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Keywords:

  • auxin;
  • IAA;
  • lateral root;
  • Arabidopsis thaliana

Summary

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

Lateral root formation is profoundly affected by auxins. Here we present data which indicate that light influences the formation of indole-3-acetic acid (IAA) in germinating Arabidopsis seedlings. IAA transported from the developing leaves to the root system is detectable as a short-lived pulse in the roots and is required for the emergence of the lateral root primordia (LRP) during early seedling development. LRP emergence is inhibited by the removal of apical tissues prior to detection of the IAA pulse in the root, but this treatment has minimal effects on LRP initiation. Our results identify the first developing true leaves as the most likely source for the IAA required for the first emergence of the LRP, as removal of cotyledons has only a minor effect on LRP emergence in contrast to removal of the leaves. A basipetal IAA concentration gradient with high levels of IAA in the root tip appears to control LRP initiation, in contrast to their emergence. A significant increase in the ability of the root system to synthesize IAA is observed 10 days after germination, and this in turn is reflected in the reduced dependence of the lateral root emergence on aerial tissue-derived auxin at this stage. We propose a model for lateral root formation during early seedling development that can be divided into two phases: (i) an LRP initiation phase dependent on a root tip-localized IAA source, and (ii) an LRP emergence phase dependent on leaf-derived IAA up to 10 days after germination.


Introduction

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

Lateral root formation is initiated close to the root tip, with anticlinal divisions in pericycle cells adjacent to the protoxylem poles, giving rise to lateral root primordia (LRP) (Dolan et al., 1993; Dubrovsky et al., 2000; Malamy and Benfey, 1997a; Malamy and Benfey, 1997b). The subsequent development of the primordia follows a series of highly ordered cell divisions that ultimately lead to the emergence of lateral roots (Malamy and Benfey, 1997a; Malamy and Benfey, 1997b). Numerous studies have shown that indole-3-acetic acid (IAA), the major endogenous auxin in plants, is necessary for both the initiation and the later development of lateral roots (Blakely et al., 1988; Celenza et al., 1995; Reed et al., 1998; Ruegger et al., 1997). LRP development may be divided into two distinct phases: (i) an early phase which lasts until the LRP are 3–5 cell layers in size when no meristem is detectable; and (ii) a later phase when the primordium develops a functional meristem and emerges from the main root. This distinction is based upon results obtained from in vitro cultured roots (Laskowski et al., 1995) indicating that LRP acquire the capacity to synthesize their own auxin at the end of the later phase. The source of auxin that drives the development of LRP until they become auxin-independent, and the signals that trigger de novo synthesis of auxin in the developing LRP. are yet to be determined.

Other factors such as nutrients also affect lateral root development (Drew, 1975; Zhang and Forde, 2000). One of the nutrients that has been studied in greater detail is nitrate, which has two opposing effects on lateral root development. While a localized concentration of nitrate stimulates the elongation of emerged lateral roots, nitrate acting systemically arrests the development of lateral roots just after emergence (Zhang et al., 1999). Elongation of lateral roots following nitrate application is an effect of nitrate acting as a signalling molecule (Zhang and Forde, 1998), but the systemic inhibition is a purely nutrient effect as it can be mimicked by other nitrogen sources. Nitrate also displays complex interaction with auxin signalling. While lateral root elongation is stimulated by the localized application of nitrate in axr2 and aux1 mutants, the axr4 mutant fails to respond to this signal. Interestingly, sucrose is able to counteract the systemic inhibitory effect of nitrate on LRP development (Zhang et al., 1999). This and other evidence indicate a role for leaf-derived signals on lateral root development, in response to either nitrogen or the nitrogen to carbon ratio.

Polar auxin transport has been shown to play an important role in the development of lateral roots (Casimiro et al., 2001; Ruegger et al., 1997). Removal of apical tissues or application of polar transport inhibitors has been shown to inhibit lateral root development (Reed et al., 1998). However, as described above, lateral root development may be divided into different stages based on auxin dependence as well as developmental features. Hence it is not clear which stages of lateral root formation depend on polar auxin transport. An additional complication arises from the discovery of two distinct auxin transport streams in the root, acropetal and basipetal (Rashotte et al., 2000). As both of these are affected by auxin transport inhibitors, it is difficult to delineate the roles of the two transport streams in regulating specific stages of lateral root development.

