Nutrient Sensing and Signalling in Plants: Potassium and Phosphorus

Nutrient Sensing and Signalling in Plants: Potassium and Phosphorus

ANNA AMTMANN,* JOHN P. HAMMOND,{ PATRICK ARMENGAUD* AND PHILIP J. WHITE{

*Plant Science Group, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom { Warwick Horticulture Research International, University of Warwick, Wellesbourne, Warwick CV35 9EF, United Kingdom

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Potassium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Potassium Nutrition of Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Transcriptional Responses to K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. K Perception and Cellular Signalling Events . . . . . . . . . . . . . . . . . . . . . . D. Systemic Signalling of Plant K Status. . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Phosphorus Nutrition of Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Transcriptional Responses to P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Phosphorus Perception and Cellular Signalling Events . . . . . . . . . . . . . D. Systemic Signalling of Plant P Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Potassium and phosphorus are important macronutrients for crops but are often deficient in the field. Very little is known about how plants sense fluctuations in K and P and how information about K and P availability is integrated at the whole Advances in Botanical Research, Vol. 43 Incorporating Advances in Plant Pathology Copyright 2006, Elsevier Ltd. All rights reserved.

0065-2296/06 $35.00 DOI: 10.1016/S0065-2296(05)43005-0

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plant level into physiological and metabolic adaptations. This chapter reviews recent advances in discovering molecular responses of plants to K and P deficiency by microarray experiments. These studies provide us not only with a comprehensive picture of adaptive mechanisms, but also with a large number of transcriptional markers that can be used to identify upstream components of K and P signalling pathways. On the basis of the available information we discuss putative receptors and signals involved in the sensing and integration of K and P status both at the cellular and at the whole plant level. These involve membrane potential, voltage‐dependent ion channels, intracellular Ca and pH, and transcription factors, as well as hormones and metabolites for systemic signalling. Genetic screens of reporter lines for transcriptional markers and metabolome analysis of K- and P-deficient plants are likely to further advance our knowledge in this area in the near future.

I. INTRODUCTION Management of mineral nutrients is one of the biggest challenges that plants face during their life span. In contrast to animals, which can move or migrate to open up new food resources, plants are confined to a particular patch of soil and the minerals that are available from it. In a natural ecosystem, endogenous plant species and microbes are adapted to the nutrient diet on oVer and they form communities to optimally exploit and recreate resources. By contrast, in agricultural systems, high‐density monoculture crops deplete soil minerals quickly and therefore rely on external supplies for most of their major nutrients, particularly nitrogen (N), potassium (K), and phosphorus (P). A balanced supply of mineral nutrients is crucial for both quantity and quality of the crop, but is rarely achieved in the field. Compound fertilisers, containing N, P, and K, have a long tradition in Europe. In developing countries, the introduction of high‐yield crop varieties has been accompanied by a steep increase in N fertilisation, whereas application of P and K fertilisers has often been neglected (Gething, 1993; Laegreid et al., 1999). In many areas this has led to the depletion of arable land for these two nutrients. Even in Europe K and P can be limiting due to leaching or soil structure and chemistry (Syers, 1998). Furthermore, if uptake rates are high, depletion zones can form in the plant rhizosphere (Marschner, 1995). An insuYcient supply of K and P leads to ineYcient usage of nitrate, which can leak into the groundwater and cause environmental problems (Laegreid et al., 1999). To achieve an optimal relationship between the costs and the benefits of fertiliser usage, it is not only important to monitor and improve arable soils continuously, but also to fully understand the role of individual nutrients for plant growth and development, the interaction between diVerent nutrients, and the way plants respond to impaired nutrient balance in the soil. The study of mineral nutrition has a long tradition in the plant sciences. Pioneering work was carried out during the 19th and early 20th century by

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Saussure, Sachs, Knop, Lipman, and others to determine requirements for individual nutrients, resulting in a list of macro‐ and micronutrients essential for plants (Arnon and Stout, 1939; Epstein 1965, 1972). During the second half of the 20th century the scientific focus of research into plant nutrition moved towards studying pathways for nutrient transport (e.g., Epstein, 1973; Epstein et al., 1963), and 50 years of experimental research employing isotope fluxes, electrophysiology, and molecular biology have resulted in a vast, although still incomplete, body of data on ion channels and transporters that mediate the uptake of mineral nutrients and their redistribution within cellular compartments and plant tissues (see compilations by Blatt, 2004; Williams, 2000). In parallel, biochemical analysis has provided us with knowledge of how mineral nutrients are assimilated and metabolised (reviewed by Crawford et al., 2000; Kochian, 2000; Plaxton and Carswell, 1999). More recently, plant scientists have started to investigate how plants sense nutrients and integrate information on the availability of diVerent nutrients. Much of this research has focussed on carbon and nitrogen (Filleur et al., 2005; Moore et al., 2003; Rolland and Sheen, 2005; Zhang and Forde, 1998), whereas less is known about the perception and signalling of other nutrients. In this context it is important to remember that not only the supply of mineral nutrients, but also the plant’s demand for them, fluctuate diurnally and over a plant’s life span and depend on other environmental factors. This review focuses on plant responses to varying supplies of potassium (K) and phosphorus (P). Both nutrients are required in significant amounts for plant growth and development. Whereas P is an important component of many structural macromolecules, metabolites, and signalling molecules (Plaxton and Carswell, 1999; Vance et al., 2003), K is not assimilated into organic matter. Nevertheless, K also plays an important role in metabolism as it functions as a cofactor of many enzymes and is required for charge balancing and transport of metabolites (Marschner, 1995; Wyn Jones and Pollard, 1983). Two types of acclimatory responses can be distinguished when plants encounter nutrient shortage. The first response is linked to nutrient uptake and homeostasis. Under deficiency plants increase their capacity and affinity for nutrient uptake and activate transport processes that assist the remobilisation and redistribution of stored nutrients to support growing and metabolically active tissues, often at the cost of older and less crucial cell types. Nutrient homeostasis involves both transcriptional and posttranslational regulation of a diverse set of ion transporters in diVerent membranes and diVerent cell types (Amtmann et al., 2004; Rausch and Bucher, 2002; Smith et al., 2000b; Tester and Leigh, 2001). Although many of these

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transporters have been characterised with respect to their expression pattern, membrane localisation, biophysical properties, and regulation, very little is known about how their function and regulation are integrated at the cellular or whole plant level. The second response involves the reprogramming of plant growth, development, and metabolism with the aim of using limited resources optimally. Research into the signal pathways underlying these responses and their causal relationships is in its infancy. In order to respond to fluctuating nutrient supply, plants must sense changes in the soil environment, generate signals both at the cellular and at the systemic level, perceive and integrate these signals at specific sites of action, and translate them into a concatenated whole plant response. Many responses to K and P deficiency have been characterised in detail and several sites of action have been identified. There is also some evidence as to which signalling pathways may be involved in nutrient signalling, but there is a general lack of knowledge about primary receptors of nutrient stress. In most cases it is not known which stimulus is perceived: the nutrient itself, a physicochemical parameter linked to nutrient concentration (e.g., ionic strength, membrane potential, pH), or a change in specific metabolites? Cellular ion homeostasis aims to maintain constant levels of K and P in the root cytoplasm, and it is therefore generally assumed that the site of perception resides at the soil/root interface, i.e., in the plasma membrane of root epidermal and cortical cells. However, even this notion is questionable, as current methods to measure cytoplasmic K and P concentrations in individual cells are not sensitive enough to resolve small fluctuations of these ions in the cytoplasm. Constant improvements in in vivo nuclear magnetic resonance and imaging techniques are promising and there is hope that we will soon be able to obtain a dynamic picture of nutrient and metabolite concentrations in tissues, individual cell types, and even cellular compartments. The study of signalling pathways usually takes a reverse approach. Rather than starting the search for molecular components of nutrient sensing at the level of receptors and primary signals, the experiments commence with a well‐defined response and work backwards by identifying molecular processes that are necessary to obtain this response. Physiological and metabolic responses have only limited value for such an experimental design as they usually represent the end point of a complex interplay between diVerent signalling pathways or reflect secondary responses to defective biochemical processes. Current knowledge suggests that most plant responses manifest themselves through changes in gene expression. The relative ease with which to measure transcript levels of a large number of genes has contributed to much of the recent progress in the identification of signalling pathways.

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These experiments are often combined with a forward genetics approach, where a mutagenised population of transgenic plants carrying a promoter– reporter construct for a nutrient‐regulated gene is screened for mutant lines that lack the transcriptional response or have a constitutive response. Although individual genes that are regulated by nutrient stress have been identified and used in signalling studies, the development of full genome microarrays (transcriptomics) has allowed us to identify a large number of transcriptional markers for nutrient stresses, many of which will be an invaluable help for future studies into nutrient sensing. In particular, in the case of K, very few genes that respond to varying K supply had been identified prior to microarray technology. Much of this review is therefore dedicated to the transcriptional responses to K and P stress discovered in recent microarray studies. The review then proceeds by summarising current knowledge on perception and signalling events located upstream of the observed responses. Although transcripts are the most commonly used markers for stress signalling in plants, it should be emphasised that other responses can also reveal useful information on plant nutrient sensing. For example, the characterisation of biophysical properties of ion channels has deepened our understanding of signalling pathways, linking external K concentrations with K homeostasis. Another area of potential progress in our understanding of nutrient signalling is metabolism. Primary metabolites of nitrogen assimilation and carbon fixation have emerged as potential signals for the downstream integration of carbon–nitrogen metabolism in response to an external imbalance between nitrogen availability and photosynthesis (Coruzzi and Bush, 2001; Stitt and Fernie, 2003). Similarly, we can assume that some metabolites might play important roles as systemic signals for K and P deficiency. Advanced high‐resolution technology for measuring a large number of metabolites in individual tissue samples (metabolomics) will facilitate the identification of potential metabolic signals involved in plant responses to fluctuating P and K supply.

