Phosphatidylinositol phosphate 5-kinase genes respond to phosphate deficiency for root hair elongation in Arabidopsis thaliana.

Accepted Article

Received Date : 09-Feb-2014

Revised Date

: 22-Nov-2014

Accepted Date : 25-Nov-2014

Article type

: Original Article

Phosphatidylinositol Phosphate 5-Kinase Genes Respond to Phosphate Deficiency for Root Hair Elongation in Arabidopsis thaliana

Yukika Wada1, Hiroaki Kusano2, Tomohiko Tsuge1, and Takashi Aoyama1*

1 Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

2 Department of Technology and Science, Tokyo University of Science, Shinjuku, Katsushika-ku, Tokyo, 278-8510, Japan

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/tpj.12741 This article is protected by copyright. All rights reserved.

Accepted Article

e-mail:

(Y.W.)

[email protected]

(H.K.)

[email protected]

(T.T.)

[email protected]

(T.A.)

[email protected]

Running title: Pi-deficiency response via PIP5K genes

Keywords: phosphate deficiency, root hair elongation, transcriptional regulation, phosphatidylinositol phosphate 5-kinase, phosphatidylinositol 4,5-bisphosphate, cis-element, target gene

*Correspondence should be addressed: (T.A.)

e-mail: [email protected]

tel: +81-774-38-3262

fax: +81-774-38-3259

SUMMARY

Plants drastically alter their root system architecture to adapt to different underground growth conditions. During phosphate (Pi) deficiency, most plants including Arabidopsis thaliana enhance the development of lateral roots and root hairs, resulting in bushy and hairy roots. To elucidate the signal pathway specific for the root hair elongation response to Pi deficiency, we

This article is protected by copyright. All rights reserved.

Accepted Article

to Pi deficiency, strongly suggesting that transcriptional activation of the PIP5K genes via P1BS is a major requirement for the root hair response. Interestingly, the P1BS and sequences similar to the root hair cell-specific cis-element (RHE) (Kim et al., 2006) in the PIP5K3 upstream intergenic region are conserved in Brassicaceae genome sequences that are available in public databases (Figure S3). It has been proposed that Brassicaceae plants take advantage of rapid root growth and root hair formation rather than mycorrhizal symbiosis as a strategy for nutrition uptake (Francis and Read, 1994; Lambers et al., 2008). PIP5K3 might play a crucial role in enhancing the root hair elongation response to Pi deficiency in Brassicaceae, especially at young seedling stages, when macro-scale architectures of the root system are still immature.

Prolonged Pi deficiency for more than 5 DAG significantly enhanced root hair elongation even in the pip5k3pip5k4 double mutant seedlings. Because the pip5k3 mutant allele pip5k3-3 (SALK_000024) used in this study is not null, its residual function might be responsible for the enhancement under prolonged Pi deficiency. However, another mutant allele, pip5k3-4 (SALK_026883), an expected protein product of which has been shown to have no enzymatic activity in vitro (Stenzel et al., 2008), caused almost the same degree of defects as pip5k3-3 in root hair elongation responses in both pip5k3 single and pip5k3pip5k4 double mutant seedlings at 3 and 7 DAG (Figure S4). This leads to the idea that PIP5K genes other than PIP5K3 and PIP5K4 or genes for other phosphoinositide-metabolizing enzymes are possibly involved in the response to prolonged Pi deficiency. In 7-DAG wild-type roots grown


Accepted Article

SPX1 acts as an inhibitor of PHR1 through Pi-dependent binding to PHR1 (Puga et al., 2014), indicating that Pi deficiency can activate PHR1 cell-autonomously. Taken together, a total process from the sensing of Pi deficiency to the promotion of root hair elongation is presumable to be cell-autonomous in root hair cells.

In this study, we focused on the function of PIP5K genes in promoting root hair elongation, and found that, at least at young seedling stages, the Arabidopsis PIP5K3 and PIP5K4 genes respond to Pi deficiency via P1BSs in their promoters and promote root hair elongation. As for immediate upstream factors of PHR1, SPX1 and the SUMO E3 ligase SIZ1 have been reported to modulate the PHR1 function post-translationally (Miura et al., 2005; Puga et al., 2014), whereas the total signal transduction pathway in the upstream of PHR1 remains to be elucidated. Besides the pathway of PHR1, those involving RSL4 (Yi et al., 2010) and PER2/AL6 (Chandrika et al., 2013) have been reported to enhance root hair elongation under Pi-deficient conditions, suggesting that root hairs respond to Pi deficiency at various levels of root hair development. To elucidate the feature of each pathway and interactions among them will help us understand the plant strategy for surviving Pi starvation.

EXPERIMENTAL PROCEDURES

Plant materials and growth conditions

Arabidopsis thaliana (L.) Heynh Columbia ecotype (Col) was used as the wild type and the parental line for transgenic plants. The T-DNA insertion lines SALK_000024 (pip5k3-3),


Accepted Article

than simply by the status of systemic Pi availability. Genetic studies have identified several genes that are involved in the primary root response (Ticconi et al., 2004; Reymond et al., 2006; Sanchez-Calderon et al., 2006; Svistoonoff et al., 2007). Of those, the multicopper oxidase genes LOW PHOSPHATE ROOT 1 and 2 (LPR1 and LPR2), and the endoplasimic reticulum (ER)-localizing P5-type ATPase gene PHOSPHATE DEFICIENCY RESPONSE 2 (PDR2) have been suggested to constitute a signaling cascade in the ER (Ticconi et al., 2009). In addition, the response in lateral root development is likely to be mediated mainly by auxin signals in roots, whereas the mechanism for the conversion between Pi and auxin signals is unknown (Lopez-Bucio et al., 2002; Al-Ghazi et al., 2003; Nacry et al., 2005; Perez-Torres et al., 2008; Miura et al., 2011; Gonzalez-Mendoza et al., 2013). Interestingly, the primary and lateral root responses occur depending on accessions of Arabidopsis (Chevalier et al., 2003; Reymond et al., 2006), suggesting that these responses are genetically programmed to reflect the individual Pi-deficient circumstances of each accession.

In the micro-scale architecture of roots, Pi deficiency and deficiencies in other nutrients with poor mobility in soil remarkably enhance root hair development (Bates and Lynch, 1996; Ma et al., 2001; Muller and Schmidt, 2004). In Arabidopsis, root hairs normally emerge from specific root epidermal cells, known as trichoblasts, which contact two underlying cortical cell files (for review, see; Tominaga-Wada et al., 2011; Grebe, 2012; Lin and Aoyama, 2012). Pi deficiency increases the number of root cortical cell files, and hence the number of possible positions for root hair cells (Ma et al., 2001). In addition, it leads to the development of


Accepted Article

ectopic root hairs from atrichoblasts, root epidermal cells in non-root hair positions (Muller and Schmidt, 2004). These changes, together with the impaired root cell elongation, contribute to the massive increase in root hair density under Pi-deficient conditions (Sanchez-Calderon et al., 2005). Phytohormones including auxin, ethylene, and gibberellin play important roles in these root hair responses to Pi deficiency (Bates and Lynch, 1996; Zhang et al., 2003; He et al., 2005; Jiang et al., 2007; Lei et al., 2011; Gonzalez-Mendoza et al., 2013). However, it remains unclear how the Pi-deficiency signals connect to the phytohormone signals.

Genetic studies have identified a number of mutants that exhibit defects in root hair elongation during Pi deficiency (for reviews, see; Rouached et al., 2010; Abel, 2011; Chiou and Lin, 2011; Niu et al., 2013). In most cases, however, they are commonly defective in other responses to Pi deficiency, and it is unclear how the Pi-deficiency signal connects to the root hair elongation response. For example, the Arabidopsis G2-like MYB transcription factors PHOSPHATE STARVATION RESPONSE 1 (PHR1) and its paralog PHR1-LIKE 1 (PHL1) are redundantly involved in the root hair elongation response, as well as a wide variety of phenomena caused by Pi deficiency (Bustos et al., 2010). Although they are known to transcriptionally activate Pi-deficiency-responsive genes via the PHR1-binding sequence (P1BS), it remains unknown which of their target genes are involved in the root hair response (Rubio et al., 2001; Bustos et al., 2010).


