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DEVELOPMENT AND HORMONE ACTION

The rib1 Mutant of Arabidopsis Has Alterations in Indole-3-Butyric Acid Transport, Hypocotyl Elongation, and Root Architecture¹

Department of Biology, McGill University, Montreal, Quebec, Canada H3A 1B1 (J.P., C.S.W.); and Department of Biology, Wake Forest University, Winston-Salem, North Carolina 27109 (A.M.R., G.K.M.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Polar transport of the auxin indole-3-butyric acid (IBA) has recently been shown to occur in Arabidopsis (Arabidopis thaliana) seedlings, yet the physiological importance of this process has yet to be fully resolved. Here we describe the first demonstration of altered IBA transport in an Arabidopsis mutant, and show that the resistant to IBA (rib1) mutation results in alterations in growth, development, and response to exogenous auxin consistent with an important physiological role for IBA transport. Both hypocotyl and root IBA basipetal transport are decreased in rib1 and root acropetal IBA transport is increased. While indole-3-acetic acid (IAA) transport levels are not different in rib1 compared to wild type, root acropetal IAA transport is insensitive to the IAA efflux inhibitor naphthylphthalamic acid in rib1, as is the dependent physiological process of lateral root formation. These observed changes in IBA transport are accompanied by altered rib1 phenotypes. Previously, rib1 roots were shown to be less sensitive to growth inhibition by IBA, but to have a wild-type response to IAA in root elongation. rib1 is also less sensitive to IBA in stimulation of lateral root formation and in hypocotyl elongation under most, but not all, light and sucrose conditions. rib1 has wild-type responses to IAA, except under one set of conditions, low light and 1.5% sucrose, in which both hypocotyl elongation and lateral root formation show altered IAA response. Taken together, our results support a model in which endogenous IBA influences wild-type seedling morphology. Modifications in IBA distribution in seedlings affect hypocotyl and root elongation, as well as lateral root formation.

In vivo studies on the function of IBA are rather limited (Ludwig-Muller, 2000; Bartel et al., 2001). IBA has been identified in a number of plant species from Zea mays and Pisum sativa to Arabidopsis, and concentrations of free IBA approach the levels of free IAA in a number of plants (Ludwig-Muller, 2000). IBA, like IAA, is also found in conjugated forms, yet at significantly lower levels than IAA (Ludwig-Muller et al., 1993). IBA and IAA can be interconverted (Bartel et al., 2001), which has led to the suggestion that IBA may act as a precursor to IAA. Arabidopsis mutants whose roots have reduced sensitivity to growth inhibition by IBA but normal sensitivity to IAA have been isolated recently (Bartel et al., 2001), and many of these have defects in -oxidation, which is the pathway by which IBA is thought to be converted to IAA (Zolman et al., 2001a, 2001b). These findings support a role for IBA as an IAA precursor.

Other lines of evidence suggest that IBA might also act directly as an auxin, rather than solely being an auxin precursor. First, IBA is the preferred auxin for the induction of root formation, as it is much more potent than IAA or synthetic auxins (Ludwig-Muller, 2000). Several studies have demonstrated that internal IBA levels, not IAA levels, increase and stay elevated during IBA-induced root formation (Nordstrom et al., 1991; van der Krieken et al., 1992). The occurrence of several IBA-resistant, IAA-sensitive mutants that do not have defects in -oxidation also suggest that IBA could act directly, and not necessarily through conversion to IAA (Poupart and Waddell, 2000; Zolman et al., 2000).

Polar auxin transport is a specialized delivery system that moves IAA from its point of synthesis in young apical tissues to the rest of the plant in a highly regulated manner (Muday and DeLong, 2001). IBA and IAA are transported in a polar fashion in roots and hypocotyls of Arabidopsis at similar rates and amounts, while in the inflorescence stem IBA transport is much lower than IAA transport (Rashotte et al., 2003). The transport of IBA is regulated differently than that of IAA; IBA transport is not sensitive to inhibition by IAA efflux inhibitors, and the agr1/eir1/pin2/wav6 and aux1 mutants, which are defective in IAA transport, have wild-type levels of IBA transport (Rashotte et al., 2003). These results suggest that IBA is transported by a pathway distinct from the IAA transport pathway and support the hypothesis that IBA has activities beyond action as a precursor to IAA. Identification of mutants with altered IBA transport will be an important step in understanding the biochemical mechanisms and physiological significance of IBA transport.

Proper regulation of auxin transport is important for plant growth and development. Disruption of IAA polar transport using IAA efflux inhibitors, such as naphthylphthalamic acid (NPA) and triiodobenzoic acid (TIBA), results in a variety of phenotypes including defects in embryo and vascular tissue patterning, inhibition of lateral root, leaf primordia, and floral organ formation, reduced hypocotyl elongation in the light, and altered response in the root to gravistimulation (Okada et al., 1991; Muday and Haworth, 1994; Hadfi et al., 1998; Jensen et al., 1998; Mattsson et al., 1999; Sieburth, 1999; Reinhardt et al., 2000). Many mutations that disrupt auxin transport and affect the above-mentioned aspects of plant development have been isolated, and proteins involved in auxin transport have been identified through analysis of such mutants. Three different families of putative carrier proteins have been identified that are products of the AUX, PIN, or MDR gene families (Noh et al., 2001, 2003; for review, see Parry et al., 2001; Luschnig, 2002; Friml, 2003). Regulatory proteins have also been identified, such as those encoded by RCN1 or PID, which mediate reversible protein phosphorylation (Deruère et al., 1999; Christensen et al., 2000; Benjamins et al., 2001; Rashotte et al., 2001; Friml et al., 2004), and flavonoids, which have been suggested to act as endogenous regulators of auxin efflux (Brown et al., 2001; Buer and Muday, 2004; Peer et al., 2004). The current model is that expression of different carriers and regulatory proteins in specific cells allows precise regulation of auxin transport in a tissue- and cell-specific manner (Friml, 2003). It has also been suggested that different transporters could have different substrate specificities or affinities (Luschnig, 2002).

