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Summary

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

Plant growth responds rapidly to developmental and environmental signals, but the underlying changes in cell division activity are poorly understood. A labile cyclin-GUS reporter was developed to facilitate the spatio-temporal analysis of cell division patterns. The chimeric reporter protein is turned over every cell cycle and hence its histochemical activity accurately reports individual mitotic cells. Using Arabidopsis plants transformed with cyclin-GUS, we visualized patterns of mitotic activity in wounded leaves which suggest a role for cell division in structural reinforcement.


Introduction

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

Cell division plays an important, but poorly understood, role in plant growth control. Precise regulation of proliferation is crucial for root and shoot organ development, and adaptive growth responses to environmental changes may be directly driven by graded changes in the rate of mitotic activity (Doerner et al. 1996). However, inconvenient conventional assays have made the spatio-temporal analysis of changes in mitotic activity in response to environmental and developmental signals a daunting task. Therefore, we sought to develop a reporter that permits the unequivocal identification of mitotic activity in space and time.

Cyclin-dependent kinases (CDK) and cyclins are key cell division regulators and potential reporters of mitotic activity. Mitotic cyclins are under stringent cell-cycle control, accumulating just prior and through mitosis, followed by selective proteolysis (Evans et al. 1983; Glotzer et al. 1991). The Arabidopsis mitotic cyclin CycB1;1 is expressed only around the G2/M transition (Doerner et al. 1996; Shaul et al. 1996). Mitotic cyclin turnover requires a short peptide motif known as the ‘destruction box’ (King et al. 1996). Transcriptional and post-translational regulation together restrict the accumulation of mitotic cyclins to late G2 and M. Therefore, mitotic cyclins are excellent markers for cells undergoing mitosis. Here, we describe a labile chimeric mitotic cyclin::uidA reporter that accurately reports patterns of mitotic activity in cell cultures and plant tissues.

Results

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

Construction of a cyclin::uidA gene fusion

To recapitulate the regulation of the 48 kDa cyclin encoded by Arabidopsis CycB1;1 (p48CycB1;1), we translationally fused 1.8 kb of the CycB1;1 gene to Escherichia coli uidA, generating pCDG (Fig. 1a). The genomic fragment comprised 1148 bp of putative promoter sequences and coding sequence corresponding to the N-terminal 116 amino acids of p48CycB1;1, including a candidate mitotic destruction box (MDB). pCDG was introduced into the tobacco BY2 cell line (An 1985; Nagata et al. 1992) and Arabidopsis plants (Bechtold et al. 1993) by Agrobacterium-mediated transformation.

image

Figure 1. Schematic of the CycB1;1::uidA reporter construct (pCDG).

(a) The upper panel highlights functional motifs of cyclin CycB1;1. The lower panel shows the 1.8 kb genomic fragment, including approximately 1.2 kb of promoter sequence and the first three exons of CycB1;1 coding sequence up to amino acid 116, translationally fused to the E. coli uidA gene. Thick lines represent promoter sequences or introns, boxes represent exons.

(b) The CycB1;1::uidA reporter is expressed in the G2/M phase of the cell cycle. DNA synthesis and mitosis in synchronized tobacco BY2 cells were monitored by 3H-thymidine incorporation, and calculating the percentage of cells in metaphase, anaphase and telophase [mitotic index (MI)], respectively. Cyclin expression was measured by RNA blot analysis (At cyc =  CycB1;1::uidA; Nt cyc =  Nicta;cycB1;2, a tobacco cyclin used as control (Setiady et al. 1995)).

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Histochemical activity of the chimeric reporter accurately reflects cyclin regulation in synchronized cell cultures and root meristems

To determine whether the cyclin–GUS chimera encoded by pCDG was useful to report mitosis with high fidelity, we analysed RNA levels and GUS activity in synchronized BY2 [pCDG] cultures: CycB1;1::uidA RNA accumulation anticipated mitotic activity (Fig. 1b), and was very similar to the previously reported expression kinetics of CycB1;1 (Shaul et al. 1996). Histochemically detected GUS activity paralleled RNA abundance throughout the time course (Figs 1b and 2a–d), indicating that the chimera was progressively degraded as cells exited mitosis (Fig. 2d). To more precisely delineate the correlation of cyclin–GUS activity with mitotic activity, we analysed nuclear morphology in synchronized, histochemically stained cells (Fig. 3). Between 6 and 8 h after the cells were released from aphidicolin arrest, the majority of stained cells had an interphase nuclear morphology and were probably in late G2. At subsequent time points, increasing fractions of the stained cell population revealed nuclear morphologies characteristic for prophase, metaphase, anaphase and telophase chromosomes, respectively. The decline of mitotic activity (t=  13–15 h) was accompanied by a transient increase of the proportion of stained cells with interphase nuclear morphology, presumably due to slow degradation of cyclin–GUS.

