Ethylene–auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana

Authors


*(fax +1 541 737 3573; e-mail ivanchem@science.oregonstate.edu).

Summary

Plant root systems display considerable plasticity in response to endogenous and environmental signals. Auxin stimulates pericycle cells within elongating primary roots to enter de novo organogenesis, leading to the establishment of new lateral root meristems. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in root branching are not well characterized. We find that enhanced ethylene synthesis, resulting from the application of low concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), promotes the initiation of lateral root primordia. Treatment with higher doses of ACC strongly inhibits the ability of pericycle cells to initiate new lateral root primordia, but promotes the emergence of existing lateral root primordia: behaviour that is also seen in the eto1 mutation. These effects are correlated with decreased pericycle cell length and increased lateral root primordia cell width. When auxin is applied simultaneously with ACC, ACC is unable to prevent the auxin stimulation of lateral root formation in the root tissues formed prior to ACC exposure. However, in root tissues formed after transfer to ACC, in which elongation is reduced, auxin does not rescue the ethylene inhibition of primordia initiation, but instead increases it by several fold. Mutations that block auxin responses, slr1 and arf7 arf19, render initiation of lateral root primordia insensitive to the promoting effect of low ethylene levels, and mutations that inhibit ethylene-stimulated auxin biosynthesis, wei2 and wei7, reduce the inhibitory effect of higher ethylene levels, consistent with ethylene regulating root branching through interactions with auxin.

Introduction

The pattern of lateral root formation is a complex developmental process that is tightly regulated to achieve efficient nutrient and moisture acquisition from the soil. In most eudicot plants only primary roots are formed in the embryo, and emerge during seed germination. After germination, pericycle cells in the root become competent to undergo a precise series of divisions, and form lateral root primordia. Lateral roots emerge through the tissues of the parent root, elongate, and in turn undergo branching, thereby allowing plants to elaborate their root systems and to explore large volumes of soil. Environmental cues appear to play distinct roles in lateral root initiation and emergence, effectively optimizing the distribution of roots in the soil (Malamy, 2008).

In Arabidopsis, lateral root initiation is restricted to specific pericycle cell files adjacent to a xylem pole. A limited number of pericycle cells undergo transverse division, each generating a small group of short cells in the same file: referred to as stage-I primordium (Malamy and Benfey, 1997). The newly divided cells expand laterally, and some divide periclinally giving rise to two cell layers (stage II). From stage II through to stage VI, a predictable pattern of cell division and expansion generates a dome-like structure, with a radial organization similar to that of the mature root tip. Growth continues as a result of the elongation of the basal cells, and the primordium emerges through the outer layers of the parent root. At the time of emergence, the primordium forms an active meristem and becomes a new lateral root.

The principles that govern the longitudinal positioning and spacing of lateral root primordia are not yet understood (Malamy, 2005, 2008). However, the earliest divisions in lateral root initiation are seen approximately 1.4 mm from the primary root tip (Beeckman et al., 2001), and form less than 14 h after xylem-adjacent pericycle cells leave the meristem (Dubrovsky et al., 2000). Dubrovsky et al. (2006) estimated that pericycle cells remain competent for lateral root formation during a relatively narrow developmental window: between 10 and 16 h in Arabidopsis thaliana. If pericycle cells are inhibited during this time window, no lateral root primordia will form later in the corresponding portion of the root (Gladish and Rost, 1993). Interestingly, using the DR5:GUS auxin reporter line, De Smet et al. (2007) have reported that an oscillating auxin response maximum in the basal region of the meristem seems responsible for priming pericycle cells for lateral root initiation. This suggests that early events in the life of a pericycle cell might affect its future competence for lateral root formation.

Numerous observations suggest a tight correlation between auxin and lateral root formation. Elevated levels of auxin, either by exogenous application or by enhanced synthesis, increase lateral root formation (Boerjan et al., 1995; Celenza et al., 1995). Exogenously applied auxin overrides the developmental window requirement, and induces lateral root initiation along the entire xylem-adjacent pericycle (Himanen et al., 2002; Laskowski et al., 1995): supporting evidence that auxin regulates the placement and frequency of lateral root primordia. Furthermore, plants with mutations that reduce auxin signaling have severe defects in lateral root formation. The slr1 (solitary root1) mutant of Arabidopsis forms no lateral roots, even in the presence of auxin, because of a gain-of-function defect in the SLR/indole-3-acetic acid 14 (IAA14) protein (Fukaki et al., 2002). AUX/IAA proteins are repressors that dimerize with a family of auxin response factors (ARFs), thereby inhibiting auxin-induced transcription. SLR/IAA14 interacts with ARF7 and ARF19 in yeast two-hybrid assays, suggesting that the stabilized SLR/IAA14 protein represses the initiation of lateral root primordia by suppressing ARF7 and ARF19 function (Fukaki et al., 2005; Wilmoth et al., 2005). Auxin acts by binding to an F-box protein, TIR1, leading to the targeted degradation of repressor AUX/IAA proteins and the subsequent expression of auxin-induced genes (Dharmasiri et al., 2005; Kepinski and Leyser, 2005).

The gaseous plant hormone, ethylene, has been implicated in diverse developmental processes, such as germination, fruit ripening, root hair formation and senesce, and may function in concert with auxin in some of these processes (reviewed in Reid, 1995). Ethylene synthesis in response to stress inhibits organ growth (Achard et al., 2006), whereas ethylene production in response to flooding stimulates adventitious root emergence in wetland plants (Lorbiecke and Sauter, 1999; Steffens et al., 2006; Visser et al., 1996). Root elongation is synergistically inhibited by auxin and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) (Růžička et al., 2007; Stepanova et al., 2007; Swarup et al., 2007), thereby demonstrating crosstalk between ethylene and auxin in root growth. One mechanism for this synergy is the positive regulation of auxin synthesis by ethylene. Stepanova et al. (2005, 2007) have demonstrated that the WEI2 and WEI7 (WEAK ETHYLENE INSENSITIVE) genes of Arabidopsis, involved in Trp-dependent auxin biosynthesis, are upregulated by ethylene, thereby suggesting a mechanism for ethylene-regulated auxin synthesis. The ethylene inhibition of root elongation also requires functional auxin signaling pathways. Mutants bearing stabilized forms of AXR2 (auxin resistant 2)/IAA7 and AXR3/IAA17 proteins show ethylene-resistant root growth, whereas those stabilized in SHY2 (short hypocotyl 2)/IAA3 and SLR/IAA14 are strongly resistant to auxin, but not to ethylene (Růžička et al., 2007; Swarup et al., 2007). The arf7 and arf19 single and double mutants may exhibit ethylene-resistant root elongation (Li et al., 2006; Ruzicka et al., 2007).