In addition to endogenous factors, exogenous factors also affect auxin transport, such as light and gravity stimulation (Muday, 2001; Blancaflor and Masson, 2003). Redistribution of auxin following tropic stimulation has been shown by the activation of an auxin-inducible reporter gene on one side of stems (Li et al., 1991) and roots (Rashotte et al., 2001; Ottenschläger et al., 2003), coinciding with differential growth. Light quality, intensity, and direction can all affect auxin response and transport (Swarup et al., 2002). Dim red light was shown to cause an increase in polar auxin transport in cucumber (Cucumis sativus) hypocotyls (Shinkle et al., 1992). Steindler and coworkers (1999) have shown that shade-induced hypocotyl elongation was dependent on auxin transport, as it can be inhibited by NPA, and the auxin response mutant axr1-12 is defective in this response. Arabidopsis hypocotyls grown in low light, but not in the dark, are sensitive to growth inhibition by NPA (Jensen et al., 1998). Finally, hypocotyl basipetal transport of IAA is inhibited by NPA only in low-light conditions and not in the dark (Rashotte et al., 2003). Together these results suggest light can affect auxin transport. The cloning of the BIG gene also supports a link between auxin transport and light response. Surprisingly, it was determined that two Arabidopsis mutants, doc1 (dark overexpressor of chlorophyll a/b-binding protein), which was isolated because it expresses abnormally high levels of light-induced genes in the dark, and tir3 (transport inhibitor response), which is defective in auxin transport and in NPA binding, are both mutations of the BIG gene (Gil et al., 2001; Kanyuka et al., 2003; Cluis et al., 2004).

Links between auxin and photomorphogenesis in seedlings have also been supported by publications showing that mutants defective in auxin transport or response have defects in photomorphogenesis (Morelli and Ruberti, 2002). For example, dominant mutations in the AXR2/IAA7, AXR3/IAA17, and SHY2/IAA3 auxin response genes all cause short hypocotyls, expanded cotyledons, and leaf production in the dark, phenotypes that are characteristic of photomorphogenesis and not seen in etiolated wild-type seedlings (Leyser et al., 1996; Tian and Reed, 1999; Nagpal et al., 2000; Tian et al., 2002). HY5 is a bZIP protein that is suggested to act as a positive regulator of light signaling. hy5-1 mutants have defects in hypocotyl elongation in the light, but also in root gravitropism and lateral root formation (Oyama et al., 1997; Cluis et al., 2004). These auxin-related phenotypes in light response mutants further suggest a functional link between the light and auxin-signaling pathways.

In this study, we studied IAA and IBA transport in different tissues of the resistant to IBA (rib1) mutant. rib1 was isolated in a screen for mutants with defects in root gravitropism and shown previously to have an altered response when the root is reoriented with respect to the gravity vector (Poupart and Waddell, 2000). Further characterization of the mutant revealed it was less sensitive to root elongation inhibition by the auxins IBA and 2,4-D, but had a wild-type response to IAA and the synthetic auxin NAA. rib1 did not show resistance to other classes of plant hormones tested, but did show resistance to the IAA efflux inhibitors NPA, TIBA, and 9-hydroxy fluorene carboxylic acid. Phenotypically, rib1 seedlings could be distinguished from wild type by their shorter primary root and increased number of lateral roots. Adult rib1 plants, however, were indistinguishable from the wild type in terms of shoot length and secondary inflorescence formation. Based on its phenotypes and the resistance of rib1 to IAA efflux inhibitors, we had hypothesized that this mutant could be defective in IBA transport or response (Poupart and Waddell, 2000).

This study shows that rib1 is defective in IBA transport but has wild-type levels of IAA transport. To the best of our knowledge, this is the first demonstration of such a phenotype. These defects in IBA transport can be correlated to defects in root elongation, lateral root formation, and hypocotyl elongation. Our results also suggest a role for IBA in hypocotyl response to light signals and that this response could be modulated by Suc. Finally, we characterize the responses of rib1 and wild-type seedlings to exogenous IAA and IBA, and to IAA transport inhibitors NPA and TIBA. Together these results suggest RIB1 could be a regulator of IBA transport important for defining root architecture and in hypocotyl elongation response to light and Suc.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Transport of Auxin in rib1

In seedlings, both IAA and IBA are transported in three different flows: from the shoot apical meristem at the tip of the hypocotyl down toward the root (hypocotyl basipetal transport), from the root/shoot junction at the base of the root to the tip (root acropetal transport), and from the root tip back up toward the root base over a short distance (root basipetal transport; Muday and DeLong, 2001; Rashotte et al., 2003). We measured IAA and IBA transport in rib1 and wild-type seedlings to directly assess the impact of the rib1 mutation on auxin transport (Table I). Significant changes in IBA transport are seen in all three seedling transport streams. In rib1 seedlings, hypocotyl basipetal transport of IBA is reduced to approximately 60% of the value of wild type, while IBA root basipetal transport is reduced to approximately 75% of wild type. Opposite to the decreases observed for basipetal transport, root acropetal IBA transport in rib1 is increased relative to wild-type levels by approximately 180% of wild-type levels.