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Figure 2. Histochemical staining in cells and plants transformed with pCDG.

(a–d) Synchronized BY2 cells were histochemically stained at different time points after release from aphidicolin arrest. (a) t=  0 h, MI 0.3%; (b) t=  6 h, MI 0.3%; (c) t=  12 h, MI 16.5%; (d) t=  16 h, MI 5.8%.

(e–f) Histochemical analysis of roots from FA4C Arabidopsis plants. (e) Individual cells in the root meristem with strong GUS activity (20 × ). (f) Higher magnification (100 × ) of the root apex shows staining of a single cell.

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image

Figure 3. Cyclin–GUS reports mitotic activity.

Cells were stained with DAPI following the histochemical assay and the cell-cycle phase of stained cells was analysed. At every time point, >  1000 cells were evaluated to determine the percentage of cells in prophase, metaphase, anaphase and telophase, respectively, and plotted as mitotic index [MI].

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The spatial pattern of GUS activity in 10 distinct Arabidopsis lines was very similar and the homozygous line FA4C was selected for further analysis. Asynchronous cell division activity in meristems is reflected in stochastic patterns of single mitotic cells in Arabidopsis root apical meristems. Histochemical analysis revealed, on average, 24 individual stained cells per root meristem (n=  34) (Fig. 2e,f), suggesting that the chimeric reporter was rapidly degraded in planta. This pattern is consistent with the single-cell accumulation of CycB1;1-homologous RNA observed by in situ hybridization in root cross-sections in radish, a close relative of Arabidopsis (Doerner et al. 1996). GUS staining was detected in all cell layers, indicating that the substrate penetrated throughout the root.

Thus, by the criteria of accurate timing of induction (Figs 1b, 2a–c and 3), progressive reduction of GUS activity upon exit from M-phase (Figs 2d and 3) and appropriate single-celled expression in growing roots (Fig. 2e,f), we conclude that the cyclin–GUS recapitulates key aspects of mitotic cyclin regulation. Therefore, the CycB1;1 promoter-driven labile cyclin–GUS chimera is a valid tool to accurately report spatio-temporal patterns of mitosis.

Activation of cyclin expression in response to wounding

Following mechanical injury, cell division is ectopically activated in many plants, resulting in wound periderm formation and occasionally wound callus. We used the FA4C line to examine cell division activation in relation to stress-responses following wounding of leaves in Arabidopsis(Fig. 4a).

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Figure 4. Induction of cell division and GUS activity at a wound site.

(a) Schematic of the wound site; left: triangular incision of the leaf blade. Right: side view of the incision with epidermal cells on the leaf surface and palisade cells underneath. (b,d) Visualization of UV fluorescence along the edge of the lesion after wounding (5 × ). (b) t=  0 h; (c) t=  24 h; (d) t=  48 h. (e,f) Histochemical staining of leaves at 24 and 48 h, respectively, after wounding (5 × ). (g) RT–PCR analysis of CycB1;1 RNA induction in two independent leaf samples. Top row: products generated with CycB1;1-specific primers using cDNA generated from tissue excised from the wound periphery at t=  0 h (left panel) and from tissue excised at t=  48 h (right panel). Bottom row: products generated with eIF4a;At-specific primers with cDNA from tissue excised from the wound periphery at t=  0 h (left panel) and from tissue excised at t=  48 h (right panel). (h) Magnified view of wound. The arrowhead points to a newly inserted anticlinal cell walls (10 × ).

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Immediate–early responses to trauma are not transcription-dependent and involve the oxidative cross-linking of cell wall material including the formation of fluorescent phenolic compounds (Brady & Fry 1997). Visualization of the wound site by UV fluorescence indicates that this process is rapidly completed (Fig. 4b–d). Mitotic activity is not apparent at 24 h, indicating that it plays no role in early responses to trauma (Fig. 4e). However, 48 h following the incision, histochemical staining revealed strong CycB1;1-directed GUS activity in individual mesophyll cells along the perimeter of the wound (Fig. 4f). Cell division activity diminishes thereafter and no GUS activity was detected 4 days after wounding. RT–PCR analysis of tissue flanking the wound confirmed that induction of GUS activity was due to the activation of CycB1;1-transcription (Fig. 4g). Examination of the wound perimeter at higher magnification revealed newly formed cell walls (Fig. 4h, arrowhead) perpendicular to the leaf surface and parallel to the plane of the wound surface (Fig. 4a), suggesting a function in mechanical reinforcement.

