Cell-cycle regulation of hydroxyproline-rich glycoprotein HRGPnt3 gene expression during the initiation of lateral root meristems

The Plant Journal (1994) 6(5), 717-727

Cell-cycle regulation of hydroxyproline-rich glycoprotein HRGPnt3 gene expression during the initiation of lateral root meristems

Pablo Vera t, Chris Lamb and Peter W. Doerner* Plant Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA

Summary

Analysis of transgenic tobacco plants containing a tobacco hydroxyproline-rich glycoprotein HRGPnt3 gene promoter-~glucuronidase (GUS) gene fusion (HRGPnt3-uidA) showed that this promoter is active not only in the early stages of initiation of lateral roots as previously described, but also in the initiation of adventitious roots, with similar selective expression in a subset of pericycle cells. HRGPnr3 is also induced during initiation of hairy roots following transformation by Agrobacterium rhizogenes. The auxin indole acetic acid (IAA) induces an increase in the number of characteristic discrete sites of HRGP- nt3 expression. It is shown that these sites are destined to form new root primordia from pericycle cells of both adventitious and main roots. Dose-dependent induction of root meriatems by auxin overcomes the limitations of this naturally stochastic process and makes lateral root initiation amenable to biochemical analysis. Quiescent pericycle cells, which are developmentally arrested in the G2 phase of the cell cycle, rapidly progress into M phase upon mitogenic stimulation. Colchicine and nocodazole, which block completion of mitosis, inhibited the activation of the HRGPnt3 promoter but did not block auxin Induction of parA, a marker for de-differentiation in leaf mesophyll cell-derived protoplasts. Hydroxyurea, which inhibits cell-cycle progression at the G1/S-phase transition and also blocks lateral root initiation, did not inhibit HRGPnt3 induction. Thus, HRGPnt3 induction precedes completion of the first cell division during primordium formation, and is one of the Initial steps in a sequential program of gene expression activated

Received 8 February 1994; revised 11 July 1994; accepted 20 July 1994. *For correspondence (fax + 1 619 558 6379; e-mail peter.doemer @ qm. salk.edu). tpresent address: Departmento de Biotecnologia, Universidad Politecnica de Valencia, E-46022, Spain.

upon stimulation of cell division for the development of a new meristem during lateral root Initiation.

Introduction

In contrast to shoots, where the pattern of leaves and other lateral appendages is established in the apical meristem, lateral roots are generated by de novo activation of meristems from discrete subsets of quiescent pericycle cells distant from the root apex (Steeves and Sussex, 1989). Lateral root induction is an infrequent and stochastic event, therefore, previous investigators have taken advantage of auxins to promote initiation of primordia at a high rate (Blakely et al., 1988; Torrey, 1950).

In previous studies we have identified the tobacco HRGPnt3 gene, which encodes a novel hydroxyproline- rich glyceprotein, as a potential molecular marker for the initiation of lateral root formation (Keller and Lamb, 1989). Thus, histochemical analysis of transgenic tobacco plants containing an HRGPnt3 proomoter-~-glucuronidase gene fusion (HRGPnt3-uidA) suggested that the HRGPnt3 promoter was specifically induced in the discrete group of cells involved in the initiation of lateral roots. This selective, transient pattern of expression, in cells apparently destined to form the tip of the emerging lateral root, indicates that the encoded cell wall protein may have a specialized structural function, possibly in the initial cell divisions and also during subsequent mechanical penetration of the cortex and epidermis, and that the HRGPnt3 promoter responds to an early morphogenetic signal for lateral root induction.

In the present paper we further investigate the relationship between HRGPnt3 expression and root initiation, and examine the induction of the HRGPnt3 promoter in relation to cell-cycle control during the establishment of a new meristem. The HRGPnt3 promoter is activated not only during lateral root induction but also in an absolutely cor- responding pattern during other forms of root induction such as adventitious and hairy root formation. We demon- strate that localized HRGP-uidA expression in pericycle cells invariably predicts the site ~of a lateral root. Further- more, we show that during IAA-induced lateral root formation, HRGPnt3 is expressed prior to completion of the first division of meristem founder cells and that HRGPnt3 expression is controlled by a cell-cycle checkpoint active during mitosis.

717

718 Pablo Vera, Chris Lamb and Peter W. Doerner

Results

Adventitious roots

We have previously described the histochemical localization of HRGPnt3-uidA in small subsets of cells involved in the initiation of lateral roots (Keller and Lamb, 1989). To study further the involvement of this gene in root development, we examined whether HRGPnt3-uidA was also expressed in transgenic tobacco plants during adventitious root formation. In tobacco, adventitious roots are sto- chastically initiated by the onset of cell divisions in the cell layers immediately surrounding the stem vascular tissue, in a process resembling lateral root organogenesis. HRGPnt3 expression during adventitious root development was examined by histochemical staining of GUS activity using the chromogenic substrate X-Gluc (Jefferson, 1987). In situ GUS staining of intact stems

showed that HRGPnt3-uidA expression occurred at discrete sites immediately adjacent to the stem vascular tissue (Figure la). In stem cross-sections, GUS activity was localized to small regions on the outer perimeter of the vascular system at positions analogous to the pericycle cell layer of the primary root (Figure lb). These regions appeared as small, hemispherical bumps, representing pdmordia of the new adventitious roots. In contrast to the invariant positioning of primordia across from the protoxylem poles of the stele during lateral formation in tobacco roots, adventitious root primordia were initiated along the whole perimeter of the stem vascular tissue. Strong GUS staining continued as the number of cells in these primordia increased during subsequent penetration of the cortical layers and epidermis (Figure lc and d), but only weak or no GUS staining was observed once the adventitious root tip had emerged from the shoot (Figure le and f).