A more accurate comparison is the amount of IBA and IAA basipetal transport in the inflorescence tissue, as those assays are done in similar ways by assessing transport in inverted inflorescence segments. The amount of IBA transport is extremely low and at the levels of background. At most, the amount of IBA transport in the inflorescence is 4% of the levels of IAA transport performed in identical assays, consistent with a previous report (Rashotte et al., 2003). The rib1 mutation has no effect on inflorescence IBA transport (Table I). These results are consistent with the wild-type appearance of adult rib1 plants (Poupart and Waddell, 2000).

Effect of NPA on IAA Transport in Wild Type and rib1

We have examined the effect of NPA on IBA transport and have never found evidence of NPA inhibition of root basipetal or acropetal IBA transport, or on basipetal transport of IBA in the hypocotyls. These experiments have been extensively repeated in wild type (Rashotte et al., 2003), and preliminary experiments in rib1 also show no regulation of IBA transport by NPA in all three transport streams (data not shown). While our transport studies indicate that IBA transport is not regulated by NPA, the roots of rib1 are altered in sensitivity to NPA.

To explore the regulation of auxin transport in rib1, IAA transport was examined in the presence of NPA, as shown in Table II. Overall levels of IAA transport are unaffected by the rib1 mutation under the conditions of our assays (Table I). Table II shows that all flows of IAA transport are significantly reduced by NPA in wild-type seedlings and adult plants, but with the magnitude of reduced transport much greater in the inflorescence stems. Likewise, IAA basipetal transport in the inflorescence stem, hypocotyl, and root are inhibited by NPA in rib1. Analysis of the effects of NPA on IAA transport in rib1 reveals one difference in regulation of IAA transport relative to wild type. NPA does not inhibit root acropetal IAA transport in rib1. This finding is interesting in light of the fact that the only IBA transport stream elevated in rib1 is this acropetal auxin transport in the roots.

As rib1 exhibits a significant decrease in hypocotyl basipetal transport of IBA, we examined whether hypocotyl elongation was also affected in the rib1 mutant under several light intensities and two Suc concentrations, as shown in Figure 1. Under the conditions used for the transport assays, low white light (5 µmol m^–2 s^–1) and 1.5% Suc, rib1 hypocotyls are significantly longer than wild-type hypocotyls (Fig. 1). In the dark on the same media, rib1 hypocotyls are also significantly longer, but with a greater difference (20% as compared to 35%, respectively). However, there is no difference in hypocotyl length under high white-light conditions (95 µmol m^–2 s^–1) and 1.5% Suc. This last result was surprising as we had previously noted that rib1 had a long hypocotyl under different growth conditions, i.e. high white light with a 16-h-day/8-h-night cycle and 1% Suc. This prompted us to further examine the effects of light on rib1 hypocotyl length.

View larger version (22K): [in this window] [in a new window]   Figure 1. Hypocotyl length in wild-type and rib1 seedlings grown in different light and Suc conditions. Wild-type and rib1 seedlings were plated on media containing 1.5% Suc or Suc-free media. Plates were placed in dark, low white (LW), high white (HW), red (R), far red (FR), or blue (B) light, and hypocotyl length was measured after 5 d (with Suc) or 7 d (without Suc) of growth. Average length and SE of 138 to 209 individuals from seven to nine trials are presented for conditions with Suc. Average length and SE of 32 to 143 individuals are presented from one representative trial without Suc. Similar results were obtained in one to five additional trials. Asterisks indicate P values from two-tailed Student's t test comparing wild type to rib1 under each condition. *, P value < 0.05; ***, P value < 0.001; actual values range from 3.9 x 10–5 to 2.5 x 10–32.

Effect of Exogenous Auxin on Hypocotyl Elongation

The effects of exogenous IAA and IBA on hypocotyl elongation were investigated in the dark and in two different light conditions, including the low white-light condition used for the hypocotyl basipetal transport assays (Fig. 2). Media with 1.5% Suc was used, as this is the media used in auxin transport assays. In the dark, both IAA (Fig. 2A) and IBA (Fig. 2B) inhibit hypocotyl elongation in rib1 and wild type. Small differences in rib1 response to IAA under dark conditions were detected (Fig. 2A). More notably, the dose-response curve of rib1 to IBA is clearly shifted toward higher concentrations, indicating a reduced sensitivity of rib1 to IBA under these conditions (Fig. 2B). Exogenous auxin also inhibits hypocotyl elongation under low-light conditions. Under these light conditions, the response of rib1 to both IAA (Fig. 2C) and IBA (Fig. 2D) requires higher concentrations relative to the wild-type curve, indicating that hypocotyl elongation in rib1 is also less sensitive to inhibition by both auxins in these conditions. A comparison of the concentrations of auxins that lead to a 50% inhibition (IC₅₀) of hypocotyl elongation confirms that rib1 dramatically affects the response to IBA in the dark (5.1 versus 11.5 µM for No-0 and rib1, respectively), while the response to IAA is only marginally affected (2.0 and 2.8 µM for wild type and rib1, respectively). In low-light conditions, however, both IAA and IBA responses are affected by rib1 with the IC₅₀ for rib1 hypocotyls being 4.4-fold higher for IAA and 2.6-fold higher for IBA than wild type.

View larger version (34K): [in this window] [in a new window]   Figure 2. Dose response of hypocotyl elongation to auxins in different light conditions in the presence of 1.5% Suc. Wild-type and rib1 seedlings were plated on media containing the indicated amounts of IAA (A, C, and E) or IBA (B, D, and F). Plates were placed in dark (A and B), low-light (C and D), or high-light (E and F) conditions, and hypocotyl length was measured after 5 d of growth. Average and SE is presented as the percent of elongation in the absence of auxin for each genotype. Error bars smaller than the symbols are not visible. The average of six to 26 seedlings from one representative trial is shown. Similar results were obtained in two or three additional trials.

