COPLBI-558; NO OF PAGES 5 Available online at www.sciencedirect.com Phosphate starvation signaling: a threesome controls systemic Pi homeostasis Peter Doerner Systemic signaling between roots and shoots is required to maintain mineral nutrient homeostasis for optimal metabolism under varying environmental conditions. Recent work has revealed molecular components of a signaling module that controls systemic phosphate homeostasis, modulates uptake and transport in Arabidopsis. This module comprises PHO2, a protein that controls protein stability, the phloem-mobile microRNA-399 and a ribo-regulator that squelches the activity of miR399 towards PHO2 by a novel mechanism. This advance is a significant step for the design of future rational approaches to improve crop phosphate use efficiency. Addresses Institute of Molecular Plant Science, School of Biological Sciences, Daniel Rutherford Building, King’s Buildings, University of Edinburgh, Edinburgh EH9 3JH, Scotland, United Kingdom Corresponding author: Doerner, Peter ([email protected]) Long-distance and systemic signaling When roots grow into a low Pi patch, or the root system overall encounters Pi-deficient conditions, Pi must be provided to sustain meristematic and physiological activities in this organ. This is because plants can only acquire new Pi resources by growth that brings roots into contact with previously unexploited soil. In the metabolically highly active cells of the root apex, Pi levels must be maintained at high levels. Steady state cytoplasmic concentrations between 5 and 15 mM have been reported [3]. This is not primarily because large amounts of Pi are consumed, i.e. incorporated into newly synthesized DNA, RNA and membranes; but rather because Pi functions analogously to a currency, and is required for most biochemical transactions in the cell to proceed. During the early stages of an episode of Pi starvation, this demand is satisfied by Pi translocation from shoot tissues via the phloem. Current Opinion in Plant Biology 2008, 11:1–5 This review comes from a themed issue on Cell signalling and Gene regulation Edited by Jason Reed and Bonnie Bartel 1369-5266/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2008.05.006 Introduction Phosphate [Pi] is an essential macronutrient for plants that is generally in short supply as it is difficult to assimilate. Pi is distributed very heterogeneously in the soil matrix, which conditions specific behavioral patterns for root growth. These have been recently reviewed [1] and will not be discussed in detail here. Phosphate distribution within the plant is also heterogeneous at different scales and follows cellular patterns of physiological demand; as a generalization, under Pi replete conditions: shoot tissues accumulate more Pi than root tissues, metabolically active cells more than less active cells, and the vacuole and plastids more than the cytoplasm [2,3]. Such non-uniform distribution requires extensive and sophisticated regulatory mechanisms that allow plants to maintain Pi homeostasis required for efficient metabolism, specifically in non-equilibrium conditions when acquisition rates vary. This review will focus on recent advances in our understanding of systemic Piresponsive signaling mechanisms and highlight some of the major unresolved questions. www.sciencedirect.com Precise regulation of net Pi flux from shoot to root in these conditions is critical as careful budgeting of limiting resources has consequences for competitive success vis a` vis other plants competing for the same limiting resources, and overall fitness to permit completion of the individual’s lifecycle. Recent work has uncovered a regulatory module involved in the systemic control of Pi allocation (Figure 1) that hard-wires this cost–benefit analysis. A family of phloem-mobile microRNAs (miR399a-f), their target gene PHO2, and a family of regulatory, non-coding RNAs, the IPS/At4-like genes, form a circuit in which the balance of microRNA and ribo-regulator abundance fine-tune PHO2 activity. Regulated proteolysis in Pi signaling Phosphate 2 (PHO2) was first identified by mutational approaches, by virtue of the phenotype of the pho2 mutant, which overaccumulates Pi in shoots of plants grown in Pi-replete conditions [4]. PHO2 encodes an E2 ubiquitin conjugase-related enzyme (UBC24), and is expressed