II. POTASSIUM A. POTASSIUM NUTRITION OF PLANTS

Potassium is the most abundant inorganic cation in plants, comprising up to 10% of a plant’s dry weight (Leigh and Jones, 1984). Potassium is an important macronutrient for plants, which carries out vital functions in metabolism, growth, and stress adaptation. These functions can be classified into those that rely on high and relatively stable concentrations of K in

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certain cellular compartments or tissues and those that rely on K movement among diVerent compartments, cells, and tissues. The first class of functions includes enzyme activation, stabilisation of protein synthesis, neutralisation of negative charges on proteins, and maintenance of cytoplasmic pH homeostasis (Marschner, 1995). The optimal K concentration for enzyme activation and protein synthesis is of the order 100 mM (Wyn Jones and Pollard, 1983). Thus for optimal metabolic activity, cells rely on controlled potassium concentrations of around 100 mM in metabolically active compartments, i.e., the cytoplasm, the nucleus, the stroma of chloroplasts, and the matrix of mitochondria. Other roles of potassium are linked to its high mobility. This is particularly evident where K movement is the driving force for osmotic changes as, for example, in stomatal movement, light‐driven and seismonastic movements of organs, or phloem transport. In other cases, K movement provides a charge‐balancing counterflux, essential for sustaining the movement of other ions. Thus energy production through Hþ‐ATPases relies on overall Hþ/K exchange (Tester and Blatt, 1989; Wu et al., 1991), and transport of sugars, amino acids, and nitrate is accompanied by K fluxes (Marschner, 1995). The directed movement of potassium is also required for growth. Accumulation of potassium in plant vacuoles creates the necessary osmotic potential for cell extension. Rapid cell extension relies on high mobility of the used osmoticum and therefore only few other inorganic ions (e.g., Naþ) can replace K in this role (Reckmann et al., 1990). Once cell growth has come to a halt, maintenance of osmotic potentials can be carried out by less mobile molecules such as sugars, and K ions can partly be recovered from vacuoles (Marschner, 1995; PoVenroth et al., 1992). Plants have mechanisms to accumulate potassium from very low external concentrations, and because of its high mobility, potassium can be redistributed quickly between diVerent compartments and tissues under fluctuating external potassium conditions. Plants therefore grow well over a wide range of external K supplies (approximately 10 M to 10 mM). As for all nutrients, critical concentrations for starvation or toxicity depend on other environmental factors. In particular, stress conditions that are linked directly to potassium availability, such as drought and salinity, narrow the window for potassium suYciency. Due to the vital role that potassium plays in plant growth and metabolism, potassium‐deficient plants show a very general phenotype, which is characterised by reduced growth, especially of aerial parts. A halt in lateral root growth has been described for K‐starved Arabidopsis thaliana plants grown on agar plates (Armengaud et al., 2004). Physiological symptoms of potassium deficiency include reduced photosynthesis and impaired regulation of transpiration. A study on rice varieties that diVer in their K use

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eYciency showed that K‐eYcient genotypes had a greater relative tillering and grain‐filling rate in low K compared to K‐ineYcient genotypes (Yang et al., 2004). These parameters were correlated with leaf K concentrations, net photosynthetic rate, stomatal conductance, and Rubisco activity. Although these findings can be explained easily with known roles of K in stomatal function and enzyme activation, it is diYcult to determine which K‐dependent process is the most crucial one in creating deficiency symptoms. For example, a lack of K will impede the establishment of Hþ gradients, inhibit the activity of photosynthetic enzymes, and disturb source‐sink transport of sugars; all these factors have an impact on photosynthetic rates. For many plants, a decrease in chlorophyll levels under K deficiency has been reported. Interestingly, when K‐starved red beet plants were supplied with nontoxic levels of Na (which can replace K in its osmotic role), chlorophyll levels were still lower than in K‐suYcient plants, but no diVerence in photosynthetic rates was observed (Subbarao et al., 2000). Inhibition of phloem transport in K‐starved plants has been suggested as the reason for low starch content in storage organs such as potato tubers (Marschner, 1995). However, a study in alfalfa roots found that other sugars accumulate in roots during K deficiency, and the authors suggest that reduced starch synthesis in these plants is a result of direct K dependency of the starch synthase enzyme rather than a lack of available sugars (Volenec et al., 1996). K deficiency also has an impact on nitrogen metabolism. Low levels of total N were measured in K‐starved alfalfa roots, together with low concentrations of soluble protein and vegetative storage proteins (Volenec et al., 1996). This could be due to K dependency of N‐metabolising enzymes or to K dependency of nitrate and amino acid transport. Surprisingly, interactions between N and K, although well established in the field, have not yet been studied at the molecular level. In contrast to well‐described K deficiency symptoms, acclimation responses of K‐starved plants are less well documented. An increase in root surface area to increase the nutrient uptake potential, which is a typical response to P deficiency, has not been reported for K‐starved Arabidopsis plants. However, a recent study involving a number of crop species showed an increase in root surface areas under low K, which was solely due to an increase in root hair length (Høgh‐Jensen and Pedersen, 2003). Root hair growth was correlated positively with K accumulation and was more pronounced in rape and cereals (rye, ryegrass, and barley) than in legumes (lucerne, red clover, and pea). The question whether this response is lacking in Arabidopsis or whether it can only be observed in specific conditions is important, as root hair mutants could provide a useful means to identify K receptors and signalling pathways.

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An increase of the diamine putrescine is a well‐established feature of K‐ deficient plants (Boucherau et al., 1999; Richards and Coleman, 1952). The exact role of putrescine in K stress acclimation remains to be elucidated, but it has been suggested that polyamines act as metabolic buVers by binding to charged macromolecules during stress (Altman and Levin, 1993). Polyamines also regulate ion channels and, therefore, could be involved in K homeostasis. A clear distinction between detrimental stress symptoms and stress acclimation responses is diYcult and will only be achieved through the identification and characterisation of mutants. B. TRANSCRIPTIONAL RESPONSES TO K

1. Responses linked to ion transport Several microarray studies have been carried out with the aim to identify transcriptional targets of varying external K supply. Maathuis et al. (2003) employed a custom‐designed Arabidopsis membrane transporter microarray (AMT chip) carrying 50‐mer probes for some 1000 known and putative membrane transporter genes to identify transcriptional responses of Arabidopsis roots during plant acclimation to low K, low Ca, and increased NaCl. In contrast to Ca and salt stress, which generated diVerential expression in a large number of genes, K starvation led to only a small number of genes showing diVerential expression between starved and resupplied plants. Even more striking was the lack of transcript changes for genes encoding known and putative K transporters. Because the study was carried out on mature plants, which had been grown in K‐suYcient medium for several weeks prior to the experiments (lasting 3 to 96 h), the lack of transcriptional response could be explained by the fact that the plants had accumulated suYcient K to complete their life cycle, without the need for further uptake. Nevertheless, K redistribution between diVerent tissues must have taken place in these plants as a small but significant reduction in total shoot K levels was observed. It can be concluded that regulation of K transporters for tissue K homeostasis occurs mostly at the post-translational level, a notion that is supported by other studies that observed transcriptional regulation of K channels by several plant hormones and stresses but not in response to K starvation (Pilot et al., 2003). The only known K channel that shows a significant transcriptional response to K starvation and resupply is SKOR1, an outward rectifying K channel, which is localised in the xylem parenchyma and thought to be involved in the delivery of K to the shoot (Gaymard et al., 1998; Pilot et al., 2003). The observed downregulation of SKOR1 during K starvation might be a means of retaining K in the roots under these conditions (Maathuis et al., 2003). Amongst the other

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transporters, vacuolar proton pumps and aquaporins were transcriptionally regulated by K stress, but they also responded to Ca and NaCl stress, indicating a general role in ion homeostasis. A downregulation of both the V‐type ATPase and the pyrophosphates in response to decreasing supply of K and Ca suggests that the proton gradient across the tonoplast is an important parameter in regulating ion uptake and release from the vacuole during fluctuating K and Ca supply, thus supporting intracellular homeostasis of these ions. K starvation also induced the downregulation of several genes encoding water channels (aquaporins). This response also occurred during Ca starvation, and because neither of the two treatments created a change in external water potential, it is likely that the observed regulation of aquaporins does not reflect an osmotic adaptation. The authors discussed the possibility that a change in transmembrane water permeability might change the ratio of apoplastic to symplastic ion flow, which could be important for root‐shoot allocation of K and Ca (Maathuis et al., 2003). A weak transcriptional response to K starvation in mature Arabidopsis plants was also reported by Gierth et al. (2005), who used AVymetrix full genome arrays to monitor transcript levels in roots after plant exposure to low K for 6 h to 7 days. By contrast, a study by Armengaud et al. (2004) with spotted full genome arrays based on the Qiagen probe set identified a large number of K‐regulated genes. Several diVerences between this and the other studies might explain the discrepancy. First, Armengaud and colleagues (2004) used much younger plants for their experiments. Not only did these plants require much higher rates of K uptake for extensive growth, but they were also grown from germination in low K conditions and therefore had no opportunity to build up significant K stores. Second, the identification of K‐regulated genes by Armengaud et al. (2004) was not based solely on starvation treatments but included resupply of K after long‐term starvation. This experimental design allowed the scientists to monitor rapid responses of K‐depleted plants to external K. Third, data analysis in the Armengaud study employed a rank‐based procedure (Breitling et al., 2004), which is better suited to identify subtle but significant changes in transcript levels than fold‐change cutoVs applied in the other studies. Nevertheless, Armengaud et al. (2004) also found a general lack of transcriptional responses of K transporters to external K. Only HAK5, a member of the KUP/HAK family of K transporters, was consistently found to be upregulated in roots of K‐starved plants both on microarrays (Armengaud et al., 2004; Gierth et al., 2005; Hampton et al., 2004) and in real‐time polymerase chain reaction analysis (Ahn et al., 2004). Not only does this point to an important role of HAK5 in high‐aYnity K uptake (Gierth et al., 2005), but it also identifies HAK5 as a useful marker to study K sensing at the root

