Phosphorus in plants exists either as the free inorganic orthophosphate form (Pi) or as organic phosphate esters. The Pi concentration of tissues generally reflects the Pi supply (White & Hammond, 2008). This is true in crop plants, such as Brassica napus and Cucurbita maxima (Pant et al., 2008), pasture plants, such as Medicago truncatula (Branscheid et al., 2010), and undomesticated species, such as Hakea prostrata (Shane et al., 2004). Tissue Pi is separated into two physiologically separate pools. The metabolically active Pi pool, of the order of 0.1–0.8 mg P g−1 dry weight (DW) (Bieleski, 1968; Rébeilléet al., 1983; Rouached et al., 2011), is located in the cytoplasm and is kept within fairly narrow limits (Bieleski, 1968; Mimura et al., 1996; Mimura, 1999). Cellular Pi in excess of current cytoplasmic need is stored in the vacuole and is used to buffer the Pi demands of the cytoplasm, and is accordingly the most variable P fraction. For example, barley (Hordeum vulgare) grown in the absence of added Pi had no detectable vacuolar P, as measured using 31P-NMR (Foyer & Spencer, 1986). As Pi was withheld from barley, pre-grown in nutrient solution containing 1.0 mM Pi, the vacuolar Pi concentration decreased sharply, while the cytosolic Pi concentration was less affected. Using protoplasts and vacuoles isolated from intact barley leaves, it was shown that the vacuolar Pi concentration declined from 145 to 5 mM, when P supply declined from 40 to 0 mM, whereas that of the cytoplasm (cytosol plus mitochondria and plastids) declined from 35 to 26 mM (Mimura et al., 1990). Similarly, for soybean (Glycine max) having just reached full-flowering development, the vacuolar Pi concentration decreased from 8.0 to < 0.05 mM as P supply declined, whereas the cytosolic Pi concentration declined considerably less markedly, from 8.3 to 0.23 mM; the hexose monophosphate concentration declined less than that of Pi, from 7.7 to 0.54 mM (Lauer et al., 1989). Pratt et al. (2009) reported that Pi concentrations in the cytosol of sycamore (Acer pseudoplatanus) and Arabidopsis suspension cells were much lower than those in the cytoplasm. In summary, there is significant compartmentalization of Pi within cells, and Pi concentrations are most variable in the vacuole, showing its buffering function. Differences in P uptake by plants result in large variability of Pi concentration, making it a sensitive indicator of plant P status (Bollons & Barraclough, 1999).
The main pools for esterified P are nucleic acids, phospholipids, phosphorylated water-soluble low relative molecular mass (Mr) metabolites (commonly referred to as P-esters) and phosphorylated proteins. Phosphorylation of proteins serves a regulatory function, so phosphorylated proteins will not be considered further. In the following sections, opportunities for increased P efficiencies in the most significant P pools will be explored.
Fig. 1 summarizes available data on inorganic and organic P fractions in photosynthetic tissue of a range of species. Observations of total P concentrations ([P]) > 5 mg g−1 DW are generally from plants growing at very high P supply. Optimal concentrations for most crops are < 4 mg P g−1 DW (McLachlan et al., 1987; Föhse et al., 1988; Rodríguez et al., 2000). For wheat (Triticum aestivum), Bollons & Barraclough (1999) reported critical concentrations for total P and Pi of approx. 3 and 0.6 mg g−1 DW, respectively. In order of size, and ignoring stored (vacuolar) Pi, P pools are usually RNA > P-lipid > P-ester > DNA > metabolically active Pi. Averaged for all cases where total [P] < 4 mg g−1 DW (n = 21 species), and taking P-ester = 1, there are 1.3 units of lipid P, 2.0 units of nucleic acid P (mainly RNA) and 1.4 units of inorganic P.
