport indeed induces extra vein formation, which may partially inhibit viral systemic transport.

RPN9 Silencing Alters BR Signaling

RPN9 plants had curled crinkly leaves with increased xylem and reduced phloem in the vascular bundles. The silenced leaves were dark green and underwent early senescence initiated from the veins (Fig. 4B). These phenotypes were not observed in NPA-treated plants and cannot all be explained simply by reduced auxin transport, but point to the likelihood of the involvement of the BR-signaling pathway in RPN9-mediated regulation. BRs are involved in xylem cell differentiation by regulating secondary wall formation and the programmed cell death that occurs during TE differentiation (Yamamoto et al., 1997). Increased endogenous BRs or increased BR perception promotes xylem and suppresses phloem formation (Szekeres et al., 1996; Yamamoto et al., 1997; Choe et al., 1999; Nagata et al., 2001; Cano-Delgado et al., 2004) and induces secondary wall formation and programmed cell death (Yamamoto et al., 1997). To test the effect of BR on plant growth, we grew N. benthamiana on Murashige and Skoog medium containing 100 nm brassinolide (BL; the most biologically active BR). The cotyledons and leaves of the BL-treated plants curled down at the sides (Fig. 6A), which is similar to the leaves of RPN9 plants (Fig. 4B). Exogenous IAA did not alter cotyledon and leaf morphology (Fig. 6A).

Figure 6.
BR signaling is involved in RPN9-mediated regulation. A, N. benthamiana cotyledon curled down under BL treatment. Seedlings were grown on Murashige and Skoog medium with 100 nm BL or 10 μm IAA. Pictures were taken at 10 d postgermination. B, ...

BRs are perceived by the receptor-like kinases BRI1, BRL1, and BRL3, which then inactivate the negative regulator BIN2 and stabilize its downstream transcription factors BZR1 and BES1 to promote BR-regulated gene expression (Cano-Delgado et al., 2004; Nemhauser and Chory, 2004; Vert and Chory, 2006). BRL1 and BRL3 are specifically expressed in vascular cells and a loss-of-function mutation in BRL1 causes reduced xylem and increased phloem differentiation (Cano-Delgado et al., 2004). Conversely, overexpression of BRI1 results in increased procambial activity leading to increased xylem differentiation. These results suggest the existence of a vascular cell-specific BR perception system. The N. benthamiana ortholog of the vascular-localized BRL1 was clearly induced in RPN9 leaves (Fig. 6B), whereas the mRNA levels of NbBRI1 and NbBIN2 remained unchanged in RPN9 plants (data not shown). Gene expression analyses were carried out on samples collected 8 DPS, a time when the expression of RPN9 was clearly suppressed (Fig. 1B), but the plants appeared morphologically normal. Thus, the observed changes in gene expression are likely to reflect direct effects of RPN9 silencing rather than secondary effects from morphological alterations. Our results indicate a possible increase of BR perception in vascular tissues in RPN9 plants. Loss of function in BRL1 causes increased phloem and reduced xylem differentiation (Cano-Delgado et al., 2004), which correlates perfectly with our data that RPN9 plants have elevated BRL1 expression and display increased secondary xylem and reduced phloem.

As a subunit of the 26S proteasome, RPN9 is likely to function through protein degradation. To determine whether RPN9 regulates degradation of many proteins or only a small number of targets, we analyzed total polyubiquinated proteins. If RPN9 affects global protein degradation, we would expect to see an accumulation of polyubiquinated proteins in RPN9 plants, as was observed in the rpn10 mutant in Arabidopsis (Smalle et al., 2003). We did not detect obvious differences in the level of total polyubiquinated protein between control and RPN9 plants (Fig. 6C). Therefore, we hypothesize that RPN9 regulates vascular development by targeted degradation of specific proteins.

Previous studies have shown that a downstream transcription factor of the BR-signaling pathway, BZR1, is regulated by 26S proteasome-mediated degradation (Wang et al., 2002). To determine whether BZR1 is a possible target of RPN9, we transiently expressed Arabidopsis BZR1-CFP in the RPN9 and control leaves and detected increased BZR1-CFP accumulation in RPN9 leaves (Fig. 6D). In contrast, we did not detect differences in BRI1-GFP accumulation between control and RPN9 plants (Fig. 6D). Thus, BZR1 is likely a target of RPN9-mediated protein degradation.

