Root-derived auxin contributes to the phosphorus-deficiency-induced cluster-root formation in white lupin (Lupinus albus)

Transcription

1 Physiologia Plantarum 148: Copyright Physiologia Plantarum 2012, ISSN Root-derived auxin contributes to the phosphorus-deficiency-induced cluster-root formation in white lupin (Lupinus albus) Zhi Bin Meng a, Xue Di You a, Dong Suo a,d, Yun Long Chen a, Caixian Tang b,jianliyang a and Shao Jian Zheng a,c, a Key Laboratory of Conservation Biology for Endangered Wildlife of the Ministry of Education, College of Life Sciences, Zhejiang University, Hangzhou , China b School of Life Sciences, La Trobe University, Melbourne, Bundoora, VIC 3086, Australia c State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou , China d Department of Biology, University of Virginia, Charlottesville, VA , USA Correspondence *Corresponding author, sjzheng@zju.edu.cn Received 13 August 2012; revised 1 October 2012 doi: /j x Formation of cluster roots is a typical morphological response to phosphorus (P) deficiency in white lupin (Lupinus albus), but its physiological and molecular mechanisms are still unclear. We investigated the role of auxin in the initiation of cluster roots by distinguishing the sources of auxin, measuring the longitudinal distribution patterns of free indole-3-acetic acid (IAA) along the root and the related gene expressions responsible for polar auxin transport (PAT) in different developmental stages of cluster roots. We found that removal of shoot apex or primary root apex and application of auxin-influx or -efflux transport inhibitors, 3-chloro-4-hydroxyphenylacetic acid, N-1- naphthylphthalamic acid and 2,3,5-triiodobenzoic acid, to the stem did not affect the number of cluster roots and the free-iaa concentration in the roots of P-deficient plants, but when these inhibitors were applied directly to the growth media, the cluster-root formation was greatly suppressed, suggesting the fundamental role of root-derived IAA in cluster-root formation. The concentration of free IAA in the roots was higher in P-deficient plants than in P-adequate ones, and the highest in the lateral-root apex and the lowest in the mature cluster roots. Meanwhile the expression patterns of LaAUX1, LaPIN1 and LaPIN3 transcripts related to PAT was consistent with concentrations of free IAA along the lateral root, indicating the contribution of IAA redistribution in the cluster-root development. We proposed that root-derived IAA plays a direct and important role in the P-deficiency-induced formation of cluster roots. Introduction Phosphorus (P) is one of the major limiting nutrients for plant growth. Many plants develop various strategies to enhance the uptake of phosphate (Pi) from soil by altering root architecture and increasing Pi mobility by releasing organic acid anions or phosphatases (Neumann and Römheld 1999, Lambers et al. 2006). The formation of cluster roots is an effective adaptation mechanism. Cluster roots are a root structure of densely Abbreviations CHPAA, 3-chloro-4-hydroxyphenylacetic acid; DMSO, dimethyl sulfoxide; IAA, indole-3-acetic acid; NAA, 1-naphthaleneacetic acid; NPA, N-1-naphthylphthalamic acid; PAT, polar auxin transport; Pi, phosphate; TIBA, 2,3,5-triiodobenzoic acid. Physiol. Plant. 148,

2 formed rootlets with determinate length along certain area of a lateral root in most Proteaceae species and some other species (Lamont 2003). They can be characterized into four stages: initiation, determinate growth, exudative burst and associated physiology (Skene 2003). Basically, morphological changes are followed by physiological changes. The initiation of the cluster roots is the very first and most important step. A cluster primordium initiates from the pericycle and opposite protoxylem poles, and breaks through the root epidermis into determinate length (Skene 2000b). The primordia form the clustered initiation zone in a longitudinal pattern along the lateral root. It is hypothesized that there would be pulses of signals or altered local sensitivities in a longitudinal pattern (Watt and Evans 1999, Skene 2000b), but direct evidence is still lacking. Plant response to P deficiency is a sophisticated system including Pi sensing and regulatory mechanisms that integrate signals with long- and short-distance, external and internal, general and local transport (Lin et al. 2008, Liu et al. 2009, Yang and Finnegan 2010). In white lupin (Lupinus albus), shoot-derived long-distance signals are expected to be upstream during cluster-root initiation and physiological changes. For instance, shoot-derived signals, especially