in shoot and root tissues [5,6]. In shoots, histochemical analysis indicated expression in leaf vasculature, and in roots, expression was observed in all vascular cell types, except the mature xylem [5]. PHO2 is thus the second genetic function involved in responses to Pi-starvation that is mechanistically linked to ubiquitin or similar modification systems. The SIZ1 gene, shown earlier to modify the putative transcription factor Phosphate starvation Response 1 (PHR1) in vitro, encodes a SUMO E3 ligase [7]. However, SIZ1 is not specific for Pi Current Opinion in Plant Biology 2008, 11:1–5 Please cite this article in press as: Doerner P, Phosphate starvation signaling: a threesome controls systemic Pi homeostasis, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.05.006 COPLBI-558; NO OF PAGES 5 2 Cell signalling and Gene regulation signaling, as it is involved in regulating responses to many types of abiotic stress. Reciprocal grafting studies showed that a pho2 genotype in roots is sufficient to mimic the whole plant pho2 Figure 1 phenotype [6], suggesting that UBC24 primarily acts in root tissues. When phosphate is available, low PHO2 activity leads to increased expression of two rootexpressed phosphate transporter genes, Pht1;8 and Pht1;9 [5,6] and also of at least two members of the IPS/At4 gene family [6]. Overexpression of the two high-affinity transporter genes is most likely responsible for most of the pho2 phenotype, as RNAi-mediated inhibition of their expression in the pho2 background suppresses the accumulation of excess shoot phosphate [6]. It appears likely that UBC24 protein functions by targeting genes involved in controlling the expression of the two transporters and other affected genes for degradation. Negative control of Pi-responsive gene expression has previously been reported [8], and could involve the degradation of a transcriptional repressor or, alternatively, control the stability of a transcriptional activator for Pht1;8 and Pht1;9 expression in the presence of Pi. The mechanisms by which UBC24 affects systemic Pi distribution remain to be elucidated in detail. microRNA 399 as long-distance signal Several microRNAs have recently been shown to be involved in regulating plant mineral nutrient homeostasis. These include: miR395, which is regulated by sulfur [9]; miR398, regulated by copper [10], which may also play an indirect role in the growth response to perceived Pi limitation, as a multi-copper oxidase plays a critical role in this response in Arabidopsis [11]; and miR399, which is regulated by phosphate [12]. Schematic illustration of the regulatory module that controls systemic Pi homeostasis. The regulatory module, comprising PHO2, miR399 and IPS/At4 genes controls the activity of PHO2 in roots to regulate the allocation of Pi to the shoot. High PHO2 activity (1) which occurs when the plant perceives adequate Pi supply, results in low levels of Pht1;8 and Pht1;9 phosphate transporter expression. Likewise, levels of miR399 expression (2) and IPS/At4 (3) expression are low in high phosphate, but in contrast to miR399, IPS/At4 expression is suppressed further by UBC24 activity in the presence of high Pi [6]. This keeps the regulatory module in a highly Pi-limitation responsive state. In a high Pi environment, phosphate is transported to mature and young leaves. When the plant, specifically the shoot, experiences Pi limitation, miR399 expression is strongly stimulated, particularly in shoot tissues (4). Mature miR399 is then translocated via the phloem to the root system. Here (5), binding of cognate sites in the 50 UTR of PHO2 transcripts to miR399charged silencing complexes leads to the degradation of PHO2 mRNA, resulting in low UBC24 protein levels. This leads to increased Pht1;8 and Pht1;9 expression, facilitating increased Pi uptake and transport to the shoot. In low phosphate, Pi in shoot tissues is mobilized from mature leaves to young leaves (4) and also to roots to sustain root meristem activity for the longest time possible. As the Pi starvation response progresses, expression of IPS/At4 is induced. These ribo-regulators (5) inhibit the action of miR399-charged silencing complexes on PHO2 mRNA, and thus allow UBC24 levels to adjust to the dynamic balance of supply and demand in the plant more rapidly. Current Opinion in Plant Biology 2008, 