Few reports in the literature have examined the role of ethylene in lateral root formation. The Neverripe (Nr) mutant of tomato has reduced ethylene response caused by a dominant negative mutation in one of the tomato ethylene receptors (O’Malley et al., 2005). Nr has an increased underground root mass resulting from the inhibition of ethylene signaling (Clark et al., 1999). Reciprocally, the polaris (pls) mutant of Arabidopsis, which displays enhanced ethylene signaling, has reduced lateral root formation (Chilley et al., 2006). The adjoining report by Negi et al. (2008) examines lateral root formation in eto1, (ethylene overproducing 1), ein2 (ethylene insensitive 2), etr1 (ethylene resistant 1) and ctr1 (constitutive triple response 1), and shows that ethylene negatively regulates lateral root formation in Arabidopsis, and alters polar auxin transport.

Here, we examined the hypothesis that ethylene and auxin interact to regulate the initiation of lateral root primordia in the pericycle, as well as lateral root emergence and elongation. We find a complex dose response for lateral root initiation, with ACC stimulating this process at low doses, and inhibiting it at higher physiological doses. We also find that auxin–ethylene crosstalk occurs near the growing tip of the root, and regulates the lateral root frequency in a manner correlated with the rate of root growth. This interaction is dependent at least in part on ethylene-stimulated auxin biosynthesis, and Aux/IAA and ARF protein function.

Results

Ethylene inhibits lateral root initiation acting at the tip of the growing primary root, although at very low concentrations ethylene promotes lateral root initiation

Ethylene has been shown to strongly inhibit the elongation of root cells that form in the presence of ethylene, but does not affect root length in root regions where cells had already completed cell wall formation before the rise in the level of ethylene (Le et al., 2001). We examined the effect of ethylene treatment on lateral root initiation and elongation, considering these two developmental regions independently. We define the proximal root portion as the root that formed before the ACC treatment, and the distal root portion as the root that formed in the presence of ACC after the transfer (Figure 1a). To raise the levels of ethylene we treated Columbia seedlings with the ethylene precursor ACC, and examined lateral root branching over a wide (0.02–5 μm) range of ACC concentrations. We observed no significant changes in the number of lateral root initiation events in the root formed prior to transfer onto ACC (designated as the proximal portion) (Figure 1a,b). In the primary root formed after transfer onto ACC (designated the distal portion), the total number of lateral root initiation events increased at 0.04 μm ACC, but at higher doses of ACC the total number of lateral root initiation events decreased in a concentration-dependent manner, reaching a plateau at approximately 1 μm (Figure 1b). Thus, ethylene affected lateral root initiation strictly in the distal primary root portion that elongated during the treatment. Root length was unaffected by the ACC treatment in the proximal root portion formed prior to the treatment, whereas elongation of the newly formed distal root portion decreased with increased ACC doses in wild type, as shown in Table 1.

Figure 1.

 Ethylene inhibits lateral root initiation and primary root growth in Arabidopsis thaliana.
(a) Five-day-old control seedlings and ethylene-overproducing mutants, eto1, were transferred to media containing the indicated level of ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and were grown for an additional 4.5 days. The arrowhead indicates the position of the root tips at the beginning of the treatment.
(b) The number of initiated lateral roots in the proximal and distal root portions was quantified in cleared roots using differential interference contact (DIC). The means ± SE are reported for 15–18 roots. The statistical differences between untreated and treated roots and between wild-type and eto1 roots were determined by a Student’s t-test; *≤ 0.01.
(c) The number of initiated lateral roots at each indicated developmental stage was determined in distal root portions in cleared roots using DIC. The means ± SE are reported for 14–17 roots. The statistical differences between untreated and treated roots or between wild-type and eto1 roots were determined by a Student’s t-test; ≤ 0.02.
(d) Roots were treated with 0.2 μm ACC for 10 days and DR5:GUS staining was performed prior to clearing the roots. A group of four closely spaced lateral root primordia expressing DR5:GUS in the distal portion of roots are shown. Arrowheads indicate cell walls between adjacent cortical cells. Scale bar: 25 μm.

Table 1.   Effect of ethylene on primary root growth, cortex cell length and lateral root initiation in Arabidopsisa
Growth conditionsRoot growth parametersa
Growth increment (mm)Cell length (μm)Rate of cell production (cells/day)Lateral root densitybLateral root frequencyc
  1. aFor growth conditions and treatment, see Figure 1. The means ± SE are reported with n = 14–17 for each group (*≤ 0.02).

  2. bNumber of initiation events per mm.

  3. cNumber of initiation events per 100 cortical cells.

0.00 (MS med.)33.0 ± 3.1170.9 ± 10.248.3 ± 1.10.58 ± 0.110.0 ± 1.1
0.04 μm ACC25.3 ± 2.3141.3 ± 15.1*44.8 ± 1.20.92 ± 0.1*13.1 ± 1.2*
0.08 μm ACC21.8 ± 1.9*120.5 ± 17.6*45.2 ± 1.00.80 ± 0.2*9.7 ± 1.1
0.20 μm ACC14.1 ± 2.4*108.5 ± 20.2*32.5 ± 1.1*0.68 ± 0.17.4 ± 0.9*
1.00 μm ACC12.7 ± 1.7*93.1 ± 19.3*32.8 ± 1.1*0.60 ± 0.15.6 ± 1.0*
5.00 μm ACC10.4 ± 0.5*88.4 ± 15.6*29.4 ± 0.9*0.62 ± 0.15.5 ± 1.0*
eto112.9 ± 2.9*115.2 ± 18.2*22.5 ± 3.3*0.44 ± 0.25.4 ± 1.9*

The Arabidopsis mutant eto1 displays ethylene overproduction as a result of dominant negative stabilization of the ETO1/ACS5 protein (Chae et al., 2003). In eto1, lateral root initiation was similar to that in the Columbia seedlings treated with 1 μm or higher ACC concentrations (Figure 1, Table 1). Because eto1 has elevated ethylene levels throughout development, it shows fewer lateral root initiations along the entire root.

