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First published online April 4, 2008; 10.1105/tpc.107.057737 The Plant Cell 20:969-981 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Phosducin-Like Protein 3 Is Required for Microtubule-Dependent Steps of Cell Division but Not for Meristem Growth in Arabidopsis[W],[OA]Department of Cell and Developmental Biology, John Innes Centre, Norwich, NR4 7UH, United Kingdom 1 Address correspondence to robert.sablowski{at}bbsrc.ac.uk.
Given the central role of cell division in meristems, one might expect meristem growth to be regulated by mitotic checkpoints, including checkpoints for correct microtubule function. Here, we studied the role of two close Phosducin-Like Protein 3 homologs from Arabidopsis thaliana (PLP3a and PLP3b) in the microtubule assembly pathway and determined the consequences of inhibiting PLP3a and PLP3b expression in the meristem. PLP3 function is essential in Arabidopsis: impairing PLP3a and PLP3b expression disrupted microtubule arrays and caused polyploidy, aneuploidy, defective cytokinesis, and disoriented cell growth. Consistent with a role in microtubule formation, PLP3a interacted with β-tubulin in the yeast two-hybrid assay and, when overexpressed, increased resistance to drugs that inhibit tubulin polymerization. Inhibition of PLP3 function targeted to the meristem caused severe mitotic defects, but the cells carried on cycling through DNA replication and abortive cytokinesis. Thus, we showed that PLP3 is involved in microtubule formation in Arabidopsis and provided genetic evidence that cell viability and growth in the meristem are not subordinate to successful completion of microtubule-dependent steps of cell division.
Cell division is a highly regulated process that includes mechanisms to ensure genome integrity, such as the spindle assembly checkpoint, which delays mitosis until all chromosomes are correctly attached to the mitotic spindle (Musacchio and Hardwick, 2002  ). In animals, cells that cannot satisfy the spindle checkpoint are often eliminated by senescence or apoptosis, reducing the risk of tumorigenesis due to aneuploidy (Rieder and Maiato, 2004; Weaver and Cleveland, 2005). It is not known whether this type of quality control plays a significant role during plant development.
In plants, most cell division occurs in the meristems and in the early stages of organ development (Inze and De Veylder, 2006
The behavior of microtubules in all these processes is highly dynamic, with constant polymerization and depolymerization of
More recently, a novel step in tubulin folding has been described in yeast. Phosducin-Like Protein 1 (PLP1) was found to interact with CCT and to modulate the efficiency of β-tubulin and actin folding, at an early step distinct from the prefoldin complex (Lacefield and Solomon, 2003 Here, we show that PLP3 function is essential for microtubule-related processes during Arabidopsis development: inhibition of the function of two close PLP3 homologs disrupted microtubule arrays in vivo and interfered with microtubule-dependent functions, including nuclear division, cytokinesis, and oriented cell expansion. When inhibition of PLP3 function was targeted to the shoot meristem, gross defects in cell division were seen but the cells continued to cycle through DNA replication and abortive cytokinesis. Thus, in spite of the central role that cell division plays in meristem function, cell viability and growth in the meristem did not appear to be subordinate to a checkpoint for correct microtubule-dependent functions.
PLP3a Is Developmentally Regulated Here, we refer to Arabidopsis PhLP3 (Blaauw et al., 2003 ) as PLP3a and to a closely related gene, which encodes a protein 90% identical and 97% similar to PLP3a, as PLP3b. PLP3a initially attracted our attention because microarray experiments indicated that this gene was preferentially expressed in the inflorescence meristem (using whole seedlings as the baseline) and was activated by ectopic expression of the meristem development gene SHOOT MERISTEMLESS (STM), suggesting that PLP3a could be relevant to meristem function (C. Woodward and R. Sablowski, unpublished data). RNA in situ hybridization confirmed the expression of PLP3a in the inflorescence and floral meristems but also showed expression in floral organ primordia and to a lower level throughout the inflorescence tip (Figures 1A and 1B ).