Effect of Exogenous Auxin on Lateral Root Formation

Lateral root formation in response to exogenous auxin was investigated in wild-type seedlings grown in low light (5 µmol m^–2 s^–1) and the presence of 1.5% Suc. Figure 3 shows a dose-response curve of lateral root formation in response to IAA and IBA in wild-type and rib1 seedlings. The graph indicates that IBA is more potent at inducing lateral roots. Lateral root induction occurs at lower concentrations of IBA than IAA and the slope of the IBA graph is steeper.

View larger version (21K): [in this window] [in a new window]   Figure 3. Comparison of IAA and IBA effects on lateral root formation in wild-type and rib1 seedlings. Four-day-old seedlings were transferred to media containing the indicated amounts of auxin. After 6 d of growth in the presence of auxin the number of emerged lateral roots was counted from 17 or 18 seedlings in each trial. The averages and SEs from two combined trials are shown.

In order to examine the statistical significance of the differences between lateral root numbers in wild type and rib1 in the presence of these two auxins at a range of concentrations, a three-way factorial ANOVA was performed. The differences between genotype (F_1,974 = 120.9, P < 10^–3), between auxin used (F_1,974 = 183.3, P < 10^–3), and concentration (F_6,974 = 599.7, P < 10^–3) were all significant. The two-way interactions of auxin type and genotype, auxin type and concentration, and genotype and concentration were also significant (F_1,974 = 10.0, P < 0.002; F_6,974 = 55.1, P < 0.001; F_6,974 = 24.1, P < 0.001, respectively). These statistical tests allow us to conclude that wild type and rib1 have significantly different responses to each auxin and each dose of auxin. Yet, the precise comparison that should be made is whether rib1 and wild type are significantly different at each auxin concentration used. This statistical comparison was performed using a Neuman-Keuls post-hoc comparison of means. This post-hoc analysis is justified based on the statistical differences in genotype by auxin type and genotype by auxin concentration revealed in the two-way interactions identified by ANOVA, as described above. For these comparisons, the differences between wild type and rib1 become significant at IBA doses of 1 µM and greater and for IAA at doses of 3 µM or greater (degrees of freedom = 974; P < 0.007). Therefore, the rib1 mutation reduces the sensitivity to both IBA and IAA, but with a significant difference in response detected at a lower concentration of IBA than IAA and with a greater magnitude of effect with IBA.

Effect of Auxin Transport Inhibitors on rib1 Seedlings

The rib1 mutant has been shown to be less sensitive not only to IBA and 2,4-D but also to the IAA efflux inhibitors NPA, TIBA, and 9-hydroxy fluorene carboxylic acid by root elongation assays (Poupart and Waddell, 2000). Figure 4 quantifies the effects of the IAA efflux inhibitors NPA and TIBA on lateral root formation in wild-type and rib1 seedlings. The concentration of NPA used (0.1 µM) inhibits lateral root formation in wild type to approximately 20% of the numbers in nontreated wild-type seedlings. In contrast, NPA reduces lateral root formation in rib1 much less, to about 77% of nontreated levels. rib1 is also less sensitive to the effects of TIBA; at 0.1 µM, TIBA reduces the number of lateral roots formed in wild type to approximately 45% and in rib1 to 71% of nontreated controls. Interestingly, in wild type NPA is significantly more effective in inhibiting lateral root formation than TIBA (20% versus 45%; Student's t test, P = 6 x 10^–3), while the difference between the effect of 0.1 µM NPA or TIBA is not significant in rib1 (77% versus 71%; P = 0.66). Taken together with the previous report (Poupart and Waddell, 2000), these results show that rib1 is less sensitive to IAA efflux inhibitors for both primary root elongation and lateral root formation responses. Additionally, the reduced ability of NPA to inhibit lateral root development in rib1 parallels the insensitivity of acropetal IAA transport to NPA, further linking these two processes.

View larger version (16K): [in this window] [in a new window]   Figure 4. Effect of auxin transport inhibitors on lateral root formation in wild-type and rib1 seedlings. Wild-type (gray bars) and rib1 (white bars) seedlings were grown for 4 d on control media and then transferred to media containing 0.1 µM NPA or TIBA. After 6 d of growth in the presence of auxin, the number of emerged lateral roots was counted. Data are expressed as a percentage of the number of lateral roots formed in the absence of inhibitor. Average and SE of 36 individuals from two assays (NPA) or 54 individuals from three assays (TIBA) are presented.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Basipetal IAA transport has been implicated in the control of Arabidopsis root growth and gravitropism (Rashotte et al., 2000; Blancaflor and Masson, 2003). Blocking root basipetal IAA transport by localized application of auxin transport inhibitors, or by mutations in genes encoding IAA transport proteins such as AGR1/EIR1/PIN2/WAV6 or AUX1, results in defects in gravity-induced reorientation and root elongation (Maher and Martindale, 1980; Bell and Maher, 1990; Chen et al., 1998; Rashotte et al., 2000). rib1 has a slowed response to gravity and a shorter primary root (Poupart and Waddell, 2000), indicating a defect in elongation growth. rib1 has wild-type levels of root basipetal IAA transport but has significantly reduced root basipetal IBA transport, suggesting a role for IBA transport in controlling root elongation growth and gravitropic curvature.

