AUX1 Promotes Lateral Root Formation by Facilitating Indole-3-Acetic Acid Distribution between Sink and Source Tissues in the Arabidopsis Seedling

The aux1 Mutant Initiates a Reduced Number of LRP

The auxin-resistant aux1 mutant has been reported to form reduced numbers of lateral roots compared with wild-type seedlings (Hobbie and Estelle, 1995). To establish the developmental basis for the effect of the aux1 mutation on lateral root development, the number of lateral roots originating in aux1 and wild-type roots were quantified between 5 and 13 DAG (Figure 1) . Seedling roots were first cleared to enable LRP from stage III onward to be visualized and counted. Mutant aux1 plants were observed to form ∼50% fewer lateral roots compared with the wild type, and this difference was maintained throughout the study period (Figure 1). This finding is in agreement with a previous observation made at a single time point (Hobbie and Estelle, 1995).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

aux1 Forms Fewer Lateral Roots Than Does the Wild-Type throughout Development.

Time course of lateral root number in wild-type and aux1-7 seedlings. Wild-type (Columbia ecotype) and aux1-7 seed were germinated on Murashige and Skoog (1962) agar and allowed to grow vertically. At 2-day intervals between 5 and 13 days, the numbers of lateral roots were counted using a dissecting microscope. The numbers shown are means of 15 seedlings ±sd.

Two possible explanations exist for why aux1 forms fewer lateral roots than does the wild type. The aux1 mutant root may form a reduced number of LRP. Alternately, a wild-type number of primordia may form but a subset may arrest before stage III. To test these models, sections were made of the primary root between 2.5 and 20 mm from the root tip of 9-day-old wild-type and aux1 seedlings to observe the number and developmental stages of primordia formed (Table 1). The first 20 mm of both the wild-type and aux1 primary root contains examples of primordia at all seven stages of development (Table 1) (Casimiro et al., 2001). However, the stage distribution of primordia in the aux1 mutant is altered, with an increased proportion at stage I in aux1 (33%) compared with the wild type (8%). Nevertheless, a summation of all primordia at each developmental stage in aux1 roots was found to be approximately half that observed in wild-type roots (Table 1). The latter observation correlates very closely with the reduction in the number of emerged lateral roots formed by aux1 plants compared with the wild type (Figure 1) (Hobbie and Estelle, 1995). Hence, we conclude that the greater number of stage I primordia reflects a delay rather than the developmental arrest of primordia in the transition from stage I to stage II.

AUX1 Is Expressed in LRP before the First Periclinal Division

To gain further insight into the role of AUX1 during lateral root development, transgenic seedlings expressing an AUX1 promoter–driven β-glucuronidase (GUS) reporter were examined (Marchant et al., 1999). Previous studies have demonstrated that the promoter fragment selected was sufficient to drive the expression of the AUX1 coding sequence to fully complement several aux1 mutant primary root phenotypes (Bennett et al., 1996). We have observed that the same AUX1 genomic fragment also was capable of rescuing the aux1 lateral root defect, restoring wild-type numbers of lateral roots (data not shown).

AUX1 promoter–driven GUS reporter expression was detected initially in the primary root (Figure 2A) , followed by the shoot apical meristem (Figure 2B), and then at various stages of lateral root development (Figures 2C to 2F). Closer examination of 4- to 10-day-old seedlings revealed that between 5 and 20% of stage I primordia showed detectable levels of GUS (Table 2, Figure 2C), indicating that AUX1 expression was induced at a late phase of stage I primordium development, before the first periclinal division. In contrast, GUS activity was detected in all lateral primordia at later stages of development (Figures 2D and 2E). After lateral root emergence, GUS expression became localized to the apical tip extending to or just beyond the elongation zone, with weak staining in the vascular cylinder extending to the junction with the primary root (Figure 2F). In summary, the AUX1 promoter–driven GUS reporter was detected initially in all cells associated with the developing LRP but subsequently became limited to the root apex and differentiating vascular tissues at the base of the emerged lateral root.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

AUX1 Is Expressed within the Shoot Apical Meristem and the Primary Root Tip throughout Lateral Root Development.