In addition, in approximately 10% of roots treated with ACC for 4–6 days, and in 50% of roots treated for 10 days, we observed groups of closely spaced lateral root primordia separated by between one and three cortical cells (Figure 1d). In untreated roots, two lateral root primordia are rarely found at such close proximity (not shown). This suggests that ethylene can change the spacing between lateral root primordia.

Ethylene alters the ability of pericycle cells to participate in lateral root initiation

We closely examined the morphology of lateral root primordia in ACC-treated Columbia seedlings and in eto1 roots to determine the developmental stage that was sensitive to ethylene. There were no major histological abnormalities or signs of primordia arrest in the Columbia seedlings treated with up to 5 μm ACC, and nor were any major changes observed in eto1 (not shown). The proportion of early I–III-stage primordia increased at the low ACC dose, but decreased at ACC doses of 0.2 μm and above. The number of developing and emerging primordia is relatively constant over this range of ACC doses (Figure 1c), which is consistent with ethylene affecting pericycle cell participation in primordia initiation, rather than inhibiting primordia development. Expression of the auxin-responsive DR5:GUS reporter was examined as a secondary and indirect measure of primordia development in Columbia in response to ACC. In particular, expression of this auxin reporter marks cells of lateral root primordia, and establishes a gradient at primordia tips, whereas abnormal primordia development is accompanied by defects in DR5 activity and distribution (Benkováet al., 2003). Columbia roots treated with 0.2 μm ACC had a correctly established DR5 gradient at the primary root tip, consistent with normal primordium development (Figure S1b). In addition, the DR5 activity appeared slightly increased in primordia cells upon treatment with ACC (Figure S1b), consistent with ethylene being able to moderately upregulate auxin biosynthesis (Růžička et al., 2007; Stepanova et al., 2007; Swarup et al., 2007) and transport (Negi et al., 2008). In contrast, 25 μm ACC inhibited DR5:GUS expression (data not shown), and strongly suppressed primary and lateral root growth, acting apparently above the optimal range for root development.

Decrease in the overall lateral root number with increasing ethylene concentrations could simply reflect the shorter length of the primary root. Indeed, the number of initiation events calculated per mm of primary root length first increased at 0.04–0.08 μm ACC, and then remained relatively constant in the distal root portion over a 25-fold ACC concentration range (Table 1), consistent with this simple explanation. However, we also found that both cortical cell length and the cortical cell production rate were modified by ethylene in the distal root portion (Table 1), suggestive of a more complex interconnection between root length and lateral root initiation frequency at higher ACC doses. To obtain more accurate information on the cellular frequency of primordia initiation, we calculated the number of lateral root initiation events per root length corresponding, on average, to 100 cortical cells in a file. We refer to this parameter as lateral root frequency, as opposed to the lateral root density that is measured simply based on root length. Measured on a cellular basis, the frequency of lateral root initiation increased at 0.04 μm ACC, and then significantly decreased, reaching a plateau at 1 μm ACC (Table 1). In eto1, the lateral root frequency was reduced to 55% of the level in untreated Columbia, which was similar to seedlings treated with 1–5 μm ACC. This shows that ethylene can modify the lateral root initiation frequency in a concentration-dependent manner.

Auxin acts synergistically with ethylene to inhibit lateral root initiation on new growth

Auxin is generally considered to have a positive effect on Arabidopsis lateral root initiation and elongation (Laskowski et al., 1995), with enhanced root formation at IAA concentrations of 0.1 μm and above (Poupart et al., 2005), whereas ethylene negatively regulates lateral root initiation in the root portion formed during the treatment, as described above. We therefore asked whether auxin and ethylene acted antagonistically on root initiation when added together. Negi et al. (2008) determined that the positive effect of 1 μm IAA on lateral root formation dominated the negative effect of 1 μm ACC in a combination treatment, but examined the effect without examining the root portions formed prior to and during the treatment separately. This experiment was performed here with a 20-fold lower IAA concentration, and a 5-fold lower ACC concentration, to reduce the strong inhibitory effects of these compounds on root elongation, and because of the effects of these hormones in mature and newly formed root tissues. At 0.05 μm, IAA inhibited elongation of the newly formed distal root portion by reducing cell length, and slightly enhanced the lateral root inhibition in the proximal root formed prior to the transfer to IAA. ACC at 0.2 μm reduced both cortex cell length and the cortex cell production rate, and significantly reduced the lateral root initiation, but only in the distal portion formed during the treatment. In the combined treatment, the root grew on average 8.6 mm over the 5-day treatment period. Cortical cell length was synergistically reduced by IAA and ACC in the combination treatment, whereas the cell production rate was unaffected by the IAA added in addition to ACC (Table 2). IAA increased the number of lateral root initiation events in the proximal root portion formed prior to the transfer, even in the presence of ACC (Figure 2). In contrast, in the distal root portion formed after the transfer, a strong synergistic amplification of the inhibitory ACC effect was observed in the presence of IAA (Figure 2), with a decrease in the lateral root initiation frequency down to 1.9 ± 0.02 initiation events per 100 cortical cells from 7.9 ± 0.23 initiation events per 100 cortical cells, which is equivalent to 23% of the lateral root frequency in distal root portions treated with ACC alone (Table 2). Even non-inhibitory ACC doses became inhibitory to lateral root initiation when combined with IAA at this low concentration (Figure S2). Furthermore, IAA applied alone did not promote, but rather slightly reduced, the lateral root initiation frequency in the distal root portion (Table 2). The strong synergistic effect of IAA and ACC in the distal primary root portion formed during the treatment suggests that ethylene and auxin interact to suppress lateral root initiation in root regions, where they inhibit growth.

