Phosphate Deprivation in Maize: Genetics and Genomics

Plants acquire phosphorus (P) from the soil in order to reach the intracellular concentrations necessary for growth and reproduction. To prevent P starvation, both monocotyledonous and dicotyledonous plants have evolved well-regulated systems for inorganic phosphate (Pi) scavenging, acquisition, and recycling. Phosphate starvation responses (PSR) in plants include release of organic acids and hydrolytic enzymes to scavenge soil Pi from organic and inorganic sources, metabolic modifications to bypass ATP requirements, recycling and mobilization of internal P resources, modifications of root system architecture to increase soil exploratory capacity, and, for many plant species, the establishment of symbiotic relations with arbuscular mycorrhizal fungi (Bucher, 2007). Maize (Zea mays) is one of the most widely cultivated crop plants, for both staple food and industrial usage, in tropical and temperate soils worldwide. Under cultivation, especially in acidic and alkaline soils, large quantities of P fertilizer are applied to maize fields in order to maximize yields. P fertilizers, however, are becoming increasingly costly owing to dwindling natural reserves of rock Pi and an increase in the costs of extracting what remains; it is projected that we have already moved beyond the point of peak production (Cordell et al., 2011) and that reserves will be exhausted before the end of the century (Khasawneh et al., 1986). Recent progress in maize genetics and genomics has furthered characterization of the molecular basis of maize PSR, with the potential to provide important information in the development of maize varieties with enhanced P use efficiency. The most pertinent questions in the study of maize PSR are as follows: How similar is the maize response to what is known in other plant species? And how is the maize response typical of cereals in general? Exhaustive microarray analyses performed in the model plant Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), together with a small but increasing number of reports concerning the molecular basis of PSR in maize as well as other cereals, are beginning to shed light on these questions. Furthermore, the recent completion of maize genome sequencing (Schnable et al., 2009; Vielle-Calzada et al., 2009) has opened new possibilities for the comparative study of maize and other model species. In Table I, we summarize current knowledge of maize PSR and compare this with what is known from Arabidopsis. Sequencing of the maize genome has revealed maize gene families to typically be more expanded than their counterparts in Arabidopsis, and we have consistently found multiple maize genes that exhibit close homology to any given Arabidopsis sequence. This level of gene duplication raises the possibility for paralog subfunctionalization and, consequently, both fine-tuning and greater plasticity in maize PSR. Gene models from Arabidopsis (The Arabidopsis Information Resource version 10), rice (release 6.1), maize (release 4a.53, filtered set), Brachypodium (release 1.0), and sorghum (release 1.4) were considered for identification of the putative orthologs by using the OrthoMCL pipeline (Li et al., 2003) with default parameters except for miR399, At4, and IPS1. Adapted from Lin et al. (2009). ND, No data; NPOI, no putative ortholog identified. Gene models from Arabidopsis (The Arabidopsis Information Resource version 10), rice (release 6.1), maize (release 4a.53, filtered set), Brachypodium (release 1.0), and sorghum (release 1.4) were considered for identification of the putative orthologs by using the OrthoMCL pipeline (Li et al., 2003) with default parameters except for miR399, At4, and IPS1. Adapted from Lin et al. (2009). ND, No data; NPOI, no putative ortholog identified. Several key regulatory components involved in the regulation of P deprivation response have been characterized in Arabidopsis, including transcription factors such as PHOSPHATE STARVATION RESPONSE1 (Rubio et al., 2001), PHR1-LIKE1 (Bustos et al., 2010), WRKY75 and ZAT6 (Devaiah et al., 2007a, 2007b), IPS1/At4 (Shin et al., 2006; Franco-Zorrilla et al., 2007), the microRNA miR399 (Bari et al., 2006; Chiou et al., 2006; Pant et al., 2008), and members of the PHO family (Aung et al., 2006; Stefanovic et al., 2007). In addition, global gene expression studies in Arabidopsis have revealed that the responses to P deprivation include alterations in many biochemical and signaling pathways, regulated by a complex network involving members of a range of transcription factor gene families (Hammond et al., 2004; Misson et al., 2005; Morcuende et al., 2007). Microarray analysis and large-scale sequencing projects in a number of species have reinforced this idea and have widened our knowledge and uncovered new strategies exploited by plants to cope with P starvation, revealing how particular species have evolved specific mechanisms. Valuable genetic and genomic information has been obtained from Arabidopsis, rice, bean (Phaseolus vulgaris), and lupine (Lupinus albus), for which large-scale studies have permitted a detailed characterization of genetic responses to P starvation (Uhde-Stone et al., 2003; Wasaki et al., 2003; Misson et al., 2005; Hernández et al., 2007; Morcuende et al., 2007). For maize, using a microarray platform to characterize a P starvation-tolerant maize genotype, a comprehensive view of P starvation responses in maize roots was obtained, showing that the steady-state level of over 1,100 transcripts (2% of those analyzed) is modulated by P availability, and among those, at least 33% do not have a significant match with an ortholog in the Arabidopsis genome (Calderon-Vazquez et al., 2008). Such analysis allowed the identification of some biochemical pathways either not reported previously as P responsive in other plant species or at least not as responsive as in maize roots, including a putative shift in nitrogen (N) metabolism to preserve N-containing metabolites, phenylpropanoids, lignin, and fatty acid biosynthesis, in parallel with an increase in the β-oxidation (Calderon-Vazquez et al., 2008). Furthermore, a proteomic analysis of root tips identified changes in protein accumulation following long-term P starvation (Li et al., 2007), indicating modifications in carbohydrate metabolism, amino acid and nucleotide synthesis and degradation, and secondary metabolism. Thus, to date, it is known that coordinated gene expression is necessary to acquire and utilize P efficiently in maize and that a number of regulatory components are also P regulated. In this Update, recent findings and opportunities for further understanding the regulation of maize PSR are summarized. The wide diversity of maize germplasm includes many varieties adapted to grow in low-P soils, although the basis of this adaptation remains to be studied in detail. Commercial maize breeding efforts and large-scale public programs have placed relatively little emphasis on Pi relations per se; efficient N use and drought tolerance remain the major goals of targeted efforts to alleviate abiotic stress. Nonetheless, a number of screens of diverse plant material have been performed, taking an important step toward the development of varieties better able to acquire and use available Pi resources. Furthermore, screens of this type provide the starting point for a characterization of the genetic architecture of maize responses to different Pi availabilities. Given the high level of phenotypic variation present in maize, there may be difficulties in detecting relevant genetic variation on the basis of a direct comparison of performance under low-Pi stress (Sawers, 2009). For this reason, many studies have made a relative comparison of performance between low- and high-Pi conditions. A standard screening methodology is well illustrated by the study of Da Silva and Gabelman (1992). In that work, seedling performance was assessed in a panel of maize inbred lines, and a substantial range of performance was observed under both low- and high-Pi availability. Furthermore, the ranking of the lines differed between the two growth conditions, indicative of a specific effect related to Pi, as opposed to more general variation in plant performance. Such studies provide strong evidence that significant variation exists in the ability of maize to exploit the P in its environment, a prerequisite for selection-based improvement. Performance screens are not by themselves informative with regard to the number of genes that underlie the variation observed or the nature of their effects. Consequently, researchers have used quantitative trait loci (QTL) analyses to provide further information on the genetic architecture of variation in Pi response. A number of studies have employed a B73-Mo17 recombinant inbred line mapping population to investigate variation in overall performance (Kaeppler et al., 2000) and specific root developmental traits (Zhu et al., 2005). Significantly, when analyses have been performed under low- and high-Pi availability, certain QTL have been common to both conditions, while others have been specific to one or the other environment. A further QTL study using a population derived from the Chinese varieties 082 and Ye107 examined a broader range of traits, including root characteristics, vegetative growth, and the quantity of plant exudates (Chen et al., 2008). A number of chromosomal regions were associated with QTL for multiple traits, indicative of tight linkage of causative genes or pleiotropy. Such QTL are candidates for general regulators of PSR. Collectively, QTL analyses support the existence of multiple loci linked to variation in Pi relations. Although, to date, none of the genes underlying one of these QTL has been identified, a combination of further mapping and transcriptome analysis may facilitate future gene identification and further our understanding of the relationship between Pi availability and maize performance. When comparing P starvation responses among plants, we can define a basic set of shared responses. This “core” is constituted by the modification of the mobilization, uptake, and transport of P. These mechanisms were first identified in dicotyledonous species, and they have also been observed in maize genotypes tolerant to P starvation. Here, the current knowledge on how different genes may act during Pi starvation is contrasted to what is hypothesized in maize. Typical plant PSR include release of Pi from organic and inorganic sources by increasing the synthesis and secretion of organic acids, acid phosphatases, and ribonucleases from roots, in parallel with the up-regulation of genes encoding high-affinity Pi transporters (Bariola et al., 1994; Li and Anderson, 1997; Daram et al., 1998; Liu et al., 1998; del Pozo et al., 1999; Neumann and Römheld, 1999; Raghothama, 1999; Baldwin et al., 2001; Chiou et al., 2001; Li et al., 2002). Several genes that code for Pi-responsive transporters have been described in rice (Paszkowski et al., 2002), barley (Hordeum vulgare; Schünmann et al., 2004a), wheat (Triticum aestivum; Tittarelli et al., 2007; Miao et al., 2009), and maize (Nagy et al., 2006), and it is known that in maize and rice roots, the up-regulation of P transport- and P recycling-related genes was the most extensive adaptation under Pi starvation, as reflected by the up-regulation of several putative