shoot P status, are demonstrated to be the key regulators generally controlling the development of cluster roots because the formation is suppressed in both P-deficient ( P) and P-sufficient (+P) root halves in a root-split experiment (Shane et al. 2003). In addition, exogenous sucrose is transported from shoot to root to induce formation of cluster roots and expression of genes encoding phosphate transporter and acid phosphatases (Liu et al. 2005, Zhou et al. 2008), so sucrose has been suggested to be one of the systemic signals in long-distance signaling under P deficiency (Yang and Finnegan 2010). However, it is still an open question whether there is any other systemic signal involved. Auxin, a phytohormone involved in the root architecture establishment, is a promising player in the shoot-to-root signaling cascades (Robert and Friml 2009). Application of 1-naphthaleneacetic acid (NAA), an auxin analogue, either by foliar feeding or root feeding, increases the number of cluster roots under +P conditions in white lupin (Gilbert et al. 2000, Skene and James 2000). However, the citrate exudation is not detected in the auxin-analogues-induced cluster roots (Hocking and Jeffery 2004), and NAA can only induce cluster roots in Lupinus species (Skene and James 2000). In Arabidopsis, early emergence of lateral roots depends on shoot-derived auxin (Bhalerao et al. 2002). However, the source of auxin is likely dependent on the growth stage. For example, auxin derived from the shoot is the major source of auxin in the root within the first 10 days after germination in Arabidopsis (Bhalerao et al. 2002). However, in plants older than 10 days, Indole-3-acetic acid (IAA) is synthesized in all organs including the roots (Ljung et al. 2001). IAA is transported from source to sink tissues via polar auxin transport (PAT), which requires auxin influx and efflux in cells mediated by influx (AUXs) and efflux carriers (PINs) (Marchant et al. 2002, Benkov et al. 2003, Blilou et al. 2005). In PAT-defective mutants, most effects of P starvation are altered by the localized modifications of auxin concentration, indicating that the auxin redistribution is important for the performance of P-deficiency-induced responses (Nacry et al. 2005). In white lupin, there are reports on the mechanism of PAT in etiolated hypocotyls (Inés et al. 2004, 2007), but none on cluster roots. It is also known that there are auxin carriers responsible for auxin influx LaAUX1, and efflux LaPIN1 and LaPIN3 in stele (Oliveros-Valenzuela et al. 2007). However, little is known about the relative importance of different sources of auxin and its PAT in cluster-root initiation. Here, white lupin was used as a test-plant species to examine the source of auxin and its PAT during the formation of cluster roots. We hypothesized that if the auxin polar transport from the shoot to root plays a determinative role in cluster-root formation, the cluster-root formation should be greatly affected by blocking the transport pathway and decapitation of the shoot. Moreover, if there is no PAT along the lateral roots with different stages of cluster-root development, free IAA and expression of genes involved in PAT should be evenly distributed. Materials and methods Plant materials and growth condition Seeds of white lupin (L. albus cv Kiev Mutant) were sterilized using 75% ethanol for 1 min. The seeds were thoroughly rinsed with deionized water, and germinated in a solution containing 1 mm CaCl 2 and 5 μm H 3 BO 3 for 3 days at 25 C in the dark. Two uniform seedlings were transformed to each 1-l container containing continuously aerated nutrient solution of following composition (in μm): 600 K 2 SO 4, 200 MgSO 4, 600 CaCl 2, 100 NH 4 NO 3, 700 Ca(NO 3 ) 2 10 FeNaEDTA, 0.2 CoSO 4,0.03Na 2 MoO 4,5H 3 BO 3,0.75ZnSO 4,0.75 MnSO 4 and 0.2 CuSO 4. The solution ph was adjusted daily to 6.0 with 1 M KOH or HCl. The +P plants were supplied with 50 μm KH 2 PO 4 in the nutrient solution, while the P plants were deprived of P. The nutrient solution was renewed every other day. Plants were grown in a controlled environment with a 14 h/20 ± 1 C day and 10 h/18 ± 1 C night cycle, a light intensity of μmol m 2 s 1 and a relative humidity of 482 Physiol. Plant. 148, 2013