Root acropetal transport of IAA has been shown to be important for lateral root formation, as inhibition of this flow of transport results in a reduction of lateral root formation (Reed et al., 1998) and movements of free IAA into the root are timed to drive lateral root elongation (Bhalerao et al., 2002). Four characteristics of rib1 are consistent with altered acropetal IBA transport also being linked to the control of lateral roots. First, when seedlings are grown under high light, there are also elevated levels of IBA transport in rib1 relative to No-0 that occur specifically in the root acropetal transport flow; acropetal IAA transport is similar in the two genotypes. Second, rib1 mutant seedlings have approximately 60% more lateral roots than wild type at 14 d when grown in high light (Poupart and Waddell, 2000), consistent with the higher root acropetal IBA transport under similar light levels. Third, the regulation of root acropetal IAA transport is modified in rib1 and is no longer sensitive to NPA. This is reflected in the reduced sensitivity of seedlings to inhibition of lateral root formation by NPA. Fourth, lateral roots are induced by application of exogenous auxin, either IBA or IAA, and rib1 exhibits a higher level of insensitivity to IBA than to IAA. These results support the hypothesis that root acropetal transport of IBA modulates lateral root formation, like the acropetal transport of IAA. The root phenotypes of rib1 (increased number of lateral roots in light-grown seedlings, shorter primary root, and slowed gravity response) are therefore suggested to be the result of IBA transport disruptions, indicating that IBA, in addition to IAA, has an important role in defining root architecture.

One point that is still not clear about these results is the relationship between the action of auxin transport inhibitors and the rib1 mutation in modulation of root growth and transport. NPA and TIBA don't directly affect IBA transport, yet rib1 is less sensitive to the effect of NPA on acropetal IAA transport. rib1 is also less sensitive to the inhibitory effect of both TIBA and NPA on lateral root formation. rib1 also shows resistance to NPA inhibition of hypocotyl and primary root elongation (Poupart and Waddell, 2000). This scenario partially parallels the rcn1 mutant, which has a defect in a gene encoding a regulatory subunit of protein phosphatase 2A and which shows a similar phenotype: though root acropetal IAA transport in this mutant is normal, it shows defects in regulation of this transport by NPA (Rashotte et al., 2001). By analogy, RIB1 could encode a protein that regulates IBA transport, directly affecting IBA transport and affecting the response of some flows of IAA to NPA. Alternatively, RIB1 could have a more direct role in the regulation of root acropetal IAA transport. In the presence of RIB1, acropetal IAA transport occurs through an NPA-sensitive pathway, while in the absence of RIB1 acropetal IAA transport occurs through a second pathway, which is NPA insensitive. This second pathway would be the transport stream normally utilized by IBA. A third possibility is that the rib1 mutation affects IBA specificity in a protein that normally functions in both IAA and IBA transport. The mutated form of the protein may no longer recognize or respond to IBA, but retains most of its wild-type IAA activity with some limited changes in the regulation of IAA transport and subtly altered responses to IAA (see below).

An additional link between the altered IBA transport in rib1 and lateral root formation is identified in assays that examine lateral root induction in response to exogenous auxin. IBA is a more potent inducer of lateral roots than IAA in wild-type seedlings grown under the low-light conditions used for our physiological assays. These results contrast with the results of Zolman et al. (2000) who found that IAA induced more lateral roots than IBA when both auxins were used at similar concentrations. Many factors vary between our studies and these factors have been shown to affect lateral root formation in Arabidopsis: age of seedlings, Suc and nitrogen salt composition of media, length of hormone treatment, ecotype, and light conditions (Forde, 2002; Malamy, 2005). For example, in an earlier study comparing lateral root formation of rib1 and No-0, in which plants were grown in high light with lower Suc levels, rib1 had elevated lateral root formation (Poupart and Waddell, 2000), while under the lower light and higher Suc conditions used in this study, rib1 and No-0 have similar levels of lateral roots. As noted earlier, rib1 is less sensitive than wild type to lateral root induction by IBA and IAA, but the magnitude of the insensitivity to IBA is greater. Nonetheless, these experiments revealed one physiological condition (low light and 1.5% Suc) in which rib1 roots (and as discussed below, rib1 hypocotyls) display some modest resistance to IAA and suggest either cross talk or direct interaction between the two auxins.

Hypocotyl basipetal transport of IBA is reduced in the rib1 mutant, while IAA transport is unaffected in rib1 hypocotyls. We also found hypocotyl elongation to be affected by the rib1 mutation in a range of conditions. In the absence of Suc, rib1 hypocotyls are longer than wild type in white and red light, not significantly different in the dark or blue light, and shorter in far-red light. In the presence of Suc, rib1 hypocotyls are longer than wild type in dark- and low-light conditions, but not different in high-light conditions. Our data therefore suggest IBA transport has a role in hypocotyl elongation both in the light (without Suc) and in the dark (with Suc). Our results also suggest interaction between light and Suc signaling on hypocotyl elongation of rib1. Exactly where and how RIB could be integrated in this complex regulatory web is unclear at present, but it would affect IBA transport in response to light. Light has been shown previously to affect auxin transport in cucumbers (Shinkle et al., 1992), but also to specifically affect IBA transport in Arabidopsis (Rashotte et al., 2003).