In this study, we have identified the sources of auxin that are likely to be involved in the regulation of lateral root development. Our results indicate that, following seed germination, free IAA is formed in a process that is regulated by light. Later on in development, auxin synthesis is initiated in the first developing true leaves and we postulate that this auxin is required for the first emergence of LRP. As the root system becomes more developed, the emergence of the latter primordia depends to a lesser degree on leaf-derived auxin, as the root system gains competence to synthesize its own auxin.

Results and discussion

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

Emergence of the first lateral roots correlates with a rapid transient increase in root IAA

A time course study of IAA levels in Arabidopsis roots was performed along with analysis of lateral root emergence in order to assess the role of IAA in controlling specific stages of LRP development. The primary root experienced a pulse of high IAA levels in the root 5–7 days after germination (DAG) (Figure 1a) which could be correlated with the timing of emergence of the first lateral root primordia (Figure 1b). Dark-grown seedlings, in contrast to those grown under long-day (LD) conditions, displayed a nearly constant level of IAA in the root (Figure 1c) and few emerged lateral roots (Figure 1d).

image

Figure 1. IAA content, root length and number of LRP in germinating wild-type seedlings.

IAA levels in the aerial parts (●) and root (○) of seedlings grown in LD (a) and darkness (c). Root length (●) and number of emerged LRP (○) in seedlings grown under LD conditions (b) and darkness (d). Error bars indicate SD. The number of replicates was 4–6. FW, fresh weight.

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The initial high level of IAA in the aerial parts of light-grown seedlings 1–3 DAG (Figure 1a) could result from the release of IAA from conjugate hydrolysis or alternatively from de novo IAA biosynthesis. The data presented here do not allow us to distinguish between the two possibilities raised above. In support of IAA synthesis is the observation that the NIT2 gene encoding a nitrilase is expressed in the mature embryos and during early seedling germination. This gene encodes a nitrilase that could potentially be of use in generating IAA via indoleacetonitrile (IAN) (Vorwerk et al., 2001). Also, the presence of large quantities of hydrolysable IAA conjugates offers the growing seedling the possibility of deriving IAA from conjugate hydrolysis until the seedling can initiate its own de novo synthesis in the cotyledons and first developing leaves (Bialek and Cohen, 1992; Epstein et al., 1980; Ljung et al., 2001a; Ljung et al., 2001b). In either case, the finding that high levels of free IAA are detected in the aerial tissues of seedlings grown under LD conditions (Figure 1a), whereas the levels in comparable tissues of dark-grown seedlings are much lower (Figure 1c), indicates that the process of IAA formation, whether derived from conjugate breakdown or via biosynthesis, is light-dependent.

The increased auxin levels in the roots result from transport of auxin originating from the first true leaves

The increased root IAA levels of seedlings between 5 and 7 DAG could result from IAA transported from the aerial tissues. Alternatively, it has been suggested that LRP that are 3–5 cell layers in size are capable of IAA synthesis (Laskowski et al., 1995), leading to the possibility that the increased level of IAA in 5–7 DAG roots is a result of de novo synthesis in developing LRP. To differentiate between these two possibilities, the IAA levels were measured in 5 mm segments of roots from wild-type and the axr4 aux1 double mutant. The axr4 aux1 double mutant forms only 10% of the wild-type number of mature lateral roots (Hobbie and Estelle, 1995), and no stage III or later LRP that may be able to undergo de novo IAA biosynthesis were found in the roots of 5–7-day-old seedlings under the growth conditions described here. The 5 mm root segment proximal to the hypocotyl junction was the first part of the root that showed an increase in IAA levels in wild-type (Figure 2a), supporting the hypothesis that IAA is transported from the shoot to the root. The axr4 aux1 double mutant showed a similar increase in IAA levels to the wild-type in the 5 mm segment proximal to the hypocotyl (Figure 2b). These results strongly indicate that the pulse of IAA in the roots of 5–7-day-old seedlings must be derived from the shoot and not from IAA that is synthesized locally in the LRP. It is also important to note that the IAA pulse reaches the root tip at 7–8 DAG (Figure 2a,b).

image

Figure 2. The distribution in different parts of germinating wild-type and axr4aux1 seedlings. 

