Ethylene Modulates Stem Cell Division in the Arabidopsis thaliana Root

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Science  27 Jul 2007:
Vol. 317, Issue 5837, pp. 507-510
DOI: 10.1126/science.1143409


The construction of multicellular organisms depends on stem cells—cells that can both regenerate and produce daughter cells that undergo differentiation. Here, we show that the gaseous messenger ethylene modulates cell division in the cells of the quiescent center, which act as a source of stem cells in the seedling root. The cells formed through these ethylene-induced divisions express quiescent center-specific genes and can repress differentiation of surrounding initial cells, showing that quiescence is not required for these cells to signal to adjacent stem cells. We propose that ethylene is part of a signaling pathway that modulates cell division in the quiescent center in the stem cell niche during the postembryonic development of the root system.

The bodies of multicellular land plants are derived from populations of dividing cells called meristems, which contain stem cells. Clonal analysis revealed that the ultimate source of cells in the Arabidopsis thaliana root meristem is the quiescent center (QC), a group of four cells that divides infrequently and can give rise to cells in all tissue systems of the root (1). The QC cells are surrounded by initials that divide to regenerate themselves and produce cells that contribute to the root body and have also been considered to be stem cells (2, 3). Because initials can be replaced by QC cells, the former may be considered to be short-term stem cells, whereas the latter are long-term stem cells. QC cells produce signals that promote division of the abutting initial cells and repress initial cell terminal differentiation (4). Together, the QC cells and the surrounding initial cells constitute a stem cell niche (5, 6).

To identify genes that control cell proliferation in the stem cell niche, we screened our collection of root mutants for plants with defective QC cellular organization resulting from deregulated QC cell division. We identified two mutants, E6263 and E4510, in which the cells of the QC divide (Fig. 1, B to F). In the E6263 mutant shown in Fig. 1F, one of the QC cells has divided transversely, resulting in a three-celled QC when viewed in longitudinal section. We determined the time course of the onset of the mutant QC phenotype by quantifying cell divisions for 12 days after germination. QC organization is identical in both wild type and mutants (E6263, E4510) at 2 days. By 4 days after germination, 96% of mutant roots have undergone QC divisions, whereas none was observed in wild type (n = 21) (Fig. 1, E and F). By 8 and 12 days, QC divisions had occurred in all mutant plants examined (n > 20) (Fig. 1, G to J). Thus, the gene that is defective in the E6263 and E4510 mutants is required normally to repress division in the QC during wild-type development and is usually active during or soon after germination.

Fig. 1.

Identification of mutants in which the QC cells divide. (A) Wild-type stem cell niche organization (schematic). (B) Wild-type, (C) E6263, and (D) E4510 root morphologies. Cellular organization of the stem cell niche region (a pair of cells in longitudinal section) in (E) 4-day-old, (G) 8-day-old, and (I) 12-day-old wild-type roots, as revealed by propidium iodide staining. Red lines show the outline of the two QC cells that are visible. Cellular organization in (F) 4-day-old, (H) 8-day-old, and (J) 12-day-old E6263 mutant roots showing supernumerary divisions in the QC (red arrowheads). Scale bars, 25 μm.

We determined by positional cloning that the defective gene in these mutants is At3g51770 (fig. S1). At3g51770 encodes ETHYLENE OVERPRODUCER1 (ETO1), and plants homozygous for loss-of-function eto1 mutations produce excessive amounts of ethylene (7, 8). ETO1/At3g51770 is a ubiquitin E3 ligase that controls the rates of ethylene synthesis by modulating the levels of aminocyclopropane carboxylic acid synthase 5 (ACS5), a protein that catalyzes the rate-limiting step in ethylene biosynthesis (8). ETO1 is expressed throughout the plant (fig. S1). Mutations in E6263 (hereafter eto1-11) and E4510 (hereafter eto1-12) result in G→E and G→R at amino acid positions 457 and 786, respectively. Moreover, crosses between plants that are homozygous for the recessive eto1-11 mutation and the previously characterized recessive eto1-1 mutation produce mutant F1 offspring with deregulated QC cell divisions, which indicates that mutation of the ETO1 gene is responsible for the observed QC phenotype.

