The specific overexpression of a cyclin-dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth

The size of tomato fruit results from the combination of cell number and cell size, which are respectively determined by the cell division and cell expansion processes. As fruit growth is mainly sustained by cell expansion, the development of fleshy pericarp tissue is characterized by numerous rounds of endoreduplication inducing a spectacular increase in DNA ploidy and mean cell size. Although a clear relationship exists between endoreduplication and cell growth in plants, the exact role of endoreduplication has not been clearly elucidated. To decipher the molecular basis of endoreduplication-associated cell growth in fruit, we investigated the putative involvement of the tomato cyclin-dependent kinase inhibitor SlKRP1. We studied the kinetics of pericarp development in tomato fruit at the morphological and cytological levels, and demonstrated that endoreduplication is directly proportional to cell and fruit diameter. We established a mathematical model for tissue growth according to the number of divisions and endocycles. This model was tested in fruits where we managed to decrease the extent of endoreduplication by over-expressing SlKRP1 under the control of a fruit-specific promoter expressed during early development. Despite the fact that endoreduplication was affected, we could not observe any morphological, cytological or metabolic phenotypes, indicating that determination of cell and fruit size can be, at least conditionally, uncoupled from endoreduplication.     

Cell division and endoreduplication: doubtful engines of vegetative growth

Currently, there is little information to indicate whether plant cell division and development is the collective effect of individual cell programming (cell-based) or is determined by organ-wide growth (organismal). Modulation of cell division does not confirm cell autonomous programming of cell expansion; instead, final cell size seems to be determined by the balance between cells formed and subsequent tissue growth. Control of growth in regions of the plant therefore has great importance in determining cell, organ and plant development. Here, we question the view that formation of new cells and their programmed expansion is the driving force of growth. We believe there is evidence that division does not drive, but requires, cell growth and a similar requirement for growth is detected in the modified cycle termed endoreduplication.     

Cell and leaf size plasticity in Arabidopsis: what is the role of endoreduplication?

Leaf area expansion is affected by environmental conditions because of differences in cell number and/or cell size. Increases in the DNA content (ploidy) of a cell by endoreduplication are related to its size. The aim of this work was to determine how cell ploidy interacts with the regulation of cell size and with leaf area expansion. The approach used was to grow Arabidopsis thaliana plants performing increased or decreased rounds of endoreduplication under shading and water deficit. The shading and water deficit treatments reduced final leaf area and cell number; however, cell area was increased and decreased, respectively. These differences in cell size were unrelated to alterations of the endocycle, which was reduced by these treatments. The genetic modification of the extent of endoreduplication altered leaf growth responses to shading and water deficit. An increase in the extent of endoreduplication in a leaf rendered it more sensitive to the shade treatment but less sensitive to water deficit conditions. The link between the control of whole organ and individual cell expansion under different environmental conditions was demonstrated by the correlation between the plasticity of cell size and the changes in the duration of leaf expansion.     

Inhibition of maize endosperm cell division and endoreduplication by exogenously applied abscisic acid

Abscisic acid (ABA) is thought to play a role in inhibiting or aborting kernel growth during water deficit. To test the responsiveness of early endosperm development to ABA concentrations, cylinders containing (±)ABA in a buffered agar medium were applied to the apical pericarp surface of kernels on intact, well‐watered maize ( Zea mays L. cv. Pioneer Brand 3925) plants from 5 to 11 days after pollination (DAP). Endosperm nuclei were analyzed by flow cytometry to assess effects on cell division and endoreduplication. ABA treatments of ≥ 100 µM substantially decreased endosperm cell numbers and fresh weight accumulation, but did not affect average cell size. ABA at ≥ 300 µM decreased the proportion of nuclei in the size classes ≥ 12C, indicating that the rate of transition to endoreduplication status was inhibited, and decreased the progressive advance from 12C to 24C to 48C, indicating that the rate of S‐phase cycling of endoreduplicating cells was inhibited. We conclude that cell division was more responsive to ABA concentrations than were endoreduplication or cell expansion growth.     