For a wider view of PLP3a expression, we used reporter genes that contained the complete intergenic region upstream of PLP3a and the complete coding sequence and introns, fused with the β-glucuronidase (GUS) or green fluorescent protein (GFP) reporters inserted in frame just before the stop codon. Three independent GUS reporter lines were examined in detail and showed the same pattern. In the inflorescence, GUS expression was detected in the meristem and in young floral buds and subsequently remained strong in parts of the carpels and stamens (Figure 1C). During the vegetative phase, GUS was initially expressed throughout the mature embryo, including the hypocotyl and cotyledons (Figure 1D). In seedlings, expression was highest in the shoot meristem and leaf primordia, in the primary and lateral root meristems, and at the base of the hypocotyls; lower levels were seen in the elongating zone of the root, in the hypocotyls near the shoot apex, and in the vasculature of cotyledons (Figures 1E and 1G); in leaves, the initial expression in the primordia disappeared as the leaves began to expand (Figure 1F). This pattern was consistent with the expression of PLP3a in different organs as determined by quantitative RT-PCR (Figure 1H) and reported in publicly available expression array data (https://www.genevestigator.ethz.ch/at/).
The two independent GFP reporter lines analyzed showed the same expression pattern as the GUS lines and were used for a more detailed analysis at the cellular level. Optical sections through the inflorescence apex showed GFP throughout the meristem and floral buds (Figure 1I); in the root meristem, GFP was seen in all meristem cells and in the elongation zone (Figure 1J). In both cases, the GFP fusion protein was dispersed in both the cytoplasm and in the nucleus (Figure 1K), similar to the localization reported for PhLP3 in C. elegans (Ogawa et al., 2004 In summary, expression of PLP3a was highest in tissues with active cell division and decreased as cells differentiated but was not exclusively associated with cell division, as it was also seen in the vasculature and in mature reproductive organs.
Loss of PLP3 Function Led to Cell Division Defects, Aneuploidy, and Polyploidy
As PLP3b expression overlapped with PLP3a (Figure 1H), these genes could be functionally redundant. To test this, we crossed the 35S:PLP3a(RNAi) lines with a PLP3b insertional mutant (plp3b-1) caused by a Ds element insertion in the first exon. The plp3b-1 mutation abolished expression of PLP3b (see Supplemental Figure 1 online), but on its own did not cause any visible phenotypes. However, when each of the three independent 35S:PLP3a(RNAi) lines was combined with plp3b-1 (with both transgene and mutation confirmed by PCR genotyping), a novel phenotype was seen (Figures 2C to 2F). The cotyledons and hypocotyls of 35S:PLP3a(RNAi) plp3b-1 seedlings appeared rough and deformed, cotyledon number was frequently abnormal (1 to 3), the seedlings failed to produce more than one or two leaves, and in the most severe cases growth of the primary root was also inhibited. A range of phenotypic severity (Figure 2C) was seen in each line, with more severe defects in the lines with the strongest reduction in PLP3a expression (lines 1 and 2, Figure 2A). As often seen for RNAi, the penetrance of the abnormal phenotype in populations of 35S:PLP3a(RNAi) plp3b-1 seedlings was not complete, with the frequency of deformed and arrested seedlings in different lines ranging from 25% (line 1, n = 337) to 73% (line 2, n = 185). Nevertheless, the fact that these phenotypes were seen only when independent 35S:PLP3a(RNAi) lines were crossed into the plp3b-1 background confirmed the specificity of RNAi inhibition in these lines (since a knockout of the most closely related gene was still required) and showed that PLP3b was indeed able to cover for partial loss of PLP3a function during seedling development. Closer examination of the deformed cotyledons in 35S:PLP3a(RNAi) plp3b-1 plants revealed that they contained enlarged and misshapen cells, sometimes forming a sector within the cotyledon (Figures 2D to 2F). Partial cell walls (arrows in Figures 2E and 2F) indicated defects in cytokinesis (see also Figure 7H). 4',6-Diamidino-2-phenylindole (DAPI) staining showed that the nuclei of the abnormal cotyledon cells were larger than in the wild type and contained multiple nucleoli, instead of the single nucleolus seen in wild-type cells (cf. insets in Figures 2G and 2H). In the 35S:PLP3a(RNAi) plp3b-1 seedlings, pairs of cells that appeared to derive from a recent mitosis often, but not always, had their nuclei very closely juxtaposed (cf. pairs of cells indicated by arrows in Figure 2H with the wild-type controls in 2G).