To determine whether RPN9 silencing affects other proteins regulated by 26S proteasome degradation, we examined the protein level of ICE1 (Chinnusamy et al., 2003), a MYC transcription factor regulated by ubiquitin/26S proteasome-mediated proteolysis (Dong et al., 2006). We transiently expressed the AtICE1-GFP fusion and detected the degradation of ICE1-GFP after 16 h at 0°C in both RPN9 and control leaves (Fig. 6E), which indicates that ubiquitin/26S proteasome-mediated ICE1 degradation is not affected by RPN9 silencing.


Many plant viruses rely on vascular structure, primarily the phloem, for their systemic transport. The vascular system is also essential for plant development. However, the molecular regulatory mechanisms of plant vascular development are still poorly understood. From a viral transport suppressor screen using VIGS in N. benthamiana, we have identified RPN9, a regulatory subunit of the 26S proteasome. Silencing RPN9 inhibits the systemic transport of two taxonomically distinct viruses, which leads to the finding that RPN9 regulates plant vascular development. Silencing of RPN9 induced extra vein formation with increased xylem and reduced phloem, which may largely contribute to the inhibition of broad-spectrum viral systemic transport. This genetic evidence indicates that plant vascular development is regulated at the proteolysis level via a specific 26S proteasome subunit. RPN9 is a subunit of the 19S RP lid. Limited studies suggest that different subunits of 19S RP have well-differentiated functions (Glickman et al., 1999). On the basis of recent studies on RPN12a (Smalle et al., 2002), RPN10 (Smalle et al., 2003), and RPN1 (Brukhin et al., 2005) in plants that function specifically in cytokinin-, ABA-, and embryogenesis-signaling pathways, respectively, we hypothesize that RPN9 regulates vascular development via selective degradation of specific target proteins.

The extra vein formation in the immediate vicinity of the existing veins led us to the finding that auxin transport is reduced in RPN9 plants. The down-regulation of PINOID may contribute at least in part to the reduction of auxin transport in RPN9 plants. The roles of EMB30/GN and VAN3 in auxin transport and auxin signaling strongly indicate the involvement of vesicle transport in leaf vein formation (Geldner et al., 2003; Koizumi et al., 2005). Their roles in RPN9-mediated regulation have yet to be determined. Thus, we propose that RPN9 is likely to regulate auxin transport through the selective degradation of a negative regulator of PINOID that regulates polar localization of PIN proteins or components that regulate the vesicle transport of PIN proteins. Future protein accumulation analysis via a proteomic approach will help identify such proteins.

BRs have been shown to regulate plant vascular development. Up-regulation of the vascular-specific BR receptor BRL1 and the distinctive phenotype resulting from RPN9 silencing strongly indicate the involvement of BR signaling in RPN9 regulation. BRL1 is specifically expressed in vascular tissues and the brl1 knockout mutant displays increased phloem and reduced xylem (Cano-Delgado et al., 2004). The elevated expression of BRL1 observed in RPN9 plants is likely to increase BR perception and activate the BR-signaling pathway in the vascular system, which results in increased xylem and reduced phloem and early senescence initiated from the veins. BRL1 is likely to mediate a vascular-specific BR perception system. The phosphorylation and proteolytic degradation of BR-signaling transcription factors BZR1 and BES1 are critical steps of BR signal transduction pathways. Our results show that BZR1 is selectively stabilized in RPN9 plants, whereas degradation of ICE1 is not affected. Immunoblot analysis of total proteins with antiubiquitin antibody revealed no obvious difference between RPN9 plants and controls in accumulation of ubiquitin-conjugated proteins. These data suggest that RPN9 silencing does not perturb the function of the 26S proteasome globally, but rather affects the RPN9-related functions specifically by accumulating a specific subset of protein targets, such as BZR1. Thus, we provided evidence that RPN9 specifically regulates vascular development at least in part by the mechanism of regulating auxin transport and BR signaling.

Several recent studies address cross talk and overlap between BR and auxin responses and indicate a complex interdependency between the two phytohormones (Mussig et al., 2002; Goda et al., 2004; Nemhauser et al., 2004). BRs and auxin were shown to act synergistically in the control of hypocotyl elongation and lateral root growth (Mandava, 1988; Bao et al., 2004; Nemhauser et al., 2004). Our results suggest there may be an interaction between auxin transport and BR signaling in plant vascular development.