FZR2/CCS52A1 mediated endoreduplication in Arabidopsis development

Plant organogenesis generally involves three basic processes: cell division, cell expansion and cell differentiation. Endoreduplication, a process of genome replication without intervening mitosis, often occurs during cell expansion and cell differentiation. The switch from the mitotic cell cycle to the endocycle, however, is still poorly understood in plants. We have recently demonstrated that FIZZY-RELATED2 (FZR2) is a factor controlling endoreduplication in Arabidopsis. fzr2 mutants lacked gross morphological defects but showed a general decrease of endoploidy level in trichomes and other leaf cells, while expression of FZR2 under constitutive or tissue specific promoters induced extra or ectopic endoreduplication in all tissues examined. We also showed that decrease of leaf cell size in fzr2 mutants could be compensated by increased cell proliferation. In this addendum, we discuss additional phenotypes of FZR2 misexpression, including apparent mosaic leaf sectors in which local cell overexpansion due to 35S::FZR2 appears to be compensated by reduced cell expansion in neighboring tissues.Key words: Arabidopsis, fizzy-related, CCS52A1, endoreduplication, embryo, mosaic analysis, compensation mechanismPlants begin vegetative development as single zygotes and reach final sizes million-fold bigger, with complex tissues and cell types. During development, plants undergo three basic processes: cell division, cell expansion and cell differentiation. While cell division is dependent on mitotic cell cycle, cell expansion and cell differentiation are often coupled with a modified cell cycle called endoreduplication.¹ Endoreduplication enables a cell to increase its ploidy by replicating its genome without subsequent chromosomal and cellular division. This endocycle, widespread in eukaryotes but especially common in plants, may provide individual cells with the gene-expression capacity to reach larger sizes.² In the well-studied dicotyledon model Arabidopsis thaliana, endoreduplication occurs in most of the differentiated cell types, such as trichoblasts and trichomes, or cells with very high metabolic activity, such as the endosperm.³ The switch from mitotic cycles to endocycles requires cells to start another round of DNA replication without intervening mitosis. Therefore, a cell must induce re-entry into S-phase after G₁-phase while inhibiting M-phase. The regulatory mechanisms mediating the G₁ to S transition in endocycles share components of the mitotic pathways,⁴ with the CDK/CYCLIN B complex influencing DNA replication.⁵ Much evidence in yeast, fly and plants has pointed to the involvement of a WD-40 protein, Fizzy-Related/Ccs52, in triggering the switch to endoreduplication by controlling the degradation of Cyclin B.^6,7Using reverse genetics in Arabidopsis, we investigated FIZZY-RELATED2 loss-of-function mutants.⁸ fzr2 plants showed reduced endoreduplication and cell size in both pavement cells and trichomes.⁸ When FZR2 was misexpressed with the CMV 35S promoter, transgenic plants showed a range of phenotypes such as retarded growth, supernumerary trichome branches and distorted roots, with ectopic endoreduplication induced in all examined tissues. When expressed under control of the petal- and stamen-specific APETELA3 promoter, FZR2 caused great increases in the cell and nuclear sizes of petal and stamen cells, which normally endocycle little or not at all in Arabidopsis.⁸Since AP3 also drives gene expression in pollen, and pollen mother cells undergo two rounds of meiosis to generate haploid sperm cells,⁹ the effects of FZR2 expression on male gametogenesis seemed particularly interesting. Microscopic analysis showed larger pollen grains in AP3::FZR2 plants relative to wildtype, whereas DAPI staining revealed a concomitant increase in sperm cell nuclear size (Fig. 1A–D). These results suggested that endoreduplication had been induced in these pollen grains. Although these polyploid sperm cells proceeded through double fertilization, the corresponding embryos failed to complete development. Examination of cleared embryos with Nomarski microscopy showed that about half of them stopped growth at the torpedo stage (Fig. 1G and H), possibly due to abnormal endosperm development. When endosperm cellularization was completed in wildtype seeds (Fig. 1E), there were only 2 to 3 bubble-like structures at the chalazal poles of developing AP3::FZR2 seeds (Fig. 1F). This phenotype was similar to that of developing seeds derived from fertilization of a diploid plant with pollen from an hexaploid plant,¹⁰ further supporting the conclusion that AP3::FZR2 sperm cells underwent