(A) Expression within the primary root apex.

(B) AUX1::uidA expression within the shoot apical meristem.

(C) Expression within a stage I primordium.

(D) Expression within a stage III primordium.

(E) Expression within a stage VIII primordium.

(F) Expression within a mature lateral root.

Bars = 50 μm.

The aux1 Mutant Exhibits Altered IAA Distribution in Young Leaf Primordia

Although the loss of AUX1 expression at a late phase of stage I (Figure 2C) is likely to be a basis for the observed delay in stage I to stage II transition of aux1 lateral primordium development (Table 1), it cannot account for the reduced number of lateral roots being initiated in the mutant. Instead, this mutant phenotype must be caused by the absence of AUX1 expression elsewhere within the developing seedling, such as in the developing leaf primordia. Mutations that disrupt the redistribution of IAA between its aerial source and root target tissues, such as stm1 (Casimiro et al., 2001) and tir3 (Ruegger et al., 1997), modify lateral root development. A disruption in the redistribution of IAA within and between the shoot and root organs also may account for the reduced level of lateral root initiation in aux1 (Table 1). To test our impaired auxin redistribution model, IAA was quantified directly in a variety of aux1 and wild-type tissues using gas chromatography–selected reaction monitoring–mass spectrometry (GC-SRM-MS) (Casimiro et al., 2001). IAA abundance was measured initially in aerial tissues that express the AUX1::uidA reporter (Figure 2B) in aux1 and wild-type seedlings at 4, 6, or 8 DAG (Figure 3A) . Aerial tissues exhibited a peak of IAA at 6 DAG that decreased by more than twofold by 8 DAG in both wild type and aux1. The level of IAA in aux1 aerial tissues was higher than that in the wild type, with the greatest difference occurring at 6 DAG (P = 0.01). Our results suggest that the movement of IAA from aerial source tissues may be impaired in the aux1 mutant.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3.

The aux1 Mutant Accumulates Increased Levels of Leaf IAA.

(A) The levels of IAA within aerial tissues shown to express AUX1 were measured for wild-type and aux1 seedlings at 4, 6, and 8 DAG. Results are expressed as pg/mg tissue ±sd.

(B) The aux1 mutant shows a defect in the export of IAA from leaves 3 and 4. Leaves were dissected from wild-type and aux1 seedlings at 2 DAG. Leaves 1 + 2 and 3 + 4 were dissected separately from wild-type and aux1 seedlings at 4, 6, and 8 DAG. IAA levels were measured for each sample and expressed as pg/mg tissue ±sd. Results for the wild type are indicated by the blue bars, and those for aux1 are indicated by the red bars. The histograms are overlaid on images of wild-type seedlings expressing AUX1::uidA.

To obtain greater resolution of the IAA levels in wild-type and aux1 aerial tissues, leaves 1 and 2, and then leaves 3 and 4, of 4-, 6-, and 8-DAG seedlings were microdissected and IAA levels measured using GC-SRM-MS (Figure 3B). The levels of IAA in 4-DAG leaves 1 and 2 were similar for wild type and aux1, but there was a slightly higher level in aux1 leaves 3 and 4 compared with those of the wild type. In 6-day-old seedlings, the difference between IAA levels in leaves 3 and 4 of wild type and aux1 was significant (P = 0.01). However, after 8 days of growth, the levels of IAA in leaves 3 and 4 of both wild type and aux1 had decreased to similar levels. The strong accumulation of IAA in the developing leaves of the aux1 mutant at day 6 is correlated to the developmental stage at which leaves start to export newly synthesized IAA (Ljung et al., 2001). Our results suggest that mutations in AUX1 cause a delay in the export of IAA from its site of biosynthesis in the developing leaves.