Our data suggest that ethylene promotes cell division in the QC. An alternative hypothesis is that ETO1 E3-ligase controls QC division by regulating the degradation of other proteins that play no role in ethylene biosynthesis. To distinguish between these alternatives, we determined whether QC division could be controlled by experimentally manipulating ethylene biosynthesis or signaling. If the QC phenotype of eto1 mutants were the result of ethylene overproduction, the inhibition of ethylene biosynthesis by 2-aminoethoxyvinyl glycine (AVG) should suppress the supernumerary divisions in the QC of the eto1 mutant. We observed no extra QC cell division in eto1 mutants grown in media supplemented with 0.5 μM AVG (Fig. 2, A to C). Similarly, if supernumerary cell divisions in the QC of eto1 mutants were ethylene dependent, it should be possible to phenocopy eto1 by exposing wild-type plants to ethylene. Growing wild-type plants in the presence of ethylene precursor 1-amino-1-cyclopropane carboxylic acid (ACC) (50 μM) induces QC cell division (Fig. 2D). Furthermore, the eto2 mutant carries a dominant mutation in an ACC synthase (ACS) gene that results in the overproduction of ethylene (9, 10). In these mutants, the QC cells also undergo supernumerary cell divisions, as they do in eto1 mutants (Fig. 2E). These data support the conclusion that the overproduction of ethylene is responsible for the supernumerary divisions that occur in the QC divisions in eto1 roots.

Fig. 2.

Ethylene promotes QC cell division. Stem cell niche organization of (A) wild-type, (B) eto1-11 mutant, (C) eto1-11 treated with AVG (0.5 μM), (D) wild-type treated with ACC (50 μM), (E) eto2 mutant, and (F) ctr1 mutant roots. Red arrowheads, extra cell division in the QC. Scale bars, 50 μm.

To further verify that ethylene promotes QC cell division, we determined the QC phenotype of plants with defective ethylene signaling. The CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) protein is a negative regulator of the ethylene signal transduction cascade, and loss-of-function mutations in the CTR1 gene results in constitutive activation of ethylene signaling (9). If ethylene were responsible for the stimulation of QC division in the eto1 mutant, we predict that QC cells should also divide frequently in the ctr1 mutant. An increased frequency of cell divisions in the QC of ctr1 mutants results in a cellular organization that is indistinguishable from that observed in eto1 mutants (Fig. 2, B and F).

We quantified the numbers of extra QC cell divisions induced by ethylene by quantifying the number of QC cells in medial longitudinal sections of roots. Although such numbers underestimate the exact numbers of cell divisions, they allow comparison of the numbers of cell divisions induced in the QC in these different backgrounds and treatments. Treatment of wild-type seedlings with ACC increased the number of extra cells from 0.022 ± 0.003 (SD) to 1.25 ± 0.05 (SD). Similarly, eto1 [1.35 ± 0.025 (SD)] and ctr1 [1.50 ± 0.001 (SD)] mutants had more QC cell divisions than wild type [0.022 ± 0.003 (SD)]. These increases correspond to an average of at least one extra cell division per 4-day-old seedling (n = 50). These genetic and pharmacological data indicate that ethylene promotes the division of cells in the QC.

To determine the identity of the cells that result from the ethylene-induced division of the QC, we determined the expression of a number of QC-expressed genes in eto1 mutants and in wild-type plants treated with 50 μMACC. The QC25 enhancer trap gene is expressed in QC cells of untreated wild-type roots (11) (Fig. 3, G and I). Wild-type roots treated with ACC express the QC25 enhancer trap in the supernumerary cells that result from QC division, indicating that the new cells have QC identity (Fig. 3, H and J). SCR is a transcription factor that accumulates in the QC and is required for its development (12, 13) (Fig. 3A). In the eto1 mutant and in wild-type roots treated with ACC, SCR is expressed in cells derived from QC division, which confirms their QC identity (Fig. 3, B and C). Furthermore, we examined the expression of the enhancer trap J0571, which is expressed in the QC of wild type (Fig. 3 D). J0571 is expressed in the QC cells and their derivatives in wild-type plants treated with ACC and in eto1 mutants, indicating that the cells that form as a result of QC division have QC identity (Fig. 3, E and F). Together these data indicate that, although QC division is controlled by ethylene, the identity of these cells is unaffected by this hormone.

Fig. 3.