Figure 1. Expression of HRGPnt3-uidA during adventitious root formation. To observe adventitious root development, cuttings of tobacco plants transformed with HRGPnt3-uidA were grown axenically for 8 weeks. Whole stems (a, c and e) or stern cross-sections (b, d and f) were histochemi- cally stained for GUS activity to illustrate prqgressive stages of adventitious root development.

Hairy roots

The hairy root syndrome, caused by Agrobacterium rhizogenes infection of many dicotyledonous plants, is charac- terized by the proliferation of adventitious roots at the site of bacterial infection (De Cleene and De Ley, 1981; Riker eta/. , 1930). We examined whether HRGPnt3 was induced during A. rhizogenes-induced root formation. A. rhizogenes infection of stem or leaf segments excised from HRGPnt3-GUS transgenic tobacco plants leads to the development of the hairy root syndrome, with the appearance of many new roots in the infected tissue 15 days after bacterial inoculation (Figure 2a and b). Fluorometric assay of GUS activity in tissue extracts of transformed tissue revealed a 15- to 20-fold increase compared with the basal level in control extracts from HRGPnt3-uidA transgenic plants that had not been transformed by A. rhizogenes (Figure3). This marked increase in GUS activity was correlated with the accumulation of a

Gene regulation in root initiation 719

1.7kb transcript in the transformed tissue that hybridized to HRGPnt3 sequences (Figure 3, inset). This transcript could not be detected in the absence of bacterial inoculation. These observations suggested that the HRGPnt3 gene was transcriptionally activated during the emergence of hairy roots following transformation by A. rhizogenes.

Analysis of the spatial pattern of GUS activity by histochemical staining showed that in transformed stems HRGPnt3--uidA expression was localized to emerging root primordia (Figure 2c), and likewise, HRGPnt3-uidA expression could be observed at very early stages of root development in transformed leaf tissue (Figure 2d). Thus, even in roots generated following neoplastic transformation by A. rhizogenes, the HRGPnt3 promoter exhibited spatial and temporal patterns of activity remarkably similar to those observed during the normal development of lateral or adventitious roots in untransformed tissue.

Figure 2. A. rhizogenes-induced hairy roots. Expression of HRGPnt3-uidA in hairy roots induced by A. rhizogenestransformation of stem (a) and leaf tissues (b), respectively. Histochemical visualization of GUS activity in a lateral root primordium (arrow) developing from a hairy root growing out of transformed stem tissue (c). Histo chemical detection of GUS activity (arrow) in transformed leaf tissue at the onset root primordium formation (d).

720 Pablo Vera, Chris Lamb and Peter W. Doemer

Figure 3. Stimulation of HRGPnt3-uidA expression in hairy roots. Wild-type (wt) or HRGPnt3-uidA transgenic (nt3) stem segments were incubated on MS-agar medium for 18 days after transformation with A. rhizogenes (rhz) or mock inoculation with buffer (mock). Tissue samples were extracted when the transgenic stem segments transformed with A. rhizogenes (nt3-rhz) had developed the hairy root syndrome and GUS activity was determined fluorometrically. Inset: Northern blot analysis of total RNA isolated from A. rhizogenes induced stem tissue (nt3-rhz) or untransformed stem tissue (nt3-mock). Hybridization was performed with a probe specific for the 1.7 kb HRGPnt'3 mRNA.

Auxin regulation

Initiation of lateral and adventitious roots during development occurs at a relatively low frequency and such stochastic events are not well suited for detailed biochemical or molecular analysis of the underlying mechanisms. However, treatment of roots with the auxin indole acetic acid (IAA) markedly stimulates the initiation of lateral roots (Blakely et al., 1988; Torrey, 1950). Moreover, hairy root syndrome following the integration and expression of T-DNA from the A. rhizogenes Ri-plasmid is caused by perturbations of hormone physiology and increased sensitivity towards auxins in transformed cells (Cardarelli et aL, 1987; Shen et al., 1988). Therefore, we examined the effect of exogenous IAA on HRGPnt3 promoter activity in roots of transgenic tobacco containing the HRGPnt3-uidA gene fusion to determine whether this might provide a system suitable for mechanistic analysis of HRGPnt3 expres-

sion in relation to meristem activation and root initiation. For these experiments we used a hydroponic system

for plant growth. Application of IAA to hydroponically grown roots of 6-week-old plants caused a marked increase in extractable GUS activity (Figure 4a). Stimulation of GUS activity was observed within 2 h of IAA treatment, and GUS levels continued to increase for at least 48 h (Figure 4a). HRGPnt3--uidA expression was observed at IAA concentrations as low as 0.1 pM, with optimal induction at 10 pM (Figure 4b). Moreover, the frequency of primordium formation followed a similar dose response (Blakely et al., 1988; data not shown). Higher concentrations of IAA did not further stimulate GUS activity (Figure 4b).