Table 2.   Comparison of the effects of 1-aminocyclopropane-1-carboxylic acid (ACC), indole-3-acetic acid (IAA) and N-1-naphthylphthalamic acid (NPA) on primary root growth, cortex cell length, and lateral root initiation in Arabidopsisa
Growth conditionsRoot growth parametersa
Growth increment (mm) ± SECell length (μm) ±SERate of cell production cells/day ± SELateral root densitybLateral root frequencyc
  1. aFor growth conditions and treatments see Figure 2. The means ± SE are reported with n = 12–14 per group (*≤ 0.02).

  2. bNumber of initiation events per mm.

  3. cNumber of initiation events per 100 cortical cells.

No additions48.8 ± 3.5175.1 ± 15.346.4 ± 1.00.59 ± 0.1410.4 ± 0.41
0.2 μm ACC21.9 ± 0.8*108.1 ± 17.2*33.8 ± 1.2*0.73 ± 0.107.9 ± 0.23*
0.05 μm IAA25.3 ± 0.7*91.1 ± 15.1*46.3 ± 0.91.01 ± 0.029.2 ± 0.15*
ACC + IAA8.6 ± 0.6*44.2 ± 10.2*32.4 ± 1.1*0.42 ± 0.041.9 ± 0.02*
1.00 μm NPA41.3 ± 2.3181.3 ± 12.4*38.0 ± 0.90.35 ± 0.026.4 ± 0.20*
ACC + NPA25.1 ± 3.1*108.1 ± 13.0*38.7 ± 1.2*0.30 ± 0.023.2 ± 0.03*
Figure 2.

 Comparison of the effects of 1-aminocyclopropane-1-carboxylic acid (ACC), indole-3-acetic acid (IAA) and N-1-naphthylphthalamic acid (NPA) on root growth and lateral root initiation.
(a) Seedlings treated with ACC, IAA and NPA for 5 days. The arrowhead indicates the position of the root tips at the beginning of the treatment.
(b) The number of initiated lateral roots was quantified in cleared roots, and was reported separately in the distal and proximal portion. The means ± SE of 15–18 roots are reported; *P ≤ 0.01, Student’s t-test.

The effect of ACC was also examined in combination with an auxin efflux inhibitor, N-1-naphthylphthalamic acid (NPA). NPA inhibits polar auxin transport from the shoot into the root, resulting in the inhibition of lateral root formation (Casimiro et al., 2001; Reed et al., 1998). At 1 μm, NPA mildly reduced primary root growth, resulting from reduced cell production rather than from reduced cell length (Figure 2a, Table 2), which is consistent with similar results reported elsewhere (Rahman et al., 2007). This treatment also profoundly reduced the number of lateral root initiations in the distal root portion. In a combination treatment with ACC, no cumulative effect was observed either in cell length or in cell production rate. Combined treatment with NPA and ACC reduced lateral root initiations in the distal portion formed during the treatment to a greater extent than did either treatment alone (Figure 2b, Table 2), suggesting that their effects might be partially cumulative.

The DR5:GUS reporter expression pattern was examined in roots treated as described above as an indirect measure of how these treatments may be changing the auxin distribution or response. ACC-treated and IAA-treated samples exhibited an increased expression in the central cylinder of the primary root apex (Figure S1a), with a pattern very similar to that reported elsewhere (Negi et al., 2008; Růžička et al., 2007; Stepanova et al., 2007), which is further enhanced when both compounds are added together. In contrast, NPA restricted the DR5:GUS expression mainly to the quiescent center and columella cells, and both ACC and IAA reversed the effect of NPA, as reported previously (Růžička et al., 2007). These pattern changes are consistent with ACC acting synergistically with IAA, whereas the role of auxin transport in the ACC effect is more complex, as NPA did not prevent the ACC induction of DR5 expression.

Ethylene promotes the emergence of existing lateral root primordia, although at higher levels it inhibits lateral root growth

We also asked whether ethylene promotes lateral root emergence in Arabidopsis, prompted by the fact that it promotes emergence of adventitious roots in wetland plants in response to flooding (Lorbiecke and Sauter, 1999; Steffens et al., 2006; Visser et al., 1996). Numbers of emerging lateral roots were scored in the proximal root portion, where primordia number does not change significantly with time or ACC treatment (Figure 1b), thereby allowing us to quantify emergence, when initiation events are relatively constant. The number of emerging lateral roots per day was determined in plants still growing on agar under a dissecting microscope, and is reported in Figure 3a for day 3 of the observation, when the ACC effect was most obvious. The proportion of emerged primordia was also determined versus the total number of initiation events in cleared roots, and was found to be increased in the ACC-treated roots (Figure 3b). In roots detached from the shoot, both lateral root emergence and DR5 expression in primordium cells were suppressed, and ACC did not promote lateral root emergence (not shown), suggesting that shoot-derived auxin is involved in the ethylene-dependent promotion of lateral root emergence. Altogether, these results indicate that ethylene accelerates lateral root emergence in Arabidopsis, possibly via an auxin-dependent mechanism. Consistent with this prediction, DR5:GUS expression is enhanced in lateral root tips treated with low doses of ACC (Figure S1b).

Figure 3.

 Effects of ethylene on lateral root emergence and root system growth.
(a) Five-day-old seedlings were transferred to medium with 1-aminocyclopropane-1-carboxylic acid (ACC): the number of lateral roots emerging in the proximal root portion during the treatment was scored every day; numbers are presented for the roots emerging on day 3. Control versus treatment: *≤ 0.02, Student’s t-test (= 14).
(b) Five-day-old seedlings were treated with ACC for 3 days, the roots were cleared and the percentage of emerged lateral roots was determined versus the total number of initiation events. Control versus treatment: ≤ 0.01, Student’s t-test (= 10).
(c) Alterations in the overall root architecture in seedlings exposed to ACC for 10 days. Numbers at the bottom of the plates indicate ACC concentrations in μm. Yellow arrowheads indicate root tip positions at the time of transfer to treatment plates. The black arrow indicates second order lateral roots emerging in seedlings treated with 5 μm ACC.