3 65 70%. Plants were harvested after 20 days of cultivation. Cluster roots are defined as parts of secondary lateral roots bearing brush-like rootlets with a density of >10 rootlets cm 1 (Johnson et al. 1996). Decapitation of shoot and primary root After 10-days-cultivation at 0 or 50 μm P, plant shoots were excised (the apical shoot meristem was excised and the first pair of true leaves were retained) or primary roots excised (the apical root was excised and 13 cm from the root base was retained). The wound was trimmed every other day, after the decapitation of shoots. Lanolin paste treatment of auxin analogue and auxin-transport inhibitors Stock solutions (0.1 M) of auxin analogue α-naphthalene acetic acid (NAA) or auxin-transport inhibitors: N-1- naphthylphthalamic acid (NPA), 2,3,5-triiodobenzoic acid (TIBA) and 3-chloro-4-hydroxyphenylacetic acid (CHPAA) were prepared in dimethyl sulfoxide (DMSO) and 200 μl of each stock solution was added to a pre-warmed (50 C) lanolin paste with 2.5% (v/v) paraffin and 0.01% (v/v) Triton X-100 (500 μl) (Reinhardt et al. 2000). IAA lanolin paste was prepared by 0.4 ml IAA (1 M in stock solution) mixed with 1 ml lanolin paste. Lanolin paste was applied at the same time of decapitation. The paste was applied horizontally as a narrow ring in the cm region below the cotyledons with a fine rod. Control plants were applied with lanolin paste without NAA and auxin-transport inhibitors. The fresh lanolin paste was added or replenished every other day. Nutrient solution treatment of auxin analogue and auxin-transport inhibitors After cultivation in 0 or 50 μm P for 14 days, white lupin plants were subjected to nutrient solution treatment of auxin analogue NAA or auxin-transport inhibitors NPA, TIBA and CHPAA. The 1 mm stock solution of NAA and 10 mm stock solutions of NPA, TIBA and CHPAA, all in DMSO (100 μl), were added into the nutrient solution and 100 μl DMSO was added into the control treatment. The ph of nutrient solution was adjusted to 6.0 with 1 M HCl or 1 M KOH. The solutions were renewed every other day. IAA measurement Various segments of cluster roots were sampled as indicated in Fig. 5A. Leaf and root samples were excised and weighed. Each sample was extracted for IAA after addition of 250 pg of [ 13 C 6 ] IAA internal standard per 10 mg FW sample. The concentration of free (unbound) IAA in extracts was determined by gas chromatography-selected reaction monitoring mass spectrometry as described in Ljung et al. (2005). The concentration of IAA was calculated based on the addition of [ 13 C 6 ] IAA internal standard. Expression analysis of auxin related genes Various segments of cluster roots were sampled as indicated in Fig. 5A. Lateral roots bearing cluster roots were chosen. The apex of 1 cm was excised as the lateralroot apex (Ap). The region of clustered primordium was excised as the juvenile section (J). The region of rootlets next to the juvenile section, where the rootlets did not reach the maximum length, was excised as the immature section (I). The region next to the immature section, where the rootlets had the maximum length, was excised as the mature section (M). The segments were excised and frozen in liquid nitrogen immediately, and were stored at 80 C. Root samples were then ground with liquid nitrogen and total RNA in plant tissues were isolated by Trizol (Invitrogen Life Technologies, Burlington, Canada). cdna was synthesized from total RNA by PrimeScript RT reagent kit (TaKaRa, Dalian, China). In quantitative PCR, SYBR Premix Ex Taq kit (TaKaRa, Dalian, China) was used following manufacturer instructions. Light Cycler 480 (Roche) was used for the PCR and the detection of the fluorescent signal. Gene-specific primers were designed from cdna clones: LaAUX1 (AM235387); LaPIN1 (AM235388); LaPIN3 (AM407405) (Oliveros-Valenzuela et al. 2007). The primer sequences are LaAUX1, 5 -CCTTTGCTTCTGCACCTGCTA-3 and 5 -ACTTGCCCATCCTCCCAATC-3 ; LaPIN1, 5 -AGG TGGAATGTTGAAATG-3 and 5 -GCAAAG ACAAATGGGACA-3 ; LaPIN3, 5 -AGTTTCCGAC CAAAGAAGTAGAGCA-3 and 5 -AGTGACCAAATGA CACCAATGAGGC-3 ; LaUbiquitin, 5 -ATGTCA AAGCCAAGATCCAAG-3 and 5 -GAACCTTCCCAGAA TCATCAA-3. The gene-specific primers generate 143 bp products for AUX1, 284 bp products for PIN1 and 253 bp products for PIN3. Three technical replicates were performed using three RNA preparations (biological replicates) from different plants. White lupin ubiquitin gene was chosen as the endogenous control to account for variability of the initial concentration and quality of the total RNA. Statistical analysis Data are shown as means ± standard deviation (SD) of four to eight replications. The Tukey s compromise test at 5% probability was used to test the differences. Physiol. Plant. 148,