AUX1 Facilitates IAA Loading into the Vascular Transport System

Although GC-SRM-MS IAA measurements have revealed quantitative differences between aux1 and wild-type leaves, this approach cannot resolve where the highest levels of IAA are localized within leaf tissues. To provide a greater level of spatial resolution, the expression of the auxin-inducible IAA2::uidA marker (Luschnig et al., 1998; Swarup et al., 2001) was examined in the wild-type and aux1 backgrounds. In wild-type leaves 1 and 2, expression was localized predominantly to higher order vascular elements toward the base of the leaf (Figure 4A) . In aux1 leaves 1 and 2, GUS staining within vascular elements was reduced greatly (Figure 4B), despite the increased level of IAA within aerial tissues (Figures 3A and 3B). The altered pattern of IAA2::uidA expression in aux1 leaves is most consistent with impaired vascular loading rather than a developmental defect, given the fact that aux1 leaves exhibited a wild-type vascular pattern (data not shown). A threshold level of IAA is likely to be required to obtain detectable IAA2::uidA expression in leaf tissues, based on the behavior of the endogenous IAA2 gene (Abel et al., 1995). Hence, defective auxin loading in aux1 leaf vascular tissues may fail to increase IAA levels within a subset of leaf cells sufficient to induce IAA2::uidA expression.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4.

IAA2::uidA Shows Staining within the Higher Order Vasculature of Wild-Type Leaves That Is Reduced in aux1.

First leaves from wild-type and aux1 seedlings expressing the IAA2::uidA marker were stained for GUS activity at 4.5, 5.5, and 7 DAG.

(A) First leaf from a wild-type seedling at 4.5 DAG.

(B) First leaf from an aux1 seedling at 4.5 DAG.

(C) First leaf from a wild-type seedling at 5.5 DAG.

(D) First leaf from an aux1 seedling at 5.5 DAG.

(E) First leaf from a wild-type seedling at 7 DAG.

(F) First leaf from an aux1 seedling at 7 DAG.

Bars = 100 μm.

The aux1 Mutation Alters Root IAA Content and Distribution

A defect in the export of IAA from aux1 aerial tissues would be expected to result in a reduction in root IAA compared with the wild type. To determine whether this was the case, 4- to 8-day-old roots of aux1 and wild type were divided into 5-mm sectors and the amount of IAA was measured using GC-SRM-MS (Figure 5) . In 4- and 5-day-old wild-type roots, there was an even distribution of IAA throughout the root tissues (49 to 55 pg/mg fresh weight) (Figure 5). Six-day-old seedlings maintained a level of ∼50 pg/mg fresh weight within the apical three sectors but showed a slight accumulation within the most basal segment. This basal accumulation of IAA became more pronounced in 7- and 8-day-old seedlings, reaching levels up to 132 pg/mg fresh weight. However, the levels within the apical sectors of 7- and 8-day-old seedlings were similar to those found in younger seedlings. In aux1 seedlings, the levels within the apical sectors were consistently lower than those found in comparable regions of the wild type (Figure 5). It also is apparent that a more extreme basal–apical gradient was established in the aux1 seedlings between days 6 and 8 compared with the wild type, indicating trapping of auxin in the basal part of the aux1 root. The reason for this accumulation in the aux1 mutant is unclear, given the lack of AUX1 expression within this tissue.

• Download figure
• Open in new tab
• Download powerpoint

Figure 5.

Mass Spectrometry Measurement of Auxin Levels within Wild-Type and aux1 Mutant Root Tissues.

The levels of IAA within 5-mm segments of the primary root were measured for wild-type and aux1 seedlings between 4 and 8 DAG.

The Lipophilic Auxin 1-Naphthylacetic Acid Is Able to Rescue the aux1 Lateral Root Phenotype

The finding that the aux1 mutant has a reduced level of free IAA within the primary root apex compared with the wild type suggests that the reduction in the number of lateral roots formed may be a direct consequence of there being less auxin present (Figure 6) (Swarup et al., 2001). To address this possibility, the ability of exogenous auxin to restore a wild-type level of lateral root initiation was tested. Wild-type and aux1 seedlings were grown in the presence of 0.01 and 0.1 μM 1-naphthylacetic acid (1-NAA) for 9 days, and the number of lateral roots per millimeter of primary root was counted. Although the untreated aux1 control exhibited 50% fewer lateral roots compared with the wild type, mutant roots treated with 0.01 μM 1-NAA formed a wild-type density of lateral roots. Growth on higher concentrations of 1-NAA resulted in an increased but equivalent number of lateral roots emerging from both wild type and aux1. It can be concluded that 1-NAA is able to complement the aux1 lateral root defect, consistent with there being insufficient IAA within critical tissues of the mutant.