Histochemical localization of GUS activity showed that the increase in extractable GUS activity in roots following IAA treatment reflected the presence of a large number of discrete sites showing strong expression of the HRGPnt3-uidA gene fusion (Figure 5 a-c). IAA caused a similar increase in the number of sites showing strong HRGPnt3-uidA expression in adventitious roots (Figure 5d). Expression in incipient lateral and adventitious root primordia remained localized to the founder cells derived from the pericycle, and no staining was observed in adjacent quiescent cortical, epidermal or vascular tissues (Figure 5c). In further developed root initials, proliferating cells of the stelar parenchyma also expressed HRGPnt3-uidA (Figure 5c). In untreated roots, GUS activity was not detectable at the apex of the main root (Keller and Lamb, 1989; data not shown). How- ever, after IAA induction GUS staining was observed in the main root apex (Figure 5e).

The number of discrete sites of HRGPnt3--uidA expression along the axis of the main root observed by histochemical staining 36-48 h after induction by IAA was directly correlated with the number of lateral root primordia that subsequently appeared after 5--6 days. To confirm this relationship, some specimens used for histochemical visualization of GUS activity following induction by IAA were not fixed, and only transiently provided with the chromogenic substrate X-Gluc prior to transfer back to MS medium. This experimental design inevitably leads to some loss of apparent cell type specificity of HRGPnt3-uidA expression because the leuko indoxyl product of the GUS enzyme reaction with this substrate diffuses considerably prior to precipitation as insoluble indigo. Lateral roots subsequently developed at every site showing very early HRGPnt3-uidA expression (Figure 5f). These data confirmed that the highly localized expression of HRGPnt3-uidA in small sets of pericycle cells was indeed at sites of lateral root initiation. Hence, expression of this gene in the mature root is not only restricted to, but is also characteristic of, the initiation of lateral root primordia.


Figure 4. Induction of HRGPnt3-uidA expression by IAA treatment of hydroponically grown roots. Roots were excised from plantlets and incubated in Edenmeyer flasks using MS medium buffered to pH 5.5 with phosphate buffer on a rotary shaker. Samples were taken at the timepoints indicated and GUS activity extracted. Kinetics of induction of GUS activity by 10 pM IAA (a). These data represent the mean of three independent experiments with two replicates each. Dose-response for IAA induction of GUS activity in HRGPnt3-uidA roots after 48 h in- cubation (b), determined in two independent experiments with two replicates each.

HRGPnt3 expression in relation to cell division

The spatial and temporal patterns of HRGPnt3-uidA expression indicate that activation of this promoter is an early event during the development of a root primordium, and we therefore examined the regulation of this gene in relation to the establishment of a new meristem. Initiation of the lateral root primordium from quiescent pericycle cells involves cytoplasmic reorganization and activation of cell division in meristem founder cells, parA is an auxin- regulated tobacco gene that has been correlated with the re-acquisition of the competence to divide in protoplasts following their isolation from leaf mesophyll cells (Takahashi et al., 1989, 1991). cdc2 encodes the p34 protein kinase component of the universal regulator of cell-cycle progression in eukaryotes (Nurse, 1990). We compared IAA induction of HRGPnt3 with the expression

of parA and cdc2 genes as markers for de-differentiation and cell division, respectively.

IAA caused marked induction of the cdc2, parA and HRGPnt3 transcripts associated with lateral root initiation (Keller and Lamb, 1989; Martinez et al., 1992; Figure 6). Increased HRGPnt3 mRNA could first be observed after 2 h, with a marked increase after 6 h and maximum levels after 12-24 h (Figure 6). In contrast, increases in cdc2 and particularly in parA transcripts in roots could first be observed as early as 1-2 h after IAA treatment and these transcripts continued to accumulate linearly through- out the time course (Figure 6). These data suggested that, although all three transcripts were induced by IAA, HRGPnt3 might not be coordinately regulated with parA and cdc2.

This hypothesis was tested by analyzing the effects of inhibitors of cell division on gene induction by IAA. Quies-


Figure 5. Spatial pattem of HRGPnt3-uidA expression in IAA-induced tissue. Main roots (a-c, e and f) and adventitious roots (d) were treated with 10 pM IAA 48 h prior to histochemical visualization of GUS activity. The arrow in (b) indi- cetes a very young primordium. In (c), a partially dissected root reveals indigo deposition in dividing founder cells dedved from the pedcycle (p), whereas in developing medstems GUS activity is also observed in dividing stelar parenchyma cells (sp), but not in quiescent endodermal (e) or cortical (c) cells. In (f), the root was removed after 18 h of induction, stained for 6 h and returned to MS medium with 10 pM IAA for a further 96 h. The specimen shown in (f) was not fixed prior to the histochemicat GUS assay.