4 Results Cluster roots were formed under both P and +P conditions from 14 days of cultivation. The number of cluster roots was higher in the P plants than +P ones during the experimental period (Fig. 1A). The concentration of free IAA in the roots of P plants showed an obvious peak on day 18 (Fig. 1B), which is corresponding to the maximum initiation rate of cluster roots from day 18 to day 20 (Fig. 1A). In the whole plants, the concentration of free IAA was highest in the lateral roots grown in the P treatment while those in the leaves and primary roots were similar between the +P and P treatments (Fig. 1B,C). To verify the effect of IAA on cluster-root formation, we applied an auxin analogue, NAA, to the growth medium and found that the number of cluster roots was greatly increased in both +P and P plants in a dose-dependent manner (Fig. 1D). There are two main sources of de novo IAA in plants, the shoot and the primary root. To investigate their relative contribution to the increased concentration of free IAA in the P lateral roots, we removed apical meristems of shoot or primary roots 10 days after P treatment (Fig. 2C F) when the first pair of true leaves emerged. On day 20, the number of cluster roots and the endogenous IAA concentration were measured. To our surprise, the excision of the shoot or the root apex produced no negative effects in the corresponding P treatments (Fig. 2). To verify the above results further, the effects of various PAT inhibitors were tested. Applying NAA to the shoot and root junction (the 1-cm region below the cotyledon), cluster-root number was increased in both +P and P plants (Fig. 2). When NPA, an auxin-efflux inhibitor, was applied on a lanolin paste (1 cm below the treated point of IAA-lanolin) at the same time, the cluster-root number was suppressed to the same level of control treatments (Fig. 2). However, the application of NPA alone to the shoot and root junction did not affect the number of cluster roots irrespective of whether the apices were intact or removed (Fig. 2A), or the concentration of free IAA in P roots except in the plants of which both shoot and root apices were removed, but it decreased the concentration of free IAA in +P roots (Fig. 3). The lanolin paste without NPA did not impact the formation of cluster roots or IAA concentration (data not shown). This set of experiments indicates that IAA derived from the shoot was not a source of the elevated free-iaa concentration in the P roots. Because both auxin-influx and -efflux transporters are involved in PAT, we further tested the effects of different auxin-influx and -efflux inhibitors on the formation of cluster roots. CHPAA is an auxin-influx A B Fig. 1. Effect of P deficiency and exogenous NAA on the number of cluster roots (CR) and free-iaa concentration in white lupin. (A) After emergence, white lupin plants were treated with 0 or 50 μm Pfor 24 days. (B) After emergence, concentrations of endogenous free IAA in the whole root system of +P and P plants were determined. (C) White lupin plants were cultivated for 20 days in +P or P nutrient solutions and free-iaa concentrations in the leaf, primary root and lateral root were determined. (D) White lupin plants were cultivated for 14 days in P-sufficient (+P) and P-deficient ( P) conditions and then treated with NAA to a final concentration of nM in nutrient solution for 6 days. The values are means ± SD (n = 8) for (A) and (C). The values are means ± SD (n = 4) for (B) and (D). Means with different letters are significantly different (P < 0.05). inhibitor while TIBA is an auxin-efflux inhibitor (Parry et al. 2001, Blakeslee et al. 2005, Kaneyasu et al. 2007). The application of CHPAA, TIBA and NPA to the shoot root junction did not significantly change the number of cluster roots either in the intact or rootdecapitated plants (Fig. 4A). All the inhibitors at the concentration of 28.6 mm did not alter the appearance of the shoots (see Appendix S1), but a higher concentration greatly inhibited leaf phototropism (data not shown). The application of lanolin paste or DMSO (used to dissolve the inhibitors) to the shoot-to-root junction without these inhibitors had no effect on cluster-root formation (data not shown). However, the addition of these inhibitors directly into the nutrient solution greatly inhibited the formation of cluster roots in the intact P plants (Fig. 4B), C D 484 Physiol. Plant. 148, 2013