11:1–5 miR399 is strongly and specifically induced by perceived Pi limitation with parallel >1000-fold induction of the primary transcript and mature miR399d and miR399f steady state levels [5,6,12,13]. miR399 is expressed in vascular tissues, specifically in companion cells and phloem [5]. PHO2 has an unusually long 50 UTR that contains five target sites for miR399 [6,12,14], and miR399 binding to these sites leads to scission of the PHO2 transcript [15]. A further predicted, but not yet validated, miR399 target is the Pi transporter, Pht1;7, which is expressed in roots and floral tissues [16]. Forced expression of miR399 phenocopies the PHO2 loss-offunction phenotype of elevated shoot Pi accumulation [6,14], suggesting that PHO2 is the primary target of the miR399 family. A recent study reported key observations that indicate how miR399 functions in systemic signaling: shootexpressed miR399 is translocated to the root, miR399 is present in phloem exudates, and high levels of shoot miR399 expression suffice to repress PHO2 in roots [13]. Together, these properties strongly indicate that miR399 functions as systemic signal to inform root cells of the perceived state of Pi homeostasis in the shoot by controlling PHO2 levels in the root. www.sciencedirect.com Please cite this article in press as: Doerner P, Phosphate starvation signaling: a threesome controls systemic Pi homeostasis, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.05.006 COPLBI-558; NO OF PAGES 5 Phosphate starvation signaling Doerner 3 A role for non-coding RNAs A decade ago, a new class of Pi starvation-induced transcripts was discovered, in tomato and Medicago, with the unusual feature that they did not encode long open reading frames [17,18]. Subsequently, related ‘Induced by Pi Starvation’ (IPS) genes were identified in Arabidopsis [19,20], but their function remained poorly understood. In low-Pi conditions, these genes are expressed in shoot and root tissues, with particularly high levels of expression in mature root vascular tissues ([20,21], Thacker and Doerner, unpublished). Two recent papers have now uncovered exciting functional features of these genes in the regulation of Pi starvation responses: the loss-of-function of one such homolog in Arabidopsis, At4, resulted in altered shoot to root Pi ratios [21], suggesting a possible involvement in the PHO2-miR399 regulatory loop. Strikingly, it was found that a 23 base sequence that is highly conserved in all IPS1-like genes was completely complementary to miRNA399, save for two to three critical bases in the middle [21,22]. This mismatch is precisely where the miRNA guided cleavage reaction occurs, and in plant microRNA targets, these bases are normally completely complementary [23]. Overexpression of IPS1 resulted in higher levels of PHO2 accumulation and lower steadystate shoot Pi levels [22], but interestingly, did not result in degradation of the IPS1 transcript. However, these effects were lost when a modified version of IPS1 was expressed that was entirely complementary to miR399 [22]. This ability to modulate the activity of miR399 suggested a novel regulatory paradigm, for which the term ‘target mimicry’ was coined: an RNA with incomplete central complementarity functions analogously to a competitive enzyme inhibitor, effectively sequestering miR399charged silencing complexes in a state in which they cannot act on PHO2 transcripts [22]. It is not yet clear what the biological significance of this additional level of control is. At4, one member of the IPS family, is induced only gradually and late after onset of perceived Pi starvation (Lai and Doerner, unpublished), raising the possibility that target mimicry functions to dampen oscillations in Pi distribution caused by the antagonism between miR399 and PHO2, to prevent the system from becoming stuck in a detrimental state of long-term low PHO2 and high miR399 activity, or perhaps to de-sensitize the response to limiting Pi in the course of adaptation in which the balance of supply and demand are dynamic [24]. These models are consistent with the observation that the expression of at least two IPS/At4 gene family members is modulated by PHO2 [6]. Evolution of signaling mechanisms It is interesting to consider the evolution of this signaling network: the genomes of single-celled algae www.sciencedirect.com (Chlamydomonas, Ostreococcus) or simple multi-cellular algae (Volvox) lack UBC genes closely-related to PHO2, while the bryophyte (Physcomitrella) and lycophyte (Selaginella) genomes have two or more closely homologous genes (Lai and Doerner, unpublished). However, their function has not yet been characterized and so it is still unclear whether they are involved in controlling Pi homeostasis in basal plants as well. PHO2 homologs have been identified in monocots and eudicots [6], but a clear gymnosperm homolog has not yet been identified. By contrast, there is no evidence for miR399 in bryophytes or lycophytes [25,26,27], but as miR399 is not expressed under all growth conditions, it could have been missed in these studies. However, miR399 has been clearly identified in core and basal eudicots and monocots [28]. IPS1like genes have been identified in core eudicots [17,18,20], but their identification in other species is more challenging, as the conserved sequences are short and they lack a canonical structure, such as the stem-loop found in miRNA precursors, which could be used to identify candidate loci. Together, these observations suggest that the basic function ascribed to PHO2 – the feedback control of Pi assimilation – evolved first, possibly as the result of a selective advantage to prevent toxic effects associated with over-accumulation of Pi [4]. However, as plants evolved vascular systems, and also extended their lifespan, feedback controls restricted to the cellular level limited the latitude with which Pi homeostasis could be regulated for the whole plant. Thus, the evolution of systemic, RNA-based communication such as miR399and IPS1/At4-mediated mechanisms to precisely and differentially regulate Pi homeostasis at the organ level, likely provided huge selective advantages. Conclusions and future challenges Recent advances indicate that control of protein stability by PHO2 plays a pivotal role in regulating systemic Pi homeostasis. Its activity in roots is regulated by the balance of miR399 abundance, which is determined by its systemic, phloem-mediated movement from shoot to root; and the abundance of ribo-regulators of the IPS1/At4 family that antagonize miR399 activity. All members of this regulatory triad are also expressed in shoots and it will be interesting to determine whether they have an immediate function in these tissues as well. Many unresolved questions must be addressed before our knowledge of these regulatory circuits can be put to use to improve crop plant nutrient use efficiency. The main focus will be on the mechanisms that function upstream and downstream of the PHO2 regulatory module. The big prize will go to the answer of the question how Pi is perceived, and how many such mechanisms exist. One or more of these mechanisms are upstream activators of miR399 and IPS1/At4 gene expression. Another important Current Opinion in Plant Biology 2008, 11:1–5 Please cite this article in press as: Doerner P, Phosphate starvation signaling: a threesome controls systemic Pi homeostasis, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.05.006 COPLBI-558; NO OF PAGES 5 4 Cell signalling and Gene regulation gap in our knowledge are the substrates of PHO2, specifically those that mediate Pht1;8 and Pht1;9 over-expression. Furthermore, although we know that miR399 is translocated in the phloem, the mechanisms that determine how it enters the phloem and whether it is transported as a naked RNA, associated with specific carriers or as miR399-primed silencing complex are not yet known. Lastly, we must examine, at the tissue and cellular level, Pi flux in source and sink tissues to better grasp the how the balance of supply and demand is achieved. Acknowledgements I thank Veronique Vitart, Gwyneth Ingram and members of the Doerner lab for critical reading of the manuscript. Research in my lab on phosphate signaling is supported by the Darwin Trust, The Leverhulme Trust, and the Royal Society. References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1. Desnos T: Root branching responses to phosphate and nitrate. Curr Opin Plant Biol 2008, 11:82-87. An excellent recent review on the mechanisms that couple the perception of low Pi and N to adaptive growth behavior. 2. Bieleski R: Phosphate pools, phosphate transport and phosphate availability. Annu Rev Plant Physiol 1973, 24:225-252. 