To examine how ethylene affects lateral root elongation, seedlings were allowed to grow on ACC for 10 days. On 0.04 μm ACC the lateral root branches appeared slightly longer than those grown on the control medium, although the primary root length was reduced by approximately 18% (Figure 3c, Table 1). On 0.2–1 μm ACC there was an obvious change in the overall root architecture, with both the primary and lateral roots becoming considerably shorter. The pattern of lateral root growth became less consistent, and some lateral roots grew longer than others without following an acropetal order. At 5 μm, lateral root growth was strongly suppressed; however, one or two lateral roots, usually positioned near the boundary between the proximal and distal root portion, continued to grow, extending beyond the tip of the primary root and seemingly taking over the function of a main root. Surprisingly, these growing lateral roots developed second-order lateral roots before similar roots emerged in control seedlings. We speculate that ethylene generates a signal for root system architectural transitions and/or root turnover.

Ethylene induces morphological alterations in root tissues involved in lateral root formation

ACC treatment reduces root cortex cell length (Tables 1 and 2), implying that cell length in the laterally adjoining cell files will also be reduced. However, xylem-adjacent pericycle cells from which lateral root primordia initiate might not be affected, because these cells continue cell division, even in the primary root differentiation zone (Beeckman et al., 2001; Dubrovsky et al., 2000), and ACC has been shown to have no effect on cell enlargement of dividing cells within the primary root meristem in Arabidopsis (Swarup et al., 2007). To examine the ethylene effect on elongation of xylem-adjacent pericycle cells, seedlings were treated with 0.2 μm ACC for 5 days, and then the region of the root between the first and the fourth mm from the tip was excised and examined, as this region contains the xylem-adjacent pericycle cells before they enter primordium organogenesis and the earliest lateral root primordia. Confocal laser scanning microscopy (CLSM) was used to visualize root primordia in propidium iodide stained roots, as shown in Figure 4a. These analyses demonstrated that ACC dramatically inhibited the elongation of the xylem-adjacent pericycle cells. In all samples, the intra- and inter-root variation in like-treated samples was significantly smaller than the variation between cells from treated and untreated samples, as summarized in Table S1.

Figure 4.

 Ethylene induces histological changes in the xylem-adjacent pericycle, lateral root primordia and emerging lateral roots.
(a) Roots were treated with 0.2 μm 1-aminocyclopropane-1-carboxylic acid (ACC) for 5 days, and root segments from between the first and fourth mm of the root tip were excised fixed and stained with propidium iodide, and were then imaged by confocal laser scanning microscopy (CLSM). The arrowheads indicate end walls of pericycle cells. c, cortex; e, epidermis; en, endodermis; p, pericycle. For cell length and statistics see Table S1.
(b) Seedlings were germinated on media with 0.04 μm ACC for 8 days. The arrows indicate the position of primordia medians. Untreated versus treated stage-II primordia: 13.8 ± 1.1 μm and 20.0 ± 0.8 μm, ≤ 0.01, Student’s t-test, = 8–11.
(c) Five-day-old seedlings were treated with 0.2 μm ACC for 2 days; all emerging lateral roots ≤ 200-μm long were used for the analysis. Basal outer layer 1 (OL1) cells and inner cells at the tip are numbered. Number of basal OL1 cells: Col versus treatment, 7.0 ± 0.5 and 7.7 ± 0.4, > 0.05, Student’s t-test, n = 25. Number of inner OL1 cells: Col versus treatment, 7.8 ± 0.7 and 12.0 ± 0.9, < 0.001, Student’s t-test, n = 22. Red arrowheads indicate a cell wall resulting from the first periclinal division in the OL1 cell layer; black arrowheads indicate a cell wall resulting from a second periclinal division of an inner OL1 cell derivative at the tip of the new root. Scale bar: 20 μm.

Because primordia grow laterally relative to the parent root, primordia emergence would depend on primordium width in the direction perpendicular to the primary root axis. The width of stage-I primordia generally increased upon treatment with 0.04 μm ACC, and the effect became statistically significant at stage II, increasing in all examined roots, apparently as a result of cell enlargement (Figure 4b). Thus, even though ethylene has no effect on the length of the apical meristem (Swarup et al., 2007), it increased the width of the lateral root primordia. This would be beneficial to primordium emergence in the proximal root portion, where ethylene cannot change the dimensions of the parent root cells.

We also examined the structure of cleared emerged lateral roots in the proximal root portion by differential interference contact (DIC), as shown in Figure 4c, to ask if there were alterations in primordia organization as a result of ACC treatment. The OL1 (outer layer 1) of a primordium when it emerges through the parent root epidermis shows a consistent number of basal cells on either side, and a consistent number of inner cells at the tip. This organization is referred to as an 8-8-8 cell pattern, with the inner cells at the tip resulting from the first periclinal cell division in OL1 (Malamy and Benfey, 1997). In ACC-treated proximal root portions, emerging or recently emerged lateral roots (defined as less than 200 μm in length) had more inner OL1 cells at the tip, resulting from anticlinal division of these cells, although the average number of basal OL1 cells was similar, as shown in Figure 4c. In addition, the second periclinal divisions of the inner OL1 cells at the tip occurred with a greater incidence in treated roots, thereby increasing the number of OL1 periclinal cell derivatives. For example, second periclinaI divisions of OL1 cells were detected in 67% (= 36) of the emerging lateral roots after treatment with 0.2 μm ACC for 2 days, and were detected in only 27% (= 30) of the lateral roots emerging without the treatment. Thus, without changing the normal pattern of cell divisions in the prospective lateral roots, ACC promoted cell proliferation near the apex of emerging primordia, apparently accelerating primordium emergence. A more detailed analysis is required to clarify the relative contribution of cell enlargement and cell division to primordium emergence, potential interconnections between changes in cell size and cell division, as well as whether emergence is promoted at all locations of the parent root.