5 A B Fig. 2. Impact of NAA and NPA treatment in lanolin paste and removal of shoot or root apex on the number of cluster roots in 20-days-old P-deficient white lupin. After germination, uniform plants were grown in 0 (A) or 50 μm P (B) for 10 days, and removal of shoot or root apex was then carried out. Plants were treated with NAA, NPA or diluted water (CK) in the lanolin paste for 10 days. (C F) Pattern of intact white lupin and the plants with shoot or root apex removed. (C) Intact: control plants; (D) DS: removed shoot apex (with only the first pair of true leaves remaining); (E) DR: removed the primary root apex (with 13-cm primary root remaining); (F) DSR: removed both the shoot apex and the primary root apex. The white arrow indicates the position of decapitation. The white bar represents 3 cm. The values are means ± SD (n = 4) and an asterisk indicates a significant difference between a treatment and the control at P < 0.05 level. strongly suggesting that the PAT within the root is related to the formation of cluster roots. Finally, the concentration of free IAA and the expression of PAT genes along the lateral roots were measured. Root segments were sampled as indicated in Fig. 5A. Concentrations of free IAA showed a longitudinal gradient with the highest concentration in the parent lateral-root apex and the lowest in the mature cluster roots (Fig. 5B). A high-expression level of LaAUX1, encoding auxin-influx carrier, was detected in the mature cluster roots and of LaPIN1 and LaPIN3, encoding auxin-efflux carriers, in both the mature and apical region (Fig. 5C). From juvenile cluster roots to mature cluster roots, IAA concentration decreased about 50% (Fig. 5B) while the accumulation of transcripts increased about threefold (Fig. 5C). Discussion Formation of cluster roots integrates signaling cascades including internal Pi status, phytohormones, sucrose Fig. 3. Concentrations of free endogenous IAA in the root tissue of 20-days-old white lupin treated with or without NPA in lanolin paste with 0 ( P) or 50μM P (+P). After germination, uniform plants were cultured in nutrient solutions of 0 or 50 μm P for 10 days, and the shoot or root apex was excised. Plants were shoot- or primary rootdecapitated or were treated with auxin-transport inhibitor (NPA) in the lanolin paste for 10 days. Free-IAA concentration was determined in the root tissue. Intact: control plants; DS: removed shoot apex (with only the first pair of true leaves remaining); DSR: removed both the shoot apex and the primary root apex. The values are means ± SD (n = 4). Means with different letters are significantly different (P < 0.05). and nitric oxide in white lupin (Skene 2000a, Wang et al. 2010). Here we found that the concentration of endogenous free IAA increased in P roots before the initiation of the rootlets (Fig. 1A,B), and applying NAA directly to the nutrient solution not only increased the number of cluster roots in P plants but also greatly promoted the formation of cluster roots in P-sufficient plants. As the PAT process requires both auxin-influx and -efflux, we further tested both the auxin-influx inhibitor CHPAA and -efflux inhibitors NPA and TIBA, and found that the formation of cluster roots was suppressed by both kinds of inhibitors in the P plants (Fig. 4) and plants with shoot-fed IAA (see Appendix S2). All these results strongly suggest the involvement of auxin and PAT in the roots in the formation of cluster roots. The validity of PAT inhibitors and decapitation in modifying auxin distribution As auxin in the roots is originated from both shoots and roots, to investigate the relative contribution of shootderived auxin, this study used the PAT inhibitors to block the PAT from the shoot to the root. The lanolinpaste treatment system is frequently employed (Reinhardt et al. 2000, Tanaka et al. 2006). We confirmed the effectiveness of this system in our experimental conditions from the following evidence. First, shoot-fed IAA enhanced the formation of cluster roots, but when Physiol. Plant. 148,

6 A B A B Fig. 4. Number of cluster roots of white lupin plants treated with auxin-transport inhibitors under P-deficient ( P) conditions. (A) Auxintransport inhibitor treatments in the lanolin paste to the shoot and root junction. Plants were cultivated for 10 days in the P solution (intact) and primary roots were excised (DR), and then treated with auxin-transport inhibitors: TIBA, NPA, CHPAA or blank lanolin (CK) in the lanolin paste for ten more days. (B) Auxin-transport inhibitor treatments in nutrient solution. Plants were cultivated for 14 days under P condition (intact) and then treated with auxin-transport inhibitors: TIBA, NPA or CHPAA to a final concentration of 1 μm or diluted water (CK) in nutrient solution for six more days. The values are means ± SD (n = 8)andanasterisk indicates a significant difference between a treatment and the control at P < 0.05 level. C PAT inhibitors were applied to stem at the same time, such an effect was obviously diminished (Fig. 2). Second, Free