Cytokinin also plays an important role in vascular development by regulating the formation of procambial cells (Mahonen et al., 2000; Fukuda, 2004). Real-time RT-PCR analysis on cytokinin receptor and signaling genes, HK3, ARR15, and ARR5, revealed essentially no difference between RPN9 and control samples. Thus, cytokinin might not play a direct role in RPN9-mediated regulation. Several transcription factors, such as MP (ARF5; Hardtke and Berleth, 1998), homeobox protein ATHB-8 (Baima et al., 2001), and APL, a Myb-like transcription factor promoting phloem differentiation and repressing xylem differentiation (Bonke et al., 2003), are involved in vascular patterning and development. We did not detect obvious alterations in expression of these genes in RPN9 plants by real-time RT-PCR (data not shown). However, RPN9 might still regulate the expression of these genes at the protein level.

Within plant, fungal, and animal kingdoms, RPN9 is the most divergent subunit of the 26S proteasome (Yang et al., 2004). It contains a PINT/PCI domain that is present in several subunits of the 19S RP lid, COP9/signalosome complex, and translation initiation factor eIF3 (Wei and Deng, 2003), reflecting a possible common evolutionary ancestry. RPN9 is not essential in yeast (Saccharomyces cerevisiae) and Caenorhabditis elegans (Takeuchi et al., 1999, 2002), but its deletion results in temperature-sensitive growth arrest, reduced 26S proteasome accumulation, and incomplete 26S proteasome assembly in yeast (Takeuchi et al., 1999; Takeuchi and Toh-e, 2001). Recently, it was demonstrated that silencing of RPN9, RPN3, and the 20S core α6-subunit activates programmed cell death after silencing (Kim et al., 2003), which implies an essential role for RPN9 in plants. This result agrees with our data that RPN9 silencing promotes xylem formation, a process of programmed cell death in plants, and induces early senescence initiating from leaf veins. Our data also suggest that RPN9 is essential in plants and specifically regulates plant vascular development and leaf vein formation. This evidence indicates that the 26S proteasome regulates plant vascular development via a specific regulatory subunit RPN9, possibly by selective degradation of regulatory components in auxin transport and BR signaling. Further identification of the regulatory protein targets of RPN9 will help elucidate the complex molecular mechanism of plant vascular development.


Plant Growth Conditions

Nicotiana benthamiana plants and Arabidopsis (Arabidopsis thaliana) plants were grown in a plant growth room at 22°C under a 16-h light/8-h dark cycle. For BL and auxin treatments, seedlings were grown on Murashige and Skoog medium with 100 nm BL or 10 μm IAA.

VIGS Screen

A subtraction cDNA library was made by suppression subtractive hybridization between cDNAs prepared from TMV-challenged N. benthamiana (mixture of 1-, 2-, 4-, 8-, 24-, and 48-h postinoculation) and unchallenged plants with a PCR-select cDNA subtraction kit (BD Biosciences). The cDNA library was cloned into the PVX-derived silencing vector pGr106 (Takken et al., 2000) and introduced into Agrobacterium GV2260. The VIGS screen was conducted by toothpick inoculation on the abaxial side of the leaves of 4-week-old plants with Agrobacterium colonies carrying silencing constructs. Agrobacterium carrying a TMV binary construct expressing GFP with a coat protein promoter was infiltrated into wild-type N. benthamiana plants for virus propagation. TMV-GFP leaf sap was extracted and inoculated on silenced leaves 14 d after toothpick inoculation for monitoring viral transport as described in Jin et al. (2002).

Histological Analysis

Leaf and stem tissues were fixed and dehydrated for SEM analysis as described (Dong et al., 2005). Dehydrated samples were critical-point dried, sputter coated, and observed under a SEM (XL30-FEG; Philips).

For light microscopy, leaf and stem tissues were fixed, dehydrated, embedded in Technovit 7100 resin (Kulzer), and sectioned at 4-μm thickness as described (Dong et al., 2005). Sections were stained with 0.5% toluidine blue or 0.01% Auromine O (fluorescence dye) for visualization under a Nikon Microphot FXA microscope (Nikon).

