Metabolic Adaptations of Phosphate-Starved Plants

Orthophosphate (Pi) is an essential macronutrient that plays a central role in virtually all major metabolic processes in plants, particularly photosynthesis and respiration. Many metabolites are Pi monoesters, whereas the phosphoanhydride bonds of compounds such as ATP function to transfer energy from the energy-yielding process of photo-, oxidative, and substrate-level phosphorylation to the energy-dependent cellular processes of biosynthesis, ion pumping, and mechanical work. The massive use of Pi-containing fertilizers in agriculture demonstrates how the soluble Pi level of many soils is suboptimal for crop growth. Accessible reserves of rock phosphate—our major source of Pi fertilizers—are projected to be exhausted by the end of this century (Vance et al., 2003). The use of Pi fertilizers is also quite inefficient with less than 20% of applied Pi being absorbed by plants during their first growing season. The remaining Pi becomes immobile in the soil or leaches into and pollutes nearby surface waters. Agricultural Pi runoff is a primary factor in the eutrophication of lakes and marine estuaries, and has also resulted in blooms of toxic cyanobacteria. With the world’s population continuing its rapid increase, mankind faces a daunting challenge to produce sufficient food crops in the face of dwindling supplies of Pi fertilizers. A more comprehensive understanding of the biochemical and physiological mechanisms of plant Pi uptake and use is leading to the development of rational strategies and molecular tools for engineering nutrient-efficient cultivars needed to reduce agriculture’s overreliance on unsustainable Pi fertilizers. The aim of this Update article is to consider the influence of Pi nutrition on plant metabolism, with a focus on adaptive metabolic responses that serve to ameliorate the negative side effects of Pi deficiency. Examples of how metabolic Pi scavenging and recycling, and the unique flexibility of plant metabolism and bioenergetics may contribute to the survival of Pi-deficient (−Pi) plants are highlighted. Plants have evolved the ability within species-dependent limits to acclimatize to extended periods of Pi deprivation by eliciting a complex array of morphological, physiological, and biochemical/metabolic adaptations collectively known as the Pi-starvation response (PSR). The PSR arises in part from the coordinated induction of hundreds of Pi-starvation inducible (PSI) genes encoding enzymes that reprioritize internal Pi use and maximize external Pi acquisition (Vance et al., 2003; Ticconi and Abel, 2004; Fang et al., 2009; Lin et al., 2009; Nilsson et al., 2010). As is well documented throughout this Focus Issue, many elements of the PSR are controlled at the transcriptional and translational level, and −Pi plants extensively remodel their transcriptome and proteome in ways that coordinate the requisite metabolic and morphological adaptations. Large collections of microarray data regarding plant, and particularly Arabidopsis (Arabidopsis thaliana), transcriptional responses to Pi starvation have: (1) shed light on the molecular identity and regulation underlying many classical biochemical and physiological adaptations to Pi deprivation, and (2) revealed that PSI gene expression is highly coordinated in a temporal and tissue-specific manner (Fang et al., 2009; Lin et al., 2009; Nilsson et al., 2010). Recent studies are also emphasizing the importance of posttranscriptional mechanisms in the control of PSI enzyme expression and activity. This is reflected by proteomic profiling of −Pi rice (Oryza sativa), corn (Zea mays), and Arabidopsis, demonstrating that transcript abundance of various genes is not always indicative of protein accumulation during Pi deprivation (Fukuda et al., 2007; Li et al., 2008a; Tran and Plaxton, 2008). One of the best-characterized examples of posttranscriptional mechanisms in the Arabidopsis PSR is the regulatory module comprising the transcription factor PHR1, PHO2 that encodes the E2 ubiquitin conjugase UBC24, the microRNA399 (miR399), and the noncoding RNA At4 (Bari et al., 2006; Fang et al., 2009; Lin et al., 2009; Nilsson et al., 2010). miR399 regulates Pi homeostasis by controlling UBC24 expression. UBC24 functions during Pi sufficiency to promote the proteolytic turnover of PSI proteins, including high-affinity Pi transporters of the plasmalemma. Pi starvation induces PHR1 and activates expression of the phloem-mobile miR399. Binding of miR399 to complementary bases of UBC24 transcripts leads to the destruction of UBC24 mRNA, resulting in low levels of UBC24’s E2 ubiquitin conjugase activity, and the consequent accumulation of its downstream protein targets (Bari et al., 2006). Shoot-derived miR399 thus serves as a long-distance signal to suppress the expression