The effect of ethylene on lateral root initiation is blocked in mutants with defects in auxin signaling and synthesis

Low ACC concentration (0.04 μm) increases the lateral root initiation frequency, and we hypothesize that this effect may be manifested through a positive feedback between ethylene and auxin signaling. Alternatively, low ethylene levels could induce lateral root initiation through an auxin-independent pathway. slr1 (Fukaki et al., 2002) single and arf19-4 nph4-1/arf7 (Wilmoth et al., 2005) double mutants, in which lateral root initiation is abolished as a result of disruption of the auxin signaling pathway, were treated with low to moderate ACC concentrations. In 8-day-old seedlings, we found none or only one stage-I lateral root primordium per 10 untreated slr1 seedlings, and no lateral root primordia in the arf19-4 nph4-1/arf7 mutant. When the mutants were grown for the same time on media supplemented with 0.02–0.08 μm ACC, we did not observe induction of lateral root initiation, although the arf19-4 nph4-1/arf7 mutants responded with inhibition of primary root elongation, as shown for 0.04 μm ACC in Table 3. This suggests that either the promoting effect of low ACC concentrations on lateral root initiation is mediated through auxin signaling, or that the absence of auxin signaling is limiting, and that ethylene is insufficient to override the absence of the auxin signal.

Table 3.   Comparison of the effects of 1-aminocyclopropane-1-carboxylic acid (ACC) on primary root growth, cortex cell length and lateral root initiation in auxin-related mutants in Arabidopsisa
BackgroundRoot length (mm)Cortical cell length (μm)Lateral root densitybLateral root frequencyc
  1. aSeedlings were germinated on media with or without ACC for 8 days. The means ± SE are reported with n = 10–12 per group.

  2. bNumber of initiation events per mm ± SE.

  3. cNumber of initiation events per 100 cortical cells ± SE.

  4. b,cDetermined for the region between the first initiated and first emerged primordium.

Columbia
 027.6 ± 1.0182.1 ± 6.20.74 ± 0.0513.5 ± 1.0
 0.04 μm ACC19.4 ± 0.5133.7 ± 3.91.26 ± 0.0716.7 ± 0.9
 1.57E-08 6.18E-07 1.15E-05 0.01
slr1
 0 0.04 μm ACC12.8 ± 1.3133.0 ± 7.1Primordia not detected
14.8 ± 1.2150.2 ± 3.3One primordium found in 11 roots
 0.13 0.03  
arf19-1 nph4/arf7
 0
 0.04 μm ACC21.4 ± 1.3159.9 ± 5.7Primordia not detected
16.0 ± 0.6122 ± 1.5Primordia not detected
 0.0005 2.76E-07  

Several reports have uncovered mechanisms for the synergistic negative regulation of root elongation by auxin and ethylene, including crosstalk between ethylene and auxin biosynthesis. We examined lateral root formation in wei2 and wei7 mutants, which have reduced ethylene-induced IAA synthesis. We found that both mutants had reduced lateral root initiation (Figure 5b). In addition, there was a significant reduction in the ability of 1 μm ACC to inhibit lateral root initiation in wei2 and wei7, consistent with ethylene-induced auxin biosynthesis being involved in the negative effect of ethylene on lateral root formation. Spatial expression of WEI2 and WEI7 genes was also analyzed. In addition to the well-characterized gene activity in the very tip of the primary root (Stepanova et al., 2005), we observed WEI2:GUS and WEI7:GUS expression in the central cylinder of mature primary root zones, including pericycle and early lateral root primordia (Figure 5a). WEI2 and WEI7 were also expressed in aerial tissue. The localization pattern of WEI2 and WEI7 suggests that ethylene might induce auxin synthesis in tissues distant from the root tip.

Figure 5.

 Effects of ethylene on the initiation of lateral root primordia in wei2 and wei7 mutants.
(a) The expression of WEI12 and WEI7 promoter-GUS fusions were examined in 10-day-old seedlings.
(b) Seedlings were treated for 5 days with 1 μm 1-aminocyclopropane-1-carboxylic acid (ACC), and the number of primordia was quantified in cleared roots (wei2 versus Col, 31.9%; wei7 versus Col 53.4%; ≤ 0.01, Student’s t-test, n = 12). wei2 and wei7 are less inhibited in lateral root initiation (treated versus untreated: Col 35.5%, wei2 50.9%, wei7 57.1%, ≤ 0.02, Student’s t-test, = 14).

Discussion

Ethylene regulates both lateral root initiation and emergence

Ethylene has been known to suppress root growth by the inhibition of root cell elongation (Le et al., 2001; Swarup et al., 2007). We observed that at low levels ethylene promotes the frequency of lateral root initiation; however, at levels that reduce the primary root length by more than approximately 30%, ethylene becomes inhibitory to lateral root initiation. The fact that these effects are only seen in root regions that are currently elongating during the ethylene exposure, indicates that ethylene affects lateral root initiation near the growing tip of the root. At the same time, ethylene promotes the emergence of lateral root primordia with a broad dose response and in mature regions of the root that have elongated prior to the exposure to ethylene.

Above a certain threshold level, ethylene clearly inhibits xylem-adjacent pericycle cell activation in primodium morphogenesis, because the frequency of lateral root initiation, estimated as the number of initiation events per 100 cortical cells, is decreased at the higher ethylene levels (Table 1). Little information from the literature suggests how ethylene could inhibit the commitment, or the activation, of xylem-adjacent pericycle cells. We did not detect inhibition of cell division in developing lateral root primordia; however, we did detect reduced cell production by the primary root meristem (Tables 1 and 2) at the higher ethylene level. Ethylene has also been shown to affect the normal cell cycle progression, and to cause DNA endoreduplication in epidermal cells of cucumber hypocotyl (Dan et al., 2003), and to induce cell death at the G2/M boundary in tobacco root cell suspension culture (Herbert et al., 2001). However, ethylene was also recently found to promote division of quiescent center (QC) cells in the Arabidopsis meristem without modifying the QC stem-cell-like properties (Ortega-Martínez et al., 2007). Thus, ethylene seems able to either inhibit or promote cell division, dependent on the developmental context. We cannot exclude the possibility that higher ethylene levels could inhibit the competence of pericycle cells for patterned cell division that leads to primordium formation. Another reason for the pericycle cell inhibition could be their reduced elongation (Figure 4a), because, as it has been suggested (Jacobs, 1997), plant cells might need to elongate sufficiently in order to be driven to divide. Finally, insufficient pericycle cell elongation might lead to insufficient polarized organelle movement, which is proposed to precede lateral root organogenesis (reviewed in Barlow et al., 2004). Molecular data that directly address such questions are scarce.