Accepted Article

Miura, K., Rus, A., Sharkhuu, A., et al. (2005) The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses. Proc. Natl. Acad.Sci. USA, 102, 7760-7765. DOI: 10.1073/pnas.0500778102 Mollier, A. and Pellerin, S. (1999) Maize root system growth and development as influenced by phosphorus deficiency. J. Exp. Bot. 50, 487-497. DOI: 10.1093/jxb/50.333.487 Muller, M. and Schmidt, W. (2004) Environmentally induced plasticity of root hair development in Arabidopsis. Plant Physiol. 134, 409-419. DOI: 10.1104/pp.103.029066 Munnik, T. and Nielsen, E. (2011) Green light for polyphosphoinositide signals in plants. Curr. Opin. Plant Biol. 14, 489-497. DOI: 10.1016/j.pbi.2011.06.007 Nacry, P., Canivenc, G., Muller, B., Azmi, A., Onckelen, H.V., Rossignol, M. and Doumas, P. (2005) A role for auxin redistribution in the responses of the root system architecture to phosphate starvation in Arabidopsis. Plant Physiol. 138, 2061-2074. DOI: 10.1104/pp.105.060061 Niu, Y.F., Chai, R.S., Jin, G.L., Wang, H., Tang, C.X. and Zhang, Y.S. (2013) Responses of root architecture development to low phosphorus availability: a review. Ann. Bot. 112, 391-408. DOI: 10.1093/aob/mcs285


Accepted Article

Root hairs elongate via highly polarized cell expansion called tip growth, in which the exocytotic events for the deposition of new plasma membrane and cell wall are strictly localized to the root hair tip (for review, see; Smith and Oppenheimer, 2005; Cole and Fowler, 2006; Cardenas, 2009; Datta et al., 2011; Gu and Nielsen, 2013). The mechanism that regulates root hair tip growth involves signaling factors including calcium ions, reactive oxygen species, Rho-related GTPases, and phospholipids. Of these, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], one of the most studied signaling phospholipids in eukaryotes, is assumed to play a pivotal role in root hair tip growth by promoting the reorganization of the actin cytoskeleton and membrane traffic, both of which are essential for exocytosis (for review see; Xue et al., 2009; Ischebeck et al., 2010; Munnik and Nielsen, 2011; Boss and Im, 2012; Saavedra et al., 2012). PtdIns(4,5)P2 localizes to the apical region of elongating root hairs in maize and Arabidopsis (Braun et al., 1999; Vincent et al., 2005; van Leeuwen et al., 2007). The Arabidopsis B-type phosphatidylinositol phosphate 5-kinase PIP5K3, an enzyme producing PtdIns(4,5)P2, is involved in root hair elongation (Kusano et al., 2008; Stenzel et al., 2008). Yellow fluorescent protein (YFP) fusions of PIP5K3 driven by the PIP5K3 promoter localized to the elongating root hair apex (Kusano et al., 2008; Stenzel et al., 2008), and the signal intensity was positively correlated with the rate of root hair growth (Kusano et al., 2008). This suggests that the PIP5K activity for producing PtdIns(4,5)P2 in the root hair apex is a quantitative factor for the rate of root hair growth.


Accepted Article

essential for root hair formation in Arabidopsis thaliana. Plant Cell, 20, 124-141. DOI: 10.1105/tpc.107.052852 Svistoonoff ,S., Creff, A., Reymond, M., Sigoillot-Claude, C., Ricaud, L., Blauchet, A., Nussaume, L. and Desnos, T. (2007) Root tip contact with low-phosphate media reprograms plant root architecture. Nature genet. 39, 792-796. DOI: 10.1038/ng2041 Taniguchi, Y.Y., Taniguchi, M., Tsuge, T., Oka, A. and Aoyama, T. (2010) Involvement of Arabidopsis thaliana phospholipase Dζ2 in root hydrotropism through the suppression of root gravitropism. Planta, 231, 491-497. DOI: 10.1007/s00425-009-1052-x

Thibaud, M.C., Arrighi, J.F., Bayle, V., Chiarenza, S., Creff, A., Bustos, R., Paz-Ares, J., Poirier, Y. and Nussaume, L. (2010) Dissection of local and systemic transcriptional responses to phosphate starvation in Arabidopsis. Plant J. 64, 775-789. DOI: 10.1111/j.1365-313X.2010.04375.x Ticconi, C.A. and Abel, S. (2004) Short on phosphate: plant surveillance and countermeasures. Trends Plant Sci. 9, 548-555. DOI: 10.1016/j.tplants.2004.09.003 Ticconi, C.A., Delatorre, C.A., Lahner, B., Salt, D.E. and Abel, S. (2004) Arabidopsis pdr2 reveals a phosphate-sensitive checkpoint in root development. Plant J. 37, 801-814. DOI: 10.1111/j.1365-313X.2003.02005.x Ticconi, C.A., Lucero, R.D., Sakhonwasee, S., Adamson, A.W., Creff, A., Nussaume, L., Desnos, T. and Abel, S. (2009) ER-resident proteins PDR2 and


Accepted Article This article is protected by copyright. All rights reserved.

Accepted Article

and GUS histochemical analysis strongly suggest that Pi-deficiency upregulates the transcription of PIP5K3 and PIP5K4 in root hair cells.

The Pi-deficiency responses of PIP5K3 and PIP5K4 are mediated by PHR1

Because PHR1 directs a major part of transcriptional responses to Pi deficiency together with its paralog PHL1 (Bustos et al., 2010), and because PIP5K3 and PIP5K4 have one and two copies of P1BS in their upstream intergenic regions, respectively (see below), it is highly probable that PHR1 directly activates the transcription of PIP5K3 and PIP5K4 under Pi deficiency. To confirm this, we examined the steady-state transcript levels of PIP5K3 and PIP5K4 in the phr1 (SALK_067629) mutant background under the Pi-sufficient and deficient conditions at the 3-DAG stage. In phr1 roots, the upregulation of the transcript level by Pi deficiency was partially suppressed for PIP5K3 and IPS1, and was eliminated for PIP5K4 (Figure 3). This result indicates that PHR1 is involved in the responses of PIP5K3 and PIP5K4 to Pi deficiency, while another transcription factor is supposed to contribute to the residual responsiveness of PIP5K3 observed in phr1 roots. It has been reported that the phr1 mutation affects the root hair elongation in seedlings grown for 12 days under Pi deficiency (Bustos et al., 2010). While wild-type and phr1 seedlings were apparently indistinguishable at the 3-DAG stage under the both Pi-sufficient and deficient conditions used in this study (Figure S1), the Pi-deficiency


Accepted Article

response in root hair elongation was partially suppressed in phr1 (Figure 4), indicating that PHR1 mediates the root hair elongation response also at young seedling stages.

PIP5K3 and PIP5K4 are redundantly required for the root hair elongation response at young seedling stages

To investigate functions of the PIP5K3 and PIP5K4 genes in the root hair elongation response under the Pi-deficient condition, we used Arabidopsis T-DNA insertion mutants, pip5k3 (SALK_000024) and pip5k4 (SALK_001138), which were thought to be hypomorphic and null alleles for the PIP5K3 and PIP5K4 gene functions, respectively (Lee et al., 2007; Kusano et al., 2008). We confirmed by RT-PCR that very low and undetectable levels of functional transcripts were derived from the pip5k3 and pip5k4 mutant genes (Figure S2). Both mutant seedlings at 3 DAG exhibited significantly shorter root hairs than the wild type under the Pi-sufficient condition (Figure 4), indicating that PIP5K3 and PIP5K4 are involved in root hair elongation. However, root hairs of both mutants retained the responsiveness to Pi deficiency (Figure 4), suggesting their redundant gene functions in the response. Then, we examined the pip5k3pip5k4 double mutant. Root hairs of the double mutant were even shorter than those of the single mutants, and not responsive to Pi deficiency at significant levels (Figure 4), indicating that PIP5K3 and PIP5K4 are redundantly required for the root hair elongation response at young seedling stages.


Accepted Article

The P1BSs in the upstream intergenic regions of PIP5K3 and PIP5K4 are not related with root hair elongation under Pi-sufficient conditions, but required for the root hair elongation response to Pi deficiency at young seedling stages.

Results described above suggest that PHR1 and its paralog(s) promote the root hair elongation response to Pi deficiency by transcriptionally activating the PIP5K3 and PIP5K4 genes via their upstream P1BSs. To examine the possibility, we constructed genes, PIP5K3g and PIP5K4g, from genomic DNA fragments encompassing upstream intergenic regions and protein-coding regions, and their modified genes, pip5k3gmp and pip5k4gmp, which contained base substitutions in their P1BSs (Figure 5a), and introduced them into pip5k3 and pip5k4, respectively. In the resulting transgenic lines, PIP5K3g and PIP5K4g were expected to complement the mutant defects totally, and pip5k3gmp and pip5k4gmp were expected to tactically complement the mutant defects except those in the responsiveness to Pi deficiency. Root hairs of the total complementation lines, pip5k3/PIP5K3g and pip5k4/PIP5K4g, were similar in length to those of the wild type under the both Pi-sufficient and deficient conditions (Figure 5b, c), indicating that those transgenes are functional for root hair elongation and its response to Pi deficiency. The tactical complementation lines, pip5k3/pip5k3gmp and pip5k4/pip5k4gmp, also exhibited root hairs of similar length to the wild type under the Pi-sufficient condition (Figure 5b, c), indicating that the modified gens had the authentic function in root hair elongation under Pi-sufficient conditions. Under the Pi-deficient condition, however, root hairs of both tactical complementation lines were significantly shorter than those of each corresponding total


Accepted Article

complementation line (Figure 5b, c), suggesting that the modified genes had a defect in the response to Pi deficiency. Root hairs of both tactical complementation lines remained responsive to Pi deficiency (Figure 5b, c) just like pip5k3 and pip5k4 mutant root hairs (Figure 4), supporting the idea that PIP5K3 and PIP5K4 redundantly contribute to the root hair elongation response.