Previous research has suggested auxin transport is not required for hypocotyl elongation in the dark in Arabidopsis (Jensen et al., 1998). This conclusion was based on the fact that an IAA efflux inhibitor reduced hypocotyl elongation in light-grown seedlings but not in dark-grown seedlings. These results can be reconciled with ours if we consider the specificities of IBA transport. IBA transport in Arabidopsis occurs in the same directions as IAA transport, but IBA movements are not inhibited by NPA. Therefore, lack of inhibition by NPA of hypocotyl elongation in the dark does not rule out a role for IBA transport in etiolated hypocotyl growth. Additionally, IAA transport itself is not completely abolished by NPA in hypocotyls, unlike what is observed in inflorescence stem, where NPA causes almost complete inhibition of IAA transport (Rashotte et al., 2003). Furthermore, IAA transport in the hypocotyl becomes completely insensitive to NPA in the dark (this study; Rashotte et al., 2003). Taken together, these results suggest that some IAA transport also occurs through an NPA-insensitive pathway, which could use the same pathway as IBA transport. Interestingly, Jensen et al. (1998) noted that NPA had a lesser effect on hypocotyl elongation in red light than in white-, far-red, or blue-light conditions. Based on Jensen's results and the results presented here, it is tantalizing to suggest that NPA-insensitive transport is more important for the phytochrome B-mediated hypocotyl elongation inhibition response.

Exogenous auxins have different effects on hypocotyl elongation depending on light intensity. Under low white-light or dark conditions, application of either IAA or IBA results in inhibition of hypocotyl elongation in wild type. rib1 is less sensitive to inhibition of hypocotyl elongation by exogenous application of both IAA and IBA in low light, but only to IBA in the dark, showing that mutating RIB1 differentially affects hypocotyl response to IAA and IBA in different light conditions. rib1 phenotypes can be correlated to changes in response to IBA in this mutant: seedlings have longer hypocotyls in conditions under which application of low concentrations of IBA were shown to inhibit elongation (dark and low white light), but show hypocotyl lengths similar to wild type under high white-light conditions, where application of the same concentration of IBA had no significant effect on elongation. Stimulation of hypocotyl elongation in high light is seen in wild type with IBA concentrations ranging from 1 to 10 µM, a response not seen under dark or low-light conditions. Stimulation of hypocotyl elongation in rib1 requires approximately 3-fold higher concentrations of IBA. The fact that exogenous IBA can stimulate hypocotyl elongation under conditions where IAA is inhibitory to hypocotyl elongation suggests that IBA has a direct role in elongation of this organ in Arabidopsis.

Interestingly, our analysis of hypocotyl elongation and lateral root formation revealed a condition in which rib1 differs from wild type in response to IAA. Under low-light conditions and in the presence of Suc, rib1 is slightly less sensitive to the inhibitory effects that exogenous IAA has on hypocotyl elongation and the stimulatory effects on lateral root formation. The subtle changes in the mutant and the lack of any alterations to IAA transport or regulation in these tissues suggest that the effect is an indirect one and likely reflects the interaction and cross talk between the two endogenous auxins under these specific growth conditions. This interaction may be at the level of IBA to IAA conversion or at the convergence of IAA and IBA signaling or transport pathways.

This report contains evidence demonstrating that IBA transport is specifically altered in the rib1 mutant while IAA transport levels are unchanged. rib1 is the first mutant in which such a change in IBA transport has been identified. The changes in IBA transport parallel changes in IBA sensitivity in control of hypocotyl elongation and lateral root development. Interestingly, both acropetal IAA transport and lateral root formation in rib1 mutant roots are altered in their response to the IAA transport inhibitor NPA, suggesting a change in the regulation of IAA transport, and cross talk between IAA and IBA in this specific transport pathway. We also have identified one specific growth condition, low light and 1.5% Suc, in which rib1 exhibits altered IAA sensitivity. In all other conditions, we find this mutant to have wild-type IAA response. Additionally, IBA sensitivity differs under different light and Suc concentrations in both wild-type and rib1 hypocotyls and roots, consistent with a complex interaction between the transport and action of IBA and Suc and light signaling.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Isolation and preliminary characterization of rib1 has been described previously (Poupart and Waddell, 2000). rib1 arose in a Ds-mutagenized population but does not carry a Ds element. The rib1 mutation is in the Nossen ecotype of Arabidopsis (Arabidopsis thaliana); Nossen is used as the wild-type control in all assays. All experiments reported here were done with rib1 homozygotes derived from the original isolate. rib1 lines backcrossed to No-0 two times retained the suite of phenotypes described by Poupart and Waddell (2000), including resistance to IBA and 2,4-D in root elongation assays, a wild-type response to IAA in root elongation assays, an elongated hypocotyl, and shorter primary root under conditions of cycling light and increased root slanting. In addition, IAA and IBA transport in the inflorescence has been examined in this line and occurs at similar levels to those reported in Table I for rib1 (data not shown). Although the backcrossed lines show no evidence of segregation of the rib1 phenotypes, given the propensity of Ds to transpose to linked sites, the possibility exists that the rib1 phenotype is the result of more than a single gene mutation. Resolution of this issue awaits cloning of the rib1 mutant gene and complementation of the mutant phenotype with the wild-type RIB1 gene.

Chemicals

3-[5(n)-³H]-IAA (27 and 25 Ci mmol^–1) was purchased from Amersham and 3-[³H(G)]-IBA (25 Ci mmol^–1) was prepared in a custom synthesis under conditions designed to label the indole ring by American Radiolabeled Chemicals. NPA was purchased from Chem Service. All other chemicals were purchased from Sigma, unless stated otherwise.

Growth Conditions for Auxin Transport Assays

Seeds were soaked in distilled water for 30 min and surface sterilized with 95% ethanol for 5 min and 20% bleach with 0.01% Triton X-100 for 5 min. After five washes in sterile distilled water, seeds were germinated and grown on 9-cm petri plates containing sterile control medium containing 0.8% agar (Sigma type M, plant tissue culture), 1x Murashige and Skoog salts, pH 6.0; 1.5% Suc; 1 µg mL^–1 thiamine; 1 µg mL^–1 pyridoxine HCl; and 0.5 µg mL^–1 nicotinic acid. Seeds were grown in vertically oriented petri dishes in continuous 90 µmol m^–2 s^–1 fluorescent light at room temperature (22°C) for root auxin transport experiments. Seedlings used in hypocotyl assays were grown in horizontally oriented petri dishes at room temperature (22°C), but exposed to only 5 µmol m^–2 s^–1 of constant fluorescent light to increase hypocotyl length. Light values indicate the amount of light on the outside of petri dishes.