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level (Shin et al., 2004). KUP3, another member of the KUP/HAK gene family, had previously reported to be induced by K starvation in roots of Arabidopsis seedlings (Kim et al., 1998), but this response was not detected in other studies (Gierth et al., 2005; Maathuis et al., 2003). By contrast, Hampton et al. (2004) reported downregulation of KUP3 in shoots of K‐starved plants. They also found K‐dependent expression of several genes encoding putative glutamate receptors (GLR1.2, GLR 1.3) and cyclic‐ nucleotide gated channels (CNGC1, CNGC13). It appears that although a few K channels and transporters respond transcriptionally to changes in the external K supply, their response is dependent on the exact growth conditions and the plant developmental stage. Interestingly, homologues of KUP3 and HAK5 were also found to be downregulated in tomato roots after the addition of nitrate to the growth medium (Wang et al., 2000). Conversely, Armengaud et al. (2004) reported that a group of three genes encoding NRT2‐type nitrate transporters was downregulated during K starvation and upregulated quickly upon K resupply. Interestingly, this response was accompanied by the transcriptional regulation of several putative amino acid and peptide transporters, as well as several genes encoding N assimilatory enzymes. A similar interplay is found for ammonium and K. On the one hand, ammonium is known to aVect K uptake (Santa‐Maria et al., 2000; Spalding et al., 1999). On the other hand, Maathuis et al. (2003) found that ammonium transporters of the AMT family were diVerentially expressed in roots of K‐deplete and K‐replete plants. These findings provide a starting point for molecular studies into K–N interactions and suggest that N metabolites might be involved in K stress signalling. Two putative Ca transporters are regulated by external K: ACA1, a root plasma membrane Ca pump, and CAX3, a putative vacuolar cation/H antiporter (Armengaud et al., 2004). Regulation of these transporters links K stress to Ca homeostasis and could be indicative of a role for Ca in replacing K as an osmoticum. Several Ca‐regulated genes were diVerentially expressed in K‐treated Arabidopsis seedlings such as calmodulins and Ca‐dependent protein kinases, which points to the possibility that Ca signalling is involved in K sensing (see later). 2. Responses in nontransporter genes In shoots of Arabidopsis seedlings, a large number of genes related to the plant hormone jasmonic acid (JA) respond to K starvation and K resupply (Armengaud et al., 2004). These include enzymes involved in JA biosynthesis, such as lipoxygenase, allene oxide synthase, and allene oxide cyclase, as well as downstream targets of JA, such as vegetative storage proteins (VSPs),

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and proteins involved in wounding and pathogen defense (e.g., plant defensins, PDFs and polygalacturonase-inhibiting proteins, PGIPs). Interestingly, very few genes related to salicylic acid, another hormone involved in pathogen defence, were found to be regulated by K. Studies in our laboratory (P. Armengaud and A. Amtmann, unpublished results) confirmed not only an increase of JA as well as other oxylipins during K stress, but also that many of the transcriptional responses to K were absent in the JA signalling mutant coi1. Thus, JA appears to take a prominent role in mediating leaf responses to K stress. Whether these responses lead to stress acclimation by assisting the plant in nutrient storage and remobilisation or have a role in protecting K‐starved plants against increased pathogen attack remains to be elucidated. Another important class of K‐regulated genes identified by Armengaud et al. (2004) comprises genes encoding cell wall proteins. Many cell wall proteins, such as extensins and xyloglucan glucosyltransferases, were upregulated quickly upon K resupply. This indicates a rapid adjustment of cell wall properties prior to the reinstatement of K‐driven cell extension and growth. Signal pathways involved in this response can now be studied in more detail using the identified K‐regulated cell wall genes as molecular markers. Cell wall localised processes could also play a role in nutrient stress perception. For example, several genes for cell wall arabinogalactan proteins (AGPs) responded to changes in external K supply. As for animal cells, an involvement of AGPs in signal transduction has been proposed for plants (Schultz et al., 1998). Peroxidases also featured strongly among K‐regulated transcripts (Armengaud et al., 2004; Shin and Schachtman, 2004). One reason for this might be their involvement in growth‐related cell wall responses (e.g., oxidative cross‐linkage of cell wall components). Another reason for transcriptional regulation of peroxidases during K stress might be related to their function in ROS detoxification. In fact, H2O2 was identified as an important signal in K stress perception (Shin and Schachtman, 2004; see later). In addition to the K‐dependent functional gene classes described earlier, many individual K‐regulated genes were found in the microarray studies cited earlier. These included several transcription factors of diverse gene families (e.g., B3, WRKY, bZIP, scarecrow‐like, C3H4‐RING finger, AP2) and various protein kinases and phosphatases (serine/threonine PK, CIPK, PP2C). Furthermore, K stress aVected the expression of many crucial enzymes of the primary metabolism, such as enzymes for N, S, and P assimilation (e.g., glutamate dehydrogenase, ATP sulfurylase, asparagine synthase, 6P‐gluconolactonase), pyruvate synthesis (PEP carboxylase, PEP carboxykinase, malic enzyme), and sugar metabolism (glucose‐6‐phophate‐dehydrogenase, starch

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synthase), as well as secondary metabolism (e.g., members of the FAD‐linked oxidoreductase family). These changes reflect both known and unknown metabolic disturbances in K‐starved plants and also suggest that extensive metabolic reprogramming is underway in K‐starved plants. Figure 1 shows an attempt to summarise the physiological, metabolic, and signalling events reflected in microarray data. This diagram [an updated version of a similar figure in Armengaud et al. (2004)] includes new findings from JA signalling mutants and metabolic profiling. For example, K‐starvation induction of the arginine decarboxylase gene (ADC2), although reported to be JA dependent (Perez‐Amador et al., 2002), was still found in the JA signalling mutant coi1 (P. Armengaud and A. Amtmann, unpublished results), and polyamine production is therefore no longer placed downstream of JA. Furthermore, biochemical studies suggest that the regulation of NRT2 nitrate transporter genes during K stress is downstream of a K eVect on N assimilation rather than vice versa (see later). It should be pointed out that although microarray data provide a useful framework to identify the molecular processes underlying plant responses to K stress, they do not reveal the reason for the observed change in transcript. The question whether a transcript is regulated because certain upstream processes are disturbed or because it is part of an acclimation response can only be answered by detailed studies at the single gene level. C. K PERCEPTION AND CELLULAR SIGNALLING EVENTS

1. Membrane potential and cytoplasmic Ca The first point of contact between the plant and its soil environment is the root apoplast. It is generally assumed that the root apoplast reflects the soil K concentration, but depending on the availability of exchangeable K in the soil and plant K uptake rates, K gradients between the site of uptake (root cell plasma membrane) and the bulk soil solution might develop when K supply is low. Conversely, in conditions of high K supply, K might accumulate outside the Casparian strip or in the symplast of the xylem parenchyma if transport to the shoot is slow. Apoplastic K concentration and membrane voltage of root cells are the most likely primary stimuli during K stress (see later), whereas cytoplasmic K concentrations appear to remain constant under varying external K (Walker et al., 1996). The two stimuli are connected, as the electrical properties of the plasma membrane are strongly dependent on external K. Potassium channels together with the proton ATPase dominate the electric conductance of the plasma membrane and, therefore, their relative conductance determines the membrane potential, which in turn determines the driving force for K movement. Voltage‐dependent K

Fig. 1. Overview of K stress responses reflected in gene expression data. Putative components of K deficiency and adaptive responses are shown in boxes. Connecting lines are based on K‐responsive genes identified by Armengaud et al. (2004) (shown in italics) and other published information (see Armengaud et al., 2004). Solid arrows indicate stimulation; dashed lines indicate inhibition. Known K deficiency symptoms are shown in white boxes. Putative components of signaling events are indicated in dark grey. Lighter grey shading marks diVerent JA‐dependent processes potentially leading to adaptive nutrient management and defense responses. For further discussion, see text.

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channels are the best established ‘‘K receptors’’ as they respond to both voltage and external K with changes in their gating (Blatt, 1988; Blatt and Gradmann, 1997; Maathuis and Sanders, 1997; Schroeder et al., 1994). In the simplest model of a membrane‐delimited regulatory loop, K channels act both as receptors and as eVectors. For example, some inward rectifying channels shift their activation potential with the K equilibrium potential (EK), thus preventing K loss under low external Kþ (Maathuis et al., 1997). There is also evidence that proton ATPase activity is stimulated directly by external K (Briskin and Poole, 1983; Hall and Williams, 1991). This leads to a more complex but still a plasma membrane‐delimited model of a regulatory loop, which involves the plasma membrane proton pump as a second receptor (Amtmann et al., 1999). Circumstantial evidence suggests that external pH could be an important parameter in this model: (1) Measurements of apoplastic pH after application of fusicoccin (Amtmann et al., 1999; Felle, 1998) showed that the cell wall pH is indeed clamped by proton pump activity. (2) Apoplastic pH regulates root K channels with a Km that is in the physiological range of cell wall pH (Amtmann et al., 1999; Zimmermann et al., 1998). Figure 2 shows a summary of receptors (membrane transporters) and system parameters (K concentration, voltage, and pH) in this model. The final output of such a loop, in terms of transmembrane K flux in response to a change in external K, is diYcult to predict, as

Fig. 2. Model of early signalling events in response to Kþ stress. The simplest scenario (1) involves voltage‐dependent plasma membrane K channels and the membrane potential ( ). An extended regulatory loop (2) includes the plasma membrane Hþ‐ATPase and external pH. A further extension (3) considers the eVect of membrane potential changes on plasma membrane Ca2þ channels, a change in cytoplasmic Ca2þ concentration, and the eVect of the latter on vacuolar K channels as well as gene expression.

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many of the caused changes are counteractive. On the one hand, a negative shift in EK in low external K concentrations will hyperpolarise the membrane and lead to channel activation. On the other hand, inactivation of the pump in low K will lead to membrane depolarisation, thus decreasing channel open probability, and this eVect will be exacerbated by an increase in external pH. Exact quantification of the eVect of a change in external K on K fluxes requires knowledge of the membrane potential, apoplastic pH, and the regulatory profiles of K channels and pumps in the specific membrane and cell type. Although membrane‐delimited regulatory systems can contribute to cellular K homeostasis, they are not suYcient to explain many of the observed downstream eVects of changes in K supply (e.g., changes in gene expression). A simple extension of the aforementioned model considers processes that will respond to changes in the membrane potential. The best‐known example for such a process is the opening of voltage‐dependent Ca channels, which will cause Ca influx into the cell, raising the cytoplasmic Ca concentration (Grabov and Blatt, 1997; Trewavas, 2000). A K clamp of the membrane voltage can be used to induce intracellular Ca waves experimentally. Thus, Allen et al. (2001) showed that they could exploit the voltage dependence of hyperpolarisation‐activated Ca channels in guard cells and create controlled oscillations of cytoplasmic Ca by repeatedly changing between a depolarising buVer (100 mM KCl) and a hyperpolarising buVer (0.1 mM KCl). Because root cells contain both depolarisation‐ and hyperpolarisation‐ activated Ca channels (Miedema et al., 2001; Thion et al., 1998), a more complex Ca signature would be expected here in response to changes in external K. Furthermore, K fluctuations in nature will be much slower than the protocol used by Allen and colleagues (2001) and therefore changes in plasma membrane Ca fluxes might be counteracted easily by intracellular Ca homeostasis. Cytoplasmic Ca signals occur in response to many environmental stimuli (Scrase‐Field and Knight, 2003), but have not yet been reported in relation to K stress. It is important to note that although membrane depolarisation occurs in response to many diVerent ions (e.g., increase of external Naþ, ammonium, phosphate) membrane hyperpolarisation to very negative membrane voltages can only be achieved by decreasing external K. The large proportion of Ca‐regulated proteins (e.g., calmodulins, protein kinases) among K‐regulated transcripts strongly suggests some involvement of intracellular Ca in the plant’s response to K stress, but it remains to be elucidated whether their role is in Ca signalling or Ca homeostasis. Cytoplasmic Ca could link the external K stimulus not only to post-translational and post-transcriptional responses via Ca‐dependent kinases, phosphatases, and transcription factors, but also to an adjustment of tonoplast K fluxes