cent pericycle cells are developmental ly arrested in G 2 (Blakely and Evans, 1979). Nocodazole and colchicine, which inhibit the assembly of the mitotic apparatus and eventually disrupt feedback controls that couple spindle function to exit from mitosis (Nishimoto et al., 1992; Rieder

and Palazzo, 1992), completely blocked subsequent IAA

induction of HRGPnt3-uidA (Figure 7) and HRGPnt3 transcripts (Figure 8) and lateral root formation, but had no

effect on the accumulation of parA transcripts (Figure 8). The inhibition of HRGPnt3-uidA and HRGPnt3 expres-

sion by nocodazole and colchicine were specific effects of these drugs since a similar t reatment with hydroxyurea,


whicli inhibits ribonucleotide reductase and causes cells to arrest in S phase (Adams and Lindsay, 1967; Tobey et al., 1990), had no effect on IAA induction of HRGPnt3-uidA (Figure 7) or HRGPnt3transcripts (Figure 8), although hydroxyurea treatment blocked lateral root initiation. Interestingly, hydroxyurea in the absence of exogenous IAA was able to induce the accumulation of parA transcripts, although HRGPnt3 transcripts were not induced (Figure 8).

Figure 6. Kinetics of HRGPnt3-uidA, par and cdc2 expression in response to treatment with 10 pM IAA. Roots were excised from hydroponically grown plants and treated with auxin. Samples were removed at the timepoints indicated and total RNA prepared (Ausubel et al., 1987). Equal amounts were separated elsc- trophoretically, transferred to a nylon membrane and hybridized with the probes indicated.

Discussion

The regulation of a tobacco HRGP promoter (HRGPnt3) during root development and meristem formation was investigated using the I~-glucuronidase (GUS) reporter system and RNA blot hybridization. It has previously been demonstrated that transgenic plants containing the HRGPnt3-uidA gene fusion accumulated GUS activity in the pericycle of primary roots, specifically during the initiation of lateral root primordia (Keller and Lamb, 1989). To gain a deeper understanding of the developmental regulation of HRGPnt3 expression during root initiation, we examined the expression of this gene in the initiation of adventitious roots, defined here as roots not originating from the primary root, and in hairy roots.

Adventitious roots constitute the bulk root system of monocotyledonous plants, dicotyledonous plants that are propagated by means of rhizomes and runners, and lower vascular plants (Esau, 1977; Fahn, 1990). Adventitious roots in the HRGPnt3-uidA transgenic tobacco plants were induced by limiting the supply of nutrients in the

Figure 7, Effect of cell-cycle inhibitors on the expression of HRGPnt3-uidA. Roots were excised from hydroponically grown plants and treated with cell- cycle inhibitors 3 h prior to induction with 10 pM IAA or induced with auxin or inhibitors alone. Inhibitors were used at the following final concentrations: colchicine (Co), 0.01% (w/v); nocodazole (No), 10 pM; hydroxyurea (Hu), 10 raM. Roots were incubated for 24 h in MS medium buffered to pH 5.5 and extracted as described previously (Jefferson, 1987). GUS activity was assayed using the fluorogenic substrate 4-methyl umbelliferylglucuronide (MUG). GUS activity was assayed in two independent experiments.

Figure 8. Effect of cell-cycle inhibitors on the accumulation of HRGPnt3 and parA mRNA. Roots were excised from hydroponically grown plants and treated with cell- cycle inhibitors 3 h prior to induction with 10 IJM IAA or induced with auxin or inhibitors alone. Inhibitors were used at the following final concentration: colchicine (Co), 0.01% (w/v); nocodazole (No), 10 pM; hydroxyurea (Hu), 10 mM. Roots were incubated for 24 h in MS medium buffered to pH 5.5 after which total RNA was prepared (Ausubel et al., 1987). Equal amounts were separated electrophoretically, transferred to a nylon membrane and hybridized with the HRGPnt3 or parA probes.


agar-based growth medium. Whole-mount visualization of GUS-stained stems revealed that HRGPnt3-uidA was expressed from the very beginning of adventitious root formation, with GUS activity restricted to those cells that con- stituted the root primordium. These primordia were located along the outer perimeter of the vascular cylinder, in close proximity to the stem phloem and xylem. In contrast to the invariant positioning of lateral root primordia across from the protoxylem poles of the stele, the geomet- rical arrangement of adventitious root primordia along the perimeter of the stem vascular system is not as stereo- typic, presumably due to the different architecture of the stem vasculature. Localized expression of HRGPnt3-uidA continues within the primordium for the duration of radial growth through the stem cortex and epidermis, but ceases after the root emerges from the epidermis. After emerging, the adventitious root behaves like a normal root, giving rise to new aerial laterals which exhibit characteristic HRGPnt3-uidA expression during primordium formation.

The expression of HRGPnt3 was also studied in hairy roots. After inoculation of leaves or stems of HRGPnt3-uidA transgenic tobacco plants with A. rhizogenes, we observed a characteristic proliferation of hairy roots. Histochemical analysis of GUS activity in these plants showed that HRGPnt3-uidA was expressed in developing root primordia from their inception. The molecular basis for hairy root disease is the transfer and integration of T-DNA sequences into the plant genome (Tepfer, 1984), resulting in altered hormone homeostasis and dramatically increased sensitivity of transformed cells towards auxins (Cardarelli et al., 1987; Shen et aL, 1988).