1. Nucleic acids – role and minimum requirements
The nucleic acid pool is typically the largest organic P pool in a plant (Fig. 1), generally containing 40–60% of the P found in the combined organic P pool (i.e. total P minus Pi). This equates to c. 0.3–2 mg P g−1 DW, depending on the species, tissue and P supply (Smillie & Krotkov, 1960; Bieleski, 1968; Tachibana, 1987). The nucleic acid pool generally contains at least 85% RNA, with the remainder being DNA (Bieleski, 1968; Tachibana, 1987). Most RNA is ribosomal RNA (rRNA): Cucurbita ficifolia seedling roots were found to contain c. 94% rRNA, 4% transfer RNA and 2% messenger RNA (Kanda et al., 1994).
There is generally a positive correlation between rRNA content, and therefore ribosome number and protein synthesis capacity, and growth rate over a range of taxa (Elser et al., 2000, 2010). As leaves develop, rRNA levels increase to accommodate the rapid synthesis of proteins, for example, Rubisco (Suzuki et al., 2010), that are needed for photosynthesis. At full expansion or shortly thereafter, net leaf protein synthesis ceases and RNA levels decrease by up to 75% (Hensel et al., 1993; Suzuki et al., 2010). It is not clear how much of the leaf RNA remains at the end of senescence. The RNA pool generally adjusts in accordance with the growing conditions, for example, RNA concentrations increased in trees in which N concentration ([N]), [P] and growth rate were enhanced through fertilization (Reef et al., 2010).
Opportunities for increasing the use efficiency of RNA and the P it contains include optimizing the protein synthesis system during plant development. The aim would be to deploy many ribosomes, fully engaged in protein synthesis, when growth needs to be rapid, but only enough ribosomes for maintenance processes when growth is complete, and no ribosomes upon senescent death. Presently, a fraction of the 40 S and 60 S ribosomal subunits that are present at any given time is not actively synthesizing protein (Mustroph et al., 2009). The role, and thus the importance, of these subunits need to be evaluated so that the pool size could be targeted for optimization. Moreover, Rubisco large and small subunit mRNA levels can decline by > 90% in fully expanded leaves, but cytosolic rRNA levels drop < 75% during this period (Hensel et al., 1993; Suzuki et al., 2010). This suggests an increased disjunction between the actual amount of protein synthesis that takes place and the protein synthetic capacity that is available. However, ribosomes and tRNA are still needed in mature leaves for protein turnover, including replacement of damaged protein, and for acclimation and programmed cell death during senescence.
The costs of P, as RNA, of protein turnover and repair are substantial (Raven, 2011, 2012). Quigg & Beardall (2003) compiled data from vascular land plants showing protein turnover at 0.04–0.42 d−1. For a relative growth rate of a crop plant of 0.2 g g−1 d−1 this would mean an increase in RNA content relative to what would be needed with no protein turnover of 20–210% in an optimally allocating plant. With RNA representing 47% of the organic P (Fig. 1, total [P] < 4 mg g−1), this would mean that an increase of 9–99% in the organic P content of the plants is needed to support protein turnover. Assuming that the protein turnover estimates are accurate (the turnover rates are not easy to measure), protein turnover would seem to be a target for saving on P. For the specific cases of photodamage to the D1 protein of photosystem II and O2 damage to nitrogenase, it is, respectively, known and probable that these proteins turn over significantly more rapidly than most other proteins (Raven, 2011, 2012). D1 protein is ubiquitous among oxygenic organisms, and the relatively small additional P cost for ribosomes used in repairing photodamage in an optimally allocating organism is already included in PUE estimates (Raven, 2011, 2012). The greater P costs of growth often found for symbiotic and free-living diazotrophically growing photosynthetic organisms than when they are growing on combined nitrogen (Vitousek et al., 2002) might be explained, at least in part, by the need for more ribosomes for synthesizing nitrogenase to replace O2-damaged nitrogenase (Raven, 2011, 2012). This possibility needs further investigation.