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for cellular K homeostasis (Fig. 2). Indeed, K‐permeable vacuolar channels (SV and VK) are activated by Ca and would release K into the cytoplasm in response to a rise of cytoplasmic Ca levels (Allen and Sanders, 1996, 1997). 2. Reactive oxygen species Reactive oxygen species such as H2O2 have been shown to be key regulators in a range of physiological processes, such as stomatal closure, hypersensitive response to pathogens, root gravitropism, and root cell elongation (Foreman et al., 2003; Laloi et al., 2004). It has been observed that K‐deficient plants produce H2O2 and that this response is required for some transcriptional responses to K starvation (Shin and Schachtman, 2004). Thus, inhibition of ROS production suppresses the transcriptional response of two peroxidases, HAK5 and the transcription factor WRKY9 to K deprivation. The transcription of these genes was no longer induced when diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, was applied. Their transcriptional response to low K was absent in the Arabidopsis rdh2 mutant, which is mutated in one of the catalytic subunits of the NADPH oxidase. Application of H2O2 to K‐replete plants was in some but not all cases suYcient to induce gene expression (Shin and Schachtman, 2004). Interestingly, the addition of H2O2 to a growth medium containing millimolar K activated a high‐aYnity K uptake component, which was otherwise only apparent in growth medium containing micromolar external K. Because H2O2 application was not suYcient to induce HAK5 expression, this component is unlikely to be carried by HAK5. If ROS are an important signal in plant acclimation to low K, the question arises as to how this signal is created, which are downstream signalling events, and what is its physiological role. ROS are produced either in the apoplast by cell wall peroxidases and amine oxidases or as a by‐product of metabolic pathways localised in diVerent cellular compartments. Although the source of ROS production during K stress has not been identified, the observed transcriptional regulation of several cell wall peroxidases might be indicative for an apoplastic oxidative burst similar to the one caused by pathogens. The fact that many disease‐related genes were present in microarray data also points to the possibility that K stress shares ROS signalling pathways with defense responses. Downstream signalling events of ROS production in Arabidopsis include the MAPK cascade as well as redox‐state‐dependent kinases, phosphatases, and transcription factors (Laloi et al., 2004). ROS also activate Ca channels, a response that is essential for root hair growth (Foreman et al., 2003). Which, if any, of these events are also involved in responses to K stress remains to be studied. The fact that the ROS synthesis mutant rdh2 is characterised by short root hairs points to a role of ROS in increasing root

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hair length to enhance K uptake. As mentioned earlier, a root hair response to K deprivation was observed in several crop species (Høgh‐Jensen and Pedersen, 2003) but has not been reported for Arabidopsis. There is some evidence that the occurrence of a root hair response in Arabidopsis depends on ammonium being present in the growth medium (R. Gaxiola, personal communication). D. SYSTEMIC SIGNALLING OF PLANT K STATUS

1. Hormonal signals The notion of long‐distance signals to communicate K deficiency seems unnecessary, as no molecule moves faster around the plant than K itself. However, similar to the cellular situation where maintenance of constant K levels in the cytoplasm requires signals to communicate the external K concentration to intracellular compartments (e.g., vacuole, nucleus), eYcient K homeostasis at the tissue level requires systemic signals between K‐deplete and K‐replete tissues. Another reason why K is unlikely to report on plant K status is that K concentrations are generally in the millimolar range and, therefore, large absolute changes are required to achieve significant relative changes. This means that K sensors are either extremely sensitive or that the plant risks considerable depletion of K before any responses occur. Short‐ term K resupply experiments performed by Armengaud et al. (2004) on K‐ starved Arabidopsis seedlings demonstrated that within 6 h of K resupply many shoot genes responded, although no significant change of overall shoot K content was recorded at this stage. However, this does not exclude the possibility that selective shoot tissues already experienced a considerable change in K concentration. A detailed analysis of the dynamic and spatial pattern of tissue K concentrations in response to a change in external K is required to understand the nature of K communication at the whole plant level. Thus, certain cell types that experience depletion or resupply of K earlier than others could send signals to other tissues that are still suYcient/ depleted. In theory, two types of systemic signalling can be distinguished. The first type involves a sensor that records the apoplastic K concentration and translates it into a signal that moves between diVerent cellular compartments, cells, or tissues. The sensory machinery, which is likely to include membrane potential, H2O2 and Ca (see earlier discussion), can be located in root cells that are in direct contact with the soil‐apoplast continuum or in leaf cells that are particularly well or particularly poorly supplied with K (e.g., xylem parenchyma and epidermis respectively, Karley et al., 2000). Abscisic acid (as free ABA or ABA conjugates, Sauter et al., 2005) is a typical root/ shoot signal for osmotic stress (Shinozaki and Yamaguchi‐Shinozaki, 2000),

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but available microarray data do not indicate the occurrence of an ABA signal during K stress, i.e., known ABA reporter genes such as COR78 and KIN2 were not aVected by a change in K supply (Armengaud et al., 2004). Microarray analysis did, however, suggest changes in auxin (Armengaud et al., 2004) and ethylene (Shin and Schachtman, 2004) in response to K stress. Significant ethylene production by Arabidopsis seedlings was detected within 6 h after K removal. Further studies are required to prove an involvement of hormonal long‐distance signals in plant K stress signalling and to identify upstream and downstream events. 2. Metabolic signals A second type of systemic signal could originate from the breakdown of cellular homeostasis and subsequent K depletion in specific cell types or tissues. It has been shown that under salt stress, cytoplasmic K levels in leaf epidermal cells can be as low as 15 mM, whereas cytoplasmic K levels in mesophyll cells are maintained around 70 mM (Cuin et al., 2003). This finding not only confirms that plants prioritise tissue allocation of K, it also shows that in nonprioritised tissues, cytoplasmic K levels can drop to values that are considerably lower than the K optimum for protein stability and enzyme activity (Wyn Jones and Pollard, 1983). Resulting changes in protein and metabolite levels could inform K‐replete tissues of the whole plant K status. Most enzymes require K (50–150 mM) for optimal function in vitro. The activity of some 60 enzymes has been shown to depend on K (Wyn Jones and Pollard, 1983). Many of these enzymes are involved in sugar and nitrogen metabolism (e.g., starch synthase, asparaginase). Pyruvate kinase (PK) was one of the first enzymes for which K dependency was discovered (Kachmar and Boyer, 1953), and the kinetic and structural properties related to K activation have since been studied in detail. Although K‐binding sites in PK are well conserved among eukaryotic enzymes, they are absent in some bacterial PKs that do not require K for activation. Replacement of a Glu117 residue in close vicinity to the K‐binding site by lysine renders the rabbit muscle PK K independent (Laughlin and Reed, 1997), thus providing evidence that genetic engineering could be used as a means to decrease K sensitivity of this enzyme. The K dependency of eukaryotic PK activity is very steep in the physiological range of cellular K concentrations (Km ¼ 14 mM, Laughlin and Reed, 1997). In plants, PK activity has been used as an indicator for cation balance in cucumber leaves (Ruiz et al., 1999). The microarray study by Armengaud et al. (2004) showed that several enzymes in the PK pathway are regulated during K stress (Fig. 3); in particular, upregulation of two genes for malic enzyme, which catalyses an alternative pathway for pyruvate synthesis, was observed. This is likely to be a direct response to decreased pyruvate levels

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in K‐depleted tissue. Because PK is a central regulator of C/N metabolism (Smith et al., 2000a), many of the observed changes in transcripts and metabolite levels under K deficiency might lie downstream of a decrease in PK activity. Similar to PK, any K‐dependent enzyme could theoretically function as a ‘‘K sensor’’ with its products and substrates acting as signals. However, it is important to remember that such a ‘‘signal pathway’’ would require a change of K in the cytoplasm or other metabolically active cellular compartment. Only dynamic and spatial fine mapping of K levels, transcripts, and metabolites in diVerent tissues during K stress will answer the question of whether the observed downstream responses are limited to K‐depleted cells or whether K‐ dependent enzymes are involved in systemic signalling between K‐deplete and K‐replete tissues. A recent analysis of amino acid levels showed that K‐starved Arabidopsis plants have increased levels of glutamine and decreased levels of glutamate (P. Armengaud and A. Amtmann, unpublished results). This finding suggests that some enzymes required for N assimilation are K dependent. Both amino acids are highly mobile between cells and tissues and are likely to act as signals for membrane transporters. Glutamine has been shown to repress the nitrate transporter AtNRT2.1 transcriptionally (Nazoa et al., 2003), and it is likely that the downregulation of NRT2 genes under K deficiency observed by Armengaud et al. (2004) is a consequence of increased glutamine levels. Glutamate might directly regulate ion channels of the putative glutamate receptor family. This large gene family in Arabidopsis (20 members) is poorly characterised in plants (Davenport 2002; Kang and Turano, 2003), but animal glutamate receptors transport a range of cations, including K, Na, and Ca. The possibility that glutamate aVects cation currents was supported by the observation that glutamate applied to root cells causes a large membrane depolarisation and a change of cytoplasmic Ca (Dennison and Spalding, 2001). Therefore, glutamate might play a role both as a cellular signal in K‐depleted cells and as a long‐distance signal informing K‐replete tissues of whole plant K status.