Real-Time RT-PCR

Samples were collected at 8 DPI and subjected to RNA extraction and cDNA synthesis as described (Jin et al., 2002). Real-time PCR was performed on an iCycler (Bio-Rad) with SYBR Green (Bio-Rad). Each sample was measured in triplicate and calculated by the standard curve method (iCycler guide). The Actin gene was used as an internal reference. Real-time primers used for N. benthamiana genes are as follows: Actin forward, 5′-CCCAAAGGCTAATCGTGAAA-3′, reverse, 5′-ACCATCACCAGAGTCCAACA-3′; PINOID forward, 5′-GCAGCGTAGCTTCGTTTGAT-3′, reverse, 5′-TTGTTGGGACCCACGAGTAT-3′; BRL1 forward, 5′-TATACGAATACATGAAATGGGGAAG-3′, reverse, 5′-AGCATTTACCAGCCTTGCCATTCC-3′; RPN9 forward, 5′-ATGGCCGCTTTAGAGTACTT-3′, reverse, 5′-TGGTACAGATCTGATAGCGTA-3′; RPN8 forward, 5′-TTTAGATCGACTGACAGAGAAC-3′, reverse, 5′-ATTCATCTATTTTAGGTACCAAG-3′, and 20S α4-subunit forward, 5′-AAGTCTACTCCCAAGCTTCAG-3′, reverse, 5′-CAGTAACAGGATCCTCAACAG-3′. Primers for quantifying RPN9, RPN8, and the 20S α4-subunit were designed from outside the silencing region. All PCR products were sequenced and confirmed to be the correct clones. All gene expression analyses were repeated at least three times on different sets of silenced plants with comparable results, and the data presented are means of triplicates for each condition in one representative experiment. Error bars are the sd of the mean.

Auxin Transport

IAA transport activity was measured as described (Okada et al., 1991). Two-centimeter lengths of leaf midveins were cut from RPN9 and control leaves of similar size and placed into inorganic Murashige and Skoog medium containing [14C]IAA at 1.45 μm (0.08 μCi/mL) with or without 60 μm NPA in upright (acropetal) and inverted (basipetal) orientations. Distribution of labeled IAA was visualized by exposing the veins to a phosphor-imaging plate after 18 h of incubation. The amount of incorporated radioactivity within the upper 1-cm pieces was measured by a liquid scintillation counter.

Polar Auxin Transport Inhibitor Treatments

For seedling assays, seeds were germinated on Murashige and Skoog medium with 25 μm NPA (Uniroyal Chemical). Leaves were taken after 10 to 14 d for leaf-clearing analysis. For the viral infection assay, 3-week-old plants were sprayed with a heavy mist of 100 or 200 μm NPA with 0.01% Silwet L-77 (a surfactant; Lehle Seeds) as described in Nemhauser et al. (2000). Mock treatments were performed with distilled water containing 0.01% Silwet L-77.

Transient Expression Assay

Agrobacterium GV2260 (OD600 = 1.0) carrying various GFP fusion constructs (BRI-GFP, BZR1-CFP, or GFP-ICE1) was infiltrated into RPN9 and control leaves at 9 DPI. Leaf discs were collected at 66 h postinoculation and subjected to western analysis with GFP antibody sc-8334 (Santa Cruz), which recognizes both GFP and cyan fluorescent protein fusions. For detecting GFP-ICE1 fusion, the total protein extract was immunoprecipitated with GFP antibody prior to western-blot analysis.

Sequences of the mRNA fragments used for silencing RPN9 (DQ226994), RPN8 (DQ226995), and the 20S α4-subunit (DQ226996) have been deposited in GenBank.


We thank Dr. Elizabeth Lord for advice on histological analysis and access to the microscopy facility in her lab; Dr. Barbara Baker for the TMV-GFP construct; Dr. Darleen DeMason for [14C]IAA, NPA, and helpful discussions; Dr. Zhiyong Wang for the BZR1-CFP construct; Dr. Joanne Chory for the BRI1-GFP construct; Dr. Jian-Kang Zhu for the GFP-ICE1 construct and helpful discussions; Dr. James Borneman for access to a Bio-Rad real-time iCycler in his lab; and Dr. Xuemei Chen, Dr. Patty Springer, and Dr. Zhenbiao Yang for critical reading of this manuscript.


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: Hailing Jin (ude.rcu@jgniliah).