3. Mimura T: Regulation of phosphate transport and homeostasis in plant cells. Int Rev Cytol 1999, 191:149-200. 4. Delhaize E, Randall PJ: Characterization of a phosphateaccumulator mutant of Arabidopsis thaliana. Plant Physiol 1995, 107:207-213. 5. Aung K, Lin SI, Wu CC, Huang YT, Su CL, Chiou TJ: pho2, a phosphate overaccumulator, is caused by a nonsense mutation in a microRNA399 target gene. Plant Physiol 2006, 141:1000-1011. Together with ref. [6], this paper reports that the PHO2 gene encodes UBC24. Over-expression of miR399 re-capitulates the pho2 phenotype, independently confirming results also obtained in ref. [6]. A detailed analysis showed that the greater Pi uptake in pho2 and miR399 overexpressors is owing to an increased Vmax. Their analysis shows that PHO2 and miR399 expression are co-localized in the vascular cylinder. 6. Bari R, Datt Pant B, Stitt M, Scheible WR: PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants. Plant Physiol 2006, 141:988-999. Together with ref. [5], this paper reports the molecular cloning of the PHO2 gene. It is shown by reciprocal crosses that a pho2 phenotype in roots is required to generate the pho2 phenotype of excess Pi accumulation in shoots. It is shown that miR399 induction in response to low Pi is significantly diminished in the phr1 mutant background, indicating that the PHO2/miR399 regulatory module is genetically downstream of this putative transcription factor. Besides extensive micro-array analysis of responses to low Pi in various genetic backgrounds, the paper also shows that PHO2 homologs are present in the genomes of various mono- and dicot species. 7. Miura K, Rus A, Sharkhuu A, Yokoi S, Karthikeyan AS, Raghothama KG, Baek D, Koo YD, Jin JB, Bressan RA et al.: The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses.. Proc Natl Acad Sci U S A 2005, 102:7760-7765. 8. Mukatira UT, Liu C, Varadarajan DK, Raghothama KG: Negative regulation of phosphate starvation-induced genes. Plant Physiol 2001, 127:1854-1862. 9. Jones-Rhoades MW, Bartel DP: Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol Cell 2004, 14:787-799. Current Opinion in Plant Biology 2008, 11:1–5 10. Yamasaki H, Abdel-Ghany SE, Cohu CM, Kobayashi Y, Shikanai T, Pilon M: Regulation of copper homeostasis by micro-RNA in Arabidopsis. J Biol Chem 2007, 282:16369-16378. 11. Svistoonoff S, Creff A, Reymond M, Sigoillot-Claude C, Ricaud L, Blanchet A, Nussaume L, Desnos T: Root tip contact with lowphosphate media reprograms plant root architecture. Nat Genet 2007, 39:792-796. Multi-copper oxidases are identified as crucial components of signaling pathways that couple the perception of limiting Pi to changes in growth behavior in plants that experience Pi starvation. 12. Fujii H, Chiou TJ, Lin SI, Aung K, Zhu JK: A miRNA involved in phosphate-starvation response in Arabidopsis. Curr Biol 2005, 15:2038-2043. The first experimental validation that expression of members of the miR399 family is induced by low Pi. The authors demonstrate that different UBC24 transcript levels in low and high Pi are post-transcriptionally mediated by the binding sites for miR399 in its 50 UTR. This paper also shows that over-expression of miR399 enhances Pi accumulation in transgenic lines. 13. Pant BD, Buhtz A, Kehr J, Scheible WR: MicroRNA399 is a long distance signal for the regulation of plant phosphate homeostasis. Plant J 2008, 53:731-738. The first demonstration that miR399 is phloem-mobile and that this movement is sufficient to reduce PHO2 steady-state mRNA levels in roots and elevate shoot Pi concentration, indicating that miR399 is a critical systemic signal for control of Pi homeostasis. 14. Chiou T-J, Aung K, Lin S-I, Wu C-C, Chiang S-F, Sua C-L: Regulation of phosphate homeostasis by microRNA in Arabidopsis. Plant Cell 2006, 18:412-421. This study, together with refs. [5,6,12] shows that overexpression of miR399 downregulates PHO2 steady state levels and enhances Pi accumulation in shoots 5–6-fold. The authors show that pho2 plants are specifically defective in mobilizing Pi from mature leaves to young and growing leaves. 15. Allen E, Xie Z, Gustafson AM, Carrington JC: microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 2005, 121:207-221. 16. Mudge SR, Rae AL, Diatloff E, Smith FW: Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters in Arabidopsis. Plant J 2002, 31:341-353. 