III. PHOSPHORUS A. PHOSPHORUS NUTRITION OF PLANTS

Phosphorus constitutes 0.1 to 1.4% of the dry matter of a typical plant (Broadley et al., 2004). It is an essential element, which is present primarily in small metabolites, nucleic acids, and phospholipids (Marschner, 1995). However, P is also required for diverse homeostatic and signal transduction

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cascades, as the activities of many proteins are regulated by phosphorylation/dephosphorylation reactions. Plant roots acquire P from the soil solution as phosphate (Pi, H2PO4 ). The concentration of Pi in the soil solution is low (2 to 10 M), which limits its diVusion to the root system and may result in Pi depletion in the rhizosphere of a rapidly growing plant. Thus, although soil P concentrations are often high, little Pi may be available for uptake by plants. For this reason, plants have evolved many strategies to increase their ability to acquire P from the soil and to cope with periods of low Pi availability (Hammond et al., 2004a; Raghothama, 2005; Vance et al., 2003). Plants that lack P increase the eVective surface area of their root systems by increasing carbohydrate partitioning to the root, accelerating the

Fig. 3. Hypothetical signalling cascades regulating transcription during P starvation. Several cell autonomous and systemic signalling cascades may regulate transcription in P‐starved plants. (1) Some immediate transcriptional changes in roots respond indiscriminately to changes in the ionic environment. These include the upregulation of genes involved in plant defence responses. (2) Some immediate transcriptional changes respond directly to Pi in the rhizosphere and result in an increase in the density and elongation of root hairs. The production of ethylene is implicated in this process. (3) Some immediate transcriptional changes in roots respond specifically to Pi withdrawal. These include the (weak) upregulation of PHR1, which encodes a MYB‐CC transcription factor whose activity might be regulated posttranscriptionally. The PHR1 transcriptional cascade upregulates genes encoding other transcription factors, riboregulators, protein kinases, Pi transporters, RNases, phosphatases, and metabolic enzymes through the P1BS cis element. (4) Reduced P transport to the shoot results rapidly in a decrease in shoot P concentration. This inhibits shoot growth before root growth is aVected and leads indirectly to an increase in the root:shoot ratio. (5) Reduced shoot Pi supply aVects [Pi]cyt and primary metabolism, both through allosteric interactions and transcriptional changes, leading to an increase in the concentrations of starch, sucrose, and organic acids in leaves. (7) Increased leaf sucrose concentrations result in (a) an increase in sucrose transport to the root, through the parallel upregulation of sucrose transporters, (b) a reduction of photosynthesis, through downregulation of many photosystem subunits and small subunits of RuBisCo, and (c) the production of anthocyanins through a TTG1‐TT8/EGL3‐PAP1/PAP2‐dependent cascade. (7) A decrease in shoot P upregulates the expression of genes encoding enzymes involved in the synthesis of sulpholipids and galactolipids, possibly through the P1BS cis element. (8) An increase in sucrose transported in the phloem to the root (a) promotes root growth and results in an increase in the root:shoot ratio and (b) acts as a systemic signal to modulate the expression of the AtPHR1 transcriptional cascade upregulating genes encoding riboregulators, Pi transporters, RNases, phosphatases, and metabolic enzymes. It also regulates the expression of other transport proteins. (9) An increased translocation of carbohydrates and organic acids in the phloem stimulates the release of organic acids from the root. (10) A reduction in cytokinin and altered auxin transport within the root initiates and promotes lateral root elongation. The location of lateral root elongation is determined by P availability in the rhizosphere, which is perceived by the root tip.

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initiation and growth of lateral (Chevalier et al., 2003; Forde and Lorenzo, 2001; Lynch and Brown, 2001; Malamy, 2005) or cluster roots (Dinkelaker et al., 1995), and by increasing the number and length of root hairs [Fig. 3, processes (2), (8), and (10); Bates and Lynch 1996; Forde and Lorenzo, 2001; He et al., 2005; Jungk, 2001]. They also form more associations with mycorrhizal fungi (Karandashov and Bucher, 2005). They increase the phosphate influx capacity of their root cells severalfold (Lee, 1993; Rausch and Bucher, 2002; Smith et al., 2003) and they secrete protons, enzymes, and organic acids into the rhizosphere [Fig. 3, processes (3), (8), and (9)] to release Pi from organic and inorganic sources in the soil (Brinch‐Pedersen et al., 2002; Dinkelaker et al., 1995; Lo´ pez‐Bucio et al., 2000; Miller et al., 2001; Narang et al., 2000). In parallel, diverse metabolic changes occur in P‐starved plants, enabling them to utilise P more eYciently. It is noteworthy that small metabolites, nucleic acids, and phospholipids contribute about equally to leaf P content. Thus, metabolism adjusts to P starvation by employing reactions that do not require Pi or adenylates (Hammond et al., 2004a; Plaxton and Carswell, 1999; Vance et al., 2003), by inducing intracellular phosphatases and nucleases that remobilise P from cellular metabolites and nucleic acids (Bariola et al., 1994; Brinch‐Pedersen et al., 2002; del Pozo et al., 1999), and by replacing phospholipids in thylakoid and extraplastidic membranes by galactolipids and sulpholipids [Fig. 3, process (7); Andersson et al., 2003; Essigmann et al., 1998; Ha¨ rtel et al., 2000; Yu et al., 2002]. Through these responses, plants acclimate to periods of P starvation. However, one unfortunate consequence of P starvation is the accumulation of sucrose and starch in leaves [Fig. 3, processes (5) and (6)]. The accumulation of sucrose reduces the expression of many genes involved in photosynthesis and eventually leads to a decline in photosynthetic performance [Fig. 3, process (6); Martin et al., 2002; Paul and Pellny, 2003; Rook and Bevan, 2003]. The characteristic accumulation of anthocyanins in response to P deficiency is thought to protect nucleic acids from UV damage and chloroplasts from the photoinhibitory damage caused by P‐limited photosynthesis [Fig. 3, process (6); Hoch et al., 2001]. In order to respond appropriately to P starvation, a plant must first perceive a lack of P and then generate signalling cascades to initiate and coordinate its acclimatory responses (Franco‐Zorrilla et al., 2004; Hammond et al., 2004a; Ticconi and Abel, 2004; Vance et al., 2003). It is assumed that these acclimatory responses result from changes in gene expression, and significant progress towards understanding the control of transcriptional responses to P starvation has been achieved through the phenotypic characterisation of mutants and the judicious application of molecular biology and transcriptional profiling technologies.

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B. TRANSCRIPTIONAL RESPONSES TO P

The expression of thousands of genes is altered when plants have insuYcient P for growth (Al‐Ghazi et al., 2003; Hammond et al., 2003, 2004a,b, 2005; Misson et al., 2005; Uhde‐Stone et al., 2003; Wang et al., 2002; Wasaki et al., 2003; Wu et al., 2003). These phosphate starvation‐responsive (PSR) genes have been grouped into ‘‘early’’ genes that respond rapidly (within hours after P withdrawal), transiently, and often nonspecifically to P withdrawal and ‘‘late’’ genes that alter the morphology, physiology or metabolism of plants upon prolonged P starvation (Hammond et al., 2003, 2004a,b). The early transcriptional responses to P withdrawal include increased expression of genes encoding general stress‐related proteins, such as chitinases and peroxidases, that respond indiscriminately to many biotic and abiotic challenges (Hammond et al., 2003, 2004b; Misson et al., 2005; Wang et al., 2002). However, they also include altered expression of genes encoding various transcription factors (e.g., zinc finger proteins, HD‐ZIP, WRKY transcription factors, MYB‐CC transcription factors, bHLH DNA‐ binding proteins; Hammond et al., 2003, 2004b; Rubio et al., 2001; Todd et al., 2004; Wang et al., 2002; Wu et al., 2003), riboregulators (e.g., At4/IPS family; Burleigh and Harrison, 1999; Hou et al., 2005; Liu et al., 1997; Martı´n et al., 2000; Mu¨ ller et al., 2004; Shin et al., 2004; Wasaki et al., 2003), and other components of intracellular signalling cascades, such as protein kinases and protein phosphatases, that respond more specifically to P withdrawal (Hammond et al., 2003, 2004b; Wang et al., 2002; Wu et al., 2003). The late transcriptional responses to P withdrawal are primarily acclimatory responses. In general, they improve the acquisition of P from the soil and/or promote the eYcient use of P within the plant (Hammond et al., 2004a; Vance et al., 2003). In roots, the expression of genes encoding members of the Pht1 Pi transporter family (Al‐Ghazi et al., 2003; Rausch and Bucher, 2002; Smith et al., 2003; Wasaki et al., 2003), intracellular and apoplastic RNases (Ko¨ ck et al., 1995; Mu¨ ller et al., 2004; Wasaki et al., 2003), and phosphatases (Baldwin et al., 2001; Berger et al., 1995; del Pozo et al., 1999; Haran et al., 2000; Miller et al., 2001; Mu¨ ller et al., 2004; Petters et al., 2002; Stenzel et al., 2003; Wasaki et al., 2003) are increased gradually during P starvation. Genes encoding enzymes of the glycolytic pathway are upregulated, leading to the production of organic acids, as are genes encoding putative plasma membrane organic acid channel proteins (Uhde‐ Stone et al., 2003; Wasaki et al., 2003). Genes encoding proteins regulating or regulated by the activity of plant hormones (Al‐Ghazi et al., 2003; Hammond et al., 2004a; Misson et al., 2005; Uhde‐Stone et al., 2003; Wu

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et al., 2003) are also aVected by prolonged P starvation. Changes include the increased expression of auxin responsive genes, such as AIR1, AIR3, AIR9, AIR12, HRGP, and LRP1, that control lateral root development (Al‐Ghazi et al., 2003; Casson and Lindsey, 2003; Uhde‐Stone et al., 2003), the expression of several genes involved in ethylene biosynthesis (ACC oxidase, methioninesynthase and S‐adenosyl methionine synthetase) and signalling (EREB2), which could mediate the transcription of ethylene‐responsive genes (Rietz et al., 2004; Uhde‐Stone et al., 2003; Wu et al., 2003), and the expression of genes encoding cytokinin oxidases, which may be involved in the breakdown of cytokinin in roots, thereby releasing the negative control cytokinins have on root development (Hammond et al., 2004a; Uhde‐Stone et al., 2003). Interestingly, root systems of diVerent plant species, and diVerent genotypes of a particular species, vary considerably in the extent of their morphological change, and their sensitivity, to reduced P supply (Chevalier et al., 2003; Hodge, 2004; Lynch and Brown, 2001; Narang et al., 2000; Robinson, 1994). This may allow the genes influencing such traits to be identified through quantitative genetic analyses (Lynch and Brown, 2001; Vreugdenhil et al., 2005). In shoots, the expression of genes encoding proteins that have an impact on both primary and secondary metabolism is influenced by P starvation. Genes encoding many photosystem subunits and small subunits of RuBisCo are downregulated, and genes encoding PEP carboxylases, sucrose synthases, fructose‐1,6‐bisphosphatases, UDP‐glucose pyrophosphorylases, and sucrose transporters are upregulated in P‐deficient plants (Ciereszko et al., 2001; Hammond et al., 2004a; Paul and Pellny, 2003; Plaxton and Carswell, 1999; Pen˜ aloza et al., 2005; Toyota et al., 2003; Uhde‐Stone et al., 2003; Wu et al., 2003). Several of these changes reflect the necessity to bypass ATP‐ and Pi‐dependent enzymes when P is scarce and the changing metabolism required to generate energy and carbon skeletons during P deficiency (Plaxton and Carswell, 1999). Genes encoding enzymes involved in anthocyanin biosynthesis and genes encoding enzymes that remobilise P from cellular molecules, such as phosphatases, nucleases, and phosphodiesterases, are also upregulated in leaves of P‐deficient plants (Hammond et al., 2003, 2004a,b; Uhde‐Stone et al., 2003; Vance et al., 2003; Wu et al., 2003). In addition, the expression of genes encoding enzymes that increase the proportion of galactolipids and sulpholipids in thylakoid and extraplastidic membranes are also increased in P‐deficient plants (Essigmann et al., 1998; Hammond et al., 2003; Ha¨ rtel et al., 2000; Misson et al., 2005; Wasaki et al., 2003; Yu et al., 2002), which enables photosynthesis to be maintained despite a reduction in phospholipid content. It is thought that many of the late transcriptional changes are induced by increasing leaf sugar concentrations,

