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Plant Signal Behav. Aug 2009; 4(8): 781–783.
PMCID: PMC2801399
Low phosphate signaling induces changes in cell cycle gene expression by increasing auxin sensitivity in the Arabidopsis root system
Claudia Anahí Pérez Torres,1 José López Bucio,2 and Luis Herrera Estrellacorresponding author1
1Laboratorio Nacional de Genómica para la Biodiversidad; Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; Irapuato, Guanajuato México
2Instituto de Investigaciones Químico-Biológicas; Universidad Michoacana de San Nicolás de Hidalgo; Morelia, Michoacán México
corresponding authorCorresponding author.
Correspondence to: Luis Herrera Estrella; Laboratorio Nacional de Genómica para la Biodiversidad; Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; 36821 Irapuato; Guanajuato, Mexico; Email: lherrera/at/ira.cinvestav.mx
Received June 4, 2009; Accepted June 8, 2009.
Lateral root development is an important morphogenetic process in plants, which allows the modulation root architecture and substantially determines the plant's efficiency for water and nutrient uptake. Postembryonic root development is under the control of both endogenous developmental programs and environmental stimuli. Nutrient availability plays a major role among environmental signals that modulate root development. Phosphate (Pi) limitation is a constraint for plant growth in many natural and agricultural ecosystems. Plants posses Pi-sensing mechanisms that enable them to respond and adapt to conditions of limited Pi supply, including increased formation and growth of lateral roots. Root developmental modifications are mainly mediated by the plant hormone auxin. Recently we showed that the alteration of root system architecture under Pi-starvation may be mediated by modifications in auxin sensitivity in root cells via a mechanism involving the TIR1 auxin receptor. In this addendum, we provide additional novel evidence indicating that the low Pi pathway involves changes in cell cycle gene expression. It was found that Pi deprivation increases the expression of CDKA, E2Fa, Dp-E2F and CyCD3. In particular, E2Fa, Dp-E2F and CyCD3 genes were specifically upregulated by auxin in Pi-deprived Arabidopsis seedlings that were treated with the auxin transport inhibitor NPA, indicating that cell cycle modulation by low Pi signaling is independent of auxin transport and dependent on auxin sensitivity in the root.
Key words: phosphate signaling, auxin transport, auxin sensitivity, roots
Phosphorus (Pi) is an essential macronutrient for numerous metabolic and developmental processes in plants. In most natural soils, Pi is often limited due to its strong affinity for cations such as Ca, Mg and Fe, and because its rapid conversion to organic forms that are not readily available for plant uptake.1 Pi deprivation results in adaptive morphological modifications such as altered root architecture that helps the plant to explore greater soil volumes for Pi acquisition.2 Important factors in root system remodeling under low Pi conditions include increased lateral root formation and root hair proliferation, which are related to the entrance of the primary root into a determinate program of growth, in which cell division is arrested and cell differentiation promoted at the root tip.3 This particular developmental program enhances the expression of genes involved in the low-Pi rescue response in the differentiating root regions as revealed by the transcriptional activation of Pi-responsive genes such as those encoding high affinity Pi transporters and phosphatases, which directly participates in Pi uptake from the soil.3
Auxin plays an important role in modulating root system architecture. Many previous studies have shown that application of exogenous auxin (IAA) increases the number of LRs.4,5 In contrast, treatment with auxin transport inhibitors such as NPA (N-1-naphthylphthalamic acid) decreases the number of LRs.6,7 Recent molecular genetic studies using Arabidopsis mutants have provided considerable information on the role of auxin biosynthesis, homeostasis, transport and signaling regulating root morphogenetic processes.810 It has been found that LR initiation and subsequent LR primordium development require both auxin transport and signaling. At low concentrations of auxin, AUX/IAA repressors inhibit the activity of AUXIN RESPONSE FACTORS (ARFs) transcription factors. When auxin concentrations exceed a certain threshold, the interaction between AUX/IAA proteins and the SCFTIR1/AFB1–3 ubiquitine ligase is promoted, thus triggering the destruction of AUX/IAA proteins by the proteosome. ARFs are then free to regulate the expression of auxin-responsive genes, such as those involved in LR formation.11
In a recent work, we investigated the role of various components of the auxin signaling pathway in root system architecture adjustment during Pi-deprivation in Arabidopsis. It was found that roots of Pi-deprived seedlings were resistant to the inhibitory effects of the auxin transport inhibitor NPA on LR formation. In addition, seedlings grown under low Pi conditions that were transferred to medium containing NPA and auxin produced more LR primordia and LRs than seedlings grown under sufficient Pi conditions transferred to media with the same concentration of NPA and auxin. These results showed that low Pi signaling makes the pericycle cells more sensitive to auxin, thus resulting in an increased LR proliferation and the formation of more branched root systems.