The localized and transient expression pattern of HRGPnt3-uidA in adventitious roots and in hairy roots corresponds to that previously observed in lateral root initials (Keller and Lamb, 1989). While most cell types can give rise to hairy root tumors after transformation with A. rhizogenes, adventitious root primordia in tobacco origi- nate from cells at positions in the stem analogous to the root pericycle. In contrast to the well-defined root pericycle, these stem tissues are histologically less distinct. However, our data indicate that these shoot tissues are none the less functionally equivalent to the root pericycle in that they, but not surrounding tissues, are competent for HRGPnt3 induction upon perception of appropriate signals. Moreover, the present data indicate that HRGPnt3 expression occurs at the inception of all de novo root meristems, regardless of the tissue and cell type of origin.

Auxin plays a key role in both lateral root formation and the hairy root syndrome (Blakely et aL, 1988; Cardarelli et aL, 1987; Shen et aL, 1988; Wightman et aL, 1980) and the dose-dependent formation of root primordia in response to auxin constitutes an experimental system suitable for molecular studies when compared with the stochastic induction observed in untreated roots.

HRGPnt3 mRNA transiently accumulates in roots within 2 h of IAA treatment. However, the kinetics of HRGPnt3 gene induction by auxin indicate that it does not belong to the class of genes that are very rapidly induced, presumably as an initial response to auxin perception (Abel et al., 1994; Theologis, 1986; Theologis and Ray, 1982; Zurfluh and Guilfoyle, 1982). The sensitivity of HRGPnt3 induction to inhibition by colchicine and nocodazole, drugs that block cytokinesis, together with the exquisite ceU-type specificity of expression, suggest that HRGPnt3 induction is a consequence of the commitment of pericycle cells to organize a new medstem, rather than a direct response to auxin treatment.

The induction of HRGPnt3 gene expression following the onset of cell division in pericycle cells was further studied using inhibitors of cell-cycle progression, to determine whether its expression was cell-cycle dependent and, if so, to identify the cell-cycle checkpoint at which its expression is regulated. Pretreatment of roots with nocodazole or with colchicine, drugs that cause microtubule depolymer- ization and thus block spindle assembly (de Brabander et al., 1976; Rieder and Palazzo, 1992), blocked the induction of HRGPnt3 by auxin as well as lateral root formation. This was a specific effect of these drugs, since treatment of roots with hydroxyurea, which arrests cells at the G1/S transition (Adams and Lindsay, 1967), and completely blocked lateral root formation, had no effect on IAA induction of HRGPnt3. Pericycle cells are developmentally arrested in G 2 (Blakely and Evans, 1979), and, when meristem founder cells are stimulated to proliferate, commence proliferating synchronously (Blakely et al., 1982). Inhibition of HRGPnt3 induction by nocodazole and colchicine but not by hydroxyurea suggests that HRGPnt3 expression is a component of a program of gene activation controlled by a checkpoint acting during or after mitotic spindle apparatus assembly, but prior to the G1/S transition. Furthermore, the rapid accumulation of HRGPnt3 mRNA upon IAA stimulation and its insensitivity towards inhibition by hydroxyurea, indicates that activation of HRGPnt3 expression occurs upon the initial commitment to meristem formation and does not require continued cycles of cell division within the incipient primordium. In contrast, parA expression in tobacco and cdc2 expression in Arabidopsis, which are not inhibited by colchicine (Hemerly et a/., 1993), are likely to be controlled by sepa- rate regulatory networks, insensitive to checkpoint control at or after spindle assembly.

The kinetics of HRGPnt3 mRNA accumulation suggest that the p65 HRGPnt3 gene product may be mechanistically involved in cytokinesis in lateral root primordia. How might a structural protein located in the primary cell wall interact with the cytoskeleton? Components of the cell wall can control cytokinesis by feedback mechanisms. In protoplasts treated with 2,6-dichlorobenzonitrile, a

reversible inhibitor of cellulose synthesis, cytokinesis is blocked but mitosis is only slightly inhibited (Meyer and Herth, 1978), indicating that cell division is coupled to cell wall formation. Treatment of tobacco protoplasts with 3,4- dehydro-L-proline, a selective inhibitor of prolyl hydroxy- lase, inhibits cell wall assembly and cell division (Cooper et al., 1994). Furthermore, addition of HRGPs to tobacco protoplasts stabilizes the cortical microtubule network (Akashi and Shibaoka, 1991), indicating that the cytoskeleton can interpret and respond to cues from the cell wall. Functional analysis of the HRGPnt3 gene product will help to determine whether cytokinesis during lateral root formation depends on HRGPnt3 expression. It remains to be determined whether the dependence of cytokinesis on cell wall formation identifies a plant-specific cell-cycle checkpoint or whether the coupling of these two processes is required mechanistically, but not a controlling step for cytokinesis.

parA was identified as a gene of unknown function induced by 2,4-D in protoplasts following their isolation from leaf mesophyll cells (Takahashi et al., 1989) and is a marker for cells re-acquiring proliferative competence, Although pericycle cells are not fully differentiated, as indicated by their accumulation of cdc2A mRNA (Martinez et al., 1992), the observation of markedly increased cytoplasmic density and volume (Bell and McCully, 1973; Karas and McCully, 1970) in stimulated pericycle cells, suggests that cellular reprogramming occurs prior to the onset of cell division. Although parA expression has previously been detected in tobacco roots (Takahashi et aL, 1991), our data provide molecular evidence that developmental reprogramming occurs in root tissues at an early stage of lateral root meristem formation, parA mRNA increased in roots for the duration of auxin induction. This contrasts with the early and transient induction of parA in mesophyll protoplasts, suggesting that the parA gene product is only required prior to completion of the first cell division (Takahashi et al., 1991). Persistent induction of parA during lateral root induction is presumably due to the recruitment of additional cells of the pericycle and stelar parenchyma into the primordium during later stages of lateral root development (McCully, 1975).