  • Baima S, Possenti M, Matteucci A, Wisman E, Altamura MM, Ruberti I, Morelli G (2001) The Arabidopsis ATHB-8 HD-zip protein acts as a differentiation-promoting transcription factor of the vascular meristems. Plant Physiol 126: 643–655 [PMC free article] [PubMed]
  • Bao F, Shen JJ, Brady SR, Muday GK, Asami T, Yang ZB (2004) Brassinosteroids interact with auxin to promote lateral root development in Arabidopsis. Plant Physiol 134: 1624–1631 [PMC free article] [PubMed]
  • Bonke M, Thitamadee S, Mahonen AP, Hauser MT, Helariutta Y (2003) APL regulates vascular tissue identity in Arabidopsis. Nature 426: 181–186 [PubMed]
  • Brukhin V, Gheyselinck J, Gagliardini V, Genschik P, Grossniklaus U (2005) The RPN1 subunit of the 26S proteasome in Arabidopsis is essential for embryogenesis. Plant Cell 17: 2723–2737 [PMC free article] [PubMed]
  • Cano-Delgado A, Yin YH, Yu C, Vafeados D, Mora-Garcia S, Cheng JC, Nam KH, Li JM, Chory J (2004) BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis. Development 131: 5341–5351 [PubMed]
  • Carland FM, Fujioka S, Takatsuto S, Yoshida S, Nelson T (2002) The identification of CVP1 reveals a role for sterols in vascular patterning. Plant Cell 14: 2045–2058 [PMC free article] [PubMed]
  • Carland FM, McHale NA (1996) LOP1: a gene involved in auxin transport and vascular patterning in Arabidopsis. Development 122: 1811–1819 [PubMed]
  • Carland FM, Nelson T (2004) Cotyledon vascular pattern2-mediated inositol (1,4,5) triphosphate signal transduction is essential for closed venation patterns of Arabidopsis foliar organs. Plant Cell 16: 1263–1275 [PMC free article] [PubMed]
  • Cheng NH, Su CL, Carter SA, Nelson RS (2000) Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana. Plant J 23: 349–362 [PubMed]
  • Chinnusamy V, Ohta M, Kanrar S, Lee BH, Hong XH, Agarwal M, Zhu JK (2003) ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev 17: 1043–1054 [PMC free article] [PubMed]
  • Chisholm ST, Mahajan SK, Whitham SA, Yamamoto ML, Carrington JC (2000) Cloning of the Arabidopsis RTM1 gene, which controls restriction of long-distance movement of tobacco etch virus. Proc Natl Acad Sci USA 97: 489–494 [PMC free article] [PubMed]
  • Chisholm ST, Parra MA, Anderberg RJ, Carrington JC (2001) Arabidopsis RTM1 and RTM2 genes function in phloem to restrict long-distance movement of tobacco etch virus. Plant Physiol 127: 1667–1675 [PMC free article] [PubMed]
  • Choe SW, Noguchi T, Fujioka S, Takatsuto S, Tissier CP, Gregory BD, Ross AS, Tanaka A, Yoshida S, Tax FE, et al (1999) The Arabidopsis dwf7/ste1 mutant is defective in the Delta(7) sterol C-5 desaturation step leading to brassinosteroid biosynthesis. Plant Cell 11: 207–221 [PMC free article] [PubMed]
  • Christensen SK, Dagenais N, Chory J, Weigel D (2000) Regulation of auxin response by the protein kinase PINOID. Cell 100: 469–478 [PubMed]
  • Crawford KM, Zambryski PC (1999) Phloem transport: are you chaperoned? Curr Biol 9: R281–R285 [PubMed]
  • Deyholos MK, Cordner G, Beebe D, Sieburth LE (2000) The SCARFACE gene is required for cotyledon and leaf vein patterning. Development 127: 3205–3213 [PubMed]
  • Dong CH, Agarwal M, Zhang Y, Xie Q, Zhu JK (2006) The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proc Natl Acad Sci USA 103: 8281–8286 [PMC free article] [PubMed]
  • Dong J, Kim ST, Lord EM (2005) Plantacyanin plays a role in reproduction in Arabidopsis. Plant Physiol 138: 778–789 [PMC free article] [PubMed]
  • Friml J, Yang X, Michniewicz M, Weijers D, Quint A, Tietz O, Benjamins R, Ouwerkerk PBF, Ljung K, Sandberg G, et al (2004) A PINOID-dependent binary switch in apical-basal PIN polar targeting directs auxin efflux. Science 306: 862–865 [PubMed]