We next crossed pip5k3/PIP5K3g with pip5k4/PIP5K4g, and pip5k3/pip5k3gmp with pip5k4/pip5k4gmp, to obtain total and tactical double complementation lines, pip5k3pip5k4/PIP5K3gPIP5k4g and pip5k3pip5k4/pip5k3gmppip5k4gmp, respectively. Seedlings of both double complementation lines grown for 3 DAG under the Pi-sufficient condition exhibited root hairs of length similar to the wild type (Figure 5b, c). However, the total complementation line, but not the tactical complementation line, was responsive to Pi deficiency, and root hair lengths were significantly different between them under the Pi-deficient condition (Figure 5b, c). The steady-state transcript levels of PIP5K3g and PIP5K4g, but neither pip5k3gmp nor pip5k4gmp, responded to Pi deficiency in 3-DAG roots of these complementation lines (Figure 6). These results indicate that the root hair elongation response to Pi deficiency at young seedling stages requires the P1BSs, through which PHR1 is assumed to activate PIP5K3 and PIP5K4 transcriptionally.


Accepted Article

Signal pathways independent of PIP5K3 and PIP5K4 possibly mediate root hair elongation responses to prolonged Pi deficiency after germination

To investigate involvement of PIP5K3 and PIP5K4 in root hair elongation responses to prolonged Pi deficiency after germination, we observed root hairs of wild-type, pip5k3pip5k4, pip5k3pip5k4/PIP5K3gPIP5k4g, and pip5k3pip5k4/pip5k3gmppip5k4gmp seedlings grown for 3 to 10 DAG under the Pi-deficient condition. Although root hairs of neither the double mutant nor tactical double complementation line significantly responded to Pi deficiency at the stage of 5 DAG or earlier, root hairs of the both lines substantially elongated responding to Pi deficiency at 7 DAG or later (Figure 7). Pi deficiency upregulated the steady-state transcript levels of neither pip5k3gmp nor pip5k4gmp at 7 DAG (Figure 6). These suggest the existence of other regulatory pathways than that involving PIP5K3 and PIP5K4 for the root hair elongation response to prolonged Pi deficiency.

DISCUSSION

Pi deficiency affects root hair elongation through numerous signaling pathways (Rouached et al., 2010; Abel, 2011; Chiou and Lin, 2011; Niu et al., 2013). Although all the pathways are thought to have biological meanings, interactions among them possibly prevent us from understanding each single pathway. Especially, severe effects of Pi deficiency such as RAM exhaustion might indirectly impact root hair elongation. To avoid this complexity, we used


Accepted Article

young seedlings at the 3-DAG stage, where effects of Pi deficiency on the growth of both aerial organs and roots are still inapparent. As the result, we revealed that the PIP5K3 and PIP5K4 genes are responsive to Pi deficiency in root hair cells at the 3-DAG stage, and that a Pi-deficiency signal is transferred to the pathway for root hair elongation via the genes. The product of PIP5K, PtdIns(4,5)P2, is a signal activating actin cytoskeletal reorganization and membrane traffic, both of which promote exocytosis (Santarius et al., 2006; Saarikangas et al., 2010; Mayinger, 2012). The level of PtdIns(4,5)P2 on the plasma membrane positively correlates with exocytotic competence in animal cells (Di Paolo et al., 2004; Milosevic et al., 2005). Consistent with this, in the elongating root hair apex, where exocytosis actively occurs, the amount of the PIP5K3-YFP driven by the PIP5K3 promoter was well correlated with the rate of root hair tip growth (Kusano et al., 2008). Moreover, inducible expression of PIP5K3 enhanced root hair elongation (Kusano et al., 2008). These strongly suggest that PIP5K and PtdIns(4,5)P2 are quantitative factors of root hair tip growth. As for PIP5K4, its function in the polar tip growth of pollen tubes has been reported (Ischebech et al., 2008; Sousa et al., 2008). Hence, it is reasonably supposed that Pi-deficiency enhances root hair elongation through the activation of PIP5K genes including PIP5K3 and PIP5K4, while activation of other positive regulators for exocytosis might be required simultaneously.

At the 5-DAG stage or earlier, the introduction of the PIP5K3 and PIP5K4 transgenes with the authentic sequences, but not the modified transgenes lacking P1BS, could complement the defect of the pip5k3pip5k4 double mutant in the root hair elongation response


Accepted Article

to Pi deficiency, strongly suggesting that transcriptional activation of the PIP5K genes via P1BS is a major requirement for the root hair response. Interestingly, the P1BS and sequences similar to the root hair cell-specific cis-element (RHE) (Kim et al., 2006) in the PIP5K3 upstream intergenic region are conserved in Brassicaceae genome sequences that are available in public databases (Figure S3). It has been proposed that Brassicaceae plants take advantage of rapid root growth and root hair formation rather than mycorrhizal symbiosis as a strategy for nutrition uptake (Francis and Read, 1994; Lambers et al., 2008). PIP5K3 might play a crucial role in enhancing the root hair elongation response to Pi deficiency in Brassicaceae, especially at young seedling stages, when macro-scale architectures of the root system are still immature.

Prolonged Pi deficiency for more than 5 DAG significantly enhanced root hair elongation even in the pip5k3pip5k4 double mutant seedlings. Because the pip5k3 mutant allele pip5k3-3 (SALK_000024) used in this study is not null, its residual function might be responsible for the enhancement under prolonged Pi deficiency. However, another mutant allele, pip5k3-4 (SALK_026883), an expected protein product of which has been shown to have no enzymatic activity in vitro (Stenzel et al., 2008), caused almost the same degree of defects as pip5k3-3 in root hair elongation responses in both pip5k3 single and pip5k3pip5k4 double mutant seedlings at 3 and 7 DAG (Figure S4). This leads to the idea that PIP5K genes other than PIP5K3 and PIP5K4 or genes for other phosphoinositide-metabolizing enzymes are possibly involved in the response to prolonged Pi deficiency. In 7-DAG wild-type roots grown


Accepted Article

under the Pi-deficient condition, the steady-state transcript level of PIP5K6 was upregulated as well as that of PIP5K3 (Figure S5), suggesting a redundant function of PIP5K3 and PIP5K6. Although PIP5K6 lacks P1BS in its intragenic and upstream intergenic regions, a microarray analysis has shown that the transcript level of PIP5K6 in roots grown for 7 days responds to Pi deficiency depending on the PHR1 and PHL1 gene functions (transcriptomic data in Bustos et al., 2010). However, PtdIns(4,5)P2, and hence PIP5K, may have various roles other than that in root hair elongation. Even within a same cell, independent pools of PtdIns(4,5)P2 might have different functions from each other (Heilmann and Heilmann, 2013). Detailed functional analyses of PIP5K genes are needed to clarify their functions in Pi-deficiency responses. In this connection, pip5k3pip5k4 double mutant roots showed no differences in the primary root elongation response to Pi deficiency under the conditions used in this study (Figure S6), although promoter activities of the PIP5K3 and PIP5K4 upstream regions were upregulated by Pi deficiency also in the cell elongation zone (Figure 2b).

Under prolonged Pi-deficient conditions, other transcription factors than PHR1 and PHL1 might also be involved in the root hair elongation response. HRS1 is a candidate of such transcription factors because the HRS1 promoter is activated by Pi deficiency in 10-day-old root subepidermal and epidermal cells including root hair cells (Liu et al., 2009). A microarray analysis using the fusion transcription factor GR-PHR1 has shown that HRS1 is a direct target gene of PHR1 (transcriptomic data in Bustos et al., 2010). RSL4, transcripts of which are


Accepted Article

upregulated by a signal downstream of LPR1 and LPR2, might considerably contribute to the root hair elongation response under prolonged Pi deficiency, where the primary root response to Pi deficiency occurs (Reymond et al., 2006; Svistoonoff et al., 2007; Yi et al., 2010). AL6/PER2 might also be involved in response to prolonged Pi deficiency via ETC1 (Chandrika et al., 2013).