Discussion

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

Transcriptional and post-translational mechanisms together mediate a sharp peak of mitotic cyclin activity at G2/M. Here, we show that GUS activity recapitulates cell-cycle regulation of mitotic cyclin activity with high fidelity when cyclin regulatory motifs mediating post-translational control are grafted onto the heterologous GUS polypeptide. This novel chimeric gene is therefore of great utility to examine patterns of mitotic activity in growth and development.

Stimulus-dependent proteolysis of the cyclin–GUS chimera

The histochemical activity of the cyclin–GUS chimera is detectable in late G2 and M (Figs 1b, 2a–d and 3). Proteolysis of the chimera is not yet completed when cells exit from M phase, as revealed by the transient increase in histochemically stained cells with interphase nuclear morphology at late time points (Fig. 3; t=  13–15 h). Anaphase-promoting complex-dependent proteolysis of mitotic cyclins persists throughout G1 in yeast and probably also in plants: Chloramphenicol acetyltransferase (CAT) activity encoded by an N-terminal cyclin–CAT chimera accumulates only in late G2 and M, even when the chimera is constitutively expressed throughout the cell cycle (Genschik et al. 1998). Due to the lower sensitivity of histochemical versus fluorometric assays, residual cyclin–GUS activity in G1 is not visualized in our experiments. Taken together, our data show that the N-terminal domain of p48CycB1;1 is sufficient to mediate stimulus-dependent proteolysis of a heterologous protein.

In contrast to a previously described stable, transcriptional cyclin promoter–GUS construct (Ferreira et al. 1994), the labile chimeric reporter enables the quantitative analysis of mitotic patterns. For example, the analysis of changing spatial patterns of dividing cells (Fig. 2e) in meristems of roots adapting to altered growth conditions will permit a better understanding of the role of cell division in controlling organ growth.

Cell division in wounded leaves

We found that mitosis is a delayed wound response. Three lines of evidence from our experiments argue that, during wounding, cytokinesis serves to insert new walls for the structural reinforcement of the leaf:

First, the spatial organization of dividing cells is restricted to a thin sector along the periphery of the incision; second, the orientation of newly inserted walls is perpendicular to the epidermis and parallel to the wound surface; and third, division was not found to be accompanied by growth. Thus, wound-induced cell division activation described here is distinct from the formation of wound callus, in which continuing proliferation gives rise to a protective tissue layer.

Our observations raise the possibility that mechano-sensory pathways stimulated by increased strain in the compromised tissue activate cell division in wounded leaves.

A general approach to accurately recapitulate regulation of protein stability

We have used stimulus-dependent proteolysis to convert the otherwise stable GUS protein into a reporter suitable for histochemical analysis of transient processes. This approach is likely to have broader utility. For example, ubiquitin-dependent turnover of phytochrome is accelerated approximately 100-fold after photo-conversion (Vierstra 1994). Chimeric proteins incorporating diverse destabilizing modules could be useful to assay signal transients or to conditionally control the abundance of any desired protein for the engineering of synthetic biochemical switches.

Experimental procedures

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

Constructs

The CycB1;1 cDNA (formerly designated cyc1aAt (Hemerly et al. 1992), see Renaudin et al. (1996) for current nomenclature) was used to probe an Arabidopsisλ-genomic library. A 2.8 kb HindIII fragment including approximately 1.2 kb of upstream sequence and genomic sequence corresponding to the first 306 amino acids encoded by the CycB1;1 gene was isolated. 3′ deletions of this fragment generated a 1.8 kb fragment that included promoter sequence and CycB1;1 coding sequence up to amino acid 116. pSK-GUS was generated by subcloning an XbaI–SacI fragment containing the E. coli uidA gene from pBIG (Becker 1990) into pSK (Stratagene). The 1.8 kb cyclin fragment was then digested with HindIII and EagI and ligated into pSK-GUS digested with XmaI and HindIII using an adapter (5′-GGCCTCAGCATCGATACCGG-3′). The amino acid sequence at the junction was: VAAREK lerpqhryrvgqsl MLRPVE (CycB1;1 and uidA sequences in large capitals; adapter sequence in small capitals). The resulting 3.0 kb fragment was cloned into the HindIII–SacI sites of the pBIB transformation vector (Becker 1990) to give pCDG, which was introduced into A. tumefaciens GV3101 (Koncz & Schell 1986).