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as 22% of genes responding to P deficiency are also diVerentially regulated in shoots of an Arabidopsis mutant (pho3 ¼ suc2; Lloyd and Zakhleniuk, 2004) with elevated leaf sugar concentrations (J. P. Hammond, unpublished results). Interestingly, genes encoding protein kinases and phosphatases were significantly more represented amongst these genes, suggesting that they may be involved in fine‐tuning enzyme activities. It has been observed that PSR genes can be clustered into coregulated groups (regulons) that exhibit specific temporal and spatial expression patterns in response to unique sets of environmental and/or developmental stimuli in addition to P withdrawal. Thus, it has been suggested that P withdrawal elicits several diVerent regulatory cascades and that each subset of PSR genes might share a common regulatory cascade (Franco‐Zorrilla et al., 2004; Hammond et al., 2003, 2004a,b; Vance et al., 2003). The next section presents the temporal sequence, tissue specificity, and orchestration of transcriptional responses to increasing P deficiency in plants. C. PHOSPHORUS PERCEPTION AND CELLULAR SIGNALLING EVENTS

1. Membrane potential and cytoplasmic Ca When the ionic environment of a plant cell changes, the electrochemical gradients for ion movement across its plasma membrane also change. Thus, one of the immediate consequences of drastic changes in the ionic composition of the rhizosphere is altered ionic flux across the plasma membrane of root cells, which can cause changes in membrane potential (Mimura, 1999). Whether a change in the flux of a particular nutrient will aVect the membrane potential depends on the contribution of the related fluxes to the overall membrane conductance. Whereas K is a major determinant of the membrane potential, P fluxes under normal conditions are relatively small and will not make a large contribution to the membrane conductance. However, after prolonged starvation, resupply of P to the medium has been shown to cause fast and transient depolarisations, which increased with the length of prestarvation and were followed by long‐term hyperpolarisation (Dunlop and Gardiner, 1993). Several conclusions can be drawn from these findings: (1) Phosphate transport is coupled to the movement of a net positive charge (reflecting PO43 /nHþ cotransport), (2) P fluxes increase with increasing P deficiency (possibly due to transcriptional upregulation of high‐aYnity P transporters), and (3) long‐term P starvation results in decreased pump activity (possibly due to a rise in cytoplasmic pH or depletion of ATP stores). Thus removal of P is unlikely to have drastic eVects on the membrane potential, but long‐term starvation and resupply will have a strong impact on the membrane potential, which can be linked to a number

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of downstream responses as described earlier for K. Whether a cytosolic Ca2þ signal is caused by changes in external P remains to be determined, but such perturbations in cell membrane potential have the potential to alter Ca2þ influx and, thereby, cytosolic Ca2þ concentrations ([Ca2þ]cyt; White 2000; White and Broadley, 2003). This may account for some of the similarities between the ‘‘early’’ nonspecific transcriptional changes in response to P withdrawal and other nutritional, abiotic, and biotic challenges (Hammond et al., 2003). Pathogenesis‐related genes in particular are characteristically triggered by Ca2þ signals (White and Broadley, 2003). Nevertheless, in addition to the initial, nonspecific changes in gene expression, P‐specific responses are also initiated. 2. Transcription factors and promoter elements The transcriptional response of some genes in root cells to P withdrawal appears to be cell autonomous. The expression of these genes changes only a few hours after P withdrawal and, therefore, is unlikely to be regulated by a systemic signal (Hammond et al., 2003; Wang et al., 2002; Wu et al., 2003). They may also occur in roots of defoliated plants. During this period there is little change in cytoplasmic Pi concentration [Pi]cyt, which is maintained in the range 2 to 10 mM until P deficiency becomes severe (Lee et al., 1990; Mimura, 1999; Schachtman et al., 1998). Thus, any immediate changes in gene transcription observed in roots following P withdrawal are unlikely to be initiated by changes in [Pi]cyt. However, it is possible that they are initiated in response to changes in vacuolar or apoplastic Pi concentration or to changes in metabolism. The expression of several transcription factors changes rapidly in response to P stress. These include members of the WRKY, WD40, bHLH, MYB, HD‐ZIP, zinc finger, and At4/IPS families (Burleigh and Harrison, 1999; Hammond et al., 2003, 2004b; Hou et al., 2005; Liu et al., 1997; Martı´n et al., 2000; Misson et al., 2005; Mu¨ ller et al., 2004; Rubio et al., 2001; Shin et al., 2004; Todd et al., 2004; Wang et al., 2002; Wasaki et al., 2003; Wu et al., 2003). The MYB transcription factors are early components of the transcriptional cascades initiated specifically by P withdrawal. Members of the MYB‐CC subfamily appear to be essential to these transcriptional cascades (Rubio et al., 2001; Todd et al., 2004). These proteins contain a MYB DNA‐binding domain, a second domain predicted to form a coiled coil (CC) usually involved in protein–protein interactions, and a glutamate‐ rich C terminus found in transcriptional activators. There are at least 15 genes encoding members of the MYB‐CC subfamily in Arabidopsis (Fig. 4). These include AtPHR1 (At4g28610), AtPHR2 (At1g79430), AtNSR1 (At3g04030), and AtNSR2 (At5g18240). A suggested subfamily

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nomenclature of ‘‘MCC’’ (MYB‐CC) proteins is proposed here (Fig. 2). Transcription of several of these genes, including AtPHR1, AtPHR2, and AtMCC6, respond to P starvation (Hammond et al., 2005; Rubio et al., 2001; Todd et al., 2004; Wu et al., 2003). The AtPHR1, AtPHR2, and AtMCC6 proteins appear to be positive regulators of P starvation responses. The Arabidopsis phr1 mutant shows less of an increase in shoot/root ratio, reduced accumulation of anthocyanins, and lower expression of Pi‐responsive genes under P starvation than wild‐type plants (Rubio et al., 2001). The Arabidopsis AtPHR1 signalling pathway has been elucidated and appears to have been conserved during the evolution of plants. The AtPHR1 protein recognises the DNA sequence (GnATATnC), termed the P1BS element, which is present in the promoters of many ‘‘late’’ PSR genes (Franco‐Zorrilla et al., 2004; Hammond et al., 2004a; Hou et al., 2005; Misson et al., 2005; Rubio et al., 2001). In Arabidopsis, these include genes encoding transcription factors, protein kinases, Pi transporters, RNases, phosphatases, metabolic enzymes, and enzymes involved in the synthesis of sulpholipids and galactolipids (Franco‐Zorrilla et al., 2004; Rubio et al., 2001). The transcription of many of these genes is repressed in P‐starved plants by the application of phosphonate/phosphite (H2PO3 or HPO32 ), a nonmetabolised analog of Pi, implicating a Pi‐sensing mechanism in these transcriptional cascades (Ticconi et al., 2001, 2004; Varadarajan et al., 2002). Mutations in either PHR1 or the P1BS cis element reduce the expression of genes whose promoters contain the P1BS sequence during P starvation (Franco‐Zorrilla et al., 2004; Rubio et al., 2001; Schu¨ nmann et al., 2004). It is noteworthy that some of these genes encode components of biochemical signal transduction cascades and transcription factors, which may become the next amplification or selectivity step in the signalling cascade in response to P withdrawal. Curiously, however, the P1BS element is present in the promoters of 15 to 20% of Arabidopsis genes and is not preferentially contained within promoters of P‐regulated genes (Hammond et al., 2003). This suggests that the activity of AtPHR1 could be regulated post-transcriptionally in response to P withdrawal or that more than one cis element is required to eVect P‐specific transcriptional responses. It is noteworthy that promoters harbouring the P1BS element often also contain PHO‐like sequences, CACGTd and/or CdhGTGG (d ¼ G,T or A, h ¼ C,T or A), resembling a functional cis element present in the promoters of genes in the yeast PHO operon (Hammond et al., 2003, 2004a,b; Liu et al., 1997, 2005; Schu¨ nmann et al., 2004; Vance et al., 2003). However, although the PHO‐like sequence CACGTd was found to be present significantly more frequently in PSR genes

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(Hammond et al., 2003), there is no direct evidence to confirm its role in transcriptional regulation during P starvation (Schu¨ nmann et al., 2004). However, evidence shows that transcriptional repressors also regulate PSR genes, as mobility shift assays with promoter fragments from PSR genes detect DNA‐binding proteins in nuclear extracts from P‐replete plants that are absent in P‐starved plants (Mukatira et al., 2001). 3. Signals involved in root morphological adaptations Some changes in gene expression respond to the rhizosphere P concentration. These changes occur irrespective of whether another part of the root system is supplied with P. One physiological process responding directly to low rhizosphere P concentrations is the development of transfer cells with high Hþ‐ATPase activities in the rhizodermis of tomato roots (Schikora and Schmidt, 2002). Another physiological process responding to local Pi availability is the proliferation of root hairs in places with low rhizosphere P concentration (Bates and Lynch, 1996). Significantly, root hair development is unaVected in the Arabidopsis phr1 mutant (Rubio et al., 2001). It is thought that interactions between increasing concentrations of auxin and ethylene increase the abundance and length of root hairs. Auxin is thought to increase trichoblast cell file number, and ethylene is important for root hair formation (He et al., 2005; Zhang et al., 2003b). The expression of several genes involved in ethylene biosynthesis (ACC oxidase, methioninesynthase and S‐adenosyl methionine synthetase) is increased in plants lacking P (Uhde‐Stone et al., 2003; Wu et al., 2003). In Arabidopsis, AtTTG1 (a WD40 protein), in tandem with AtTTG2 (a WRKY transcription factor), is implicated in root hair initiation. The current model is that AtTTG1 interacts with specific bHLH and MYB proteins to form heteromeric complexes that control gene expression through the consensus bHLH‐binding site CAnnTG, which also resembles the PHO‐like binding site (Hammond et al., 2003). In the root epidermis, AtTTG1 forms a ternary complex with the bHLHs AtGL3 or AtEGL3 and the MYB AtWER to maintain the atrichoblast state, or AtCPC, to allow hair development (Bernhardt et al., 2003; Casson and Lindsey, 2003; Montiel et al., 2004; Zhang et al., 2003a). Fig. 4. (A) Phylogenetic tree of Arabidopsis MYB‐like transcription factors homologous to AtPHR1 that contain both a SHAQKYF myb‐like DNA‐binding domain and a domain predicted to form a coiled coil (CC) structure implicated in protein–protein interactions. Protein alignments were performed using the AlignX program in Vector NTI 9.0 (Invitrogen, Paisley, UK). A suggested subfamily nomenclature of ‘‘MCC’’ (MYB‐CC) proteins is proposed. (B) The predicted amino acid sequences of the conserved SHAQKYF myb‐like DNA‐binding domain and CC domains present in the 15 Arabidopsis MYB‐CC proteins. Conserved amino acid residues are marked with an asterisk.