  • Fukuda H (2004) Signals that control plant vascular cell differentiation. Nat Rev Mol Cell Biol 5: 379–391 [PubMed]
  • Galweiler L, Guan CH, Muller A, Wisman E, Mendgen K, Yephremov A, Palme K (1998) Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue. Science 282: 2226–2230 [PubMed]
  • Geldner N, Anders N, Wolters H, Keicher J, Kornberger W, Muller P, Delbarre A, Ueda T, Nakano A, Jurgens G (2003) The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell 112: 219–230 [PubMed]
  • Glickman MH, Rubin DM, Fu HY, Larsen CN, Coux O, Wefes I, Pfeifer G, Cjeka Z, Vierstra R, Baumeister W, et al (1999) Functional analysis of the proteasome regulatory particle. Mol Biol Rep 26: 21–28 [PubMed]
  • Goda H, Sawa S, Asami T, Fujioka S, Shimada Y, Yoshida S (2004) Comprehensive comparison brassinosteroid-regulated of auxin-regulated and brassinosteroid-regulated genes in Arabidopsis. Plant Physiol 134: 1555–1573 [PMC free article] [PubMed]
  • Hamann T, Mayer U, Jurgens G (1999) The auxin-insensitive bodenlos mutation affects primary root formation and apical-basal patterning in the Arabidopsis embryo. Development 126: 1387–1395 [PubMed]
  • Hardtke CS, Berleth T (1998) The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development. EMBO J 17: 1405–1411 [PMC free article] [PubMed]
  • Hartmann-Petersen R, Seeger M, Gordon C (2003) Transferring substrates to the 26S proteasome. Trends Biochem Sci 28: 26–31 [PubMed]
  • Hobbie L, McGovern M, Hurwitz LR, Pierro A, Liu NY, Bandyopadhyay A, Estelle M (2000) The axr6 mutants of Arabidopsis thaliana define a gene involved in auxin response and early development. Development 127: 23–32 [PubMed]
  • Jang JC, Fujioka S, Tasaka M, Seto H, Takatsuto S, Ishii A, Aida M, Yoshida S, Sheen J (2000) A critical role of sterols in embryonic patterning and meristem programming revealed by the fackel mutants of Arabidopsis thaliana. Genes Dev 14: 1485–1497 [PMC free article] [PubMed]
  • Jin H, Axtell MJ, Dahlbeck D, Ekwenna O, Zhang SQ, Staskawicz B, Baker B (2002) NPK1, an MEKK1-like mitogen-activated protein kinase kinase kinase, regulates innate immunity and development in plants. Dev Cell 3: 291–297 [PubMed]
  • Jones AM (2001) Programmed cell death in development and defense. Plant Physiol 125: 94–97 [PMC free article] [PubMed]
  • Kim M, Ahn JW, Jin UH, Choi D, Paek KH, Pai HS (2003) Activation of the programmed cell death pathway by inhibition of proteasome function in plants. J Biol Chem 278: 19406–19415 [PubMed]
  • Koizumi K, Naramoto S, Sawa S, Yahara N, Ueda T, Nakano A, Sugiyama M, Fukuda H (2005) VAN3 ARF-GAP-mediated vesicle transport is involved in leaf vascular network formation. Development 132: 1699–1711 [PubMed]
  • Koizumi K, Sugiyama M, Fukuda H (2000) A series of novel mutants of Arabidopsis thaliana that are defective in the formation of continuous vascular network: calling the auxin signal flow canalization hypothesis into question. Development 127: 3197–3204 [PubMed]
  • Lam YA, Lawson TG, Velayutham M, Zweier JL, Pickart CM (2002) A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal. Nature 416: 763–767 [PubMed]
  • Mahonen AP, Bonke M, Kauppinen L, Riikonen M, Benfey PN, Helariutta Y (2000) A novel two-component hybrid molecule regulates vascular morphogenesis of the Arabidopsis root. Genes Dev 14: 2938–2943 [PMC free article] [PubMed]
  • Mandava NB (1988) Plant growth-promoting brassinosteroids. Annu Rev Plant Physiol Plant Mol Biol 39: 23–52
  • Mani A, Gelmann EP (2005) The ubiquitin-proteasome pathway and its role in cancer. J Clin Oncol 23: 4776–4789 [PubMed]