Phosphate deficiency responses of plants are known to be mediated by either systemic or local Pi sensing systems, reflecting the Pi status of the total plant or Pi availabilities around local tissues, respectively (Ticconi and Able, 2004; Doerner, 2008; Thibaud et al., 2010). A local Pi sensing system is thought to mediate the root hair elongation response to Pi deficiency because local changes in Pi availability influence root hair elongation regardless of the plant Pi status (Bates and Lynch, 1996). On the other hand, dissection analysis of systemic and local transcriptional responses to Pi deficiency has revealed that systematically controlled genes tend to have P1BS-enriched promoters (Thibaud et al., 2010), and that a minimal promoter with multimerized P1BS is systemically controlled (Bustos et al., 2010), suggesting that PHR1 functions downstream of a systemic signal. These aspects, however, don’t necessarily mean that the root hair elongation response to Pi deficiency is independent of the PHR1 function. In fact, a phr1phl1 double mutant study has provided evidence that PHR1 and PHL1 redundantly function in the root hair elongation response (Bustos et al., 2010). At least, given that PHR1 directly activates PIP5K3 and PIP5K4 in root hair cells, the downstream of PHR1 is thought to be local. Recently, it has been reported that the Arabidopsis nuclear protein


Accepted Article

SPX1 acts as an inhibitor of PHR1 through Pi-dependent binding to PHR1 (Puga et al., 2014), indicating that Pi deficiency can activate PHR1 cell-autonomously. Taken together, a total process from the sensing of Pi deficiency to the promotion of root hair elongation is presumable to be cell-autonomous in root hair cells.

In this study, we focused on the function of PIP5K genes in promoting root hair elongation, and found that, at least at young seedling stages, the Arabidopsis PIP5K3 and PIP5K4 genes respond to Pi deficiency via P1BSs in their promoters and promote root hair elongation. As for immediate upstream factors of PHR1, SPX1 and the SUMO E3 ligase SIZ1 have been reported to modulate the PHR1 function post-translationally (Miura et al., 2005; Puga et al., 2014), whereas the total signal transduction pathway in the upstream of PHR1 remains to be elucidated. Besides the pathway of PHR1, those involving RSL4 (Yi et al., 2010) and PER2/AL6 (Chandrika et al., 2013) have been reported to enhance root hair elongation under Pi-deficient conditions, suggesting that root hairs respond to Pi deficiency at various levels of root hair development. To elucidate the feature of each pathway and interactions among them will help us understand the plant strategy for surviving Pi starvation.

EXPERIMENTAL PROCEDURES

Plant materials and growth conditions

Arabidopsis thaliana (L.) Heynh Columbia ecotype (Col) was used as the wild type and the parental line for transgenic plants. The T-DNA insertion lines SALK_000024 (pip5k3-3),


Accepted Article

SALK_026883 (pip5k3-4), SALK_001138 (pip5k4), SALK_067629 (phr1) were identified in the collection of Salk Institute Genomic Analysis Laboratory (Alonso et al., 2003) and obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA). Homozygous lines for T-DNA insertions were established and used in subsequent experiments. Arabidopsis seeds were surface sterilized and kept at 4 ˚C in the dark for 3 days and germinated on agar medium standing vertically. After the seed germination, seedlings were grown on the vertical agar medium at 22 ˚C under continuous light conditions (50 μmol m-2 s-1; white fluorescent bulb FLR40SW; Hitachi, Ltd., Tokyo, Japan). The agar medium contains Murashige-Skoog salts, B5 vitamins, 2.5 mM MES, 1% sucrose, and 2% agar, except that 1 mM and 3 μM KH2PO4 were contained for normal and Pi-deficient conditions, respectively, and that the concentration of FeSO4 was 50 μM. The pH was adjusted to 5.7.

Construction of transgenes and transgenic plants

For the construction of PIP5K3p-GUS and PIP5K4p-GUS, DNA fragments encompassing the 981-bp and 1315-bp upstream intergenic sequences and initiation codons of PIP5K3 and PIP5K4 were amplified by PCR using primers listed in Table S1, respectively, and inserted between the PstI and BamHI sites of pBI101 in an in-frame manner to the GUS-coding sequence. For the construction of PIP5K3g and PIP5K4g, 3,784-bp and 4,241-bp genomic fragments of PIP5K3 and PIP5K4 encompassing upstream intergenic sequences, protein-coding


Accepted Article

sequences, intron sequences, and termination codons were amplified by PCR using primers listed in Table S1, respectively, and cloned into pUC19 between the PstI and SacI sites for PIP5K3g, and between the PstI and SmaI sites for PIP5K4g. For the construction of pip5k3gmp and pip5k4gmp, PIP5K3g and PIP5K4g were modified to have base substitutions in their P1BSs by site-directed mutagenesis as illustrated in Figure 5A. The constructed genes were recloned into pHPT121 (Kusano et al., 2008) between the Sse8387I and SacI sites for PIP5K3g and pip5k3gmp, and between the Sse8387I and Ecl136II sites for PIP5K4g and pip5k4gmp. Strains of Agrobacterium tumefaciens LBA4404 carrying each construct were used to transform Arabidopsis by vacuum infiltration. Multiple homozygous transgenic lines for each construct were established in their T3 generation, and examined in subsequent experiments.

Measurement of root hair length

Root hair length was measured basically as described before (Kato et al., 2013). Images of the primary root on the agar medium were captured with a CCD camera. Root hairs elongated horizontally to the agar surface in the region 4 to 6 mm from the root tip were measured on the image with the assistance of ImageJ (http://rsbweb.nih.gov/ij/). Immature root hairs or bulges with length less than 5 μm were omitted. The data shown are representative of consistent results from at least three independent experiments.


Accepted Article

RNA preparation, cDNA synthesis, and quantitative RT-PCR

Total RNA was isolated using ISOGEN (Nippon Gene Co., Ltd., Tokyo, Japan) according to the manufacturer’s protocol. The RNA was converted to first-strand cDNA using SuperScript™ III First-Strand System for RT-PCR (Life Technologies Co., Carlsbad, CA, USA) with random hexamers as primers for the reverse-transcription. Quantitative RT-PCR was performed with the cDNA and gene-specific primer sets using Lumino Ct SYBR Green qPCR ReadyMix (Sigma-Aldrich Co., St. Louis, MI, USA) and Eco™ Real Time PCR System (Illumina Inc., San Diego, CA, USA). A primer set for each gene (Table S1) was designed so that it generated a unique PCR product with a length from 60 to 150 bp, and examined for the designed performance in preliminary tests. The RT-PCR program was as follows: 95 ˚C for 20 sec, then 40 cycles of 95 ˚C for 10 sec and 60 ˚C for 30 sec, and then a melting curve cycle of 95 ˚C for 15 sec, 60 ˚C for 15 sec, and 95 ˚C for 10 sec. The RT-PCR was triplicated for each sample to obtain the threshold cycle value. To determine the amount of cDNA corresponding to the transcript of each gene, a standard curve was made simultaneously in the RT-PCR using the same primer set and clonal template DNA of determined amounts. To normalize the amounts of cDNA among preparations, the amount of cDNA corresponding to the UBC21 gene (At5g25760) transcript was used as a reference. The data shown are mean and S.D. values from multiple independent experiments.


Accepted Article

Gus histochemical analysis

GUS histochemical analysis was carried out using the basic procedure described before (Taniguchi et al., 2010). Arabidopsis seedlings were submerged in cold 90% acetone for 10 minutes at −20 ˚C. After several washes in 100 mM sodium phosphate buffer (pH 7.4), the tissues were incubated 8 h at 37 ˚C in a solution containing 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 100 mM sodium phosphate buffer (pH 7.4). The reaction was stopped by several washes in 100 mM sodium phosphate buffer (pH 7.4) and plant samples were fixed with the solution containing 35% ethanol, 3.7% formaldehyde, and 5% acetic acid.

ACKNOWLEDGEMENTS

We thank K. Yasuda for technical assistance. This work was supported by the Japanese Society of Promotion of Science (JSPS) (Research Fellowships for Young Scientists) to Y.W. and by JSPS (Grants-in-Aid for Scientific Research-B; 16370023) and the Ministry of Education, Culture, Sports, Science, and Technology, Japan (Grants-in-Aid for Scientific Research on Priority Areas; 18056012) to T.A. Authors have no conflict of interest to declare.


Accepted Article

SUPPORTING INFORMATION

Additional Supporting Information is available in the online version of this article.

Figure S1. Phenotypes of the phr1 mutant at the 3-DAG seedling stage under the Pi-sufficient and -deficient conditions.