The large nuclei with extranumerary nucleoli suggested that these cells were polyploid, so we used flow cytometry to measure ploidy in the abnormal seedlings. The 35S:PLP3a(RNAi) plp3b-1 seedlings had not only increased ploidy but also discrete peaks of nuclei with intermediate levels of DNA content, suggesting aneuploidy (Figure 3 ).
Loss of PLP3a/b Function Disrupted Microtubule Arrays The cellular defects caused by loss of both PLP3a and PLP3b function suggest a deficiency in microtubule functions, consistent with the proposed role of the closest homolog in yeast (PLP1) in an early step of β-tubulin folding (Lacefield and Solomon, 2003 ). To directly check whether PLP3a and PLP3b are required for microtubule assembly in vivo, we tested whether microtubule arrays were disrupted in the 35S:PLP3a(RNAi) plp3b-1 seedlings using Arabidopsis plants also expressing a fusion between GFP and  -tubulin (GFP-TUA6; Ueda et al., 1999). GFP-TUA6, plp3b-1 seedlings developed normally and showed a normal pattern of cortical microtubule arrays in the cotyledon epidermal cells (Figure 4A
). When GFP-TUA6 was crossed into the 35S:PLP3a(RNAi) plp3b-1 background, the seedlings again developed the enlarged cotyledon cells shown in Figures 2E and 2F. Using the same settings as for Figure 4A, GFP-TUA6 was barely visible in these cells, suggesting that lower levels of GFP-tubulin accumulated. With increased sensitivity, GFP-TUA6 could be detected, but it was not organized in microtubule arrays and sometimes appeared to be inside vesicles (Figures 4C and 4D); this pattern of fluorescence was not detected with the same settings in 35S:PLP3a(RNAi) plp3b-1 cells lacking GFP-TUA6.
To control for the possibility that loss of PLP3 function could preferentially affect the accumulation of the GFP-TUA fusion protein, but still leave enough native tubulin to form microtubule arrays that would not be tagged with GFP, we used the microtubule binding MAP4-GFP fusion as an independent microtubule marker (Marc et al., 1998 ). In agreement with the results using GFP-TUA6, the cotyledon epidermal cells of 35S:PLP3a(RNAi) plp3b-1 seedlings had only a few faint MAP4-GFP–marked microtubules, contrasting with the wild-type control that showed clear MAP4-GFP–marked microtubule arrays (Figures 4E and 4F).
Overexpression of PLP3a Increased Resistance to Inhibitors of Microtubule Assembly
Plants were generated that expressed a HA-tagged PLP3a under the 35S promoter (35S:HA-PLP3), and three independent transgenic lines were chosen in which expression of the tagged protein was confirmed by protein gel blotting (Figure 5A
). Seedlings of these lines were germinated on media containing propyzamide, oryzalin, or taxol. Both propyzamide and oryzalin are plant-specific microtubule poisons that bind rapidly and reversibly to tubulin subunits. Propyzamide inhibits the assembly of microtubules both in vivo and in vitro (Akashi et al., 1988
Propyzamide (3 µM) and oryzalin (50 nM) had similar effects on wild-type seedlings: root growth was twisted and inhibited by 65 to 70%, and root epidermal cells were grossly enlarged and protruded from the root surface (Figure 5; see Supplemental Figure 2 online). Consistent with its different mode of action, 1 µM taxol caused different growth defects: root growth was inhibited to a lesser extent (37%), and root epidermal cells did not swell, but the aerial parts were more strongly affected, showing thickened hypocotyls and inhibited cotyledon expansion (see Supplemental Figure 2 online). Increased PLP3a expression did not affect the way seedlings responded to 1 µM taxol (see Supplemental Figure 2 online) but partially suppressed the effects of propyzamide and oryzalin. In the 35S:HA-PLP3a lines, the inhibition of root elongation by 3 µM propyzamide ranged from 41 to 45% (Figure 5B), and the abnormal expansion of epidermal cells was reduced, although twisted root growth was still observed (Figures 5C to 5G). On 50 nM oryzalin, root elongation was rescued in the 35S:HA-PLP3a lines to a smaller but still significant extent, and swelling of root epidermal cells was visibly reduced (see Supplemental Figure 2 online). In conclusion, overexpression of PLP3a partially restored growth in conditions that compromise microtubule assembly, supporting the idea that PLP3 facilitates microtubule formation.