The IAA induction system used in this study has facilitated a molecular dissection of the initial events occurring in the founder cells of a new root primordium. The main conclusions of the present study are listed below: HRGPnt3-uidA is induced in all newly formed root meristems. The spatial and temporal pattern of HRGPnt3-uidA expression in auxin-induced lateral roots is indistinguishable from that observed in spontaneously formed lateral roots. The inhibition of HRGPnt3 and HRGPnt3-uidA induction by colchicine and nocodazole, but not by hydroxyurea, indicates that HRGPnt3 expression is controlled by cell-cycle progression and the in-


duction kinetics of HRGPnt3 and HRGPnt3-uidA suggest a requirement of the gene product for the first cytokinesis of founder cells.

Taken together with previous results (Martinez et al., 1992), the expression pattern, induction kinetics and response to anti-preliferative drugs of the molecular markers used in the present study define at least three regulatory pathways involved in the program of gene expression activated during stimulation of cell division in the pericycle. The first of these, which we propose controls the developmentally specified competence of pericycle cells to respond to mitogenic signals, controls the expression of cdc2 (Hemerly et aL, 1993; Martinez et aL, 1992), the universal regulator of cell-cycle progression on eukaryotes (Nurse, 1990). cdc2 mRNA accumulation in such cells is not restricted to any particular phase of the cell cycle (Hemerly et aL, 1993) and increases two- to three- fold during the time course analyzed in the present study as the number of proliferating cells increases. A second pathway, which is rapidly activated in meristem founder cells following mitogenic stimulation but prior to their commitment to the next round of cell division, controls the expression of HRGPnt3. Expression of HRGPnt3 exclu- sively in dividing cells of the incipient primordium cells suggests that this pathway itself is controlled by the program that controls cdc2 expression. Activation of this pathway is controlled by a cell-cycle checkpoint during or dependent on spindle apparatus assembly, and is not dependent on continuous proliferation, parA expression is controlled by a third pathway. The present study shows that parA mRNA accumulation is not controlled by a cell- cycle checkpoint in M or G1 phases of the cell cycle. Alternatively, parA may be independent of cell-cycle control, but its expression may be negatively feedback- regulated after cells re-enter the cell cycle. The present data are consistent with a model in which cdc2 and parA expression are controlled by two parallel pathways, while the pathway that regulates HRGPnt3 expression branches off from the former. Further molecular and genetic analysis will define the causal and temporal relationships between these pathways and their inter- actions during de novo meristem formation.

Experimental procedures

Transgenic plants

Seed obtained from selfed transgenic tobacco plants containing the HRGPnt3-uidA gene fusion (Keller and Lamb, 1989) were germinated and grown in vitro in Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) with kanamycin (100 pg m1-1) at 27°C under a 16 h light, 8 h dark regime. After about 30 days the plants were transferred to fresh hydroponic media (MS with 0.3% agar) for a further 20-30 days. Adventitious roots were analyzed from cuttings of transgenic plants grown on MS-agar

726 Pablo Vera, Chris Lamb and Peter IN. Doemer

medium with sucrose reduced to 0.3%, while plant material was grown in liquid MS medium to analyze lateral root formation.

Agrobacterium rhizogenes transformation

Agrobacterium rhizogenes (strain 9402), containing an agropine- type Ri-plasmid (kindly provided by Dr Michael A. Lawton) was grown at 28°C in YMB medium (Hooykaas et al., 1977) for 48 h. Stems from HRGPnt3-uidA transgenic tobacco plants were cut and inoculated with a few drops of the A. rhizogenes culture. Stem segments were then incubated on MS-agar plates until hairy roots appeared (15-20 days). For leaf disc inoculation, leaf segments were incubated for 24-48 h with exponentially growing bactedal cultures, washed with water, blotted and incubated on MS-agar plates supplemented with kanamycin and carbenicillin (160 pg m1-1 and 400 pg ml -~, respectively) for 10-20 days.

Root treatments

Indole acetic acid (Sigma) was prepared as a 50 mM stock and diluted in MS to the concentration indicated. Nocodazole (Sigma) was added from a 5 mg m1-1 stock solution in dimethyl sulfoxide to MS medium at a final concentration of 10 pg m1-1. Hydroxyurea (Sigma) was diluted from a I M stock in sterile water to a final concentration of 10 mM in MS. Coichicine was prepared as a 1% (w/v) solution in water and added to MS at a final concentration of 0.01%. Roots were pre-incubated for 3-5 h with inhibitors prior to the addition of IAA.