Figure S2. Relative levels of functional transcripts from the pip5k3 and pip5k4 mutant genes.

Figure S3. Conserved cis-elements in the upstream intergenic regions of Brassicaceae PIP5K3 orthologs.

Figure S4. Root hair lengths of 3-DAG and 7-DAG seedlings of the pip5k3-3, pip5k3-4, pip5k3-3pip5k4, and pip5k3-4pip5k4 mutants grown under the Pi-sufficient and deficient conditions.

Figure S5. Expression of PIP5K genes in roots of 7-DAG seedlings grown under the Pi-sufficient and deficient conditions.

Figure S6. Wild-type and pip5k3pip5k4 double mutant seedlings grown under the Pi-sufficient and deficient conditions.

Table S1. Primer sets used in this study.


Accepted Article

REFERENCES

Abel, S. (2011) Phosphate sensing in root development. Curr. Opin. Plant Biol. 14, 303-309. DOI: 10.1016/j.pbi.2011.04.007 Al-Ghazi, Y., Muller, B., Pinloche, S., Tranbarger, T.J., Nacry, P., Rossignol, M., Tardieu, F. and Doumas, P. (2003) Temporal responses of Arabidopsis root architecture to phosphate starvation: evidence for the involvement of auxin signaling. Plant Cell Envir. 26,1053-1066. DOI: 10.1046/j.1365-3040.2003.01030.x Alonso, J.M., Stepanova, A.N., Leisse, T.J. et al. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science, 301, 653-657. DOI: 10.1126/science.1086391

Bates, T.R. and Lynch, J.P. (1996) Stimulation of root hair elongation in Arabidopsis thaiiana by low phosphorus availability. Plant Cell Envir. 19, 529-538. DOI: 10.1111/j.1365-3040.1996.tb00386.x Boss, W.F. and Im, Y.J. (2012) Phosphoinositide signaling. Annu. Rev. Plant Biol. 63, 409-429. DOI: 1146/annurev-arplant-042110-103840 Braun, M., Baluska, F., von Witsch, M., and Menzel, D. (1999) Redistribution of actin, profilin and phosphatidylinositol-4, 5-bisphosphate in growing and maturing root hairs. Planta, 209, 435-443. DOI: 10.1007/s004250050746 Bustos ,R., Castrillo, G., Linhares, F., Puga, M.I., Rubio, V., Perez-Perez. J, Solano, R., Leyva, A. and Paz-Ares, J. (2010) A central regulatory system largely controls


Accepted Article

transcriptional activation and repression responses to phosphate starvation in Arabidopsis. PLoS Genet. 6, 1-15. DOI: 10.1371/journal.pgen.1001102 Cardenas, L. (2009) New findings in the mechanisms regulating polar growth in root hair cells. Plant Signal. Behav. 4, 4-8. DOI: 10.4161/psb.4.1.7341 Chandrika, N.N.P., Sundaravelpandian, K., Yu, S.M. and Schmidt, W. (2013) ALFIN-LIKE 6 is involved in root hair elongation during phosphate deficiency in Arabidopsis. New Phytol. 198, 709-720. DOI: 10.1111/nph.12194 Chevalier, F., Pata, M., Nacry, P., Doumas, P. and Rossingnol, M. (2003) Effects of phosphate availability on the root system architecture: large-scale analysis of the natural variation between Arabidopsis accessions. Plant Cell Envir. 26, 1839-1850. DOI: 10.1046/j.1365-3040.2003.01100.x Chiou, T. and Lin, S. (2011) Signaling network in sensing phosphate availability in plants. Annu. Rev. Plant Biol. 62, 185-206. DOI: 10.1146/annurev-arplant-042110-103849 Cole, R.A. and Fowler, J.E. (2006) Polarized growth: maintaining focus on the tip. Curr. Opin. Plant Biol. 9, 579-588. DOI: 10.1016/j.pbi.2006.09.014 Datta, S., Kim, C.M., Pernas, M., Pires, N.D., Proust, H., Tam, T., Vijayakumar, P. and Dolan, L. (2011) Root hairs: development, growth and evolution at the plant-soil interface. Plant Soil 346, 1-14. DOI: 10.1007/s11104-011-0845-4


Accepted Article

Desnos, T. (2008) Root branching responses to phosphate and nitrate. Curr. Opin. Plant Biol. 11, 82-87. DOI: 10.1016/j.pbi.2007.10.003 Di Paolo, G., Moskowitz, H.S., Gipson, K., Wenk, M.R., Voronov, S., Obayashi, M., Flavell, R., Fitzsimonds, R.M., Ryan, T.A. and De Camilli, P. (2004) Impaired PtdIns(4,5)P2 synthesis in nerve terminals produces defects in synaptic vesicle trafficking. Nature, 431, 415-422. DOI: 10.1038/nature02896 Doerner, P. (2008) Phosphate starvation signaling: a threesome controls systemic Pi homeostasis. Curr. Opin.Plant Biol. 11, 536-540. DOI: 10.1016/j.pbi.2008.05.006 Francis, R. and Read, D.J. (1994) The contributions of mycorrhizal fungi to the determination of plant community structure. Plant Soil, 159, 11-25. DOI: 10.1007/BF00000091 Gonzalez-Mendoza, V., Zurita-Silva, A., Sánchez-Calderon, L., Sanchez-Sandoval, M.E., Oropeza-Aburto, A., Gutierrez-Alanis, D., Alatorre-Cobos, F. and Herrera-Estrella, L. (2013) APSR1, a novel gene required for meristem maintenance, is negatively regulated by low phosphate availability. Plant Sci. 205-206, 2-12. DOI: 10.1016/j.plantsci.2012.12.015 Grebe, M. (2012) The patterning of epidermal hairs in Arabidopsis– updated. Curr. Opin. Plant Biol. 15, 31–37. DOI: 10.1016/j.pbi.2011.10.010 Gu, F. and Nielsen, E. (2013) Targeting and regulation of cell wall synthesis during tip grwoth in plants. J. Integr. Plant Biol. 55, 835-846. DOI: 10.1111/jipb.12077


Accepted Article

He, Z., Ma, Z., Brown, K.M. and Lynch, J.P. (2005) Assessment of inequality of root hair density in Arabidopsis thaliana using the Gini coefficient: a close look at the effect of phosphorus and its interaction with ethylene. Ann. Bot. 95, 287-293. DOI: 10.1093/aob/mci024 Heilmann, M. and Heilmann, I. (2013) Arranged marriage in lipid signaling? The limited choices of PtdIns(4,5)P2 in finding the right partner. Plant Biol. 15, 789-797. DOI: 10.1111/plb.12025 Hinsinger, P. (2001) Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review. Plant Soil, 237, 173-195. DOI: 10.1023/A:1013351617532 Ischebeck, T., Seiler, S. and Heilmann, I. (2010) At the poles across kingdoms: phosphoinositides and polar tip growth. Protoplasma, 240, 13-31. DOI: 10.1007/s00709-009-0093-0 Ischebeck, T., Stenzel. I., and Heilmann, I. (2008) Type B phosphatidylinositol-4phosphate 5-kinases mediate Arabidopsis and Nicotiana tabacum pollen tube growth by regulating apical pectin secretion. Plant Cell, 20, 3312-3330. DOI: 10.1105/tpc.108.059568 Jiang, C., Gao, X., Liao, L., Harberd, N.P. and Fu, X. (2007) Phosphate starvation root architecture and anthocyanin accumulation responses are modulated by the


Accepted Article

gibberellin-DELLA signaling pathway in Arabidopsis. Plant Physiol. 145, 1460-1470. DOI: 10.1104/pp.107.103788 Johnson, J.F., Vance, C.P. and Allan, D. (1996) Phosphorus deficiency in Lupinus albus. Plant Physiol. 112, 31-41. DOI: 10.1104/pp.112.1.31 Kato, M., Aoyama, T. and Maeshima, M. (2013) The Ca2+-binding protein PCaP2 located on the plasma membrane is involved in root hair development as a possible signal transducer. Plant J. 74, 690-700. DOI: 10.1111/tpj.12155

Keerthisinghe, G., Hocking, P.J., Ryan, P.R. and Delhaize, E. (1998) Effect of phosphorus supply on the formation and function of proteoid roots of white lupin (Lupinus albus L.). Plant Cell Envir. 21, 467-478. DOI: 10.1046/j.1365-3040.1998.00300.x Kim, D.W., Lee, S.H., Choi, S.B., Won, Su-Kyung., Heo, Y.K., Cho, M., Park, Y.I. and Cho, H.T. (2006) Functional conservation of a root hair cell-specific cis-element in angiosperms with different root hair distribution patterns. Plant Cell, 18, 2958-2970. DOI: 10.1105/tpc.106.045229 Kirk, G.J.D. and Van, D.L. (1997) Changes in rice root architecture, porosity, and oxygen and proton release under phosphorus deficiency. New Phytol. 135, 191-200. DOI: 10.1046/j.1469-8137.1997.00640.x Kusano, H., Testerink, C., Vermeer, J.E.M., Tsuge, T., Shimada, H., Oka, A., Munnik, T. and Aoyama, T. (2008) The Arabidopsis phosphatidylinositol phosphate