17. Burleigh SH, Harrison MJ: A novel gene whose expression in Medicago truncatula roots is suppressed in response to colonization by vesicular-arbuscular mycorrhizal (VAM) fungi and to phosphate nutrition. Plant Mol Biol 1997, 34:199-208. 18. Liu C, Muchhal US, Raghothama KG: Differential expression of TPS11, a phosphate starvation-induced gene in tomato. Plant Mol Biol 1997, 33:867-874. 19. Burleigh SH, Harrison MJ: The down-regulation of Mt4-like genes by phosphate fertilization occurs systemically and Involves phosphate translocation to the shoots. Plant Physiol 1999, 119:241-248. 20. Martin AC, del Pozo JC, Iglesias J, Rubio V, Solano R, de la Pena A, Leyva A, Paz-Ares J: Influence of cytokinins on the expression of phosphate starvation responsive genes in Arabidopsis. Plant J 2000, 24:559-568. 21. Shin H, Shin H-S, Chen R, Harrison MJ: Loss of At4 function impacts phosphate distribution between the roots and the shoots during phosphate starvation. Plant J 2006, 45:712-726. The first report that IPS/At4 genes are involved in controlling systemic phosphate allocation. at4, a loss-of-function allele of At4, accumulates excess Pi in the shoot. The authors also show that the 22–23 base sequence conserved between all IPS/At4 genes interacts with a micro RNA. 22. Franco-Zorrilla JM, Valli A, Todesco M, Mateos I, Puga MI, Rubio Somoza I, Leyva A, Weigel D, Garcia JA, Paz-Ares J: Target mimicry provides a new mechanism for regulation of microRNA activity. Nat Genet 2007, 39:1033-1037. A thorough characterization of the mechanism by which IPS/At4 genes modulate miR399 activity, thereby uncovering the novel regulatory paradigm, target mimicry. The paper conclusively shows that inhibition of miR399-charged silencing complexes, and not saturation of miR399dependent scission is the mode of IPS/At4 gene action. Artificial target www.sciencedirect.com Please cite this article in press as: Doerner P, Phosphate starvation signaling: a threesome controls systemic Pi homeostasis, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.05.006 COPLBI-558; NO OF PAGES 5 Phosphate starvation signaling Doerner 5 mimics were generated for other miRNAs, showing that this mode of regulation to fine-tune miRNA activity can be used in biotechnology for other endogenous targets. This paper extends the authors’ previous phylogenetic survey [25] to focus more on the genomes of the moss Physcomitrella patens and the lycopod Selaginella moellendorffii. 23. Jones-Rhoades MW, Bartel DP, Bartel B: MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 2006, 57:19-53. 27. Fattash I, Voss B, Reski R, Hess WR, Frank W: Evidence for the rapid expansion of microRNA-mediated regulation in early land plant evolution. BMC Plant Biol 2007, 7:13. A systematic analysis of miRNAs in the moss Physcomitrella patens uncovers novel miRNAs, including some that are differentially expressed in the haploid and diploid generations. 24. Lai F, Thacker J, Li Y, Doerner P: Cell division activity determines the magnitude of phosphate starvation responses in Arabidopsis. Plant J 2007, 50:545-556. 25. Axtell MJ, Bartel DP: Antiquity of microRNAs and their targets in land plants. Plant Cell 2005, 17:1658-1673. A phylogenetic survey was used to examine plant miRNA conservation. It was shown that many plant miRNAs, and their targets, have remained essentially unchanged since before the emergence of flowering plants. 26. Axtell MJ, Snyder JA, Bartel DP: Common functions for diverse small RNAs of land plants. Plant Cell 2007, 19:1750-1769. www.sciencedirect.com 28. Barakat A, Wall K, Leebens-Mack J, Wang YJ, Carlson JE, Depamphilis CW: Large-scale identification of microRNAs from a basal eudicot (Eschscholzia californica) and conservation in flowering plants. Plant J 2007, 51:991-1003. Using modern ultra-high throughput sequencing technologies the paper reports the identification of many conserved and some novel miRNA families from the analysis of a basal eudicot. Amongst others miR399 is identified in the California poppy Current Opinion in Plant Biology 2008, 11:1–5 Please cite this article in press as: Doerner P, Phosphate starvation signaling: a threesome controls systemic Pi homeostasis, Curr Opin Plant Biol (2008), doi:10.1016/j.pbi.2008.05.006
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