IAA transported from shoot to root is dependent on PAT process. During the vegetative growth, there are mainly two pathways involved in the IAA movement: (1) conjugated-iaa transported via phloem; (2) free- IAA transported via cell-to-cell (Robert and Friml 2009). Although the transition between bound IAA and free IAA is important in maintaining the IAA balance, the conjugated IAA is considered not to play a direct role in meristem development. Therefore, we only measured the free IAA in plants to see its relationship with clusterroot formation. NPA in lanolin effectively reduced the free IAA level in +P roots (Fig. 3). Similar results have also been reported in Arabidopsis, pea and etiolated lupin (Bhalerao et al. 2002, Inés et al. 2004, Tanaka et al. 2006). Third, we chose a maximum dose that did not exhibit visible inhibitory effects on plant growth (see Appendix S1). Besides, the biosynthesis of auxin in the shoot was further reduced after the shoot apex was removed, which has been proven to be effective in Arabidopsis (Casimiro et al. 2001). When we removed the shoot apex of white lupin, it also decreased the IAA level significantly in +P plants (Fig. 3). Therefore, in this study, both PAT inhibitors and decapitation treatment were effective to block the shoot-derived auxin. Fig. 5. The concentration of free endogenous IAA and expression of PAT genes at different development stages of cluster roots. Plants were grown in the P solution for 20 days, and the cluster roots were then excised to show developmental zones with white arrow pointing (A): Ap, apical; J, juvenile; I, immature; M, mature. White bar represents 1 cm. (B) Free-IAA concentration was determined by GC MS. The values are means ± SD (n = 4). Means with different letters are significantly different (P < 0.05). (C) Expression patterns of LaAUX1, LaPIN1 and LaPIN3 transcripts were measured by real-time quantitative PCR. Gene expression was normalized to the LaUbiquitin expression levels of each sample and the levels of expression are relative to the values of the normal root sample, which were given an arbitrary value of 1. The values are means ± SD (n = 3). The source of auxin contributing to the formation of cluster roots Since free-iaa concentration remained almost the same in roots when we removed the shoot apex of the P plants (Fig. 3), there might have been other sources that contributed to the high free IAA in P roots. To further confirm this result, we applied various auxin-influx and -efflux inhibitors to the stem just above the root system, it was clearly shown that inhibition of PAT from shoot to root did not affect the free-iaa concentration nor the number of cluster roots in the P plants (Figs 3 and 4). Therefore, it is clear that transport of IAA from shoot to root is not the major contributor of the free IAA in P 486 Physiol. Plant. 148, 2013

7 roots. In other words, the major sources of IAA in the P roots should lie in the root system itself. There are three major sources of auxin in root: apex of primary roots, apex of emerged lateral roots and the primordium zone, especially when the plants are subjected to P deficiency (Ljung et al. 2005, Nacry et al. 2005). Removal of primary root tips may disturb the distribution of rootderived auxin due to the loss of apical dominance. However, after blocking the PAT by both decapitation and PAT inhibitors, further decapitation of primary roots had no impact on the number of cluster roots or the free- IAA concentration (Figs 2 and 3). Therefore, the elevated free-iaa concentration was likely to be originated from the apex of lateral roots and their primordium zones. Redistribution of IAA and PAT within the roots play fundamental role in the formation of cluster roots Upon P deficiency, the PAT process leads to redistribution of IAA, alters the concentration of free IAA in the root tissue, increases localized IAA accumulation, and results in changes in root morphology (Nacry et al. 2005). When we applied PAT inhibitors to the nutrient solution where the roots were growing, the formation of cluster roots was significantly suppressed (Fig. 4). We showed that free IAA was not evenly distributed along the lateral roots bearing the cluster roots in the P plants, with the highest concentration in the root apex and reduced as the cluster roots became more mature (Fig. 5B). In Arabidopsis, besides IAA redistribution, altered IAA sensitivity also relates to the root morphological changes under P-deficient conditions (López-Bucio et al. 2002, Perez-Torres et al. 2008). In this study, P supply did not significantly change the