Pi transporters, phosphatase, and RNase genes (Wasaki et al., 2003; Calderon-Vazquez et al., 2008). Despite that the root architecture differs significantly between monocotyledonous and dicotyledonous plants (Hochholdinger and Zimmermann, 2008), both systems under P starvation present a set of developmental modifications that tend to increase the exploratory capacity of the plant, particularly in the P-rich upper layers of the soil. In Arabidopsis and bean, P starvation results in a modification of root hair growth (Bates and Lynch, 1996) but also in a reduction in the length of the primary root and changes in the angle of growth and the diameter of lateral roots (Bonser et al., 1996; Lynch and Brown, 2001; Williamson et al., 2001; López-Bucio et al., 2002; Hodge, 2004). In wheat and barley, root hair growth is also an adaptive modification (Gahoonia et al., 1997). In maize, it has been reported that P starvation induces alterations in the postembryonic root system, including modification in the angle, length, and number of shoot-borne and lateral roots (for review, see Calderón-Vázquez et al., 2009). However, in contrast to Arabidopsis, it appears that the primary root system in maize, which is important for early stages of development, is not affected by Pi availability, probably because cereal grains contain large quantities of the P (O’Dell et al., 1972) required during seedling establishment. Development- and hormone-related genes have been reported in Arabidopsis as mediators of changes in root system architecture in response to P starvation (López-Bucio et al., 2002; Ma et al., 2003; Pérez-Torres et al., 2009). Likewise, PHOSPHATE DEFICIENCY RESPONSE2 (PDR2) and LOW PHOSPHATE ROOT1 (LPR1) and LPR2 have been proposed as regulators of the root-meristem response to P-starvation in Arabidopsis (Ticconi et al., 2004, 2009; et al., 2006; et al., 2007), a key in root P (Ticconi et al., 2009). In maize roots, and transcription which are involved in and root and 2002; et al., exhibit expression under P starvation (Calderon-Vazquez et al., 2008). The is for the root development genes (for and et al., 2002; and transcripts responsive to acid and including members of the and gene families et al., 2007; Calderon-Vazquez et al., 2008). In the has been proposed to a number of developmental in both Arabidopsis and maize and 2007). is by the enzymes and In maize, the by the architecture et al., maize transcripts encoding enzymes have been observed to in accumulation under P starvation (Calderon-Vazquez et al., 2008). of those transcripts are similar to the Despite that there is not an ortholog of in Arabidopsis et al., 2006), the a reduction in root length and a of or its in root in Arabidopsis and 2007). the proposed of in the establishment of and by either signaling or as a in maize architecture et al., 2006), and the that but also signaling root developmental responses in Arabidopsis to P starvation and it is that may be a of root developmental responses in maize. When plants the recycling and mobilization of internal P and other metabolic during P starvation, the synthesis and of intracellular is to P and P from other in such as or et al., and a of bypass are to or enzymes et al., and In maize roots, are also between and roots and and appears to be also by that as by the high increase in expression of genes encoding and (Calderon-Vazquez et al., 2008). in their expression may the acid or provide for metabolism. the modification of metabolic pathways in maize can be at the protein and et al., 2001; Li et al., 2008), including the increase in accumulation of complex and acid to increase the of this and to organic acids for (Li et al., 2008), as reported for maize et al., and rice et al., These results to the concentrations of organic acid found in both and roots of barley plants et al., 2008). However, the that barley is a plant, which a from what has been reported for maize and rice (Wasaki et al., 2003; Li et al., 2007; Calderon-Vazquez et al., et al., 2008). is that the up-regulation of the genes has been reported in maize roots, an important reduction in the of such enzymes has been reported in of plants under P starvation in parallel with a strong in the of and in N and amino acid metabolism have been considered as an of P starvation, and it has been proposed that such changes a to the response to et al., 2007). In maize roots, N and amino acid metabolism are also regulated by P starvation. A significant of and was observed under P (Calderon-Vazquez et al., 2008). and are for maize, the general of enzymes involved in amino acid and the for synthesis of some amino acids that maize both and N at the the synthesis of secondary toward organic acid In the barley plants under P starvation to use amino acids as an especially in as proposed by et al. These results are in contrast to those reported for Arabidopsis, modifications in the of transcripts involved in amino acid metabolism are et al., 2005; Morcuende et al., 2007). In plants in it is known that are and by the release of P et al., 1998; et al., metabolism is regulated by P starvation in maize. least transcripts related to metabolism expression including genes involved in the recycling of from the A similar shift be in rice roots and in a in from an expression using an of (Wasaki et al., this not the it the in expression of particularly an increase in for genes involved in In contrast to Arabidopsis, is by regulation of and et al., 2005; et al., 2008), maize roots under P stress not present a modification in the expression of these a strong of and also related to degradation, was (Calderon-Vazquez et al., 2008). of β-oxidation in with fatty acid synthesis was also observed in