The amount of P in the DNA pool is considerably smaller than that in the RNA pool, and reductions of DNA content would seem difficult and undesirable. However, there may be useful long-term optimization strategies. A pivotal question here is whether genome size and gene copy number can be optimized. The answer will require a better understanding of the function of the so-called ‘noncoding’ regions within genomes and of the ways that genes are controlled, as well as the development of new technologies to change genome structure rationally. The common occurrence of polyploidy in agricultural and invasive species (Soltis & Soltis, 2000), suggesting competitive benefits in productive and disturbed environments, also requires further research. A high copy number of rRNA genes (a few hundred to several thousand rRNA genes per genome; Rogers & Bendich, 1987) is thought to help the plant meet the demands to produce large amounts of rRNA to support protein synthesis and growth (Elser et al., 2010). However, the actual number of rRNA genes shows considerable variation between species, between individuals of the same species and even within individuals of some species (Rogers & Bendich, 1987). The question of genome size and gene copy number applies to both nuclear and organellar genomes. As an example, there can be > 10 000 copies of chloroplast DNA per cell, depending on species, tissue, developmental stage and environmental conditions (Bendich, 1987). If such high copy numbers reflect the demands for high levels of certain transcripts, such as plastid rRNA or the transcripts for the large subunit of Rubisco (Bendich, 1987), this is in stark contrast with the low gene copy number for the small subunit of Rubisco in the nuclear genome. In future, it may be possible to engineer plant genomes in all three DNA-containing compartments to optimize gene copy numbers for minimal P content while maintaining gene function.
2. Other P pools – roles and requirements
Phospholipids are important in membranes. While it may not be feasible to reduce the total area of membranes, there may be possible efficiency gains in their synthesis and composition. Thomas & Sadras (2001) characterized the assembly process of thylakoid membrane complexes as having large excess production and subsequent breakdown of components which is energetically inefficient but allows high responsiveness to changing internal and external conditions. If the implied trade-off between efficiency and responsiveness exists, tighter regulation of biosynthesis may be a trait worth investigating for increased resource efficiency in predictable productive environments.
Sulfolipids and galactolipids, rather than phospholipids, are the major lipids in the thylakoid membrane, and to a lesser extent in the plastid envelope membranes. Phospholipids in other membranes can also be replaced by sulfolipids and/or galactolipids; either constitutively or in response to P deficiency. Data (see Supporting Information Notes S1), particularly on cyanobacteria and algae, show that this replacement can be almost complete (Van Mooy et al., 2006, 2009), and that replacement can also occur as a result of mutations. However, the information on the functional consequences of such replacement is limited to the tentative conclusion that there is no effect on proton permeability (Notes S1, Table S1), but that there may be increased membrane leakage of electrolytes which constrains chill tolerance (for Arabidopsis, see Hurry et al., 2000).
Tissue P concentration may also be decreased by lowering the content of low-Mr water-soluble Pi-esters, and the nonstorage, metabolically active Pi. Notes S2 discusses three ways by which this could be achieved for the photosynthetic CO2-assimilation pathway involving Rubisco and the photosynthetic carbon reduction cycle (Rubisco–PCRC). The conclusion is that even the most radical of the three proposed interventions, that is, replacing the Rubisco–PCRC with some other pathway involving fewer phosphorylated intermediates, is unlikely to decrease the content of the low-Mr water-soluble Pi-esters by > 10%, with an overall saving based on the allocations in Fig. 1 of 1.2%. It is important to note that phloem is cytosolic and that any decrease in Pi-esters and Pi in its metabolically active cytosol, plastid stroma, mitochondrial matrix and nucleoplasm might have impacts on P (and N) transport in phloem which is particularly relevant for remobilization from senescing tissues. Notes S3 and Table S2 deal with the phloem in more detail, showing that it is unlikely that P translocation out of senescing (or any) tissues is limited by the availability of solutes to generate turgor for phloem transport or other constraints on the composition of phloem sap.