Since the TIR1 auxin receptor is a central player in auxin perception, it was investigated whether TIR1 was responsible for the increased auxin sensitivity of Pi-deprived seedlings. Kinetic studies of TIR1:uidA expression and qRT-PCR analysis showed that TIR1 is specifically induced in response to Pi-deprivation. By examining the root growth responses of mutants defective on the TIR1/AFB1–3 family of auxin receptors under contrasting Pi availability, it was found that these genes play partially redundant roles in LR formation in response to Pi-deprivation. Interestingly, transgenic plants that overexpress TIR1 grown under Pi-sufficient conditions have a phenotype similar to that observed in Pi-deprived WT seedlings, confirming that TIR1 is indeed a limiting factor in determining auxin sensitivity and LR formation, and that small changes in its transcription level can have profound effects on root system architecture. Based on this information, it was proposed that auxin sensitivity in pericycle cells is enhanced in Pi-deprived seedlings due to the increased expression of TIR1. This in turn causes the degradation of AUX/IAA proteins, which liberates ARF transcription factors to activate the expression of genes involved in LR formation.
In Arabidopsis it has been shown that auxins regulate cell cycle gene expression during the formation of lateral roots.12,13 To elucidate whether the expression of cell cycle genes induced by auxin during the formation of LR is modified in response to Pi deficiency, we performed expression analysis of CDKA, CyCD3, E2Fa and DP-E2F genes by real-time PCR in seedlings grown on sufficient and low levels of Pi.
The expression of CDKA, E2Fa, Dp-E2F genes increased by 100% in seedlings grown under Pi deficiency, while for CyCD3 the increase in the expression was about 200% (Fig. 1). This suggests that in low Pi conditions there is an activation of cell cycle genes, resulting in an increase in cell proliferation and consequently a greater number of LRs.
Figure 1
Figure 1
Effect of Pi availability on cell cycle gene expression. Real time-PCR analysis of CDKA, E2Fa, Dp-E2F and CyCD3. Gray bars represent growth in P+, and black bars represent growth in P. Different letters are used to indicate means that differ (more ...)
To understand the cell cycle regulation mechanisms that result in the initiation of new lateral roots, detailed molecular studies are required. However, the small number of cells involved in the first lateral root initiation events seriously hampers such studies.14 Also, the lack of synchrony of the initiation events makes it very difficult to efficiently follow the development of lateral root initiation. Analyzing cell cycle progression during pericycle activation,12 established an LR inducible system in which the Arabidopsis seedlings were grown on NPA-containing medium for 72 hours after germination are transferred onto NAA-containing medium, the pericycle cells of these seedlings start synchronously to divide. This allows changes in the expression of cell cycle related genes during auxin-induced LR initiation to be monitored with the use of the marker lines and quantitative RT-PCR.12 Using this system, we evaluated the expression of cell cycle genes involved in lateral root formation. Under P+ conditions we observed that the expression of cell cycle genes is reduced 12 h after transfer to media lacking NPA and that the expression of E2Fa and Dp-E2F was slightly increase by auxin treatment (Fig. 2). In contrast, Pi deprived plants the expression cell cycle genes was slightly higher after 12 h of transfer to media lacking NPA and significantly induced by auxin treatment (Fig. 2). These results show that phosphorus deficiency induced the expression of cell cycle genes involved in the formation of RL and confirm that the roots of these plants are more sensitive to the addition of exogenous auxin (Fig. 2).
Figure 2
Figure 2
Expression pattern of cell cycle genes involved in lateral root formation in an inducible system. Wild type seedlings (Col-0) were grown for 3 days in media with NPA (1 µM) and transferred to media without NPA (with or without 10 µM NAA) (more ...)
In the referenced study, we propose a model for the increased formation of LRs under Pi-deprived conditions in which an increased expression of TIR1 in pericycle cells leads to a higher degradation rate of AUX/IAA repressors. The increase in AUX/IAA degradation under Pi deficiency allows ARF19 and probably other ARFs already linked in the AuxREs to activate auxin-responsive cell cycle, thus promoting cell division in the pericycle. This response to Pi deficiency would make pericycle cells more sensitive to auxins leading to the activation of cell cycle genes, which in turn activate the formation of additional lateral root primordia. This molecular framework is an attractive model to explain cell cycle regulation mechanism in the lateral root formation modulated by environmental signals during postembryonic developmental processes in plants.
Footnotes
Previously published online as a Plant Signaling & Behavior E-publication: http://www.landesbioscience.com/journals/psb/article/9230
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