sensitivity to IAA in white lupin, because the number of cluster roots showed a very similar responses to the exogenous NAA concentration and a significant increase occurred at the same NAA concentration (10 nm) for both P and +P plants (Fig. 1D). In the lateral root of Arabidopsis, the AUX1 and PINs facilitate auxin transport and contributed to auxin gradients. Furthermore, a high-expression level of AUX1 and PIN1 leads to the active transport of free IAA and a low-expression level leads to IAA accumulation (Marchant et al. 2002, Benkov et al. 2003, Blilou et al. 2005). To further explore the distribution pattern of free IAA along cluster roots in white lupin, we measured the expression level of the auxin-influx and -efflux transporter genes. LaAUX1, LaPIN1 and LaPIN3 displayed the same accumulation pattern, i.e. higher in the root tip and mature section and lower in the other parts (Fig. 5C). As one of the IAA sources in roots, root tips generate free IAA and transport it to basal roots in Arabidopsis (Nacry et al. 2005). The high-expression level of PAT genes implies a very active basipetal transport of IAA in the apical roots (Fig. 5). In the mature section, where cluster rootlets stopped emerging, the high level of expression indicates an enhanced transport of IAA from both the immature section and the rootlet tips in mature section to the basal root. Besides, the mature section is not the IAA source, so the active transport results in the lowest IAA concentration in this section. In the juvenile and immature section, the relatively low expression of LaAUX1, LaPIN1 and LaPIN3 favors the accumulation of IAA in these sections and may facilitate the initiation and development of cluster roots. In this study, we also found that the distribution pattern of IAA and expression pattern of PAT genes along the lateral roots with cluster roots were similar under P and +P conditions (Data not shown). All these facts indicate a close relationship between IAA distribution and the formation of cluster roots. In conclusion, by blocking the sources of auxin in the shoot through the removal of shoot apex and application of PAT inhibitors to the shoot root junction or the growth medium, we demonstrated that the root-derived auxin and its redistribution along the roots contribute to the formation of cluster roots in white lupin under P deficiency. Acknowledgements This work was supported by Chang Jiang Scholars Program, Program for Innovation Research Team in Universities (IRT1185) and the Fundamental Research Funds for the Central Universities in China. We thank Dominic Lauricella (La Trobe University) for language editing. References Benkov E, Michniewicz M, Sauer M, Teichmann T, Seifertov D, Jurgens G, Friml J (2003) Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115: Bhalerao RP, Eklöf J, Ljung K, Marchant A, Bennett M, Sandberg G (2002) Shoot-derived auxin is essential for early lateral root emergence in Arabidopsis seedlings. Plant J 29: Blakeslee JJ, Peer WA, Murphy AS (2005) Auxin transport. Curr Opin Plant Biol 8: Blilou I, Xu J, Wildwater M, Willemsen V, Paponov I, Friml J, Heidstra R, Aida M, Palme K, Scheres B (2005) The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature 433: Casimiro I, Marchant A, Bhalerao RP, Beeckman T, Dhooge S, Swarup R, Graham N, Inzé D, Sandberg G, Casero PJ, Bennett M (2001) Auxin transport promotes Physiol. Plant. 148,

8 Arabidopsis lateral root initiation. Plant Cell 13: Gilbert GA, Knight JD, Vance CP, Allan DL (2000) Proteoid root development of phosphorus deficient lupin is mimicked by auxin and phosphonate. Ann Bot (Lond) 85: Hocking PJ, Jeffery S (2004) Cluster-root production and organic anion exudation in a group of old-world lupins and a new-world lupin. Plant Soil 258: Inés LNJ, Manuel A, José S-B (2004) Role of basipetal auxin transport and lateral auxin movement in rooting and growth of etiolated lupin hypocotyls. Physiol Plant 121: Inés LNJ, Manuel A, José S-B (2007) Variation in indole-3-acetic acid transport and its relationship with growth in etiolated lupin hypocotyls. J Plant Physiol 164: Johnson JF, Vance CP, Allan DL (1996) Phosphorus deficiency in Lupinus albus (altered lateral root development and enhanced expression of phosphoenolpyruvate carboxylase). Plant Physiol 112: Kaneyasu T, Kobayashi A, Nakayama M, Fujii N, Takahashi H, Miyazawa Y (2007) Auxin response, but not its polar transport, plays a role in hydrotropism of Arabidopsis roots. J Exp Bot 58: Lambers H, Shane MW, Cramer MD, Pearse SJ, Veneklaas EJ (2006) Root structure and functioning for efficient acquisition of phosphorus: matching