Histochemical analysis

Histochemical staining of GUS activity was performed using the chromogenic substrate X-Gluc as previously described (Schmid etal., 1990). For observation of early events of lateral root formation, roots were cleared by boiling for 2 min in lactophenol (1:1:1:1 lactic acid, glycerol, phenol, H20). After rinsing in distilled water, developing initials were visualized and photographed using bright- or dark-field illumination with an Olympus SZH dissecting microscope.

Fluorometric assay

Plant tissues were extracted and GUS activity assayed using the fluorogenic substrate 4-methyl umbelliferylglucuronide (MUG) as previously described (Jefferson, 1987; Schmid et al., 1990). The reaction was carried out at 37°C and fluorescence quantitated in an AMINCO fluoro-colorimeter. Standards of freshly prepared solutions of 4-methyl umbelliferone were used to calibrate the fluorometer. Proteins in extracts were quantified (Bradford, 1976) and all GUS activities are expressed per mg protein in extracts.

Northern blot analysis

Total cellular RNA was isolated as previously described and separated on formaldehyde gels in 1.1% agarose (Ausubel et aL, 1987). Northern blots were hybridized with the following probes: an EcoRI-Psll fragment containing part of the open reading frame of the tobacco HRGPnt3gene (Keller and Lamb, 1989); the ;~U8 tobacco parA cDNA clone (Takahashi et al., 1989) (kindly provided by Dr T. Nagata, Nagoya University, Japan); or a Hindlll fragment of the coding sequence of the Arabidopsis thaliana cdc2

gene (Martinez et aL, 1982). Probes were labeled by nick trans- lation (HRGPnt3) or by random priming (parA and cdc~ and hybridization was performed at 65°C (HRGPnt3 and parA) or 60°C (cdc2) as described (Ausubel et al., 1987). Blots were washed with 0.2 x SSC (1 x SSC = 0.15 M NaCI/0.015 M sodium citrate, 1% SDS) at 60°C (HRGPnt3and parA ) or 50°C (cdc2).

Acknowledgments

We thank Cindy Doane for helping to prepare the manuscript, Dr Beat Keller, Dr Carmen Martinez and Dr Toshiyuki Nagata for plasmids, Dr Beat Keller for transgenic tobacco seeds and Dr Antonio Leyva for helpful discussions. This work was supported by a grant to C.J.L. from the Samuel Roberts Noble Foundation. P.V. was a Fellow of the Spanish Science and Education Ministry.

References

Abel, S., Oeller, P.W. and Theologla, A. (1994) Early auxin- induced genes encode short-lived nuclear proteins. Proc. Natl Acad. Sci. USA, 91,326-330.

Adams, R.L.P. and Llndsay, J.G. (1967) Hydroxyurea. J. Biol. Chem. 242, 1314-1317.

Akashi, T. and Shibeoka, H. (1991) Involvement of transmem- brane proteins in the association of cortical microtubules with the plasma membrane in tobacco BY-2 cells. J. Cell Sci. 98, 169-174.

Ausubel, F.M., Brent, R., Kingston, R.E., More, D.D., Seid- man, J.G., Smith, J.A. and Struhl, K. (1987) Current Proto- cols in Molecular Biology. New York: Greene Publishing Associates and Wiley Interscience.

Bell, J.K. and McCully, M.E. (1970) A histological study of lateral root initiation and development in Zea mays. Protoplasma, 70, 179-205.

Blakely, L.M. and Evans, T.A. (1979) Cell dynamics studies on the pericycle of radish seedling roots. Plant Sci. Lett. 14, 79-83.

Blakely, L.M., Durham, M., Evans, T.A. and Blakeley, R.M. (1982) Experimental studies on lateral root formation in radish seedling roots. I. General methods, developmental stages, and spontaneous formation of laterals. Bot. Gaz. 143, 341-352.

Blakely, L.M., Blakely, R.M., Colowit, P.M. and EIIiott, D.S. (1988) Experimental studies on lateral root formation in radish seedling roots. II. Analysis of the dose-response to exogenous auxin. Plant Physiol. 87, 414-419.

Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the prin- ciple of protein-dye binding. Anal. Biochem. 72, 246-254.

Cardarelli, M., Span6, L., Marfottl, D., Mauro, M.L., Van Sluys, M.A. and Conatatino, P. (1987) The role of auxin in hairy root induction. MoL Gen. Genet. 298, 457-463.

Cooper, J.B., Heueer, J.E. and Varner, J.E. (1994) 3,4-Dehy- droproline inhibits cell wall assembly and cell division in tobacco protoplasts. Plant Physiol. 104, 747-752.

de Brabender, M.J., Van de Vein), R.M.L., Aerts, F.E.M., Borgers, M. and Janssen, P.A.J. (1976) The effects of methyl [5-(2-thienylcarbonyl)-I H-benzimidazol-2-yl]carbamate (R 17934; NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro. Cancer Res. 36, 905-916.

De Cleene, M.D. and De Ley, J. (1981) The host range of infectious hairy root. Bot. Rev. 47, 147-194.

Esau, K. (1977) Anatomy of Seed Plants. New York: John Wiley and Sons.