of UBC24 in the roots. Expression of the ribo-regulator At4 is strongly induced during prolonged periods of Pi starvation (Fang et al., 2009; Lin et al., 2009; Nilsson et al., 2010). At4 binds to complementary bases of miR399, thereby inhibiting its silencing of UBC24 mRNA. This allows UBC24 levels to rapidly adjust to the dynamic balance of Pi supply and demand. High-throughput deep sequencing has identified additional miRNAs that control various genes involved in the Arabidopsis PSR (Hsieh et al., 2009). For example, miR165, miR778, miR827, and miRNA2111 are all highly induced in −Pi Arabidopsis, whereas miR169, miR395, and miR398 are repressed (Hsieh et al., 2009). Cross talk between these miRNAs may coordinate their expression under specific nutrient deficiencies, thereby facilitating plant survival in −Pi environments (Hsieh et al., 2009). Future challenges include the need to identify the specific targets of the various miRNAs, as well as to pinpoint the molecular mechanisms by which they control cellular Pi homeostasis. Posttranslational mechanisms of metabolic control are another important feature of the PSR and include Pi’s role as an allosteric activator or inhibitor of many key control enzymes of intermediary plant metabolism (Plaxton and Podestá, 2006). This is illustrated by the major regulatory enzyme of starch biosynthesis, ADP-Glc pyrophosphorylase, which displays potent allosteric inhibition by Pi. Transgenic potato (Solanum tuberosum) plants expressing a Pi-insensitive bacterial ADP-Glc pyrophosphorylase overproduce starch (Stark et al., 1992). The results demonstrated that allosteric control of this plastidic enzyme, not the amount of its protein subunits, is the major physiological determinant of starch biosynthesis (Stark et al., 1992). Thus, the well-documented accumulation of starch by −Pi plant cells may largely arise from the release of ADP-Glc pyrophosphorylase from allosteric inhibition by Pi, owing to the large (up to 50-fold) reductions in cytoplasmic Pi pools that accompany long-term Pi deprivation (Duff et al., 1989; Vance et al., 2003). Similarly, PSI vacuolar acid phosphatase (APase) isozymes display potent product inhibition by Pi (Bozzo et al., 2004a; Veljanovski et al., 2006; Tran et al., 2010a). The vacuolar Pi concentration of Pi-sufficient plants (>10 mm) will exert significant feedback inhibition on their APase activity. Conversely, the depletion of vacuolar Pi pools that accompanies extended Pi deprivation (Fang et al., 2009) will effectively relieve the inhibition of PSI APases by Pi, thus contributing to their enhanced activity in vivo. It is evident that a complete description of the metabolic adjustments of −Pi plants not only requires the characterization of how Pi serves to control gene expression and protein turnover, but also how physiologically relevant changes in Pi levels modulate the activities of preexisting enzymes. As discussed below, protein phosphorylation and glycosylation are emerging as essential posttranslational modifications that control the activity of and/or subcellular targeting of diverse enzymes up-regulated by −Pi plants (Gregory et al., 2009; Tran et al., 2010b). A challenging goal will be to document the functional consequences of posttranslational modifications such as reversible protein phosphorylation in the signaling and metabolic pathways involved in plant acclimation to nutritional Pi deprivation. A common feature of the plant PSR is the development of dark-green or purple shoots due to anthocyanin accumulation brought about by PSI anthocyanin biosynthetic enzymes (Vance et al., 2003; Fang et al., 2009). Anthocyanins are a class of red/purple-colored flavonoids that can protect nucleic acids from UV damage and chloroplasts from photoinhibitory damage (Zeng et al., 2010). The up-regulation of high-affinity Pi transporters of the plasma membrane is another important component of the plant PSR (Fig. 1). These transporters are energized by ATP-dependent proton efflux, and actively assimilate Pi against a steep concentration gradient, as the soluble Pi concentration in the rhizosphere can be up to 10,000-fold lower than that of root cells. High-affinity Pi transporters of Arabidopsis belong to the nine-member PHT1 family and consist of H+/Pi symporters with 12 (Fang et al., 2009; Lin et al., 2009). all are to Pi to a of tissue-specific with in and root cells are in cells of the with or plants Pi acquisition during Pi to Pi in shoots during Pi sufficiency due to its role in Pi (Fang et al., 2009; Lin et al., 2009). A various adaptive metabolic processes by that may plants acclimatize to nutritional Pi deficiency. pathways of and and vacuolar by −Pi plant cells they the on and Pi, the levels of which during Pi Large of acids by and may also be by to the (1) Pi and and (2) and its to by Pi