Accepted Article

5-kinase PIP5K3 is a key regulator of root hair tip growth. Plant Cell, 20, 367-380. DOI: 10.1105/tpc.107.056119 Lambers, H., Raven, J.A., Shaver, G.R. and Smith, S.E. (2008) Plant nutrient-acquisition strategies change with soil age. Trends Ecol. Evol. 23, 95-103. DOI: 10.1016/j.tree.2007.10.008 Lee, Y., Kim, Y.W., Jeon, B.W., Park, K.Y., Suh, S.J., Seo, J., Kwak, J.M., Martinoia, E., Hwang, I. and Lee, Y. (2007) Phosphatidylinositol 4,5-bisphosphate is important for stomatal opening. Plant J. 52, 803-816. DOI: 10.1111/j.1365-313X.2007.03277.x Lei, M., Zhu, C., Liu, Y., Karthikeyan, A.S., Bressan, R.A., Raghothama, K.G. and Liu, D. (2011) Ethylene signalling is involved in regulation of phosphate starvation-induced gene expression and production of acid phosphatases and anthocyanin in Arabidopsis. New Phytol. 189, 1084-1095. DOI: 10.1111/j.1469-8137.2010.03555.x Li, Z., Xu, C., Li, K., Yan, S., Qu, X. and Zhang, J. (2012) Phosphate starvation of maize inhibits lateral root formation and alters gene expression in the lateral root primordium zone. BMC Plant Biol. 12, 89-106. DOI: 10.1186/1471-2229-12-89 Lin, Q. and Aoyama, T. (2012) Pathways for epidermal cell differentiation via the homeobox gene GLABRA2: update on the roles of the classic regulator. J. Integr. Plant Biol. 54, 729-737. DOI: 10.1111/j.1744-7909.2012.01159.x


Accepted Article

Linkohr, B.I., Williamson, L.C., Fitter, A.H. and Leyser, H.M.O. (2002) Nitrate and phosphate availability and distribution have different effects on root system architecture of Arabidopsis. Plant J. 29, 751-760. DOI: 10.1046/j.1365-313X.2002.01251.x Liu, H., Yang, H., Wu, C., Feng, J., Liu, X., Qin, H. and Wang, D. (2009) Overexpressing HRS1 confers hypersensitivity to low phosphate-elicited inhibition of primary root growth in Arabidopsis thaliana. J. Integr. Plant Biol. 51, 382-392. DOI: 10.1111/j.1744-7909.2009.00819.x Lopez-Bucio, J., Cruz-Ramirez, A. and Herrera-Estrella, L. (2003) The role of nutrient availability in regulating root architecture. Curr. Opin. Plant Biol. 6, 280-287. DOI: 10.1016/S1369-5266(03)00035-9

Lopez-Bucio, J., Hernandez-Abreu, E., Sanchez-Calderon, L., Nieto-Jacobo, M.F., Simpson, J. and Herrera-Estrella, L. (2002) Phosphate availability alters architecture and causes changes in hormone sensitivity in the Arabidopsis root system. Plant Physiol. 129, 244-256. DOI: 10.1104/pp.010934

Lynch, J. (1995) Root architecture and plant productivity. Plant Physiol. 109, 7-13. DOI: 10.1104/pp.109.1.7 Lynch, J.P. and Brown, K.M. (2001) Topsoil foraging – an architectural adaptation of plants to low phosphorus availability. Plant Soil, 237, 225-237. DOI: 10.1023/A:1013324727040


Accepted Article

Ma, Z., Bielenberg, D.G., Brown, K.M. and Lynch, J.P. (2001) Regulation of root hair density by phosphorus availability in Arabidopsis thaliana. Plant Cell Envir. 24, 459-467. DOI: 10.1046/j.1365-3040.2001.00695.x Malamy, J.E. (2005) Intrinsic and environmental response pathways that regulate root system architecture. Plant Cell Environ. 28, 67-77. DOI: 10.1111/j.1365-3040.2005.01306.x Martín, A.C., del Pozo, J.C., Iglesias, J., Rubio, V., Solano, R., de La Pena, A., Leyva, A. and Paz-Ares, J. (2000) Influence of cytokinins on the expression of phosphate starvation responsive genes in Arabidopsis. Plant J. 24, 559-567. DOI: 10.1046/j.1365-313x.2000.00893.x Mayinger, P. (2012) Phosphoinositides and vesicular membrane traffic. Biochim. Biophys. Acta 1821, 1104-1113. DOI: 10.1016/j.bbalip.2012.01.002 Milosevic, I., Sorensen, J.B., Lang, T., Krauss, M., Nagy, G., Haucke, V., Jahn, R. and Neher, E. (2005) Plasmalemmal phosphatidylinositol-4,5-bisphosphate level regulates the releasable vesicle pool size in chromaffin cells. J. Neurosci. 25, 2557-2565. DOI: 10.1523/JNEUROSCI.3761-04.2005 Miura, K., Lee, J., Gong, Q., Ma, S., Jin, J.B., Yoo, C.Y., Miura. T., Sato. A., Bohnert, H.J. and Hasegawa, P.M. (2011) SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation. Plant Physiol. 155, 1000-1012. DOI: 10.1104/pp.110.165191


Accepted Article

Miura, K., Rus, A., Sharkhuu, A., et al. (2005) The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses. Proc. Natl. Acad.Sci. USA, 102, 7760-7765. DOI: 10.1073/pnas.0500778102 Mollier, A. and Pellerin, S. (1999) Maize root system growth and development as influenced by phosphorus deficiency. J. Exp. Bot. 50, 487-497. DOI: 10.1093/jxb/50.333.487 Muller, M. and Schmidt, W. (2004) Environmentally induced plasticity of root hair development in Arabidopsis. Plant Physiol. 134, 409-419. DOI: 10.1104/pp.103.029066 Munnik, T. and Nielsen, E. (2011) Green light for polyphosphoinositide signals in plants. Curr. Opin. Plant Biol. 14, 489-497. DOI: 10.1016/j.pbi.2011.06.007 Nacry, P., Canivenc, G., Muller, B., Azmi, A., Onckelen, H.V., Rossignol, M. and Doumas, P. (2005) A role for auxin redistribution in the responses of the root system architecture to phosphate starvation in Arabidopsis. Plant Physiol. 138, 2061-2074. DOI: 10.1104/pp.105.060061 Niu, Y.F., Chai, R.S., Jin, G.L., Wang, H., Tang, C.X. and Zhang, Y.S. (2013) Responses of root architecture development to low phosphorus availability: a review. Ann. Bot. 112, 391-408. DOI: 10.1093/aob/mcs285


Accepted Article

Osmont, K.S., Sibout, R. and Hardtke, C.S. (2007) Hidden branches: developments in root system architecture. Annu. Rev. Plant Biol. 58, 93-113. DOI: 10.1146/annurev.arplant.58.032806.104006 Peret, B., Clement, M., Nussaume, L. and Desnos, T. (2011) Root developmental adaptation to phosphate starvation: better safe than sorry. Trends Plant Sci. 16, 442-450. DOI: 10.1016/j.tplants.2011.05.006 Peret, B., Desnos, T., Jost, R., Kanno, S., Berkowitz, O., and Nussaume, L. (2014) Root architecture responses: in search for phosphate. Plant Physiol. (in press) DOI: 10.1104/pp.114.244541 Perez-Torres, C.A., Lopez-Bucio, J., Cruz-Ramirez, A., Ibarra-Laclette, E., Dharmasiri, A., Estelle, M. and Herrera-Estrella, L. (2008) Phosphate availability alters lateral root development in Arabidopsis by modulating auxin sensitivity via a mechanism involving the TIR auxin receptor. Plant Cell, 20, 3258-3272. DOI: 10.1105/tpc.108.058719 Puga, M.I., Mateos, I., Charukesi, R., Wang, Z., Franco-Zorrilla, J.M., de Lorenzo, L., Irigoyen, M.L., Masiero, S., Bustos, R., Rodriguez, J., Leyva, A., Rubio, V., Sommer, H., and Paz-Ares, J. (2014) SPX1 is a phosphate-dependent inhibitor of PHOSPHATE STARVATION RESPONSE 1 in Arabidopsis. Proc. Natl. Acad. Sci. USA, 111, 14947-14952. DOI: 10.1073/pnas.1404654111