(a,b) IAA levels in roots from wild-type and axr4aux1 seedlings. Seedlings were harvested at 4, 5, 6, 7 and 8 DAG. The roots were dissected into 5 mm sections. IAA was measured in pooled samples containing 10 sections. The mean SD for the samples was 12.8%, and the number of replicates was 3. (c,d) IAA content and size of cotyledons and young leaves in wild-type seedlings. Cotyledons, leaves 1 + 2 and leaves 3 + 4 were sampled. IAA levels were measured (c) and the average leaf length was calculated as a percentage of maximum expansion (d). Error bars indicate SD. The number of replicates was 3–5. FW, fresh weight.

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To identify the source of IAA transported to the roots, we investigated the IAA pool sizes in cotyledons, leaves 1 + 2 and leaves 3 + 4 up to 12 DAG (Figure 2c). Our results show that IAA levels in cotyledons are nearly constant at 40–50 pg per mg tissue between 4 and 6 DAG, after which there is a gradual decrease. Leaves 1 + 2 initially have high levels of IAA, which decrease steadily between 4 and 9 DAG. In contrast to leaves 1 + 2, leaves 3 + 4 have lower IAA levels initially that remain constant between 9 and 11 DAG, followed by a decrease between 11 and 12 DAG. These results show that a good correlation exists between the decrease of IAA levels in leaves 1 + 2 and the time of the increase of IAA levels in the roots. Thus, while leaves 3 + 4 may also supply IAA to roots, the initial amount transported will probably be significantly lower compared to what originates from leaves 1 + 2. This may be indicative of a reduced dependence on shoot-derived IAA for lateral roots formed later in development. There is still much that remains poorly understood about the IAA transport capacity from leaves at different developmental stages, and also whether mature leaves terminate their IAA transport to the roots.

Solely measuring IAA contents cannot identify the true sources and the dynamic fluxes of IAA. For example, roots have been shown to be capable of de novo IAA biosynthesis (Müller et al., 1998). We have therefore, in a separate study, analysed the synthesis capacity of cotyledons, young leaves, expanding leaves, and roots in 10-day-old Arabidopsis seedlings and observed that all organs can synthesize IAA, albeit with differing synthesis capacity (Ljung et al., 2001b). This shows that leaves continue to synthesize the hormone during the early stages of leaf expansion. Our interpretation is that the decrease observed in the first developing leaves reflects the fact that they switch to becoming net exporters of IAA, correlating well with the rapid increase of IAA levels in the root during this time.

Shoot-derived IAA is required for lateral root emergence during early seedling development

The close temporal and spatial correlation between a transient increase of IAA in the roots and LRP emergence indicated that this process might be dependent on shoot-derived IAA. To investigate this possibility, the shoot source of IAA was removed by excision of aerial tissues at 4 and 7 DAG. The timing of excision was chosen based upon the finding that the IAA pulse reaches the root between 5 and 7 DAG. If shoot-derived IAA is required for the lateral root emergence, then this developmental process should be blocked following the removal of aerial tissues at 4 DAG prior to the arrival of the IAA pulse. In contrast, excision of aerial tissues at 7 DAG should have no effect on LRP emergence as the IAA pulse would have already reached the root tissues. To address the question of whether IAA derived from cotyledons contributes to the IAA pulse, two sets of excisions were performed. In the first set, all of the aerial tissues, including the hypocotyl, were excised. In the second set of seedlings, only the cotyledons were removed. The excised seedlings were grown for a further 3 days, after which time the numbers and developmental stages of lateral root primordia were recorded. Intact plants harvested at 7 and 10 DAG were used as controls.