PLP3a Interacts with β-Tubulin in the Yeast Two-Hybrid Assay
Inhibition of PLP3 Function in the Shoot Meristem Disrupted Cell Division but Did Not Prevent Cell Viability and Growth The results above showed that reduced PLP3 function caused defects in cytokinesis and nuclear division, which are likely due to a general role of PLP3 in microtubule formation. Thus, inhibition of PLP3 function provided an opportunity to investigate how meristem cells respond to defects in microtubule function that would normally trigger cell cycle arrest or apoptosis in animal cells.
In 35S:PLP3a(RNAi) plp3b-1 seedlings with a severe phenotype, the shoot apex was disorganized (Figure 2C). This could be due to a requirement of PLP3a/b for meristem function, consistent with the expression of PLP3a in the meristem and organ primordia, but we could not exclude the possibility that the defective shoot apex was an indirect consequence of earlier defects in embryogenesis. To investigate the role of PLP3a/b specifically in the shoot meristem, we generated plants in which the hairpin construct used to make double-stranded RNA matching PLP3a was controlled by the Op promoter, which is activated by the artificial transcription factor LhG4 (Moore et al., 1998 As seen for the 35S:PLP3a(RNAi) plants, STMpro:PLP3a(RNAi) plants looked normal. By contrast, when two independent STMpro:PLP3a(RNAi) lines were crossed into the plp3b-1 background, a striking phenotype was seen in the inflorescence apex. In both the primary and lateral inflorescences, the floral buds at first appeared to develop normally, but a few days after bolting the meristem and young buds were replaced by what superficially looked like disorganized tissue (Figures 7A and 7B ). A closer look, however, revealed that the floral buds were still recognizable and were organized in the correct phyllotactic pattern, but contained enormously enlarged cells (Figures 7C and 7D). In the largest of the abnormal buds, these cells measured 50 to 100 µm across, in contrast with the 5 to 10 µm cells seen in buds at approximately the same position in the wild type; roughly, this corresponds to an increase in volume of several-hundred fold. DAPI staining revealed that although greatly enlarged, these cells were not highly vacuolated and retained the high ratio of nuclear-to-cytoplasmic volume that is typical of cells in the meristem and early organ primordia (Figures 7E and 7F). The nuclei, however, were also grossly enlarged and branched; because of their irregular shapes, it was not possible to accurately estimate the DNA content in individual nuclei, but the presence of multiple nucleoli indicated that they were likely polyploid. The irregularly shaped nuclei appeared to weave around partial cell walls, suggesting incomplete cytokinesis. Staining of live tissues with FM4-64 confirmed the presence of cell wall stubs (Figures 7G and 7H), which were seen unambiguously in younger buds, before the cells had enlarged to the same extent as those in Figure 7F. As the buds grew, the enlarged cells did not elongate to the well-defined shapes seen in floral organs of wild-type buds, showing that oriented cell enlargement had also been disrupted. In summary, loss of PLP3a/b function targeted to the shoot meristem caused cellular defects comparable to those seen in the cotyledons of 35S:PLP3(RNAi) plp3b-1 seedlings. Although cell division and cellular organization were severely disrupted, growth of the meristem and floral buds persisted. Partial cell walls indicated that cytokinesis had been initiated in these cells, showing that continued cell growth was not due to a switch to the endoreduplication pathway, but persisted through defective mitosis.