• Download figure
• Open in new tab
• Download powerpoint

Figure 6.

The Defect in Lateral Root Formation Shown by the aux1 Mutant Can Be Rescued by the Lipophilic Auxin 1-NAA.

The induction of lateral roots in wild-type and aux1-7 seedlings by 1-NAA. Wild-type and aux1 seed were germinated on Murashige and Skoog (1962) agar containing 0, 0.01, or 0.1 μM 1-NAA and allowed to grow vertically for 11 days. The numbers of lateral roots were counted, and the numbers shown are means ±sd of 15 seedlings.

AUX1 Facilitates IAA Transport between Leaf and Root Tissues via the Phloem

Our study has revealed that AUX1 regulates auxin transport between (and within) IAA source and sink tissues in developing seedlings. GC-SRM-MS measurements have demonstrated that AUX1 promotes the export of IAA from leaves to roots. Auxin reporter studies suggest that AUX1 facilitates the loading of IAA into vascular tissues. Several recent experimental observations have questioned whether IAA mobilization between aerial source tissues and root sink tissues is associated exclusively with polar auxin transport. First, IAA labeling studies observed that the polar auxin transport inhibitor naphthylphthalamic acid failed to block the export of IAA from young leaf tissues (Ljung et al., 2001). Second, GC-SRM-MS measurements revealed that IAA accumulated in apical tissues of naphthylphthalamic acid–treated roots (Casimiro et al., 2001), inconsistent with a model in which root apical auxin is delivered exclusively by polar auxin transport. Recent work has revealed a role for AUX1 in postphloem unloading of IAA in root apical tissues (Swarup et al., 2001).

In this study, we report that wild-type seedlings expressing the auxin-inducible IAA2::uidA marker exhibit reporter expression predominantly within the higher order vasculature of leaf tissues, the principal site for phloem loading in the leaves (Haritatos et al., 2000). The reduced IAA2::uidA staining in the higher order vasculature of aux1 is consistent with impaired loading, and hence export, of leaf IAA. These observations suggest that AUX1 facilitates phloem loading, in addition to unloading, in auxin source and sink tissues, respectively. However, expression studies of the AUX1:GUS staining pattern in developing leaves (Figure 3B) do not indicate a specific association with vascular tissues. Nevertheless, AUX1 may facilitate IAA vascular loading indirectly by mobilizing IAA from nonvascular leaf cells, whereas other members of the AUX1 gene family, such as LAX2, appear to be strongly associated with the vasculature (N. James and M.J. Bennett, unpublished results).

AUX1 Regulates LRP Development by Facilitating Auxin Transport between IAA Source and Sink Tissues

The transport of auxin from young leaf primordia, its major aerial source (Ljung et al., 2001), to target tissues in the root represents a major architectural determinant in this organ (Reed et al., 1998; Bhalerao et al., 2002; Casimiro et al., 2001). Arabidopsis mutations that disrupt this process have been observed to modify lateral root architecture (Ruegger et al., 1997; Casimiro et al., 2001). For example, the tir3 mutation causes a reduction in polar auxin transport, leading to fewer lateral roots (Ruegger et al., 1997), whereas the root architecture of the shootmeristemless mutant has lost the acropetal pattern of development (Casimiro et al., 2001) because its failure to form leaf primordia results in a loss of the transient pulse that coordinates lateral root emergence (Bhalerao et al., 2002). Mutations in the putative auxin influx carrier AUX1 also modify root architecture as a result of a 50% reduction in lateral root number (Hobbie and Estelle, 1995) (Figure 1).