deprivation vacuolar are up-regulated to Pi from Pi Similarly, Pi from Pi and nucleic acid pools for its uptake by PSI high-affinity Pi transporters of the plasma Plants also the of Pi use during Pi starvation up-regulation of a array of PSI that and Pi from and compounds (Vance et al., 2003; Fang et al., 2009; Nilsson et al., Tran et al., 2010a). Thus, Arabidopsis plants on RNA as their source of Pi as well as control plants, whereas a which is in the induction of PSI requires Pi and Abel, This that nucleic acids in the an important source of nutritional Pi that can be by −Pi The induction of and APases function in the of nucleic acids and their to Pi, which is for root uptake by high-affinity Pi transporters (Fig. Ticconi and Abel, 2004; Fang et al., 2009). is a PSI that the protein in the of −Pi Arabidopsis cells and Plaxton, 2008). PSI function in with and APases to Pi from nucleic acids and Abel, plants also and Pi by their membrane with and A in the of −Pi plant with in and membrane and are PSI enzymes for biosynthesis in −Pi Arabidopsis (Fang et al., 2009; Lin et al., 2009). Arabidopsis under −Pi et al., which that are an important for during Pi deprivation. and are PSI that in −Pi accumulation during Pi deprivation is in the of −Pi and et al., 2006). acid that can be by an APase to release Pi and and also to be involved in in root during growth. acid to serve as a that activates a protein protein phosphorylation that root growth. to function in a is for in during Pi starvation et al., 2008). A plant response to Pi deprivation is the up-regulation of and enzymes that Pi from a and of Pi with an (Fig. 1). As about of the in soil and is the of in soil APases are to function in Pi scavenging by of −Pi This is reflected by the ability of Arabidopsis and plants to and assimilate nutritional Pi from a of including and various such as and Abel, 2004; 2009; et al., 2010). PSI APases are also into the and of −Pi plant et al., et al., 2009; Tran et al., 2010a). These APases have to function in Pi from compounds from the −Pi cells et al., Similarly, PSI APases and Pi from Pi and (Fig. 1). This is by reductions in levels of cytoplasmic metabolites during extended Pi deprivation (Duff et al., 1989; Vance et al., 2003). APases the class of plant a purple or in that results from a transfer at about from the to the et al., 2010a). are APases that Pi from a of Pi a in and cells function in the of a the of the et al., 2010a). Similarly, (1) plant that significant APase activity also have activity, and (2) of a to damage during et al., and bacterial also function as a role in signal et al., 2010a). a of is highly against et al., whereas a of APases from plant have significant activity with or acids as (Bozzo et al., 2004a; Veljanovski et al., 2006). expression of resulted in and enhanced activity, that this function as a protein phosphatase involved in the control of biosynthesis et al., 2009). Many studies have on the role that isozymes in and/or Pi scavenging and during Pi The transcription PHR1, and have in the control of expression in −Pi Arabidopsis, studies have revealed PSI is controlled by posttranscriptional mechanisms et al., 2006; Tran and Plaxton, Lin et al., 2009; Tran et al., 2010b). Pi to −Pi plants PSI genes specific that and PSI (Bozzo et al., Nilsson et al., Tran et al., 2010a). and characterization of PSI is to the molecular mechanisms underlying this biochemical of the plant as well as to identify targets for crop Pi PSI have from including (Solanum and Arabidopsis (Bozzo et al., 2006; Vance et al., 2003; Veljanovski et al., 2006; Tran et al., 2010b). of an APase known as from its to Pi starvation et al., acid identity with a PSI Arabidopsis et al., 2010b). This to its by PHR1 can enhanced expression under −Pi a APase of −Pi is to a and up-regulated by −Pi Arabidopsis et al., 2006; Tran et al., 2010b). As with the is of the nutritional Pi The Arabidopsis encodes isozymes transcriptional expression is various and et al., 2010a). to the abundance of and transcript expression is on the identity and biochemical of specific isozymes that contribute to Pi scavenging by −Pi of the first PSI to be and from −Pi It as a and is induced in and of −Pi Arabidopsis et al., This that be involved in the metabolism of during than significant Pi or scavenging in −Pi The of subcellular and the that its expression has on Arabidopsis will to its during Pi deprivation or Recent biochemical and functional studies have the as the as well as a major APase up-regulated by −Pi Arabidopsis et al., 2006; et al., Tran et al., 2010b). expression of an with vacuolar et al., 2010). A a large in and and APase as well as development to Pi et al., 2010). The results demonstrated that is a to PSI APase activity, and that plays an important role in the Pi metabolism of −Pi isozymes into the by −Pi