endoreduplication.Open in a separate windowFigure 1Comparisons of pollen grain sizes, nuclear sizes and embryo development among wildtype (WT, left: A, C, E and G) and AP3::FZR2 lines (right: B, D, F and H). (A and B) Micrographs of representative pollen grains. (C and D) DAPI staining of representative pollen grain. Arrowheads in (C and D) indicate the enlarged nuclei of sperm cells. (E and F) Micrographs of heart-stage embryos. (G and H) Micrographs of torpedo-stage embryos. Arrowheads in (F and H) indicate the abnormal endosperms. In (E–H), seeds were cleared with Hoyer solution and viewed using Nomarski optics. Scale bars represent 10 µm in (A–D), and 100 µm in (E–H).Another interesting result of this study was the different manner in which stamens and petals were altered by AP3::FZR2 expression. While petal cells showed extreme increases in size and decreases in numbers, the organs became disrupted, losing their characteristic laminar shape. Conversely, AP3::FZR2 stamens maintained their cylindrical shape, despite becoming wider at the organ level and composed of larger cells.⁸ This discrepancy in the severity of petal and stamen organ-level phenotypes may be because the two tissues respond differently to FZR2 misexpression, or because the shapes of these two organs place unique constraints on the effects of cell overgrowth. Like these stamens, roots and stems of 35S::FZR2 plants also retained normal shape despite severe distortion of internal tissue architecture.⁸ Perhaps a cylindrical organ is maintained more easily due to the dynamics of biophysical forces. It is also possible that the morphogenesis of a filamentous structure makes more use of intercellular communication than a laminar structure, so the cell proliferation and cell expansion are more strictly regulated by non-cell autonomous signals such as protein movement via plasmodesmata to provide additional positional information.¹¹ The regulatory contribution of these additional signals may override the effects of FZR2 ectopic expression.Finally, the most intriguing phenotype found in fzr2-1 mutant was that the overall leaf size showed no significant difference compared with wildtype, although the average cell was smaller. This suggests that proliferation is enhanced to generate more cells in response to the decreased average cell size. A mechanism called compensation is postulated to coordinate cell proliferation and cell expansion to attain proper organ size.¹² For example, mutations or transgenes that cause decreases in leaf cell proliferation can be compensated by extra leaf cell expansion, such that the organ approaches normal size.¹³ Little is known, however, about how organs and cells respond to local perturbations of cell sizes. In a subset of 35S::FZR2 transgenic plants, the expression of FZR2 was silenced at the whole plant level, but some groups of cells escaped silencing. These sectors showed FZR2 overexpression phenotypes such as over-branched trichomes and giant pavement cells, whereas nearby sections of the same leaf contained normal-sized pavement cells and 3- or 4-branch trichomes. These mosaic sectors provided an opportunity to observe how compensation works even within an organ. Inside the sectors were overgrown pavement cells typical of some FZR2 overexpression lines (Fig. 2A). Away from the sectors, the pavement cells were wildtype in appearance (Fig. 2C and D). At the sector boundary, however, a strip of very small cells formed (Fig. 2B). The smaller cell size at the border may have came about to compensate for the abnormally large cells within the sector, although it is unclear whether this decrease in cell size was followed reduced endoreduplication or simple space limitation.Open in a separate windowFigure 2Comparisons of cell sizes inside and outside of a mosaic sector. (A) Scanning electron microscope graphs of epidermal cells from a mosaic sector of 35S::FZR2. (B) Epidermal cells at boundary region between mosaic sector (OE) and surrounding normal cells (N). Black lines highlight the band of smaller cells. (C) Normal epidermal cells outside the mosaic sector in the same leaf (N). (D) Epidermal cells from wildtype plants (WT). Scale bar represents 100 µm.By studying fzr2 mutants and misexpression lines, we showed that FZR2 is necessary and sufficient to induce endoreduplication in various cell types. Our observation that cells increase proliferation to compensate the decreased cell size in fzr2 mutants provides important evidence that cell proliferation and cell expansion are closely interconnected to regulate organ development in Arabidopsis. Further experiments such as mosaic analysis are needed to further elucidate the compensation mechanism.     