We report that the primary reason for this reduction in aux1 lateral root number is a proportionate reduction in the rate of LRP initiation (Table 1). Lateral root developmental defects in the mutant result from the disruption of AUX1-mediated transport between IAA source and sink tissues. Our study has revealed that AUX1 regulates lateral root development by facilitating the export of IAA from newly developing leaf primordia (role 1), the unloading of IAA in the primary root apex (Swarup et al., 2001) (role 2), and the import of IAA into the developing LRP (role 3).

For several reasons, the reduced number of LRP initiated by the aux1 mutant cannot be accounted for by the delayed export of IAA from leaves 1 to 4 (role 1). First, the first LRP initiates as early as 28 hr after germination (Casimiro et al., 2001), well in advance of the initiation of leaves 1 and 2. Second, the stm1 mutant initiates a similar number of lateral roots compared with the wild type (Casimiro et al., 2001). In the absence of any evidence for significant IAA biosynthesis in the primary root (Ljung et al., 2001), we conclude that the cotyledons must act as the initial source of IAA at the early stages of development, driving lateral root initiation. Indeed, Bhalerao et al. (2002) have proposed that the accumulation of IAA in root apical tissues 1 to 3 DAG originates from the light-dependent breakdown of IAA conjugates in cotyledon tissues. Nevertheless, we cannot exclude the possibility that a leaf IAA source promotes lateral root initiation in more mature seedlings. The loss of AUX1-regulated IAA uptake into aux1 LRP (role 3) would be expected to affect lateral root development only after AUX1 gene induction in LRP at late stage I. Consistent with this prediction, we have observed that aux1 LRP development is delayed from stage I to stage II, suggesting that AUX1-mediated IAA uptake is required to facilitate the first periclinal division.

IAA Level in the Root Apex Regulates the Rate of Lateral Root Initiation

The reduced rate of aux1 LRP initiation most likely results from the loss of AUX1 activity in the primary root apex (role 2). Recently, it was demonstrated that AUX1 facilitates postphloem IAA unloading and basipetal auxin transport functions in the Arabidopsis primary root apex (Rashotte et al., 2001; Swarup et al., 2001). As a result of these dual transport functions, the loss of AUX1 activity in the primary root apex leads to reduced IAA levels and blocks basipetal auxin redistribution (Rashotte et al., 2001; Swarup et al., 2001). Several observations suggest that the basipetal auxin transport defect is unlikely to be the basis for the reduced rate of aux1 lateral root initiation. First, mutations that selectively disrupt basipetal auxin transport, such as Atpin2/eir1/agr/wav6, do not cause a lateral root defect (K. Palme, personal communication). Second, the growth of aux1 roots in the presence 10⁻⁸ M 1-NAA is not sufficient to rescue gravitropism and basipetal auxin transport (Swarup et al., 2001), yet it is capable of restoring aux1 LRP development to wild-type levels (Figure 6).

The 1-NAA rescue experiment suggests that IAA is suboptimal for lateral root initiation in aux1, which reflects the mutant's reduced capacity to unload IAA from the phloem, directly impairing the accumulation of IAA in root apical tissues (Swarup et al., 2001). Bhalerao et al. (2002) recently reported that 1- to 3-DAG wild-type seedlings accumulate IAA within their root apical tissues, coincident with the induction of LRP formation (Casimiro et al., 2001). Given these observations, we propose that AUX1 promotes lateral root initiation in the newly germinated seedling by facilitating phloem unloading of cotyledon-derived IAA in the root apex. Expression studies suggest that another member of the AUX1 gene family, LAX2, facilitates phloem loading of IAA in the cotyledon (N. James and M.J. Bennett, unpublished results).

How Can IAA Abundance in the Root Apex Influence the Rate of Lateral Root Initiation?

Beeckman et al. (2001) recently reported that Arabidopsis pericycle cells opposite the xylem pole are shorter than those found in other cell files. Therefore, LRP founder cells, which are derived from the xylem pole pericycle cell files, must undergo more divisions in the root apical meristem than do equivalent cell files. Uniquely among root tissues, xylem pole pericycle cell files proceed to the G2 phase as they become more distant from the root meristem, where they receive a signal to divide and initiate a new LRP (Beeckman et al., 2001). Suboptimal auxin levels in the aux1 root apical meristem and more basal cells could influence the mitotic activity of the xylem pole pericycle cells, which may influence the cells' ability to respond to a division factor at a later developmental stage.