Arabidopsis cells identified by and as an and of and subunits, et al., 2010b). and a specific APase activity, and but activities −Pi Arabidopsis with an biochemical for scavenging Pi from external Pi in the rhizosphere or A of this that of −Pi Arabidopsis as a of that to such as and et al., 2010b). is an important posttranslational that enzyme and/or The of and at glycosylation is to the that the targeting and of the vacuolar and of −Pi Arabidopsis arise from vacuolar and are up-regulated by −Pi Arabidopsis, transcripts are and of nutritional Pi et al., 2006; et al., Tran et al., 2010b). proteomic studies documented a of and that are also controlled at the level of protein accumulation in plants to changes in Pi (Fukuda et al., 2007; Li et al., 2008a; Tran and Plaxton, 2008). This the need to transcript profiling with biochemical and proteomic of the plant as the will a more of how in gene expression may be to adaptive changes in the metabolism of −Pi of and and an has that and for of the APase activity of −Pi et al., and that they an essential role in Pi scavenging during on nucleic acids and Pi as the source of and Plaxton, or of −Pi Arabidopsis Pi from acid of Pi from acid not and be in activity has to less than of the APase activity of Arabidopsis root and Arabidopsis is to from owing to the of an 2009). is the only of the family that has to APase and activity, and to an important role in Pi from reserves during or et al., 2009). its expression is to Pi deprivation and not in root or not to function in Pi of a the and Pi use of plants on as their source of et al., 2009). plants in their or Pi nutrition in various soils 2009; Tran et al., 2010a). This that the of soil to may not be an for plants that The biochemical characterization of and PSI isozymes their role in Pi scavenging and by −Pi (Bozzo et al., cytoplasmic Pi, a of prolonged Pi starvation is by a highly specific response that temporal and tissue-specific of PSI isozymes (Bozzo et al., 2006). APase identified as by highly induced in and of −Pi plants et al., 2010). The expression of in cells that is into the et al., 2010). with ATP as a that function in acclimation to Pi starvation the use of ATP as a Pi source from the and plant cells ATP into the and ATP is essential for plant et al., 2010a). are targets for engineering as they a role in Pi and scavenging by −Pi to activity, the of Pi fertilizers the and of thereby the amount of that is for 2009). It is of to the of Pi can be by the of such as and in crop the of −Pi in et al., 2009). The plants enhanced Pi uptake and during on −Pi This to in including a of and and a in soil et al., 2009). important component of the plant PSR is root of acids into the rhizosphere (Fig. which results in rhizosphere The for the release of acids are into their at cytoplasmic plasma membrane to be involved in plant to Pi starvation et al., This has with the up-regulation of membrane needed to such as and from root cells into the rhizosphere et al., Thus, the of acid an by a plasma membrane and the transporters that the of et al., et al., acid results in the of that Pi thus soil Pi by up to acid (1) Pi in (2) the of and its to by and may also promote the of rhizosphere that root Pi acquisition (Vance et al., 2003; Ticconi and Abel, 2004; Fang et al., 2009; 2009). The amount of as acids can be from to than of the plant (Vance et al., 2003). of acids by −Pi plants has with up-regulation of and its by reversible and (Fig. and of (Vance et al., 2003; et al., 2009). of a gene acid and in rice (Fang et al., 2009). Similarly, of in or Arabidopsis plants resulted in enhanced levels of from and of plants in Pi or in −Pi soil (Fang et al., 2009). A feature of plant metabolism is that the in a metabolic can be in a of This metabolic flexibility is reflected by engineering in which to or an enzyme to be essential and the resulting plants to and more or less (Plaxton and Podestá, 2006). an essential component of the biochemical adaptations of plants and allows to to their and As a of the large in cytoplasmic Pi levels that Pi large (up to reductions in levels of and also (Duff et al., 1989; and Podestá, 2006). This is to the enzymes of classical that are or Pi as (Fig. Pi and −Pi plants to to energy and for key pathways of and by that may promote the survival of A key component of this is the role by enzymes and metabolic Pi during Pi deprivation. that the are as are as in the or as and enzymes in to the of the membrane have to be up-regulated by −Pi plant cells and et al., 1989; et al., Vance et al., 2003; and Podestá, 2006). These PSI and vacuolar during Pi the levels of and Pi are of these to cellular ATP and Pi and enzymes such as Pi and the also promote Pi recycling, as Pi is a of their The enzyme allows −Pi plants to to the activity of becomes (Fig. and studies of and in that the of