BIN4, a novel component of the plant DNA topoisomerase VI complex, is required for endoreduplication in Arabidopsis

How plant organs grow to reach their final size is an important but largely unanswered question. Here, we describe an Arabidopsis thaliana mutant, brassinosteroid-insensitive4 (bin4), in which the growth of various organs is dramatically reduced. Small organ size in bin4 is primarily caused by reduced cell expansion associated with defects in increasing ploidy by endoreduplication. Raising nuclear DNA content in bin4 by colchicine-induced polyploidization partially rescues the cell and organ size phenotype, indicating that BIN4 is directly and specifically required for endoreduplication rather than for subsequent cell expansion. BIN4 encodes a plant-specific, DNA binding protein that acts as a component of the plant DNA topoisomerase VI complex. Loss of BIN4 triggers an ATM- and ATR-dependent DNA damage response in postmitotic cells, and this response coincides with the upregulation of the cyclin B1;1 gene in the same cell types, suggesting a functional link between DNA damage response and endocycle control.     

Elucidating the functional role of endoreduplication in tomato fruit development

Background

Endoreduplication is the major source of endopolyploidy in higher plants. The process of endoreduplication results from the ability of cells to modify their classical cell cycle into a partial cell cycle where DNA synthesis occurs independently from mitosis. Despite the ubiquitous occurrence of the phenomenon in eukaryotic cells, the physiological meaning of endoreduplication remains vague,although several roles during plant development have been proposed, mostly related to cell differentiation and cell size determination.

Scope

Here recent advances in the knowledge of endoreduplication and fruit organogenesis are reviewed, focusing on tomato (Solanum lycopersicum) as a model, and the functional analyses of endoreduplication-associated regulatory genes in tomato fruit are described.

Conclusions

The cyclin-dependent kinase inhibitory kinase WEE1 and the anaphase promoting complex activator CCS52A both participate in the control of cell size and the endoreduplication process driving cell expansion during early fruit development in tomato. Moreover the fruit-specific functional analysis of the tomato CDK inhibitor KRP1 reveals that cell size and fruit size determination can be uncoupled from DNA ploidy levels, indicating that endoreduplication acts rather as a limiting factor for cell growth. The overall functional data contribute to unravelling the physiological role of endoreduplication in growth induction of fleshy fruits.     

Nuclear DNA endoreduplication during petal development in cabbage: relationship between ploidy levels and cell size

The development of cabbage petals comprises two distinct phases: a cell division phase and a consecutive phase of cell expansion until the onset of opening. In this study, cytological changes characterizing the two phases of petal development were analysed. First, the mitotic activity and the surface area of epidermal cells during petal development were investigated. The DNA content of isolated nuclei from the different stages of petal tissues was determined by flow cytometric analysis. The results show that cell differentiation, leading to expanded cells, is characterized by endoreduplication. In the proximal part of the petal, after cell division arrest, differentiation frequently involves endoreduplication and cell enlargement. By contrast, normal diploid nuclei remained in the distal part of the lamina in the mature petal. It is suggested that the developmental programmes of the cabbage petal may be a trigger for the initiation of endoreduplication. Correlation between ploidy levels and cell size is also discussed.     

Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl

We have previously shown that endoreduplication levels in hypocotyls of Arabidopsis thaliana (L.) Heynh. are under negative control of phytochromes. In this study, the hormonal regulation of this process was analysed using a collection of A. thaliana mutants. The results show that two hormones in particular, gibberellin (GA) and ethylene, play distinct roles. Hypocotyl cells of the GA-deficient mutant ga1-11 grown in the dark did not elongate and showed a greatly reduced endoreduplication. Normal endoreduplication could be restored by supplying 10⁻⁹ M of the gibberellin GA₄₊₇, whereas the restoration of normal cell growth required 100-fold higher concentrations. The GA-insensitive mutant gai showed reduced cell elongation but normal ploidy levels. We conclude that (i) GA₄₊₇ has a global positive effect on endoreduplication and (ii) that endoreduplication is more sensitive to GA₄₊₇ than cell elongation. Ethylene had a completely different effect. It induced an extra round of endoreduplication both in light- and dark-grown seedlings and acted mainly on discrete steps rather than having a global effect on endoreduplication. The genes EIN2 and CTR1, components of the ethylene signal transduction pathway were both involved in this process. Received: 27 February 1999 / Accepted: 21 May 1999     

DELLA signaling mediates stress-induced cell differentiation in Arabidopsis leaves through modulation of anaphase-promoting complex/cyclosome activity

Drought is responsible for considerable yield losses in agriculture due to its detrimental effects on growth. Drought responses have been extensively studied, but mostly on the level of complete plants or mature tissues. However, stress responses were shown to be highly tissue and developmental stage specific, and dividing tissues have developed unique mechanisms to respond to stress. Previously, we studied the effects of osmotic stress on dividing leaf cells in Arabidopsis (Arabidopsis thaliana) and found that stress causes early mitotic exit, in which cells end their mitotic division and start endoreduplication earlier. In this study, we analyzed this phenomenon in more detail. Osmotic stress induces changes in gibberellin metabolism, resulting in the stabilization of DELLAs, which are responsible for mitotic exit and earlier onset of endoreduplication. Consequently, this response is absent in mutants with altered gibberellin levels or DELLA activity. Mitotic exit and onset of endoreduplication do not correlate with an up-regulation of known cell cycle inhibitors but are the result of reduced levels of DP-E2F-LIKE1/E2Fe and UV-B-INSENSITIVE4, both inhibitors of the developmental transition from mitosis to endoreduplication by modulating anaphase-promoting complex/cyclosome activity, which are down-regulated rapidly after DELLA stabilization. This work fits into an emerging view of DELLAs as regulators of cell division by regulating the transition to endoreduplication and differentiation.     