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Although the expression of AtTTG1 appears to be unaVected by P starvation, the expression of several key bHLH and MYB proteins is influenced by P starvation (Wu et al., 2003). The elongation of root hairs relies on the activity of hyperpolarisation‐activated Ca2þ channels (HACCs), which elevate [Ca2þ]cyt at their apex (Foreman et al., 2003; White, 1998). This enables cell expansion to proceed through the fusion of membrane vesicles with the apical plasma membrane. The activity of HACCs is increased by reactive oxygen species (Foreman et al., 2003). Thus, root hairs might be longer where rhizosphere Pi concentrations are low because Ca2þ influx through HACCs is increased or prolonged in these regions, not only because rhizosphere Ca2þ concentrations are likely to be greater, but also because Pi deficiency induces oxidative stress (Hammond et al., 2003; Jungk 2001). It is also important to recognise that lateral roots form preferentially in patches of the soil with high Pi availability (Drew, 1975; Hodge, 2004; Robinson, 1994). This phenomenon, which is more intense in P‐starved plants, suggests that lateral rooting might be regulated by (at least) two P sensors. The first, which responds to plant P concentration, predisposes a root system to lateral root formation, and the second, which responds to rhizosphere Pi concentration, determines where lateral roots are formed. This is analogous to the proliferation of cluster roots in patches of high Pi availability only when the plant has a low P status (Dinkelaker et al., 1995). Evidence suggests that plant P status is perceived in the shoot and systemic signals predispose the root system to lateral root formation. By contrast, the root tip appears to sense Pi availability in the rhizosphere, as the proliferation of lateral roots in a P‐rich patch only occurs when the root tip grows through the patch (Drew, 1975; Forde and Lorenzo, 2001; Hodge, 2004; Linkohr et al., 2002; Robinson, 1994). D. SYSTEMIC SIGNALLING OF PLANT P STATUS

1. Shoot responses When P is unavailable to plant roots, Pi transport to the xylem is reduced immediately and the xylem Pi concentration declines (Jeschke et al., 1997; Mimura, 1999). This often leads to a decrease in shoot P concentration, and a reduction in shoot growth before root growth is aVected (Clarkson and Scattergood, 1982; Cogliatti and Clarkson, 1983). This results in an increase in the plant root:shoot ratio. Thus, P acts as its own root/shoot signal. The proteins responsible for loading Pi into the xylem are unknown. However, some insight into the regulation of P transport to the shoot has been obtained from the Arabidopsis pho1 mutant. This mutant exhibits reduced

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P transport to the shoot, and it has been suggested that PHO1, which is expressed in the root stele and upregulated weakly upon P starvation, encodes a membrane protein that functions in P sensing (Hamburger et al., 2002). Ten homologs of PHO1 in Arabidopsis may be involved in coordinating diverse P starvation responses in plants. All contain an SPX domain, which is implicated in P sensing in yeast and G‐protein interactions (Wang et al., 2004). The transcription of many genes responds rapidly to P starvation in the shoot. It has been argued that changes in the expression of these genes are unlikely to be initiated by changes in [Pi]cyt because [Pi]cyt might be maintained until P deficiency becomes severe. However, because the [Pi]cyt of photosynthetic cells decreases rapidly when leaves are illuminated (Mimura, 1999), it is not inconceivable that P starvation might aVect [Pi]cyt dynamics in these cells. Such changes in [Pi]cyt may result in immediate (kinetic) changes in metabolism as a consequence of perturbed metabolite concentrations (Plaxton and Carswell, 1999). In particular, it has been suggested that reduced [Pi]cyt might promote both starch and sucrose biosynthesis, either by releasing enzymes from allosteric inhibition or through transcriptional regulation of genes encoding key enzymes (Mu¨ ller et al., 2004; Plaxton and Carswell, 1999). These changes in cell biochemistry are unlikely to reflect simply a reduced C requirement for growth during P starvation because carbohydrate metabolism returns to unstressed levels after supplying P for 2 days to P‐starved plants that have not grown significantly (Mu¨ ller et al., 2004). One of the first physiological manifestations of P starvation in shoots is the accumulation of sucrose. This may result in (1) an increase in the amount of sucrose transported to the root, (2) a reduction of photosynthesis, and (3) the production of anthocyanins (Plaxton and Carswell, 1999; Ticconi and Abel, 2004). Many genes are coregulated in shoots by increased sucrose and P starvation. These include genes encoding many photosystem subunits and small subunits of RuBisCo, which are downregulated, and enzymes involved in the production of sucrose and starch, anthocyanin biosynthesis, and sucrose transporters, which are upregulated by both increased sucrose and P starvation (Ciereszko et al., 2001; Franco‐Zorrilla et al., 2005; Lloyd and Zakhleniuk, 2004; Toyota et al., 2003). It is possible, therefore, that the regulation of some genes responding to P starvation could be aVected indirectly through changes in leaf sucrose concentration. However, because many of these responses are repressed by phosphonate in P‐starved plants (Carswell et al., 1996, 1997), a Pi‐sensing mechanism is also implicated. An increase in the transcription of sucrose transporters, in combination with an increase in leaf sucrose concentration, could account for the increase in carbohydrate transport to the root and the consequent increase in the

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root:shoot ratio observed during P starvation. The reduction of photosynthesis in P‐deficient plants is probably a direct consequence of the accumulation of sugars, as many of the genes impacting on photosynthesis are repressed by sucrose (Martin et al., 2002; Paul and Pellny, 2003; Rook and Bevan, 2003), although P deficiency may partly mitigate the sugar repression of photosynthetic genes (Hurry et al., 2000). Similarly, the production of anthocyanins during P starvation could be initiated by an increase in sucrose concentration. An interaction among WD40, bHLH, and MYB proteins is thought to control flavonoid metabolism in many plants (Irani et al., 2003). In Arabidopsis, AtTTG1 and AtTTG2 are implicated in the control of anthocyanin biosynthesis in vegetative tissue (Johnson et al., 2002; Shirley et al., 1995; Walker et al., 1999). The current model is that AtTTG1 forms a ternary complex with the bHLHs AtTT8 or AtEGL3 and the MYB proteins AtPAP1 (AtMYB75) or AtPAP2 (AtMYB90) to control flavonoid biosynthesis in leaves (Baudry et al., 2004; Zhang et al., 2003a). As in roots, the expression of AtTTG1 appears to be unaVected by P starvation (Hammond et al., 2003, 2004b). However, the expression of AtPAP1 and AtPAP2 is upregulated not only by P starvation, and in the P‐deficient shoots of the pho1 mutant (J. P. Hammond, http://aVymetrix.arabidopsis. info/narrays/experimentpage.pl?experimentid¼102), but also by high sucrose concentrations (Lloyd and Zakhleniuk, 2004). 2. Shoot–root signals The transcription of many genes in roots reflects the shoot, not the root, P concentration. This is clearly observed when Pi is available to only a portion of the root system and gene expression is monitored both in roots supplied with Pi and those that are not. Evidence from such experiments suggests that systemic signals regulate the transcription of many genes involved in acclimation to P starvation in plant roots. Several systemic signals have been proposed that may initiate genetic responses to P starvation. These include reduced Pi translocation in the phloem (Drew and Saker, 1984; Jeschke et al., 1997; Mimura, 1999), increased sucrose translocation in the phloem (Liu et al., 2005), reduced cytokinin, and/or increased auxin translocation to the root (Hammond et al., 2004a; Hou et al., 2005; Martı´n et al., 2000; Nacry et al., 2005). Although the activity of the primary transcriptional regulator implicated in responses to P starvation, AtPHR1, is thought to respond rapidly to immediate environmental signals (Rubio et al., 2001), the expression of many genes that may be regulated through AtPHR1 appears to be also regulated by systemic signals. For example, members of the At4/IPS gene family are

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downregulated systemically when Pi is available to only a portion of the root system (Burleigh and Harrison, 1999; Hou et al., 2005; Liu et al., 1997). This is consistent with the elevated expression of At4 in roots of the Arabidopsis pho1 mutant, despite their high P status (Burleigh and Harrison, 1999). These observations suggest that a systemic signal controlled by shoot P status represses these genetic responses to P withdrawal. Another key physiological response to P starvation that may be regulated by both AtPHR1 and a systemic signal is Pi uptake by root cells. The ability of roots to take up Pi is correlated with their expression of genes encoding high‐aYnity, plasma membrane Pi transporters of the Pht1 subfamily (Dong et al., 1999; Shin et al., 2004; Smith et al., 2003). Transcripts from many members of the Pht1 subfamily increase rapidly in roots of P‐starved plants (Karandashov and Bucher, 2005; Karthikeyan et al., 2002; Mu¨ ller et al., 2004; Rausch and Bucher, 2002; Schu¨ nmann et al., 2004; Shin et al., 2004; Smith et al., 2003). When only a portion of the root system is supplied with Pi or when Pi is supplied by mycorrhizal associations, the transcription of Pht1 genes in root cells, and their Pi uptake, is not determined by their P concentration but by the P status of the shoot (Chiou et al., 2001; Drew and Saker, 1984; Rausch et al., 2001; Smith et al., 2003). This observation originally led researchers to postulate that Pi uptake was determined by Pi loading into the xylem and that this might be regulated by shoot P status (or shoot P demand) through the retranslocation of Pi in the phloem, which decreases during P starvation (Drew and Saker, 1984; Jeschke et al., 1997; Mimura, 1999). This interpretation is consistent with the increased expression of genes encoding Pi transporters and Pi uptake by roots of Zn‐deficient plants (Huang et al., 2000; Marschner and Cakmak, 1986) and the Arabidopsis pho2 mutant (Dong et al., 1998), which accumulate excessive Pi in the shoot and retranslocate less Pi from the shoot to the root. However, evidence shows that the expression of Pi transporters in Arabidopsis and lupin roots is upregulated during the light period and in response to the addition of sucrose to the nutrient solution bathing the roots (Lejay et al., 2003; Liu et al., 2005; Raghothama, 2005). This suggests that an increase in sucrose transport from the shoot to the root in response to shoot P starvation could regulate the expression of these genes. It might also explain why, when growth is limited by nitrogen or sulphur, Pi transporters fail to be induced by Pi deprivation (Smith et al., 1999). Curiously, upon resupplying Pi to roots, although their Pi uptake decreases rapidly, Pi may accumulate to toxic levels in leaves, suggesting a flaw in this regulatory mechanism (Adalsteinsson et al., 1994; Clarkson and Scattergood 1982; Cogliatti and Clarkson, 1983; Green et al., 1973).