Arabidopsis PLP3a/b Are Required for Microtubule-Dependent Steps of the Cell Cycle Arabidopsis PLP3a/b belong to a family of proteins related to phosducin, which was originally described as a regulator of G-protein signaling in mammals (Bauer et al., 1992 ). There are three subfamilies of phosducin-like proteins, which had already diverged before the separation between fungi, plants, and mammals (Blaauw et al., 2003). PLP3a/b belong to the phosducin-3 family. The data available in yeast suggest that members of this subfamily are involved in the folding of cytoskeletal proteins. A mutation in PLP1 (encoding the only member of the phosducin-3 family in yeast) was isolated due to its ability to suppress the toxic effects of excess β-tubulin (Lacefield and Solomon, 2003), although loss of PLP1 function on its own caused no detectable defects (Flanary et al., 2000). The plp1 mutant did not affect the levels of β-tubulin expression but reduced its ability to form tubulin dimers (Lacefield and Solomon, 2003; Stirling et al., 2006). Consistent with the model that PLP1 participates in an early step of β-tubulin folding and in parallel with prefoldin, plp1 interacted synergistically with mutants in any of five prefoldin subunits (Tong et al., 2004).
Genes encoding phosducin-3 proteins have been less well characterized in other eukaryotes. No in vivo functional data are available from mammals and Drosophila melanogaster. Knockout of PhLP3 in D. discoideum caused no visible phenotypes (Blaauw et al., 2003
We show that differently from yeast and D. discoideum but similarly to C. elegans, phosducin-3 homologs are essential in Arabidopsis. The phenotypes caused by loss of PLP3a/b function suggest defects in at least three microtubule-dependent processes. First, aneuploidy and polyploidy suggest a failure in chromosome segregation due to a defective mitotic spindle. Our attempts to directly image mitotic spindles in cells with inhibited PLP3a/b function were not successful, but the closely juxtaposed nuclei seen in recently divided cells (Figure 2H) are noteworthy because they might result from abnormally short spindles, which have been reported for yeast and C. elegans with reduced PLP3 function (Ogawa et al., 2004
Although most of the data in other organisms also indicates that PLP3 homologs are involved in microtubule formation, mammalian PhLP3 also affected folding of actin in vitro and the yeast plp1
Given the proposed role of the yeast homolog (PLP1) in β-tubulin folding, it is reasonable to propose that PLP3a/b supports microtubule functions through the production of functional tubulin. Accordingly, PLP3a interacted with β-tubulin in yeast two-hybrid assays. At the same time, the localization of PLP3a-GFP, which was similar to that reported for the C. elegans homolog (Ogawa et al., 2004
Disruption of Microtubule-Dependent Steps of Cell Division Does Not Prevent Cell Viability and Growth in the Shoot Meristem As seen in cotyledon cells, loss of PLP3a/b function targeted to the inflorescence and floral meristems disrupted cellular processes in which microtubules play a major role, such as nuclear division, cytokinesis, and oriented cell expansion. Thus, the high expression of PLP3a/b seen in the meristems is required to support steps of cell division that are known to depend on microtubules. It must be noted, however, that PLP3a/b are also expressed in some differentiated tissues, such as vasculature and in reproductive organs, so PLP3a/b are also likely to support microtubule-dependent processes that are unrelated to cell division.