Plants for inflorescence assays were grown on a 1:1:1 mixture of perlite, vermiculite, and Sunshine mix number 1 (Sun Gro Horticulture). Plants were grown at 24°C under continuous white fluorescent light, and fertilized twice during their growth period with 0.25x Hoagland solution. Light intensity was approximately 90 µmol m^–2 s^–1.

Hypocotyl Basipetal Transport Assay

Hypocotyl transport measurements were made on 5-d-old seedlings grown under low light to elongate the hypocotyl. Seedlings were transferred to control plates and oriented vertically such that the shoot apical meristems were aligned. In this assay, mixtures containing 1% agar, 100 nM ³H-IAA, or ³H-IBA with either 100 µM NPA or dimethyl sulfoxide at the same concentration (1%) were prepared in 3-mL scintillation vials. A narrow stem transfer pipette was carefully inserted into the hardened agar mixture to produce a 1-mm-diameter cylinder of agar. This cylinder containing radioactive auxin mixture was applied such that the agar just touched the tip of the hypocotyl from which the shoot apical meristem and cotyledons were cut. Plates remained vertically oriented in the dark, to avoid auxin degradation by light (Stasinopoulos and Hangarter, 1989). Radioactive auxin transport was measured after 5 h by scintillation counting of a 5-mm segment of the base of the hypocotyl. In Table I, the average and standard error of the mean of 30 individuals from three assays are reported in fmol of auxin transported.

Root Transport Assays

Basic root auxin transport measurements were made on 6- or 7-d-old vertically grown seedlings as by Rashotte et al. (2001). In all root transport assays, seedlings were transferred to control plates and oriented vertically such that the site where radioactive auxin would be applied was aligned. Standard placement of radioactive agar lines was at the root tips for root basipetal transport and just on the root side of the root shoot junction for root acropetal transport. In each of these assays, mixtures containing 1% agar, 100 nM ³H-IAA or ³H-IBA with either 100 µM NPA, TIBA, or 1% dimethyl sulfoxide were prepared in 3-mL scintillation vials and prepared and applied as above. Auxin transport was measured after 5 h for root basipetal transport by first removing the 1 mm of tissue in contact with the agar line, then cutting a 5-mm segment from the site of application along the desired length. In root acropetal transport, measurements were made after 18 h from an application site at the root shoot junction using a 5-mm segment at the root tip. Segments were measured as above. In Table I, the average and standard error of the mean of 59 to 80 individuals from five to seven assays are reported in fmol of auxin transported.

Inflorescence Auxin Transport Assays

Inflorescence transport measurements were conducted on approximately 25-d-old plants as described previously (Okada et al., 1991; Brown et al., 2001). Care was taken to ensure wild-type and rib1 inflorescence stems of matched length were used. In this assay 333 nM ³H-IAA or ³H-IBA were applied to a 20-mm inflorescence segment, and transport into the basal 5 mm of that inflorescence segment was measured after 18 h. Each segment was placed into 2.5 mL of scintillation fluid, and the amount of radioactivity within each sample was determined using a Beckman LS6500 scintillation counter for 2 min. As rib1 segments tend to be more slender than wild-type segments, values were corrected for this weight difference. The corrected values were obtained as follows for wild type: counts for wild type were divided by the average weight of wild-type segments (=12.2 mg), to obtain counts/weight, and this value was multiplied by the average weight of wild-type and rib1 segments (=9.99 mg), to obtain counts per average weight segments. For rib1, the values were calculated in a similar fashion: [(counts for rib1)/(average weight of rib1 segments 7.8 mg)] x (average weight of wild-type and rib1 segments = 9.99 mg). In Table I, the average and standard error of the mean of 14 to 15 individuals from two or three assays are reported in pmol of auxin transported.

Calculation of Amount of Transported Auxin in All Assays

Transported tritiated auxin in each segment was determined by scintillation counting. The dpm transported in each sample was converted to fmol or pmol of auxin using the specific activity of the auxin. For seedling assays, both hypocotyl and root assays, the amount of auxin is reported in fmol. The amount of transport is comparable to previously published results (Rashotte et al., 2000, 2001, 2003), but in those previous papers the units of auxin transported in roots and hypocotyls were mislabeled as pmol and should have been reported as fmol. For inflorescence assays, higher levels of tritiated auxin were used (333 nM versus 100 nM) and higher counts were obtained. Inflorescence transport values are reported in pmol and were correctly reported as pmol in a previous report (Rashotte et al., 2003).