morphological and physiological traits. Ann Bot (Lond) 98: Lamont BB (2003) Structure, ecology and physiology of root clusters. Plant Soil 248: 1 19 Lin S-I, Chiang S-F, Lin W-Y, Chen J-W, Tseng C-Y, Wu P-C, Chiou T-J (2008) Regulatory network of microrna399 and PHO2 by systemic signaling. Plant Physiol 147: Liu J, Samac DA, Bucciarelli B, Allan DL, Vance CP (2005) Signaling of phosphorus deficiency-induced gene expression in white lupin requires sugar and phloem transport. Plant J 41: Liu T-Y, Chang C-Y, Chiou T-J (2009) The long-distance signaling of mineral macronutrients. Curr Opin Plant Biol 12: Ljung K, Bhalerao RP, Sandberg G (2001) Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth. Plant J 28: Ljung K, Hull AK, Celenza J, Yamada M, Estelle M, Normanly J, Sandberg G (2005) Sites and regulation of auxin biosynthesis in Arabidopsis roots. Plant Cell 17: López-Bucio J, Hernández-Abreu E, Sánchez-Calderón L, Nieto-Jacobo MF, Simpson J, Herrera-Estrella L (2002) Phosphate availability alters architecture and causes changes in hormone sensitivity in the Arabidopsis root system. Plant Physiol 129: Marchant A, Bhalerao R, Casimiro I, Eklof J, Casero PJ, Bennett M, Sandberg G (2002) AUX1 promotes lateral root formation by facilitating indole-3-acetic acid distribution between sink and source tissues in the Arabidopsis seedling. Plant Cell 14: Nacry P, Canivenc G, Muller B, Azmi A, Van Onckelen H, Rossignol M, Doumas P (2005) A role for auxin redistribution in the responses of the root system architecture to phosphate starvation in Arabidopsis. Plant Physiol 138: Neumann G, Römheld V (1999) Root excretion of carboxylic acids and protons in phosphorus-deficient plants. Plant Soil 211: Oliveros-Valenzuela M, Reyes D, Sánchez-Bravo J, Acosta M, Nicolás C (2007) The expression of genes coding for auxin carriers in different tissues and along the organ can explain variations in auxin transport and the growth pattern in etiolated lupin hypocotyls. Planta 227: Parry G, Delbarre A, Marchant A, Swarup R, Napier R, Perrot-Rechenmann C, Bennett MJ (2001) Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1. Plant J 25: Perez-Torres C-A, Lopez-Bucio J, Cruz-Ramirez A, Ibarra-Laclette E, Dharmasiri S, Estelle M, Herrera-Estrella L (2008) Phosphate availability alters lateral root development in Arabidopsis by modulating auxin sensitivity via a mechanism involving the TIR1 auxin receptor. Plant Cell 20: Reinhardt D, Mandel T, Kuhlemeier C (2000) Auxin regulates the initiation and radial position of plant lateral organs. Plant Cell 12: Robert HS, Friml J (2009) Auxin and other signals on the move in plants. Nat Chem Biol 5: Shane MW, De Vos M, De Roock S, Lambers H (2003) Shoot P status regulates cluster-root growth and citrate exudation in Lupinus albus grown with a divided root system. Plant Cell Environ 26: Skene KR (2000a) Cluster roots: their physiology, ecology and developmental biology. Ann Bot (Lond) 85: 899 Skene KR (2000b) Pattern formation in cluster roots: some developmental and evolutionary considerations. Ann Bot (Lond) 85: Skene KR (2003) The evolution of physiology and development in the cluster root: teaching an old dog new tricks. Plant Soil 248: Skene KR, James WM (2000) A comparison of the effects of auxin on cluster root initiation and development in Grevillea robusta Cunn. ex R. Br. (Proteaceae) and in the genus Lupinus (Leguminosae). Plant Soil 219: Tanaka M, Takei K, Kojima M, Sakakibara H, Mori H (2006) Auxin controls local cytokinin biosynthesis in the nodal stem in apical dominance. Plant J 45: Physiol. Plant. 148, 2013

9 Wang BL, Tang XY, Cheng LY, Zhang AZ, Zhang WH, Zhang FS, Liu JQ, Cao Y, Allan DL, Vance CP, Shen JB (2010) Nitric oxide is involved in phosphorus deficiency-induced cluster-root development and citrate exudation in white lupin. New Phytol 187: Watt M, Evans JR (1999) Linking development and determinacy with organic acid efflux from proteoid roots of white lupin grown with low phosphorus and ambient or elevated atmospheric CO 2 concentration. Plant Physiol 120: Yang XJ, Finnegan PM (2010) Regulation of phosphate starvation responses in higher plants. Ann Bot (Lond) 105: Zhou K, Yamagishi M, Osaki M, Masuda K (2008) Sugar signalling mediates cluster root formation and phosphorus starvation-induced gene expression in white lupin. J Exp Bot 59: Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. Effect of auxin-transport inhibitor treatments in lanolin paste in P-deficient white lupin. Appendix S2. Effects of IAA and auxin-transport inhibitors in lanolin paste treatments. Edited by J. Schjørring Physiol. Plant. 148,