The data presented here and in the accompanying report (Negi et al., 2008), as well as in earlier reports in tomato and Arabidopsis ethylene-related mutants (Chilley et al., 2006; Clark et al., 1999), show that ethylene has an inhibitory effect on lateral root formation above a certain threshold level. Negi et al. (2008) examined the genetic controls of the ethylene inhibition of lateral root development, demonstrating that enhanced ethylene signaling in the ctr1 Arabidopsis mutant reduces lateral root formation, whereas reduced ethylene signaling in etr1 and ein2 mutants increases lateral root formation. They also show that the reductions in root formation by ACC, or in the eto1 mutation, are reversed by treatment with the ethylene antagonist silver nitrate. These data support the notion that ethylene negatively regulates lateral root development.

A positive role of ethylene on lateral root formation was proposed long ago by Zobel (1974), who reported that low ethylene concentrations restored lateral root formation in the diageotropica (dgt) mutant of tomato, which was unable to form lateral roots in normal conditions. We could not reproduce Zobel’s experiments, even with a range of growth conditions, including a range of light exposures and media compositions (MI and GM, unpublished data). However, we detected a positive effect of ethylene on lateral root initiation in Arabidopsis thaliana, such as the increase of lateral root frequency at low levels of ethylene, and the occurrence of closely spaced lateral root primordia in some samples (Figuer  1, Table 1). Therefore, we conclude that ethylene can positively regulate the initiation of lateral root primordia, dependent on the growth conditions and the developmental context. In the experimental conditions used here, the positive effect appeared subtle, and was often overridden by the strong inhibitory effect of ethylene over a range of doses.

In addition to effects on primordia initiation at the tip of the primary root, ethylene promotes the emergence of lateral root primordia in Arabidopsis in a manner that is not root-tip constrained (Figure 3). Consistent with the observed promotion of emergence of adventitious root primordia in wetland plants (Lorbiecke and Sauter, 1999; Steffens et al., 2006; Visser et al., 1996), this demonstrates that ethylene promotes the growth of root primordia in a range of plant species under appropriate environmental conditions. Lateral root primordium morphogenesis is maintained through a consistent series of cell division and enlargement, specific for each developmental stage (Malamy and Benfey, 1997). Primordia in ACC-treated roots are significantly enlarged from early stages throughout emergence, and show an increased number of OL1 cells at their apices (Figure 4b,c): either of these morphological alterations could contribute, among other things, to the observed faster rate of primordia emergence.

Ethylene interacts with auxin to regulate lateral root formation

We find that ethylene regulates lateral root development in Arabidopsis, at least in part, by crosstalk with auxin. The stimulation of lateral root primordia initiation at low doses of ACC requires Aux/IAA and ARF protein function, as judged by the lack of response in the corresponding mutants (Table 3). Lateral root emergence is also an auxin-regulated process (Celenza et al., 1995; Laskowski et al., 1995); however, we have not explored the mechanism by which auxin is involved in the ethylene-dependent stimulation of lateral root emergence. Because ethylene stimulates emergence of lateral root primordia with a very broad dose response, whereas stimulation of primordia initiation shows a narrow dose dependence (Figures 1 and 3), and these effects are exhibited in non-overlapping tissues, ethylene might interact differentially with auxin in the two developmental contexts.

The mechanism by which low doses of auxin and ethylene together suppress pericycle cells from initiating lateral root primordia more effectively than ethylene alone (Figure 2, Table 2), is more difficult to explain. Several reports emphasized the synergistic negative regulation of root elongation by auxin and ethylene, including the upregulation of ethylene biosynthesis by auxin (Woeste et al., 1999), and the more recently demonstrated enhancement of auxin biosynthesis by ethylene in the root tip through the upregulation of the WEI2 and WEI7 genes (Růžička et al., 2007; Stepanova et al., 2005, 2007; Swarup et al., 2007). We observed that WEI2 and WEI7 function is required for the complete ethylene inhibition of lateral root initiation (Figure 5), suggesting that upregulation of auxin biosynthesis might contribute to the ethylene inhibition of the pericycle. Yet, the ability of auxin to promote lateral root formation is well known. We propose that the inhibition of root cell elongation by the combined effects of ethylene and auxin inhibits the root branching capacity, which would explain why auxin contributes to the ethylene-induced inhibition of branching. This response is restricted to the root regions near the growing tip of the root, which are sensitive to the inhibition of root elongation, whereas the stimulation of root branching by auxin in the proximal region of the root is unaffected by ACC treatment.

The accompanying report (Negi et al., 2008) shows that the ethylene inhibition of lateral root formation is dependent on auxin influx mediated by the Arabidopsis AUX1 protein, although the spatial context of this interaction is yet to be established. They also show that ethylene promotes the acropetal auxin flow from the shoot into the root, and the basipetal auxin transport that occurs at the tip of the root. They also find that ethylene-signaling mutants exhibit altered lateral root developmental inhibition in response to treatment with the auxin transport inhibitor NPA. Based on the results from Negi et al. (2008), the prediction would be that NPA and ACC should reduce and enhance transport, respectively, leading to opposite effects on lateral root initiation. In contrast, we find a cumulative inhibition of lateral root initiation, but not root elongation, caused by NPA and ACC treatments. Our experiments were performed with lower doses of ACC, which prevents us from making a direct comparison with the Negi et al. (2008) report.