Accepted Article

Reymond, M., Svistoonoff, S., Loudet, O. and Desnos, T. (2006) Identification of QTL controlling root growth response to phosphate starvation in Arabidopsis thaliana. Plant Cell Envir. 29, 115-125. DOI: 10.1111/j.1365-3040.2005.01405.x Rouached, H., Arpat, B. and Poirier, Y. (2010) Regulation of phosphate starvation responses in plants: signaling players and cross-talks. Mol. Plant 3, 288-299. DOI: 10.1093/mp/ssp120 Rubio, V., Linhares, F., Solano, R., Martín, A.C., Iglesias, J., Leyva, A. and Paz-Ares, J. (2001) A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes Dev. 15, 2122-2133. DOI: 10.1101/gad.204401 Saarikangas, J., Zhao, H. and Lappalainen, P. (2010) Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides. Physiol. Rev. 90, 259-289. DOI: 10.1152/physrev.00036.2009. Saavedra, L., Mikami, K., Malho, R., and Sommarin, M. (2012) PIP kinases and their role in plant tip growing cells. Plant Signal. Behav. 7, 1302-1305. DOI: 10.4161/psb.21547 Sanchez-Calderon, L., Lopez-Bucio, J., Chacon-Lopez, A., Cruz-Ramirez, A., Nieto-Jacobo, F., Dubrovsky, J.G. and Herrera-Estrella, L. (2005) Phosphate starvation induces a determinate developmental program in the roots of Arabidopsis thaliana. Plant Cell Physiol. 46, 174-184. DOI: 10.1093/pcp/pci011


Accepted Article

Sanchez-Calderon, L., Lopez-Bucio, J., Chacon-Lopez, A., Gutierrez-Ortega, A., Hernandez-Abreu, E. and Herrera-Estrella, L. (2006) Characterization of low phosphorus insensitive mutants reveals a crosstalk between low phosphorus-induced determinate root development and the activation of genes involved in the adaptation of Arabidopsis to phosphorus deficiency. Plant Physiol. 140, 879-889. DOI: 10.1104/pp.105.073825 Santarius, M., Lee, C.H. and Anderson, R.A. (2006) Supervised membrane swimming: small G-protein lifeguards regulate PIPK signalling and monitor intracellular PtdIns(4,5)P2 pools. Biochem. J. 398, 1-13. DOI: 10.1042/BJ20060565 Shimizu, A., Yanagihara, S., Kawasaki, S. and Ikehashi, H. (2004) Phosphorus deficiency-induced root elongation and its QTL in rice (Oryza sativa L.). Theor. Appl. Genet. 109, 1361-1368. DOI: 10.1007/s00122-004-1751-4 Smith, L.G. and Oppenheimer, D.G. (2005) Spatial control of cell expansion by the plant cytoskeleton. Annu. Rev. Cell Dev. Biol. 21, 271-295. DOI: 0.1146/annurev.cellbio.21.122303.114901 Sousa, E., Kost, B., and Malho, R. (2008) Arabidopsis phosphatidylinositol-4monophosphate 5-kinase 4 regulates pollen tube growth and polarity by modulating membrane recycling. Plant Cell, 20, 3050-3064. DOI: 10.1105/tpc.108.058826 Stenzel, I., Ischebeck, T., Konig, S., Hołubowska, A., Sporysz, M., Hause, B. and Heilmann, I. (2008) The type B phosphatidylinositol-4-phosphate 5-kinase 3 is


Accepted Article

essential for root hair formation in Arabidopsis thaliana. Plant Cell, 20, 124-141. DOI: 10.1105/tpc.107.052852 Svistoonoff ,S., Creff, A., Reymond, M., Sigoillot-Claude, C., Ricaud, L., Blauchet, A., Nussaume, L. and Desnos, T. (2007) Root tip contact with low-phosphate media reprograms plant root architecture. Nature genet. 39, 792-796. DOI: 10.1038/ng2041 Taniguchi, Y.Y., Taniguchi, M., Tsuge, T., Oka, A. and Aoyama, T. (2010) Involvement of Arabidopsis thaliana phospholipase Dζ2 in root hydrotropism through the suppression of root gravitropism. Planta, 231, 491-497. DOI: 10.1007/s00425-009-1052-x

Thibaud, M.C., Arrighi, J.F., Bayle, V., Chiarenza, S., Creff, A., Bustos, R., Paz-Ares, J., Poirier, Y. and Nussaume, L. (2010) Dissection of local and systemic transcriptional responses to phosphate starvation in Arabidopsis. Plant J. 64, 775-789. DOI: 10.1111/j.1365-313X.2010.04375.x Ticconi, C.A. and Abel, S. (2004) Short on phosphate: plant surveillance and countermeasures. Trends Plant Sci. 9, 548-555. DOI: 10.1016/j.tplants.2004.09.003 Ticconi, C.A., Delatorre, C.A., Lahner, B., Salt, D.E. and Abel, S. (2004) Arabidopsis pdr2 reveals a phosphate-sensitive checkpoint in root development. Plant J. 37, 801-814. DOI: 10.1111/j.1365-313X.2003.02005.x Ticconi, C.A., Lucero, R.D., Sakhonwasee, S., Adamson, A.W., Creff, A., Nussaume, L., Desnos, T. and Abel, S. (2009) ER-resident proteins PDR2 and


Accepted Article

LPR1 mediate the developmental response of root meristems to phosphate availability. Proc. Natl. Acad. Sci. USA, 106, 14174-14179. DOI: 10.1073/pnas.0901778106 Tominaga-Wada, R., Ishida, T. and Wada, T. (2011) New insights into the mechanism of development of Arabidopsis root hairs and trichomes. Int. Rev. Cell Mol. Biol. 286, 67-106. DOI: 10.1016/B978-0-12-385859-7.00002-1 van Leeuwen, W., Vermeer, J.E.M., Gadella, T.W.J. and Munnik, T. (2007) Visualization of phosphatidylinositol 4,5-bisphosphate in the plasma membrane of suspension-cultured tobacco BY-2 cells and whole Arabidopsis seedlings. Plant J. 52, 1014-1026. DOI: 10.1111/j.1365-313X.2007.03292.x Vance, C.P., Uhde-Stone, C. and Allan, D.L. (2003) Phosphorus acquisition and use : critical adaptations by plants for securing a nonrenewable resource. New Phytol. 157, 423-447. DOI: 10.1046/j.1469-8137.2003.00695.x Vincent, P., Chua, M., Nogue, F., Fairbrother, A., Mekeel, H., Xu, Y., Allen, N., Bibikova, T.N., Gilroy, S. and Bankaitis, V.A. (2005) A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs. J. Cell Biol. 168, 801-812. DOI: 10.1083/jcb.200412074 Ward, J.T., Lahner, B., Yakubova, E., Salt, D.E. and Raghothama, K.G. (2008) The effect of iron on the primary root elongation of Arabidopsis during phosphate deficiency. Plant Physiol. 147, 1181-1191. DOI: 10.1104/pp.108.118562


Accepted Article

Williamsons, L.C., Ribrioux, S.P.C.P., Fitter, A.H. and Leyser, H.M.O. (2001) Phosphate availability regulates root system architecture in Arabidopsis. Plant Physiol. 126, 875-882. DOI:10.1104/pp.126.2.875 Xue, H.W., Chen, X. and Mei, Y. (2009) Function and regulation of phospholipid signalling in plants. Biochem. J. 421, 145-156. DOI: 10.1042/BJ20090300 Yi, K., Menand, B., Bell, E. and Dolan, L. (2010) A basic helix-loop-helix transcription factor controls cell growth and size in root hairs. Nature genet. 42, 1-6. DOI: 10.1038/ng.529 Zhang, Y.J., Lynch, J.P. and Brown, K.M. (2003) Ethylene and phosphorus availability have interacting yet distinct effects on root hair development. J. Exp. Bot. 54, 2351-2361. DOI: 10.1093/jxb/erg250

FIGURE LEGENDS

Figure 1. Wild-type seedlings grown for 3- and 7-DAG under the Pi-sufficient and deficient conditions.

(a-c) Total plants (a), areal parts (b), and roots (c) of 3- and 7-DAG wild-type seedlings grown under the 1 mM and 3 μM Pi conditions are shown. (d) Root hairs on 3- and 7-DAG wild-type roots grown under the 1 mM (red) and 3 μM (blue) Pi conditions were measured, and their


Accepted Article

lengths (in the y-axis) are plotted against their distances from the root tip (in the x-axis). Bar = 5 mm (a), 1 mm (b), and 0.5 mm (c).