SIAMESE, a gene controlling the endoreduplication cell cycle in Arabidopsis thaliana trichomes

Cell differentiation is generally tightly coordinated with the cell cycle, typically resulting in a nondividing cell with a unique differentiated morphology. The unicellular trichomes of Arabidopsis are a well-established model for the study of plant cell differentiation. Here, we describe a new genetic locus, SIAMESE (SIM), required for coordinating cell division and cell differentiation during the development of Arabidopsis trichomes (epidermal hairs). A recessive mutation in the sim locus on chromosome 5 results in clusters of adjacent trichomes that appeared to be morphologically identical 'twins'. Upon closer inspection, the sim mutant was found to produce multicellular trichomes in contrast to the unicellular trichomes produced by wild-type (WT) plants. Mutant trichomes consisting of up to 15 cells have been observed. Scanning electron microscopy of developing sim trichomes suggests that the cell divisions occur very early in the development of mutant trichomes. WT trichome nuclei continue to replicate their DNA after mitosis and cytokinesis have ceased, and as a consequence have a DNA content much greater than 2C. This phenomenon is known as endoreduplication. Individual nuclei of sim trichomes have a reduced level of endoreduplication relative to WT trichome nuclei. Endoreduplication is also reduced in dark-grown sim hypocotyls relative to WT, but not in light-grown hypocotyls. Double mutants of sim with either of two other mutants affecting endoreduplication, triptychon (try) and glabra3 (gl3) are consistent with a function for SIM in endoreduplication. SIM may function as a repressor of mitosis in the endoreduplication cell cycle. Additionally, the relatively normal morphology of multicellular sim trichomes indicates that trichome morphogenesis can occur relatively normally even when the trichome precursor cell continues to divide. The sim mutant phenotype also has implications for the evolution of multicellular trichomes.     

The cell cycle-associated protein kinase WEE1 regulates cell size in relation to endoreduplication in developing tomato fruit

Tomato fruit size results from the combination of cell number and cell size which are respectively determined by cell division and cell expansion processes. As fruit growth is mainly sustained by cell expansion, the development of pericarp and locular tissues is characterized by the concomitant arrest of mitotic activity, inhibition of cyclin-dependent kinase (CDK) activity, and numerous rounds of endoreduplication inducing a spectacular increase in DNA ploidy and mean cell size. To decipher the molecular basis of the endoreduplication-associated cell growth in fruit, we investigated the putative involvement of the WEE1 kinase (Solly;WEE1). We here report a functional analysis of Solly;WEE1 in tomato. Impairing the expression of Solly;WEE1 in transgenic tomato plants resulted in a reduction of plant size and fruit size. In the most altered phenotypes, fruits displayed a reduced number of seeds without embryo development. The reduction of plant-, fruit- and seed size originated from a reduction in cell size which could be correlated with a decrease of the DNA ploidy levels. At the molecular level downregulating Solly;WEE1 in planta resulted in the increase of CDKA activity levels originating from a decrease of the amount of Y15-phosphorylated CDKA, thus indicating a release of the negative regulation on CDK activity exerted by WEE1. Our data indicated that Solly;WEE1 participates in the control of cell size and/or the onset of the endoreduplication process putatively driving cell expansion.     

Water deficit in developing endosperm of maize: cell division and nuclear DMA endoreduplication

Water deficit severely decreases maize (Zea mays L.) kernel growth; the effect is most pronounced in apical regions of ears. The capacity for accumulation of storage material in endosperms is thought to he partially determined by the extent of cell division and endoreduplication (post-mitotic nuclear DNA synthesis). To gain a better understanding of the regulatory mechanisms involved, we have examined the effect of water deficit on cellular development during the post-fertilization period. Greenhouse-grown maize was subjected to water-limited treatments during rapid cell division [from 1 to 10days after pollination (DAP)] or rapid endoreduplication (9 to 15 DAP). The number of nuclei and the nuclear DNA content were determined with flow cytometry. Water deficit from 1 to 10 DAP substantially decreased the rate of endosperm cell division in apical-region kernels, but had little effect on middle-region endosperms. Rewatcring did not allow cell division to recover in apical-region endosperms. Water deficit from 9 to 15 DAP also decreased cell division in apical-region endosperms. Endoreduplication was not affected by the late treatment in either region of the car, but was inhibited by the early treatment in the apical region. In particular, the proportion of nuclei entering higher DN A-content size classes was reduced. We conclude that cell division is highly responsive to water deficit, whereas endoreduplication is less so. We also conclude that the reduced proportion of nuclei entering higher DNA-content size classes during endoreduplication is indicative of multiple control points in the mitotic and endoreduplication cycles.     