In the case of aerial tissue removal at 4 DAG, the relative number of emerging and fully developed lateral roots decreased drastically compared to the intact seedlings (Figure 3). Seedlings where only the cotyledons were removed 4 DAG had fewer lateral roots in later developmental stages than the controls, although the reduction was much less pronounced than for seedlings where all aerial tissues were excised. In contrast, removal of all aerial tissues or cotyledons at 7 DAG did not have any effect on LRP stage distribution compared to intact plants. In addition, applying IAA in sterile agar to the cut surfaces of seedlings that were dissected at 4 DAG restored the relative numbers of LRP at different stages by 7 DAG (data not shown). These results clearly indicate that IAA derived from the aerial tissue is specifically required for the emergence of LRP during early seedling development, and that the cotyledons contribute to this process only to a minor degree (Figure 3).

image

Figure 3. Relative numbers of early compared to late stages of lateral root primordia in intact and dissected wild-type seedlings.

Either only the cotyledons or all of the aerial tissues were excised from wild-type seedlings at 4 or 7 DAG. They were then left to grow for an additional 3 days. Intact seedlings were used as a control. At 7 or 10 DAG, the roots were cleared and the primordia counted and classified. The diagrams show the relative number of early LRP (1–4 cell layers; black segment) compared to late LRP (≥ 5 cell layers; white segment). The numbers given are a percentage of total LRP number ± SD; 121 ≤ n ≤ 1152.

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A sharp basipetal IAA concentration gradient is established in the root tip before the leaf-mediated IAA pulse reaches the tip

If the shoot-derived IAA is primarily required for the emergence of LRP, then what is the source of IAA for their initiation? Earlier results have shown that DR5 expression maxima and LRP initiation sites are distinct (Casimiro et al., 2001). This has led to the suggestion that high levels of IAA exist in the root tip and that basipetal transport from this localized IAA pool to the site of LRP initiation triggers the formation of the primordium. Here we present evidence for the existence of such an IAA source that spatially coincides with DR5 expression maxima. IAA levels were measured in 1 mm sections of the root tip at different days after germination (Figure 4). Our results show that IAA distribution is asymmetrical, with high levels in the first millimetre that decline rapidly further away from the root tip. Interestingly, both the absolute IAA levels as well as the distribution pattern undergo distinct changes as the root develops. A steep concentration gradient is established between 3 and 6 DAG that is followed by a further increase of the IAA level in the first millimetre of the tip at 7 DAG. The expression of the DR5-GUS reporter is in agreement with this observation, showing an increase in expression in the first millimetre of the root tip between 4 and 10 DAG (data not shown).

image

Figure 4. IAA levels in the root tip of wild-type seedlings.

IAA was measured in 1 mm sections starting from the root tip at 3, 6 and 7 DAG. Samples contained 50 pooled sections. Error bars indicate SD. The number of replicates was 3.

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It is possible that the root tip gains the ability early during development to synthesize sufficient IAA to contribute to the formation of a basipetal concentration gradient. Establishment of this root tip gradient is likely to be independent of the apically derived IAA as it is already formed at 6 DAG, prior to IAA from the aerial tissues reaching the root tip (Figure 2a,b). However, the lack of a basipetal gradient at 3 DAG may alternatively be explained by high levels of auxin remaining in the root from the conjugated IAA pools that are likely to be present in the seed to function as a supply until the seedling initiates de novo IAA synthesis. If the root tip gains the capacity to synthesize IAA early, the gradient will be masked by IAA released from conjugated sources in the apical part of the plant. Our previous experiments clearly demonstrated that the initial stages of LRP formation can proceed independently of the leaf-mediated IAA pulse. It is interesting to observe that the basipetal concentration gradient is established before this leaf-derived IAA flux reaches the root tip. It is thus tempting to speculate that the localized IAA pool in the root drives the initial stages of LRP formation. Some support for this suggestion comes from the observation that the aux1 mutant of Arabidopsis displays reduced IAA levels in the root tip and also exhibits a reduction in the number of LRP initiated (Marchant et al., 2002;Swarup et al., 2001).

As described before, LRP emergence is significantly reduced in dark-grown seedlings, indicating that there is a control by light on the supply of IAA to the root. By dividing the root tip into two 3 mm sections and then determining the IAA levels in the sections in seedlings grown either under LD conditions or in darkness, we observed that the root tip gradient is not established in the dark (data not shown). Growth in light, which increases the overall development, may require increased production of LRP to meet the nitrogen requirements and to co-ordinate root and leaf development. This could be dependent on a signal from the leaves increasing the IAA levels in the roots, leading to an induction of LRP emergence.