  • Mattsson J, Sung ZR, Berleth T (1999) Responses of plant vascular systems to auxin transport inhibition. Development 126: 2979–2991 [PubMed]
  • Mayor TLJ, Graumann J, Smith GT, Deshaies RJ (2005) Analysis of polyubiquitin conjugates reveals that the rpn10 substrate receptor contributes to the turnover of multiple proteasome targets. Mol Cell Proteomics 4: 741–751 [PubMed]
  • Moon J, Parry G, Estelle M (2004) The ubiquitin-proteasome pathway and plant development. Plant Cell 16: 3181–3195 [PMC free article] [PubMed]
  • Mussig C, Fischer S, Altmann T (2002) Brassinosteroid-regulated gene expression. Plant Physiol 129: 1241–1251 [PMC free article] [PubMed]
  • Nagata N, Asami T, Yoshida S (2001) Brassinazole, an inhibitor of brassinosteroid biosynthesis, inhibits development of secondary xylem in cress plants (Lepidium sativum). Plant Cell Physiol 42: 1006–1011 [PubMed]
  • Nemhauser JL, Chory J (2004) BRing it on: new insights into the mechanism of brassinosteroid action. J Exp Bot 55: 265–270 [PubMed]
  • Nemhauser JL, Feldman LJ, Zambryski PC (2000) Auxin and ETTIN in Arabidopsis gynoecium morphogenesis. Development 127: 3877–3888 [PubMed]
  • Nemhauser JL, Mockler TC, Chory J (2004) Interdependency of brassinosteroid and auxin signaling in Arabidopsis. PLoS Biol 2: 1460–1471 [PMC free article] [PubMed]
  • Obara K, Fukuda H (2003) Programmed cell death in xylem differentiation. In J Gray, ed, Programmed Cell Death in Plants. Sheffield Academic Press, Sheffield, UK, pp 131–151
  • Okada K, Ueda J, Komaki MK, Bell CJ, Shimura Y (1991) Requirement of the auxin polar transport system in early stages of Arabidopsis floral bud formation. Plant Cell 3: 677–684 [PMC free article] [PubMed]
  • Ruiz-Medrano R, Xoconostle-Cazares B, Lucas WJ (2001) The phloem as a conduit for inter-organ communication. Curr Opin Plant Biol 4: 202–209 [PubMed]
  • Sachs T (1991) Cell polarity and tissue patterning in plants. Development (Suppl) 1: 83–93
  • Santa Cruz S (1999) Perspective: phloem transport of viruses and macromolecules—what goes in must come out. Trends Microbiol 7: 237–241 [PubMed]
  • Scarpella E, Marcos D, Friml J, Berleth T (2006) Control of leaf vascular patterning by polar auxin transport. Genes Dev 20: 1015–1027 [PMC free article] [PubMed]
  • Scholthof HB (2005) Plant virus transport: motions of functional equivalence. Trends Plant Sci 10: 376–382 [PubMed]
  • Sieburth LE (1999) Auxin is required for leaf vein pattern in Arabidopsis. Plant Physiol 121: 1179–1190 [PMC free article] [PubMed]
  • Sieburth LE, Deyholos MK (2006) Vascular development: the long and winding road. Curr Opin Plant Biol 9: 48–54 [PubMed]
  • Smalle J, Kurepa J, Yang PZ, Babiychuk E, Kushnir S, Durski A, Vierstra RD (2002) Cytokinin growth responses in Arabidopsis involve the 26S proteasome subunit RPN12. Plant Cell 14: 17–32 [PMC free article] [PubMed]
  • Smalle J, Kurepa J, Yang PZ, Emborg TJ, Babiychuk E, Kushnir S, Vierstra RD (2003) The pleiotropic role of the 26S proteasome subunit RPN10 in Arabidopsis growth and development supports a substrate-specific function in abscisic acid signaling. Plant Cell 15: 965–980 [PMC free article] [PubMed]
  • Smalle J, Vierstra RD (2004) The ubiquitin 26S proteasome proteolytic pathway. Annu Rev Plant Biol 55: 555–590 [PubMed]
  • Szekeres M, Nemeth K, Koncz-Kalman Z, Mathur J, Kauschmann A, Altmann T, Redei GP, Nagy F, Schell J, Koncz C (1996) Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85: 171–182 [PubMed]
  • Takahashi M, Iwasaki H, Inoue H, Takahashi K (2002) Reverse genetic analysis of the Caenorhabditis elegans 26S proteasome subunits by RNA interference. Biol Chem 383: 1263–1266 [PubMed]