Effects of Light on Hypocotyl Elongation

Hypocotyl length was determined by growing seedlings on horizontally oriented plates containing either Suc-free growth media (GM) solidified with 0.7% Difco agar or GM with 1.5% Suc and 0.8% Noble agar. GM consists of 1x Murashige and Skoog basal salts, 1% Suc, 0.5 g/L MES, 1 mg thiamine, 0.5 mg L^–1 pyridoxin, 0.5 mg L^–1 nicotinic acid, 100 mg L^–1 myo-inositol, with pH adjusted to 5.7 with 1 N KOH (Valvekens et al., 1988). Seedlings were germinated and grown in continuous dark, high white light (approximately 90 µmol m^–2 s^–1), low white light (approximately 5 µmol m^–2 s^–1), red light, or blue light. Light values indicate the amount of light on the outside of petri dishes. White light was provided by cool-white (Sylvania) fluorescent tubes; red light was provided by F40 gold (Sylvania) fluorescent tubes filtered by Rohm and Haas red plexiglas number 2423 (3.18-mm thick, Cadillac Plastic); and blue light was provided by F40 blue (Sylvania) fluorescent tubes filtered by Rohm and Haas blue plexiglas number 2424 (3.18-mm thick, Plastiques Marcon). The far-red light experiments were done in the laboratory of Professor X.-W. Deng at Yale University using F48T12/232/VHO red (Sylvania) fluorescent tubes filtered by far-red plexiglas FrF700 (3.18-mm thick, Westlake Plastics). Low white-light conditions were achieved by placing plates away from the light source, and plates received mostly indirect light in this case. Hypocotyl length was determined on 7-d-old seedlings grown in the absence of Suc or on 5-d-old seedlings grown on 1.5% Suc. Hypocotyls were measured either directly with a ruler or by tracing magnified seedlings using an overhead projector. The tracings were then digitally scanned, and measured using the public domain National Institutes of Health (NIH) Image program (developed at the U.S. NIH and available on the Internet at http://rsb.info.nih.gov/nih-image/). Data presented for hypocotyl elongation in the absence of Suc is from one representative assay; similar results were obtained in one to five other trials. Data presented for hypocotyl elongation with Suc is the average of seven or nine trials, as indicated.

Effects of IAA and IBA on Hypocotyl Elongation

Seeds were surface sterilized by vapor phase sterilization (Clough and Bent, 1998), and then stratified 4 to 7 d in the dark at 4°C before being germinated. IAA and IBA were dissolved in 1 N NaOH and diluted in water to a final stock concentration of 1 mg mL^–1 and filter sterilized. Appropriate amounts of the sterile stocks were added to media after autoclaving to obtain the different concentrations required.

Hypocotyl elongation assays were performed on horizontally oriented GM plates containing 0.8% (w/v) Difco agar. After stratification, seeds plated directly on auxin-containing plates or control media were placed either in dark, high constant white-light conditions (90 µmol m^–2 s^–1) or low-light conditions (5 µmol m^–2 s^–1). Wild-type and rib1 seedlings were plated to each of the two halves of the same plate, to ensure that they were being exposed to exactly the same conditions. Hypocotyl length was determined on 5-d-old seedlings by tracing magnified seedlings (approximately 5-fold) using an overhead projector. A transparent ruler placed beside the hypocotyls was also traced for use as a scale bar. The tracings were then digitally scanned, and measured using the NIH Image program. Similar results were obtained in three separate trials for each light condition. Data from a single representative trial are presented.

Lateral Root Formation

Lateral root formation assays were performed as described by Rashotte et al. (2001) with some modifications. Seedlings were germinated on vertically oriented plates containing GM with 1.5% Suc and 0.8% Noble agar under low white-light conditions (5 µmol m^–2 s^–1). After 4 d, seedlings were transferred to the same media containing the indicated amounts of auxins or transport inhibitor. The number of lateral roots on the whole primary root was counted under a dissecting microscope after six more days of growth; all lateral roots that had emerged from the primary root were counted. Each value is the average for 17 or 18 individual seedlings. Similar results were obtained in three separate trials. Figure 3 shows combined data from two trials for IAA and IBA. Figure 4 shows combined data from two (NPA) or three (TIBA) trials.

For all hypocotyl elongation and lateral root formation assays, wild-type and mutant seedlings were placed on two halves of the same plate to ensure exposure to identical conditions.

Effect of NPA on Seedling Growth

Sterile and stratified wild-type and rib1 seeds were plated on a horizontal line across two halves of a petri dish containing GM with 1% Suc and 0.1 µM NPA, and grown under high, white light for 14 d. A picture was taken on a Leica stereomicroscope at a magnification of 3.15x.

Statistics

The data were analyzed by two-tailed Student's t tests for equal variance when comparing wild type and rib1 using Microsoft Excel. A three-way factorial ANOVA was performed using Statistica v5.5 (Statsoft). A Neuman-Keuls post-hoc comparison of means was performed to determine for each auxin, whether there were significant differences between wild type and rib1 at each concentration of IAA and IBA that was used.

	ACKNOWLEDGMENTS

 
We wish to thank Dr. X.-W. Deng (Yale University) for use of his facilities for far-red experiments and R. Fry (Deng Laboratory, Yale University) for help with performing these experiments; Dr. M.L. Tierney (University of Vermont) for help with initial hypocotyl elongation experiments and use of her facilities; Marianne Marcoux (Waddell laboratory) for technical help; Dr. Dave Anderson (Wake Forest University) for help with statistical analyses; and Shari Brady (Muday laboratory) for help with Figure 3.

Received July 5, 2005; returned for revision August 23, 2005; accepted August 24, 2005.

	FOOTNOTES

 
¹ This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (to C.S.W.), the J.W. McConnell McGill University Majors Fellowship (to J.P.), and grants from the National Aeronautics and Space Administration Specialized Center for Research and Training (North Carolina State University; to A.M.R. and G.K.M.) and the National Aeronautics and Space Administration (grant no. NAG2–1507 to G.K.M).

² Present address: Department of Biology, University of North Carolina, Chapel Hill, NC 27599.

³ Present address: Department of Plant Cellular and Molecular Biology, Ohio State University, Columbus, OH 43210.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Gloria K. Muday (muday{at}wfu.edu).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.067967.

^* Corresponding author; e-mail muday{at}wfu.edu; fax 336–758–6008.

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