The root system gains competence to synthesize IAA between days 3 and 10

The emergence of LRP gradually becomes independent of shoot-derived auxin. This shift in dependence prompted us to investigate the possibility that the developing root system itself gains increased competence to synthesize IAA. Therefore, we compared the synthesis capacity of the root system at 3 and 10 DAG by incubating seedlings for 24 h in medium containing 30% deuterated water and 40 µm NPA. Deuterated water was used to track de novo IAA synthesis by high-resolution mass spectrometry analysis of isotopomer clusters. Naphthylphtalamic acid (NPA) was included to trap newly synthesized IAA in the tissues where the synthesis occurs and to block transport of IAA from other synthesizing tissues. Even if NPA is blocking polar IAA transport, there could be other transport routes for this hormone that contribute to the newly synthesized IAA detected in the root system. In separate experiments with deuterated water feeding, we have been able to demonstrate that the root system at day 10 has significantly enhanced capacity to synthesize IAA even without aerial tissues (Ljung et al., 2001b). This is in agreement with results presented by Müller et al., 1998) indicating that Arabidopsis roots are able to synthesize labelled IAA from labelled precursors.

Only 4% of the free IAA pool in the roots of 3-day-old plants was newly synthesized after 24 h of incubation in deuterated water, compared to 37% in roots of 10-day-old plants (Figure 5). The difference in total IAA content between day 3 and 10 is of course huge due to the massive difference in size, while the IAA concentration in the root was only 40% higher at 10 DAG compared to 3 DAG. This means that the IAA pool in a 10-day-old root system is turned over almost 10 times faster than in a 3-day-old root; in reality this demonstrates a 10-fold increase in the biosynthetic capacity of IAA by day 10 compared to day 3. These results are clearly indicative that the 10-fold increase in synthesis capacity mainly reflects a massive increase in developing lateral roots at day 10 compared to day 3.

image

Figure 5. The relationship between newly synthesized IAA and the total IAA pool in wild-type seedling roots.

Seedlings were incubated in 30% deuterated water and 40 µm NPA, starting at (a) 3 DAG or (b) 10 DAG. The diagrams show how much of the total IAA pool was newly synthesized (black sections), compared to the total IAA pool (white sections) after 24 h of incubation. The number given in (a) is the mean of two measurements (20 seedlings per sample). The number given in (b) is the mean of three measurements (10 seedlings per sample).

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Conclusion

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

The results presented in this paper indicate that developmental and environmental control of auxin metabolism exists during early seedling development and also that the sources of auxin that influence specific stages of lateral root development vary through development. We also conclude that:

  • (i)
     the increased auxin levels that accompany seed germination are regulated by light acting on conjugate hydrolysis and/or auxin synthesis;
  • (ii)
     roots experience an increase in auxin levels at about 5–7 DAG which coincide with the emergence of lateral roots;
  • (iii)
     two separate sources of auxin influence specific stages of lateral root development – root tip-localized auxin is important for primordia initiation, while leaf-derived auxin is critical for primordial outgrowth; and
  • (iv)
     the root system gradually reduces the dependence on apical tissue-derived auxin as evidenced by a major enhancement of its capacity to synthesize auxin by 10 DAG.

Experimental procedures

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

Chemicals and isotopically labelled substrates

[13C6]-IAA and 70% deuterated water were obtained from Cambridge Isotope Laboratories (Andover, Massachusetts, USA). The MS medium was from Duchefa (Harlem, the Netherlands), and all other chemicals were from Sigma (St Louis, Missouri, USA) unless stated otherwise.