Expression of genomic AtCYCD2;1 in Arabidopsis induces cell division at smaller cell sizes: implications for the control of plant growth

The Arabidopsis (Arabidopsis thaliana) CYCD2;1 gene introduced in genomic form increased cell formation in the Arabidopsis root apex and leaf, while generating full-length mRNA, raised CDK/CYCLIN enzyme activity, reduced G1-phase duration, and reduced size of cells at S phase and division. Other cell cycle genes, CDKA;1, CYCLIN B;1, and the cDNA form of CYCD2;1 that produced an aberrantly spliced mRNA, produced smaller or zero increases in CDK/CYCLIN activity and did not increase the number of cells formed. Plants with a homozygous single insert of genomic CYCD2;1 grew with normal morphology and without accelerated growth of root or shoot, not providing evidence that cell formation or CYCLIN D2 controls growth of postembryonic vegetative tissues. At the root apex, cells progressed normally from meristem to elongation, but their smaller size enclosed less growth and a 40% reduction in final size of epidermal and cortical cells was seen. Smaller elongated cell size inhibited endoreduplication, indicating a cell size requirement. Leaf cells were also smaller and more numerous during proliferation and epidermal pavement and palisade cells attained 59% and 69% of controls, whereas laminas reached normal size. Autonomous control of expansion was therefore not evident in abundant cell types that formed tissues of root or leaf. Cell size was reduced by a greater number formed in a tissue prior to cell and tissue expansion. Initiation and termination of expansion did not correlate with cell dimension or number and may be determined by tissue-wide signals acting across cellular boundaries.     

利用流式细胞仪研究拟南芥叶发育过程中细胞周期的调控

叶的形态建成依赖于细胞不断地分裂增殖和不同类型细胞的特化。在叶发育早期,叶细胞主要通过旺盛的有丝分裂来增加原基中细胞的数目。随着叶片的生长,叶细胞自顶部向基部逐渐退出有丝分裂进入内复制来增加细胞的倍性,同时伴随细胞的扩展和分化。本文介绍利用流式细胞仪研究双子叶模式植物拟南芥叶发育过程中细胞周期调控的方法和具体研究实例。我们发现至少存在3种类型的细胞周期异常的拟南芥叶发育突变体。此外,我们还介绍利用流式细胞仪测定DNA复制效率的方法。     

Synchronously developing collet hairs in Arabidopsis thaliana provide an easily accessible system for studying nuclear movement and endoreduplication

Early Arabidopsis thaliana seedling growth includes the highly synchronous development of hairs from every epidermal cell of the collet (the root-hypocotyl transition zone). The dynamics of collet hair growth, and accompanying nuclear movement and endoreduplication, were followed using a combination of different fluorescent probes for time-lapse imaging and flow cytometry. Using laser-scanning confocal microscopy on the double-transgenic Arabidopsis hybrid line NLS-GFP-GUS × YPM, there appeared to be a correlation between nuclear position and the cell tip during growth of the collet hair cells, as occurs in asynchronously developing root hairs. However, disruption of nuclear movement in the growing collet hairs using low concentrations of cytoskeletal inhibitors demonstrated that nuclear positioning close to the tip of the cell is not essential for tip-directed growth of the hair. Nuclear DNA content increases from 4C to 16C during development of the collet hairs. Following cessation of growth, nuclei moved to the base of the hairs and then their movement became asynchronous and limited. Co-visualization of RFP-highlighted prevacuolar vesicles and GFP-labelled nuclei showed that, whereas small vesicles allowed unimpeded nuclear movement within the hair, transient stops and directional reversals coincided with the presence of larger vesicles in close proximity to the nucleus. Arabidopsis collet hairs provide a robust, easily accessible, naturally synchronized population of single tip-growing cells that can be used as a model cell type for studying nuclear movement and endoreduplication.