QC cell identity and function are maintained in eto1 mutants. SCRpro::GFP expression is present in (A) the QC cells of wild type, (B) the new cells derived from the extra divisions (white arrow) in the QC of ACC-treated wild type, and (C) eto1-11 mutant. The enhancer trap J0571 is expressed in (D) the QC cells in wild-type, (E) the extra QC cells that develop in wild-type roots treated with ACC (50 μM), and (F) eto1-11 mutant. The QC-25 marker line expresses β-glucuronidase (GUS) activity (blue) in the QC, and lugol staining marks the differentiated columella cells. (G and I) In wild type, there is a single nonstaining layer of initials (arrow) between the QC and the columella. (I) is a higher-magnification view of (G). (H and J) The presence of functioning initials is revealed by the absence of lugol staining (arrow) in wild type treated with ACC (50 μM), where there is an increase in the number of blue-stained QC cells (arrowheads). (J) is a higher-magnification view of (H). Scale bars, 50 μm [(G) and (H)], 25 μm [(I) and (J)].

To test whether the extra cells that form in eto1 mutants function as QC cells, we determined whether they negatively regulated differentiation in the surrounding initial cells (3). Ablation of QC cells in wild-type roots results in the precocious differentiation of columella initials, which can be monitored by lugol staining of the starch grains present in columella cells but not columella initials (3, 14). We stained both eto1 and wild-type roots grown in the presence of ethylene with lugol and found a single layer of unstained cells below the supernumerary cells in the eto1 and ethylene-treated roots (Fig. 3, H and J). Therefore, the supernumerary cells produced in the QC of eto1 mutants display both QC identity and QC function.

Because high levels of ethylene induce cell divisions of the QC, we hypothesized that cell division would be repressed in plants that produce less ethylene than wild type and in plants that are ethylene insensitive. The extra divisions that occur in eto1 mutant roots result in the formation of more columella layers [6.09 ± 0.06 (SD)] than wild type [5.0 ± 0.06 (SD)] (fig. S2). Therefore, we used cell layer number in the columella as a proxy measure of cell division activity in the QC. Plants overexpressing ETO1 (CaMV35S::ETO1) produce less ethylene and have fewer layers [4.16 ± 0.11 (SD)] (Fig. 4C) than wild type (Fig. 4A) (8). Similarly, wild-type plants grown in the presence of AVG develop fewer columella layers [3.8 ± 0.11 (SD)] (Fig. 4B). ethylene insensitive2 (ein2) mutant plants insensitive to ethylene because they have a block in ethylene signal transduction also developed fewer cell layers than wild type (7, 15) (Fig. 4D). Together, these data support the conclusion that cell division in cells of the QC is regulated by ethylene.

Fig. 4.

Ethylene stimulates cell division leading to the formation of additional columella cell layers. (A) Phenotype of wild-type root. There is a decrease in the number of columella layers (red dots) in (B) wild-type roots that have been treated with AVG, (C) plants harboring 35S::ETO1, and (D) ein2 mutants. Scale bars, 50 μm.

Auxin regulates the development of the stem cells in the A. thaliana root, and ethylene and auxin interact to control developmental processes (1618). Therefore, it is possible that the ethylene-induced QC cell divisions observed in eto1 mutants or in ethylene-treated wild-type roots result from the activation of an auxindependent cell division pathway in the QC. No extra divisions were observed in the QC cells of wild-type seedlings that were treated with 0.1 μM of naphthaleneacetic acid compared with untreated controls (fig. S3, E and J). This suggests that auxin itself is not sufficient to induce cell division in the QC of seedling roots. (Further supporting evidence is presented in fig. S3, A to I.)

We have shown that ethylene determines the balance between proliferation and quiescence of stem cells in the root. Our data also indicate that mitotic quiescence is not required for the cell-to-cell signaling function carried out by these cells during the root development. Given the role that ethylene plays in the perception and transmission of environmental cues, it is likely that this hormone impacts on aspects of postembryonic development that are regulated by both endogenous developmental and exogenous environmental signals. This ethylene-mediated integration of endogenous and exogenous signals to control QC division is an attractive molecular framework that explains how the environment modulates stem cell division and activity during the postembryonic stage of the plant life cycle.

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Materials and Methods

Figs. S1 to S3


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