AUX1 Facilitates Lateral Root Initiation and Emergence

Close parallels can be drawn between the two-step models of Celenza et al. (1995), Laskowski et al. (1995), and Bhalerao et al. (2002). All of these authors have recognized that IAA was required for at least two stages of lateral root formation, to initiate cell division in the pericycle (stage 1) and to maintain cell division to ensure the formation of a viable LRP (stage 2). However, Bhalerao et al. (2002) placed these developmental stages into a whole-plant context. Given that IAA is a non–cell-autonomous signaling molecule, Bhalerao et al. (2002) suggested that the cotyledons and leaves represent sources of IAA for lateral root initiation and emergence, respectively.

Our study has revealed that AUX1 regulates lateral root development by facilitating the export of IAA from newly developing leaf primordia, IAA unloading in the primary root apex, and import of IAA into developing LRP. These observations provide genetic evidence to support the two-phase model of Bhalerao et al. (2002), in which AUX1 promotes IAA accumulation in the root apex, thereby influencing the rate of initiation of LRP (phase 1), and later facilitates lateral root emergence (phase 2) through IAA export from newly formed leaves and/or uptake in LRP. Recent expression studies have revealed that a distinct member of the AUX1 gene family, named LAX2, is expressed in Arabidopsis cotyledon vascular tissues (N. James and M.J. Bennett, unpublished results), suggesting that LAX2 (rather than AUX1) supplies the newly germinated seedling root with cotyledon-derived IAA. The isolation of dSpm insertions in every member of the AUX1 gene family will facilitate research into the functional importance of each protein during Arabidopsis development.

Growth of Seedlings

Arabidopsis thaliana seed was surface sterilized (Forsthoefel et al., 1992) and plated on Murashige and Skoog (1962) agar (4.3 g/L Murashige and Skoog salts [Sigma], 1% Suc, and 1% bacto agar, pH 6.0, with 1 M KOH) with or without the addition of hormones or test compounds as appropriate. The plates were placed at 4°C for 48 hr and then in constant white light at 22°C. Throughout this study, wild type (Columbia ecotype) and the aux1-7 mutant allele were used.

Staining for β-Glucuronidase Activity

Seedlings were stained according to the published protocol of Malamy and Benfey (1997) with the following modifications: 10% methanol was included in the staining solution, and the 10× buffer contained 1.6 mg/mL K₃Fe(Cn)₆. Samples were incubated at 37°C for 3 to 4 hr before destaining and mounting (Berleth and Jurgens, 1993).

Root Sections

Samples were fixed for 3 hr at 20°C (4% glutaraldehyde, 4% formaldehyde, and 50 mM Na phosphate buffer, pH 7.2). A serial ethanol dehydration was then performed (30, 50, 70, 90, and 95% [twice]) at room temperature for 1 hr at each step. Samples were embedded in Technovit 7100 resin (Heraeus Kulzer, Wehrheim, Germany) according to the manufacturer's instructions. Sections (4 μm) were cut, dried onto glass slides, and stained for 8 min in an aqueous 0.05% ruthenium red solution. The samples were mounted in DePeX (Merck, Lutterworth, UK) before photography.

Mass Spectrometry Measurement of Indole-3-Acetic Acid

Leaves and root tissue were weighed and frozen in liquid nitrogen. Five-millimeter root sections and the first developing leaves from 20 to 50 plants were dissected under a stereo microscope, collected in cold extraction buffer, and frozen in liquid nitrogen. The samples were extracted, purified, and analyzed by gas chromatography–selected reaction monitoring–mass spectrometry (GC-SRM-MS) as described by Edlund et al. (1995). Calculation of isotopic dilution was based on the addition of 50 to 100 pg of ¹³C₆–indole-3-acetic acid (IAA)/mg tissue. Samples were prepared in an ultraclean environment, and random blank samples were analyzed to detect possible contamination problems associated with the analysis of extremely low amounts of IAA.