Figure 2. Expression of PIP5K genes in roots of 3-DAG wild-type seedlings grown under the Pi-sufficient and deficient conditions.

(a) Transcript levels of PIP5K genes and the IPS1 gene in roots of 3-DAG wild-type seedlings were determined by RT-PCR. Relative transcript levels (mean + S.D., n = 3) under the 1 mM (white box) and 3 μM (gray box) Pi conditions are shown with the mean value of PIP5K3 under the 1 mM Pi condition arbitrarily set as 1. Asterisks indicate significant increases in the level compared with that of the 1 mM Pi condition (p < 0.02, Student’s t-test). (b) Promoter activities of the upstream intergenic regions of PIP5K3 (PIP5K3p-GUS) and PIP5K4 (PIP5K4p-GUS) were histochemically analyzed in roots of 3-DAG seedlings grown under the 1 mM and 3 μM Pi conditions using a GUS reporter gene. Bar = 0.5 mm.

Figure 3. Contribution of the PHR1 gene function to the Pi-deficiency response in transcript levels of the PIP5K3 and PIP5K4 genes.

Transcript levels of the PIP5K3, PIP5K4, and IPS1 genes in roots of 3-DAG wild-type (wt) and phr1 seedlings were determined by RT-PCR. Relative transcript levels (mean + S.D., n = 3) under the 1 mM (white box) and 3 μM (gray box) Pi conditions are shown with the mean value under the 1 mM Pi condition arbitrarily set as 1. Asterisks indicate significant increases in the level compared with that under the 1 mM Pi condition (p < 0.02, Student’s t-test).


Accepted Article

Figure 4. Root hair lengths of 3-DAG wild-type and mutant seedlings grown under the Pi-sufficient and deficient conditions.

(a) Root hairs of 3-DAG wild-type (wt), phr1, pip5k3, pip5k4, and pip5k3pip5k4 seedlings (five or more for each) were measured. Root hair lengths (mean + S.D., n=100) under the 1 mM (white box) and 3 μM (gray box) Pi conditions are shown. Asterisks indicate significant increases in the length compared with that under the 1 mM Pi condition (p < 0.01, ANOVA and Tukey’s HSD test). Hashes indicate significant decreases in the length compared with that of the wild type under the same Pi condition (p < 0.01, ANOVA and Tukey’s HSD test). Crosses indicate significant decreases in the length compared with those of both pip5k3 and pip5k4 mutants under the same Pi condition (p < 0.01, ANOVA and Tukey’s HSD test). (b) Roots of 3-DAG wild-type (wt) and mutant seedlings grown under the 1 mM and 3 μM Pi conditions are shown. Bar = 0.2 mm.

Figure 5. Root hair lengths of 3-DAG total and tactical complementation seedlings grown under Pi-sufficient and deficient conditions.

(a) The structures of the genes introduced for total and tactical complementation of the pip5k3 and pip5k4 mutants are shown schematically. Boxes and lines correspond to coding and non-coding regions, respectively. Vertical arrows indicate the sites of T-DNA insertions in pip5k3 (SALK_000024) and pip5k4 (SALK_001138). The sites and sequences of P1BSs and altered P1BSs are shown with substituted bases underlined in altered P1BSs.


(b) Root hairs of

Accepted Article

3-DAG seedlings of the wild-type (wt), and total (pip5k3/PIP5K3g, pip5k4/PIP5K4g, and pip5k3pip5k4/PIP5K3gPIP5K4g) and tactical (pip5k3/pip5k3gmp, pip5k4/pip5k4gmp, and pip5k3pip5k4/pip5k3gmppip5k4gmp) complementation lines (five or more seedlings for each) were measured. Root hair lengths (mean + S.D., n = 100) under the 1 mM (white box) and 3 μM (gray box) Pi conditions are shown. Asterisks indicate significant increases in the length compared with that under the 1 mM Pi condition (p < 0.01, ANOVA and Tukey’s HSD test). Hashes indicate significant decreases in the length compared with that of each corresponding total complementation line under the 3 μM Pi condition (p < 0.01, ANOVA and Tukey’s HSD test). (c) Roots of 3-DAG wild-type (wt), and total and tactical complementation seedlings grown under the 1 mM and 3 μM Pi conditions are shown. Bar = 0.2 mm.

Figure 6. Pi-deficiency responses in transcript levels of the PIP5K3 and PIP5K4 genes, and the introduced genes for total and tactical complementation in roots of 3- and 7-DAG seedlings.

Transcript levels of the PIP5K3 and PIP5K4 genes in roots of 3- and 7-DAG wild-type (wt) seedlings, the PIP5K3g and PIP5K4g genes in roots of 3- and 7-DAG pip5k3pip5k4/PIP5K3gPIP5K4g seedlings, and the pip5k3gmp and pip5k4gmp genes in roots of 3- and 7-DAG pip5k3pip5k4/pip5k3gmppip5k4gmp seedlings were determined by RT-PCR. Relative transcript levels (mean + S.D., n = 3) under the 1 mM (white box) and 3 μM (gray box) Pi conditions are shown with the mean value under the 1 mM Pi condition arbitrarily set as 1.


Accepted Article

Asterisks indicate significant increases in the level compared with that under the 1 mM Pi condition (p < 0.02, Student’s t-test).

Figure 7. Root hair lengths of wild-type, pip5k3pip5k4 double mutant, and total and tactical double complementation seedlings grown under prolonged Pi-deficient conditions after germination

(a) Root hairs of 3-, 5-, 7-, and 10-DAG wild-type (wt), pip5k3pip5k4, pip5k3pip5k4/PIP5K3gPIP5K4g, and pip5k3pip5k4/pip5k3gmppip5k4gmp seedlings (three or more for each) were measured. Root hair lengths (mean + S.D., n = 50) under the 1 mM (white box) and 3 μM (gray box) Pi conditions are shown. Asterisks indicate significant increases in the length compared with that under the 1 mM Pi condition (p < 0.05, ANOVA and Tukey’s HSD test). (b) Roots of 7-DAG wild-type (wt), pip5k3pip5k4, pip5k3pip5k4/PIP5K3gPIP5K4g, and pip5k3pip5k4/pip5k3gmppip5k4gmp seedlings grown under the 1 mM and 3 μM Pi conditions are shown. Bar = 0.2 mm.









Uncovering genes and ploidy involved in the high diversity in root hair density, length and response to local scarce phosphate in Arabidopsis thaliana.

Phosphatidylinositol 4-phosphate negatively regulates chloroplast division in Arabidopsis.

Arabidopsis PHOSPHATE TRANSPORTER1 genes PHT1;8 and PHT1;9 are involved in root-to-shoot translocation of orthophosphate.

Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis.

Multiple phytohormones promote root hair elongation by regulating a similar set of genes in the root epidermis in Arabidopsis.

Differentiating phosphate-dependent and phosphate-independent systemic phosphate-starvation response networks in Arabidopsis thaliana through the application of phosphite.

AtOPR3 specifically inhibits primary root growth in Arabidopsis under phosphate deficiency.

Distinct sensitivities to phosphate deprivation suggest that RGF peptides play disparate roles in Arabidopsis thaliana root development.

phosphate translocator GPT2 directs metabolic acclimation.

A vacuolar phosphate transporter essential for phosphate homeostasis in Arabidopsis.

Arabidopsis thaliana RALF1 opposes brassinosteroid effects on root cell elongation and lateral root formation.

Exposure of iron nanoparticles to Arabidopsis thaliana enhances root elongation by triggering cell wall loosening.

SABRE is required for stabilization of root hair patterning in Arabidopsis thaliana.

Recent Advances in Understanding the Molecular Mechanisms Regulating the Root System Response to Phosphate Deficiency in Arabidopsis.

The histone deacetylase HDA19 controls root cell elongation and modulates a subset of phosphate starvation responses in Arabidopsis.

Systematic characterization of novel lncRNAs responding to phosphate starvation in Arabidopsis thaliana.

Formation of phosphatidylinositol 3-phosphate by isomerization from phosphatidylinositol 4-phosphate.

Abscisic acid regulates root elongation through the activities of auxin and ethylene in Arabidopsis thaliana.

SHR pathway in Arabidopsis thaliana.

Natural variations in expression of regulatory and detoxification related genes under limiting phosphate and arsenate stress in Arabidopsis thaliana.

Low phosphate activates STOP1-ALMT1 to rapidly inhibit root cell elongation.

Differential induction of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase genes in Arabidopsis thaliana by wounding and pathogenic attack.

HDG11 upregulates cell-wall-loosening protein genes to promote root elongation in Arabidopsis.

Increased root hair density by loss of WRKY6 in Arabidopsis thaliana.