Hypothesis for the function of ethylene in lateral root formation

Based on our observations, we propose a model of how ethylene could participate in the root system establishment in A. thaliana, as shown in Figrue  6. At low doses, ethylene stimulates auxin biosynthesis, which promotes lateral root initiation through the primary auxin response mechanism that involves Aux/IAA and ARF proteins. At higher doses, ethylene inhibits lateral root initiation: this may be a mechanism by which stress, such as mechanical impedance to penetrating the soil or flooding, may increase ethylene synthesis through changes in the stability of the ETO1/ACS5 protein. In response, root growth is suppressed, and the rate of root cell production is reduced. Root cells, including xylem-adjacent pericycle cells, which give rise to lateral root primordia, undergo insufficient elongation, leading to the inhibition of the root capacity to form new lateral branches. However, existing lateral root primordia in other regions of the root are promoted to emerge, inducing an architectural alteration in the root system, and increasing its exploratory potential in locations more optimal for root growth. This could be related to the ability of ethylene to induce lateral enlargement of the lateral root primordia cells, and to increase the rate of cell division in these cells.

Figure 6.

 Model for the participation of ethylene in lateral root formation in Arabidopsis.
At low levels, ethylene promotes auxin biosynthesis and/or response, and promotes lateral root initiation in young root portions. Upon an increase in the level of ethylene, ethylene interacts with auxin in the tip of the primary root and suppresses root growth. This inhibits lateral root initiation in root regions with inhibited growth. Simultaneously, ethylene promotes the emergence of existing lateral root primordia.

Auxin, shoot derived or locally synthesized, interacts synergistically with ethylene in the tip of the primary root to regulate root cell elongation, and proteins like WEI2/ASA1 and WEI7/ASB1 provide a feedback control between the levels of the two hormones and the extent of their interaction. Ethylene also promotes the acropetal polar auxin transport from the shoot into the root, and the basipetal auxin flow from the root tip towards the root elongation zone, and affects auxin unloading into cells through the AUX1 protein (Negi et al., 2008). These findings provide additional layers of complexity in the crosstalk between auxin and ethylene in the root apex, which controls root elongation, and in pericycle cells, which controls root branch formation. However, although the mechanisms are not yet clear, the results presented provide strong evidence in support of a role of ethylene in regulating both the initiation and elongation of lateral roots, through crosstalk with auxin.

Experimental procedures

Plant material and growth conditions

The Arabidopsis wei2 and wei7 (Stepanova et al., 2005), eto1 (Chae et al., 2003), slr1 (Fukaki et al., 2002) and arf19-4 nph4-1/arf7 (Wilmoth et al., 2005) mutants, and WEI2/ASA1:GUS, WEI7/ASB1:GUS (Stepanova et al., 2005) and DR5:GUS (Ulmasov et al., 1997) reporter lines have been previously reported. All plant material was in the Columbia background. Seeds were sterilized in 20% commercial bleach, stored for 2 days at 4°C in the dark, and were then plated on 0.5x MS medium with 2% sucrose and 0.8% agar, tissue-culture grade, pH 5.7–5.8. Seedlings were grown on vertical plates at 22°C with 25 μmol m−2 sec−2 light density under long-day (16-h light, 8-h dark) conditions. At day 5 after sowing, seedlings were transferred to vertical plates containing hormones or other supplements, or were germinated in the presence of supplements (as indicated in the figure legends), and were grown for the indicated period of time. The ethylene precursor ACC was purchased from Calbiochem (http://www.emdbiosciences.com/html/CBC/home.html), the IAA efflux inhibitor NPA was purchased from Chem Service, Inc. (http://www.chemservice.com) and the IAA was purchased from Sigma-Aldrich (http://www.sigmaaldrich.com). Measurements of root growth increments or cell length were performed using Image-Pro Plus (Media Cybernetics, http://www.mediacy.com).

Root samples and microscopy

Root portions that were formed before or during the treatments were harvested separately for analysis. All initiation events, including emerged lateral roots and lateral root primordia, were quantified in cleared roots. To observe WEI2/ASA1:GUS, WEI7/ASB1:GUS and DR5:GUS expression, roots were stained for GUS before clearing. Root clearing and GUS staining were performed as described previously (Ivanchenko et al., 2006). Usually, between eight and 16 seedlings were used per sample in each experiment that was repeated between two and four times. Statistical analysis was performed using the Student’s t-test. Values are shown as means ± SE. Length of at least 10 cortical cells was measured for each root along the entire root growth increment, so as to exclude the possibility of differential response to the treatment. The average root length corresponding to 100 cortical cells in a file was estimated for each root, based on the elongation increment and average cortex cell length of the root. The rate of cell production was estimated for roots at day 3 of treatment, based on the growth increment and the average cortical cell length.

Sample analysis was performed using a Zeiss Axiovert microscope (http://www.zeiss.com) with DIC optics. Images were acquired using a SPOT CCD camera and optics (Diagnostic Instruments, http://www.diaginc.com).

Confocal laser scanning microscopy

For pericycle cell measurements and imaging, root fragments were excised from the region between the 1st and the 4th mm of the root tip, and were incubated with 10 μg ml−1 propidium iodide for 10 min before being used for CLSM. For imaging of lateral root primordia, entire roots were fixed with 4% formaldehyde in phosphate buffer, pH 6.8, supplemented with 5 μg ml−1 propidium iodide for 6 h at 4°C, washed briefly, mounted in the buffer and were then observed. Microscopy was performed using a confocal laser scanning Zeiss Axiovert LSM 510 Meta microscope, using the 543-nm excitation line of a He/Ne laser.

Acknowledgements

We gratefully acknowledge J. Fowler for providing laboratory space, S. Napsucialy-Mendivil, A. Saralegui, A. Ocadiz Ramírez and N. Doktor for their excellent technical assistance, S. Negi, R. Cole and M. Miller for their critical reading of the manuscript, J. Alonso for the wei2 and wei7 Arabidopsis mutants and the WEI2:GUS and WEI7:GUS lines, J. Kieber for the eto1 mutant, H. Fukaki for the slr1 mutant, and J. Reed for the arf19-4 nph4-1/arf7 mutant. This work was supported by the USDA National Research Initiative Competitive Grants Program (grant 2006-03434 to MGI, 2006-03406 to GKM), and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, DGAPA, UNAM (grant IN225906), and the Mexican Council for Science and Technology, CONACyT (grant 49267) to JGD.

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