In spite of the severe disruption of cell division in 35S:PLP3a(RNAi) plp3b-1 plants, meristem growth and cell viability persisted. A similar, but milder phenotype has been described for the tso1 mutant, which affects a nuclear protein that has been proposed to control the transcription of genes required for cell division (Liu et al., 1997
The continued growth of the meristem and organ primordia in spite of severe cell division defects has two wider implications. First, it adds support to the idea that the mechanism that promotes growth in the meristem is not subordinate to correct cell division. Meristem growth is tolerant to several types of defects in cell division. The ton and fass mutants showed that the orientation of cytokinesis is not critical for meristem function and organ growth (Torres-Ruiz and Jurgens, 1994
The apparent tolerance of meristem cells to defective mitosis also has implications for the maintenance of genome stability in plants. In animal cells, microtubule poisons disrupt the mitotic spindle and cause a delay in M-phase; if the cells eventually overcome this delay and exit mitosis without having corrected the spindle defect, they often withdraw permanently from the cell cycle or undergo apoptosis (Rieder and Maiato, 2004
Plant Material Arabidopsis thaliana (Landsberg erecta [Ler] or Columbia) was grown in vermiculite:soil:sand mix at 18°C, on a 16-h-light/8-h-dark cycle. Plants were transformed by the floral dip method (Clough and Bent, 1998 ) with the constructs described below, using Agrobacterium tumefaciens strain C58C or strain GV3103 RK9 containing pSOUP (for pGREEN derivatives).
The plp3b-1 mutation (Ler background) has a Ds insertion in the first exon of At5g66410 and was obtained from the CSHL Genetrap collection (http://genetrap.cshl.org, line GT9152). The STMpro:LhG4 line (Ler) was a gift from Yuval Esched (Weizmann Institute, Tel Aviv, Israel). Arabidopsis lines containing GFP-TUA6 (Ueda et al., 1999
DNA Constructs
To create 35S:PLP3a(RNAi), which contains the 35S promoter directing expression of inverted repeats of PLP3a, the coding sequence for PLP3a was amplified from floral cDNA using the primers (5'-ATGGACCCAGATGCAGTCAAATCGACTCTC-3') and (5'-CAACTGCTTGATCAGTCAGAATC-3') and cloned into pcr2.1-TA (Invitrogen). The PLP3a cDNA was subcloned into pENTR4 (Invitrogen) and recombined using the Gateway system (Invitrogen) into pJawohl17-RNAi (gift from Imre Somsich, Max Planck Institute, Koeln, Germany). To create Op:PLP3a(RNAi), the PLP3a inverted repeats from 35S:PLP3a(RNAi) were cloned into the SalI site of pW49 (gift from Phil Wigge, John Innes Centre), modified from pU-6Op To generate 35S:HA-PLP3a, full-length PLP3a cDNA was amplified using the primers (5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGTCAGAATCAGAGTCCAGATTCTCTG-3') and (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGATCCAGATGCAGTCAAATC-3') and recombined into pDONR201 (Invitrogen), then into pGWB15 (gift from Tsuyoshi Nakagawa, Shimane University, Japan), which allows HA tagging of proteins under the 35S promoter.
Microscopy Techniques
Quantitative RT-PCR
Flow Cytometry
Treatments with Microtubule Poisons
Yeast Two-Hybrid Screen
Accession Numbers
Supplemental Data
We thank Alfonso Muñoz-Gutierrez for help with experiments on the interaction between PLP3a and β-tubulin, John Doonan, Brendan Davies, Imre Somssich, Ian Moore, Tsuyoshi Nakagawa, Yuval Eshed, Phil Wigge, and Karim Sorefan for materials, and John Doonan, Peter Shaw, Michael Lenhard, Crisanto Gutierrez, and Henrik Buschmann for helpful discussions. M.M.C. received a long-term EMBO fellowship and an Intra-European Marie-Curie fellowship (MEIF-CT-2003-503985). Work in the R.S. lab is funded by the Biotechnology and Biological Sciences Research Council.
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.plantcell.org) is: Robert Sablowski (robert.sablowski{at}bbsrc.ac.uk).
[W] Online version contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.107.057737 Received December 21, 2007; Revision received February 21, 2008. accepted March 14, 2008.
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(gift from Ian Moore, Oxford University, UK). The resulting Op:PLP33a(RNAi) construct contained six copies of the Op sequence and the tobacco mosaic virus 