to the metabolism of in −Pi cells in et al., for the of this in −Pi by the rapid release of from compounds from and et al., of and enzyme and/or activity are up-regulated in response to Pi deprivation in a of plant (Vance et al., 2003; and Podestá, 2006). Recent have the molecular of the response to Pi in Arabidopsis and rice and which the up-regulation of specific and protein isozymes under the control of transcription The induction and in phosphorylation of the at its phosphorylation to be an component of the Arabidopsis PSR (Gregory et al., 2009). this the first that regulatory enzyme phosphorylation to the plant and is with the that the PSI genes of Arabidopsis include the protein genes and (Fang et al., 2009; et al., 2009; Lin et al., 2009; Nilsson et al., 2010). is a transcription factor that functions as a negative of PSI genes in Arabidopsis, including and et al., of additional of the signal pathways that nutritional Pi with the control of and transcription and as well as and and turnover will be a for is a of a of biosynthetic including the involved in the of The large of during biosynthesis are not always but can be by various as well as the plant to the of cellular to the plant soluble and thus of up to plant levels are to such as or Pi starvation that significant reductions in cellular ATP pools (Duff et al., 1989; and Podestá, 2006; et al., 2008). The mechanisms of homeostasis in plant cells and the of be under such as or Pi to be during the biosynthesis of essential needed to and/or to that have processes may be a of the metabolic adaptations of plants to that to ATP not pools is by the significant up-regulation of pyrophosphorylase, and the by or extended Pi starvation (Duff et al., 1989; et al., and Podestá, 2006; et al., 2008). As Pi allosteric effects on the activity of and and Podestá, the large in cytoplasmic Pi pools of −Pi plants promote the in activity of that of This has by a of plants that that the of and under of Pi et al., of and a that can the survival of plant cells that have ATP owing to such as Pi starvation or This has by studies of or plants levels of enzymes and For (1) a in root levels of potato plants a bacterial soluble resulted in plant and during lower ATP and an ability to of et al., (2) a role for in in by of in activity et al., and of the resulted in plants that to nutritional Pi deprivation et al., As in and the induction of enzymes may plants to nutritional Pi deprivation (1) (2) cellular pools of Pi from The significant reductions in Pi and levels that extended Pi deprivation will the at the of ATP the of pathways of a can be under the of and/or Pi are during Pi Plants to Pi by the up-regulation and/or of the and/or pathways of the and and and Podestá, 2006). This allows of the acid and with ATP and may thereby contribute to the survival of −Pi This has by the and metabolism of −Pi to a functional et al., of under Pi with levels of with It that a adaptive by which plant cells can modulate their response to Pi and that also has in cellular and balance et al., The of transcript and proteome profiling has to the effects of Pi on the expression of many genes and in plants such as Arabidopsis, and the enhanced expression of enzymes as pyrophosphorylase, and and as that not Pi or as has (Plaxton and Podestá, 2006; et al., 2007; Li et al., 2008a; Fang et al., 2009; Nilsson et al., 2010). The regulation of genes involved in primary metabolism demonstrates the of genes involved in the and and metabolism to ATP and during Pi deficiency. into the plant PSR have that have revealed hundreds of PSI genes encoding that are to −Pi plants reprioritize internal Pi use and maximize Pi acquisition from the soil (Vance et al., 2003; Ticconi and Abel, 2004; Fang et al., 2009; Lin et al., 2009; Nilsson et al., 2010). these results be by the that transcript abundance not protein and that transcript profiling about the subcellular of gene posttranslational modifications that may be essential for their and Thus, a understanding of metabolic of the PSR will the of transcript profiling and tools with and a understanding of the to which and changes in enzymes and pathways influence plant is relevant to the applied goal of engineering crops to including Pi The and biochemical and functional characterization of and Pi scavenging and enzymes up-regulated by −Pi plants will also contribute to the development of rational strategies for engineering These strategies may include the of PSI high-affinity Pi and enzymes and membrane transporters that in enhanced acid by during nutritional Pi deficiency. crops are needed to of unsustainable and Pi fertilizers for and of have various of the metabolic adaptations of plants to nutritional Pi deprivation. is also to have to studies of −Pi plants, particularly and

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