Arabidopsis and tobacco BY2 transformation

A. tumefaciens GV3101 [pCDG] was used to transform Arabidopsis (ecotype Col-0) (Bechtold et al. 1993) and tobacco BY2 cells (An 1985). Ten homozygous Arabidopsis lines were selected on MS medium (Gibco BRL) containing 50 μg ml− 1 of kanamycin. After co-cultivation with A. tumefaciens GV3101 [pCDG] for 72 h, transformed BY2 cells were selected (Kodama & Komamine 1995) with 100 μg ml− 1 of kanamycin. Individual calli (n= 25) were dispersed in liquid BY2 medium and cultured under selective conditions for at least four further culture cycles prior to analysis.

Cell-cycle-specific analysis of pCDG

To establish a synchronized cell population, N. tabacum BY2 [pCDG] cells were arrested in S phase with aphidicolin (Kodama & Komamine 1995). DNA synthesis and progression through mitosis were monitored by measuring 3H-thymidine incorporation and by establishing the mitotic index (MI), respectively. After visualization by DAPI (4′6,-diamidino-2-phenylindole) staining, cells in metaphase, anaphase and telophase were scored to calculate the MI at each datum point (n≥  500). Following removal of aphidicolin, samples were withdrawn every hour for RNA blot analysis and the GUS histochemical assay. RNA blots were successively probed with the CycB1;1 cDNA (Hemerly et al. 1992) and a 393 bp Nicta;CycB1;2 cDNA fragment (Setiady et al. 1995) generated with the following primers, 29T: 5′-GGTCTATGC-TGCTCGACACACC-3′ and 29B: 5′-CCTAGTAGTAGTGCTCTTG-CAT-3′.

Histochemical analysis of BY2 cells and Arabidopsis plants

Arabidopsis plants were grown in a 16 h light/8 h dark cycle at 24°C, except in wounding experiments (9 h light/15 h dark). Tissue was histochemically assayed as described (Jefferson 1987). For shoot tissues, the assay buffer was vacuum-infiltrated for 10 min. Synchronized BY2 cells were fixed in 50 mm NaPO4 buffer (pH 7.5) with 0.03% formaldehyde for 20 min prior to histochemical analysis.

Cyclin induction during wounding

Cyclin RNA levels after wounding of approximately 3-week-old rosette leaves were analysed by an RT–PCR assay. RNA was isolated from six individual leaves by excising wedge-shaped tissue sections encompassing the wound in 2 day post-wounding samples and samples of equivalent size for non-wounded controls. cDNA synthesis was performed at 50°C using SuperScript II RT (Gibco BRL) and gene-specific primers for CycB1;1 (5′-ACCACAAGCAGCCTTGCTTC-3′) and Arabidopsis eukaryotic initiation factor 4A-1 (eIF4A-1; GenBank accession number X65052) (5′-AGGGCCCTCATGACCTTCTCAAT-3′). Cyclin and control transcripts were visualized after amplification with gene-specific primers (CycB1;1: 5′-GATGATGACTTCTCGTTC- GATTGT-TCC-3′ and 5′-GTCGCTTTCTTCTTAGTAGCCTTCT-3′eIF4A-1 5′-CTCTCGCAATCTTCGCTCTTCTCTTT-3′ and 5′-TTCTCA- AAACCATAAGCATAAATACCC-3′). PCR conditions were: initial denaturation for 3 min at 95°C; followed by 28 (CycB1;1) or 24 (eIF4A-1) cycles of 95°C for 60 sec, 58°C for 30 sec and 72°C for 60 sec, with a 4 min final extension reaction.

Acknowledgements

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

We thank Dr Joanne Chory for providing the Arabidopsisλ genomic library and Dr Roger Beachy for the tobacco BY2 cell line. We thank Cindy Doane for help with preparation of the figures. We thank Don Fosket, Shalu Mittal, Michael Neff, Chris Lamb and Marco Takita for useful discussions. A.C.-C. was supported by NSF Postdoctoral Fellowship BIR-9510821. This work was supported by USDA grant no. 95–37304–2228 to P.D.

References

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