Plant material and growth conditions

Arabidopsis thaliana wild-type seeds were of the Columbia ecotype. The double mutant axr4 aux1 and the DR5::uidA transgenic line were both in the Columbia background. Seeds were sterilized for 2 min in a 70% ethanol and 0.1% Tween-20 mixture, followed by a brief wash in 95% ethanol. After drying, the seeds were plated on solidified medium containing 1 × MS, 2% sucrose, 0.7% agar, with a pH of 5.6, and the plates were sealed with parafilm (American National Can, Chicago, IL, USA). After cold treatment (4°C for 72 h) in darkness, the plates were placed vertically under LD conditions (18 h light, 6 h darkness) at a temperature of 22°C. The light was provided from fluorescent tubes (General Electrics Polylux XL840, GE Lighting AB, Stockholm, Sweden) with a light intensity of 67 µmol m−2 sec−1.

For the measurements of IAA biosynthesis, wild-type seeds were grown on horizontal soft agar plates (1 × MS, 1% sucrose, 0.5% agar, pH 5.7). Three or 10 days post-germination, plants were transferred to flasks with liquid medium (1 × MS, 1% sucrose, pH 5.7) with or without 30% deuterated water and 40 µm NPA, and incubated in constant light for 24 h with gentle shaking.

Quantification of IAA and measurement of IAA biosynthesis

For analysis of endogenous IAA levels, leaves and whole root tissue were collected at different DAG, weighed and frozen in liquid nitrogen. To be able to measure IAA levels in different parts of the root, 1–5 mm root sections from 20–50 plants were dissected under a stereo microscope, collected in cold extraction buffer and frozen in liquid nitrogen. The samples were extracted, purified and analysed by GC selected reaction monitoring MS as described by Edlund et al. (1995). Calculation of isotopic dilution was based on the addition of 50–100 pg [13C6]-IAA per mg tissue. Samples were prepared in an ultra-clean environment and random blank samples were analysed to detect possible contamination problems associated with the analysis of extremely low amounts of IAA. Extracts from feeding experiments with deuterated water require a second purification step. These samples were purified as described above, methylated and thereafter applied on conditioned 50 mg C18 SPE columns (Bond Elut, Varian, Harbor City, California, USA). The columns were washed with 20% methanol in 1% acetic acid and eluted with methanol. IAA was analysed by GC–MS in the selected ion monitoring mode with a resolution of 10 000. Incorporation of 2H from deuterated water into IAA is expressed as the ratio of deuterium-labelled IAA (m/z 203 + 204 + 205) to unlabelled IAA (m/z 202) corrected for the contribution of natural isotopic abundance and for background.

Dissection of aerial tissues from seedlings

Seedlings were dissected in sterile environment 4 or 7 days post-germination, with a sterilized pair of scissors for eye surgery (PMS, Tuttlingen, Germany) while the seedlings were still on the plates. In one part of the experiment, all of the aerial tissues including the hypocotyl were excised. In another part, only the cotyledons were excised. The plates were re-sealed and put back in the growth room for an additional 3 days. For the IAA application study, Pasteur pipettes containing sterile agar with the same composition as the growth medium were prepared. The agar in the pipettes also contained IAA in concentrations ranging from 0 to 10 µm. Directly after dissection, agar from the pipettes was applied to the cut surfaces of the seedlings. The plates were then re-sealed, and, to reduce light-induced IAA breakdown, the plates were wrapped in aluminium foil. They were then put back in the growth chamber for an additional 3 days.

Histology and histochemistry

In the GUS assays, the seedlings were stained for GUS activity for 3 h in darkness at 37°C, in a solution containing 0.5 mg ml−1 X-Gluc, 20% methanol, 2.9 mg ml−1 NaCl, and 0.16 mg ml−1 K3Fe(CN)6, in a 0.1 m Tris buffer. The roots were cleared according to Malamy and Benfey (1997a) and mounted as whole mounts in 50% glycerol on microscope slides. The developing LRP were counted and classified using a Leica MZFL III stereomicroscope. The root lengths were measured with the aid of a Leica DC100 charge-coupled device camera and Leica QWin image analysis software.

Acknowledgements

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

We thank Roger Granbom for excellent technical assistance. We also thank the Arabidopsis Biological Resource Center (ABRC) for providing seeds of the axr4 aux1 mutant. This research was funded by the Swedish Natural Science Research Council and a European Community framework IV LATIN network grant (BIO4-CT96-0487).

References

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