  • Takeuchi J, Fujimuro M, Yokosawa H, Tanaka K, Toh-e A (1999) Rpn9 is required for efficient assembly of the yeast 26S proteasome. Mol Cell Biol 19: 6575–6584 [PMC free article] [PubMed]
  • Takeuchi J, Toh-e A (2001) Genetic dissection of the yeast 26S proteasome: cell cycle defects caused by the Delta rpn9 mutation. Biochimie 83: 333–340 [PubMed]
  • Takken FLW, Luderer R, Gabriels S, Westerink N, Lu R, de Wit P, Joosten M (2000) A functional cloning strategy, based on a binary PVX-expression vector, to isolate HR-inducing cDNAs of plant pathogens. Plant J 24: 275–283 [PubMed]
  • Tamagnone L, Merida A, Parr A, Mackay S, Culianez-Macia FA, Roberts K, Martin C (1998) The AmMYB308 and AmMYB330 transcription factors from antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco. Plant Cell 10: 135–154 [PMC free article] [PubMed]
  • Ueda M, Matsui K, Ishiguro S, Sano R, Wada T, Paponov I, Palme K, Okada K (2004) The HALTED ROOT gene encoding the 26S proteasome subunit RPT2a is essential for the maintenance of Arabidopsis meristems. Development 131: 2101–2111 [PubMed]
  • Ueki S, Citovsky V (2002) The systemic movement of a tobamovirus is inhibited by a cadmium-ion-induced glycine-rich protein. Nat Cell Biol 4: 478–485 [PubMed]
  • Vert G, Chory J (2006) Downstream nuclear events in brassinosteroid signalling. Nature 441: 96–100 [PubMed]
  • Wang ZY, Nakano T, Gendron J, He JX, Chen M, Vafeados D, Yang YL, Fujioka S, Yoshida S, Asami T, et al (2002) Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev Cell 2: 505–513 [PubMed]
  • Wei N, Deng XW (2003) The COP9 signalosome. Annu Rev Cell Dev Biol 19: 261–286 [PubMed]
  • Whitham SA, Anderberg RJ, Chisholm ST, Carrington JC (2000) Arabidopsis RTM2 gene is necessary for specific restriction of tobacco etch virus and encodes an unusual small heat shock-like protein. Plant Cell 12: 569–582 [PMC free article] [PubMed]
  • Willemsen V, Friml J, Grebe M, van den Toorn A, Palme K, Scheres B (2003) Cell polarity and PIN protein positioning in Arabidopsis require STEROL METHYLTRANSFERASE1 function. Plant Cell 15: 612–625 [PMC free article] [PubMed]
  • Wolf DH, Hilt W (2004) The proteasome: a proteolytic nanomachine of cell regulation and waste disposal. Biochim Biophys Acta 1695: 19–31 [PubMed]
  • Yamamoto R, Demura T, Fukuda H (1997) Brassinosteroids induce entry into the final stage of tracheary element differentiation in cultured zinnia cells. Plant Cell Physiol 38: 980–983 [PubMed]
  • Yang PZ, Fu HY, Walker J, Papa CM, Smalle J, Ju YM, Vierstra RD (2004) Purification of the Arabidopsis 26S proteasome—biochemical and molecular analyses revealed the presence of multiple isoforms. J Biol Chem 279: 6401–6413 [PubMed]
  • Ye ZH (2002) Vascular tissue differentiation and pattern formation in plants. Annu Rev Plant Biol 53: 183–202 [PubMed]
  • Zambryski P (2004) Cell-to-cell transport of proteins and fluorescent tracers via plasmodesmata during plant development. J Cell Biol 164: 165–168 [PMC free article] [PubMed]
  • Zambryski P, Crawford K (2000) Plasmodesmata: gatekeepers for cell-to-cell transport of developmental signals in plants. Annu Rev Cell Dev Biol 16: 393–421 [PubMed]
  • Zhong RQ, Ye ZH (1999) IFL1, a gene regulating interfascicular fiber differentiation in Arabidopsis, encodes a homeodomain-leucine zipper protein. Plant Cell 11: 2139–2152 [PMC free article] [PubMed]

Articles from Plant Physiology are provided here courtesy of American Society of Plant Biologists
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...