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3. Metabolic signals Interestingly, a systemic sucrose signal has been implicated in the regulation of other genes responding to P starvation in roots. These include genes encoding transport proteins, such as AtAMT1.1, AtNrt1.1, LeNrt2, LeIRT1, and LaMATE (Al‐Ghazi et al., 2003; Lejay et al., 2003; Liu et al., 2005; Wang et al., 2002; Wu et al., 2003), and phosphatases, such as AtVSP, AtACP5, AtPAP12, and LaSAP1 (Baldwin et al., 2001; Berger et al., 1995; Li et al., 2002; Liu et al., 2005). Indeed, the Arabidopsis pho3 (¼suc2) mutant, which lacks a sucrose transporter that loads the phloem, does not secrete acid phosphatase in response to P starvation (Lloyd and Zakhleniuk, 2004). Interestingly, the promoters of many of these genes contain both the AtPHR1‐binding motif (GnATATnC) and the PHO‐like sequence (CACGTd, where d ¼ G/T) that is overrepresented in genes responding to P starvation and apparently functions as an enhancer for plant genes responding to sugars and biotic and abiotic stress (Hammond et al., 2004a,b; Liu et al., 2005). It has been speculated that proteins from the bZIP class of transcription factors might bind to the latter (Hammond et al., 2004a). Interestingly, sucrose represses the translation of several bZIP transcription factors through a highly conserved, translated, upstream open reading frame encoding a peptide of 25 to 42 amino acids in their 50 ‐untranslated region (Wiese et al., 2004). Five Arabidopsis bZIP genes harbour this sequence: AtbZIP1, AtbZIP2, AtbZIP11, AtbZIP44, and AtbZIP53. Interestingly, the expression of all these genes is aVected by P starvation and/or carbohydrate accumulation (J.P. Hammond, http://aVymetrix.arabidopsis.info/narrays/ experimentpage.pl?experimentid¼102; Lloyd and Zakhleniuk, 2004; Price et al., 2004; Wu et al., 2003). Promoter dissection of the soybean VspB gene identified two contiguous domains that mediated sucrose induction and phosphate inhibition (Tang et al., 2001). Intriguingly, the domain mediating phosphate inhibition contains a sequence (CATTAATTAG) that may bind HD‐ZIP proteins as transcriptional repressors and is found in the promoters of other genes responding to both sucrose accumulation and P starvation (Tang et al., 2001; Toyota et al., 2003). However, sucrose is not the only signal that has an impact on the expression of genes encoding At4/IPS riboregulators, Pi transporters, and acid phosphatases. The expression of all these genes decreases rapidly (within 30 min) in roots and shoots of P‐starved plants when resupplied with Pi. This apparently occurs before a change in carbohydrate concentration can be detected, but is consistent with an increase in tissue Pi concentration (Mu¨ ller et al., 2004). The repression of expression of genes encoding At4/IPS riboregulators, Pi transporters, and acid phosphatases in P‐starved plants by phosphonate has implicated a Pi sensor in their regulation (Ticconi et al., 2001, 2004; Varadarajan et al.,

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2002). However, because phosphonate can be translocated rapidly throughout the plant, the location of the Pi sensor is unknown (Guest and Grant, 1991). The AtPDR2 gene has also been implicated in this transcriptional cascade, as (1) changes in the expression of these genes are more sensitive to P starvation, and greater, in the pdr2 mutant and (2) pdr2 plants grown in the presence of phosphonate have a wild‐type phenotype (Ticconi et al., 2004). Nevertheless, it would be interesting to determine whether sucrose and starch concentrations accumulate in leaves of phosphonate‐treated, P‐starved plants. 4. Hormonal signals Cytokinin has been implicated in the control of transcriptional responses to P starvation and has also been suggested as a systemic signal. Cytokinin concentrations are lower in roots of P‐starved plants, and the exogenous application of cytokinins suppresses the expression of At4, AtIPS1, AtPT1, and AtACP5 in roots of P‐starved Arabidopsis (Martı´n et al., 2000) and OsIPS1 and OsIPS2 in roots of P‐starved rice (Hou et al., 2005). Because the AtCRE1 receptor protein has been implicated in regulating these responses in Arabidopsis, it is noteworthy that the expression of AtCRE1 is induced by cytokinins and downregulated by P starvation (Franco‐Zorrilla et al., 2002). However, cytokinin is unlikely to be the systemic signal itself because the addition of exogenous cytokinins to a portion of the root system cannot repress the transcription of PSR genes systemically (Franco‐Zorrilla et al., 2004). Exogenous cytokinins also repress lateral rooting in P‐starved plants (Franco‐Zorilla et al., 2002), and the phenotype of the Arabidopsis pho2 mutant, which has a longer primary root and fewer lateral roots than wild‐ type plants, indicates the influence of shoot P status on this trait (Chen et al., 2000; Williamson et al., 2001). Plants lacking P are also more sensitive to an auxin‐induced increase in lateral root number and density (Lo´ pez‐Bucio et al., 2002). Thus, it is thought that a decrease in cytokinin concentration releases a negative control on root development, initiation of lateral roots is promoted by increasing auxin concentrations, and interactions between increasing auxin and ethylene concentrations promote lateral root elongation in P‐starved plants (Franco‐Zorilla et al., 2004; Hammond et al., 2004a; Lo´ pez‐Bucio et al., 2005; Lynch and Brown, 2001; Malamy, 2005; Nacry et al., 2005; Ticconi and Abel, 2004; Vance et al., 2003). Further, it has been suggested that a reduction of primary root growth in P‐starved plants may initiate the development of lateral roots by altering auxin transport from the shoot through the root (Ticconi and Abel, 2004). It is clear that the expression of genes encoding proteins regulating or regulated by the activity of

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plant hormones changes appropriately in plants during P starvation (Al‐Ghazi et al., 2003; Hammond et al., 2004a; Rietz et al., 2004; Uhde‐ Stone et al., 2003; Wu et al., 2003), but little is known of their systemic regulation. However, sucrose does not appear to be the systemic signal because growing Arabidopsis plants on media containing sucrose had no eVect on the morphological responses of their root system to P starvation (Williamson et al., 2001) and when Arabidopsis plants were grown on media containing high sucrose:N ratios, lateral root initiation was inhibited almost completely (Malamy, 2005). In addition to changes in the concentrations of Pi, sucrose, and plant hormones in the phloem sap during P starvation, changes in the concentrations of other solutes are also apparent (Jeschke et al., 1997). To compensate for the decreased Pi concentration of the phloem sap, the concentrations of inorganic anions, such as chloride, amino acids, such as glutamate, and organic acids, such as malate, succinate, shikimate, and oxalate, increase in phloem sap during P starvation (Jeschke et al., 1997). The phloem sap also includes submillimolar concentrations of several organic P metabolites, such as nucleotides and hexose phosphates, whose response to P starvation is unknown. These solutes may act as systemic signals and/or contribute to acclimatory responses to P starvation. Interestingly, the presence of glutamate in the rhizosphere of the root tip inhibits primary root growth, but this eVect is not observed when glutamate is added to other parts of the root system (Filleur et al., 2005). In some situations the exudation of organic acids into the rhizosphere parallels the production of organic acids in the shoot. Because the exudation of organic acids by roots is of fundamental importance for releasing Pi from organic and inorganic sources in the soil, it is noteworthy that this may be controlled systemically through the regulation of shoot metabolism.

IV. CONCLUSIONS The ability to perceive and integrate information on nutrient availability in the soil is a fundamental prerequisite for plant survival. Enhancing our knowledge of the molecular processes involved in plant nutrient stress responses is an important contribution plant scientists can make to the development of sustainable agricultural practices. We have shown in this review that the combination of a wide range of experimental techniques has led to an impressive body of data, which provides a good foundation for future progress in this field. Transcriptional profiling of K‐ and P‐starved plants has created a wealth of novel information on plant molecular

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responses to nutrient stress and has provided us with a large number of potential molecular markers for K and P sensing in plants. Forward and reverse genetics studies are now required to identify signalling elements that lie upstream of individual marker genes and to characterise the physiological function of individual K‐ and P‐regulated genes in stress acclimation. In addition to research at the single gene level, future progress can be expected from further ‘‘omics’’ studies. One methodology that is likely to deliver exciting new insights into nutrient signalling is metabolic profiling, which not only facilitates the discovery of putative metabolite signals, but can also help us pin down primary enzymatic targets of K and P stress. To obtain a truly comprehensive picture of the signalling events and acclimation processes occurring under K and P deficiency, fine mapping of K and P levels, together with transcripts, proteins, and metabolites, in tissues and cellular compartments, as well as an increased dynamic resolution during stress, are required. Correlation of all these system parameters in time and space should allow us to outline a first systemic model of their complex causal relationships. Targeted manipulation of key elements of such a model can then be used to test the predicted links and refine the model.

ACKNOWLEDGMENTS Work by PJW and JPH was supported by the Department for Environment, Food and Rural AVairs, UK (Projects HH3501SFV and HH3504SPO). Research in the laboratory of AA and PA is supported by the Biotechnology and Biological Sciences Research Council.

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