Fahn, A. (1990) Plant Anatomy. Oxford: Pergamon Press. Hemerly, A.S., Ferreira, P., Engler, J.d.A., van Montagu, M.,

Engler, G. and Inz6, D. (1993) cdc2a expression in Arabidop- sis is linked with competence for cell division. Plant Cell, 5, 1711-1723.

Hooykaas, P.J.J., KlapwiJk, P.M., Nuti, M.P., Schilperoort, R.A. and R6rsch, A. (1977) Transfer of the Agrobacterium tumefaciens Ti plasmid to avirulent agrobacteria and to Rhizo- bium ex planta. J. Gen. Microbiol. 98, 477-484.

Jefferson, R.A. (1987) Assaying chimeric genes in plants: The GUS gene fusion system. PlantMol. Biol. Rep. 5, 387-405.

Karaa, I. and McCully, M.E. (1973) Further studies on the histol- ogy of lateral root development in Zea mays. Protoplasma, 77, 243-269.

Keller, B. and Lamb, C.J. (1989) Specific expression of a novel cell wall hydroxyproline-rich giycoprotein gene in lateral root initiation. Genes Devel. 3, 1639-1646.

Martinez, M.C., Jergensen, J.-E., Lawton, M.A., Lamb, C.J. and Doerner, P.W. (1992) Spatial pattern of cdc2 expression in relation to meristem activity and cell proliferation during plant development. Proc. Natl Acad. Sci. USA, 89, 7360-7364.

McCully, M.E. (1975) The development of lateral roots. In The Development and Function of Roots (Torrey, J.G. and Clark- son, D.T., eds). London: Academic Press, pp. 105-124.

Meyer, Y. and Herth, W. (1978) Chemical inhibition of cell wall formation and cytokinesis, but not of nuclear division, in protopiasts of Nicotiana tabacum L. cultivated in vitro. Planta, 142, 253-262.

Murashlge, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497.

Nishimoto, T., Uzawa, S. and Schlegel, R. (1992) Mitotic check- points. Curr. Opinions Cell Biol. 4, 174-179.

Nurse, P. (1990) Universal control mechanism regulating onset of M-phase. Nature, 344, 503-508.

Rieder, C.L. and Palazzo, R.E. (1992) Colcemid and the mitotic cycle. J. CellSci. 102, 387-392.

Riker, A.J., Banfleld, W.M., Wright, W.H., Keitt, G.W. and


Sagen, H.E. (1930) Studies on infectious hairy root of nursery apple trees. J. Agnc. Res. 41,507-540.

Schmld, J., Doerner, P.W., Clouse, S.D., Dixon, R.A. and Lamb, C.J. (1990) Developmental and environmental regulation of a bean chalcone synthase promoter in transgenic tobacco. Plant Cell, 2, 619-631.

Shen, W.H., Petit, A., Guam, J. and Tempe, J. (1988) Hairy roots are more sensitive to auxin than normal roots. Proc. Natl Acad. Sci. USA, 85, 3417-3421.

Steevee, T.A. and Sussex, I.M. (1989) Pattems in Plant Development. Cambridge: Cambridge University Press.

Takahashl, Y., Kuroda, H., Tanaka, T., Machida, Y., Takebe, I. and Nagata, T. (1989) Isolation of an auxin-regulated gene cDNA expressed during the transition from Go to S phase in tobacco mesophyll protopiasts. Proc. Nat/Acad. Sci. USA, 86, 9279-9283.

Takahashi, Y., Kusaba, M., Hiraoka, Y. and Nagata, T. (1991) Characterization of the auxin-regulated par gene from tobacco mesophyll protoplasts. PlantJ. 1,327-332.

Tepfer, D. (1984) Transformation of several species of higher plants by Agrobactetium rhizogene~. Sexual transmission of the transformed genotype and phenotype. Cell, 37, 959-967.

Theologla, A. (1986) Rapid gene regulation by auxin. Ann. Rev. Plant Physiol. 37, 407--438.

Theologis, A. and Ray, P.M. (1982) Early auxin-regulated polyadenylated mRNA sequences in pea stem tissue. Proc. Natl Acad. Sci. USA, 79, 418-421.

Tobey, R.A., Olshi, N. and Crisaman, H.A. (1990) Cell cycle synchronization: Reversible induction of G2 synchrony in cultured rodent and human diploid fibroblasts. Proc. Nat/Acad. Sci. USA, 87, 5104-5108.

Torrey, J.G. (1950) The induction of lateral roots by indoleacetic acid and root decapitation. Am. J. Bot. 37, 257-264.

Wightman, F., Schneider, E.A. and Thimann, K.V. (1980) Hor- monal factors controlling the initiation and development of lateral roots. II. Effects of exogenous growth factors on lateral root formation in pea roots. Physiol. Plant. 49, 304-314.

Zurfluh, L.L. and Guilfoyla, T.J. (1982) Auxin-induced changes in the population of translatable messenger RNA in elongating sections of soybean hypocotyl. Plant Physiol. 69, 332-337.

Documents

Cell-cycle regulation of hydroxyproline-rich glycoprotein HRGPnt3 gene expression during the initiation of lateral root meristems