maize, to provide for a use of in maize roots to during P starvation (Calderon-Vazquez et al., 2008). A number of P starvation responses have been identified by gene expression in maize that have not been identified in other species. A significant of the and transcripts encoding components of the were either or under P starvation. for maize the this shift in expression a in the of maize roots (Calderon-Vazquez et al., 2008). A similar of under P starvation has also been in rice, although not by of (Wasaki et al., as opposed to Arabidopsis, is efficiently by arbuscular mycorrhizal fungi (Paszkowski and 2002), and, as in other species, this significantly growth P and of under 2008). In maize, the to the plant can on the species, soil P and and et al., and 2002; et al., 2005; et al., with maize inbred lines that mycorrhizal of maize genotypes under than the with the mycorrhizal as is a key factor in (Kaeppler et al., although further analyses have that maize germplasm may significant variation in the ability to from mycorrhizal per et al., The establishment of mycorrhizal is by a molecular between and plant including the root of et al., 2005; et al., and the modification of plant et al., 2005). is a of transport systems are to facilitate the of metabolites, including a set of specific Such transporters have been reported in a number of species, including the cereals rice, barley, and maize (Paszkowski et al., 2002; et al., 2005; et al., regulatory as well as common that the biochemical and developmental responses to P starvation, have been identified among several plant species, it is that or responses have also be particularly for maize, given the of the responses to P deprivation among diverse maize in Arabidopsis has that the regulation of PSR several (for review, see Chiou and including the transcription factors and PHR1-LIKE1 et al., 2007; et al., 2010), a also proposed as a of P starvation responses including root architecture et al., an involved in P and from roots to (Bari et al., 2006), miR399, a microRNA that and Pi (Aung et al., 2006; Pant et al., 2008), the family that miR399 et al., 2007), and et al., 2007; et al., results have been obtained in by rice, barley, and wheat responses. potential rice orthologs of can be identified in the rice genome and and are involved in P availability signaling by the expression of P In addition, but not is related to modifications in root architecture in both and pathways et al., 2008). In barley, Schünmann et al. identified the in of an was previously identified as the in of genes in Arabidopsis (Rubio et al., Furthermore, P starvation expression in barley et al., in the was found in the of the gene et al., 2007; Miao et al., 2009), a Pi regulated by P starvation in roots, that be P starvation responses in The ortholog of also as an important of the response to P starvation in cereals et al., 2009). by Liu et al. has that and miR399 in the up-regulation of however, a in the regulation of P response pathways in rice et al., 2009; Liu et al., In the was reported as the ortholog of Arabidopsis et al., Pi and and was also regulated by in the as in Arabidopsis (Aung et al., 2006; et al., 2006; Pant et al., et al., in the maize genome have identified at least two similar to that the signaling may be common in both monocotyledonous and dicotyledonous species. The maize genes the possibility of subfunctionalization at the level of maize PSR developmental However, it remains to be those are a of duplication or are members of a the rice genome two both reported as involved in Pi starvation responses et al., 2008). is to the of the of not in maize but also in other gene that as a potential of P starvation in is the rice transcription factor To date, no Arabidopsis has been identified. the tolerance to P starvation and also root A microarray analysis of the lines at least genes as in their These findings that a that tolerance to P starvation et al., 2005). The maize genome at least two to in the expression of other genes involved in Pi responses in maize remains to be However, maize lines a have been reported to exhibit tolerance of low-P stress and an expression of key genes in synthesis and in including and Pi (Li et al., These results that Pi tolerance in maize can be modulated by the expression Pi-responsive genes that have been previously characterized in other plant species. other families in maize roots in response to P starvation are the and and transcripts were in maize roots, and one of similar to a of the was found among the most genes in response to Pi In the and transcription factors were also reported as regulated by P starvation (Calderon-Vazquez et al., 2008). In it is that some of the mechanisms P responses are in maize and Arabidopsis, including the microRNA maize genome at least miR399 et al., 2009). Maize is as a staple food but also as a of primary for industrial The identification of P starvation-tolerant genotypes and knowledge of how they are to of this described and are that have been used to provide a general of the maize PSR. However, more detailed and comprehensive studies that the diversity of responses in maize germplasm are The is to on a gene analysis of the maize to those that have been reported as key components in P starvation signaling pathways in other plants by and as well as studies in other model In the by taking of sequencing together with the available information on the maize genome (Schnable et al., 2009; Vielle-Calzada et al., 2009), it is to and maize genotypes with P starvation responses and P to set strategies for mapping and these we will have a understanding of the and molecular to P starvation in maize and to of Pi with genes identified by genome