Supporting Information Notes S2
Possibility of decreasing the low-molecular-mass water-soluble P esters
A significant economy in the use of P by plants could be achieved by decreasing the content of
low-Mr (relative molecular mass) water-soluble phosphate esters, which represent approximately
15–20% of total P in most plants (main text Fig. 1, inset).
How could a decrease in the low-Mr water-soluble ester P be achieved? One possibility is to alter
metabolic pathways radically by genetic modification of plants to decrease the total number of
phosphorylated water-soluble intermediates, with emphasis on eliminating reactions involving a
low-Mr water-soluble P ester present at a high concentration. A second possibility is to retain the
existing pathways of metabolism and to alter one or more enzymes in a way which, as an
emergent property, involves lower concentrations of a low-Mr water-soluble P ester. A third
possibility is the addition of reactions to a pre-existing pathway which decreases the overall use
of P. Most of the suggestions of genetic modification of plants that involve pathways with
phosphorylated intermediates concern photosynthetic CO2 assimilation.
The most radical possibility is that of altering metabolic pathways by genetic modification of
plants to decrease the total number of phosphorylated water-soluble intermediates, with emphasis
on eliminating reactions involving a low-Mr water-soluble P ester present at a high
concentration. Perhaps the best way to address this issue is to determine what the plants
themselves are capable of. Under severe Pi stress, several enzymatic bypasses for steps in
glycolysis that require P-esters or adenylates are upregulated (see; Plaxton & Podestá, 2006;
Plaxton & Tran, 2011). These bypasses do not require Pi or adenylates, allowing respiratory C-
flux through glycolysis to continue to generate energy and to produce the C-skeletons needed for
key anabolic processes under conditions where available Pi and/or adenylate charge are
decreased. Some bypass enzymes such as pyrophosphate (PPi)-dependent phosphofructokinase
and pyruvate orthophosphate dikinase use PPi to accomplish cellular work. Another enzyme that
uses PPi, a tonoplast H+-PPiase, is also induced under Pi deficiency, helping to maintain
vacuolar pH at lower adenylate charge (Palma et al., 2000). These and other enzymatic bypasses
may provide opportunities for reducing the amount of Pi locked up in P-ester pools.
The suggestion by Bar-Even et al. (2010) of replacing ribulose bisphosphate carboxylaseoxygenase (Rubisco) and the associated photosynthetic carbon reduction cycle (PCRC) by
another autotrophic CO2 assimilation pathway was not aimed at economising on low-Mr watersoluble P esters, but rather at eliminating the oxygenase activity of Rubisco with its associated
energy costs and also a decreased energy cost for CO2 assimilation while decreasing the halfsaturation value for CO2 in photosynthesis. Bar-Even et al. (2010) selected reactions from the
five known autotrophic CO2 fixation pathway other than Rubisco-PCRC which are not inhibited
by O2 and (for carboxylases) have a high CO2 affinity; from the present viewpoint the important
feature is that the five other pathways have many fewer phosphorylated intermediates than
Rubisco-PCRC (see: Raven, 2009; Bar-Even et al., 2010; Berg, 2011). Since the PCRC or the
oxidative pentose phosphate pathway are the only source of erythrose-4-P used, with
phosphoenolpyruvate, in the shikimate pathway of aromatic synthesis, some of enzymes of the
pentose cycles and their phosphorylated intermediates must be present even if the reductant
generating role of the oxidative pentose phosphate pathway is replaced by NADP+ reduction by,
for example, cytosolic NADP+-linked malate dehydrogenase or isocitrate dehydrogenase. Since
the five pathways of autotrophic CO2 assmilation other than Rubisco – PCRC all require the
glucogenetic pathway to produce sugars and 3-phosphoglycerate, the only phosphorylated
compounds associated with Rubisco –PCRC and the photorespiratory carbon oxidation cycle, but
not the other five pathways or the oxidative pentose phosphate pathway are ribulose-1.5bisphosphate (RuBP) sedoheptulose-1.7-bisphosphate (SBP) and 2-phosphoglycolate. The SBP
and 2-phosphoglycolate concentrations are generally low, while RuBP is usually a, if not the,
major component of the stromal pool of low-Mr water-soluble P ester with a significant fraction
bounds to Rubisco and associated with Mg2+ (von Caemmerer & Edmondson, 1986; Gerhardt et
al., 1987; Servaites & Geiger, 1995; Ruuska et al., 2000; Viil et al., 2001). A much smaller
fraction of 3-phosphoglycerate is bound to Rubisco, where it acts as a negative effector (von
Caemmerer & Edmondson, 1986; Woodrow & Berry, 1988; Servaites & Geiger, 1995). Rubiscosaturating concentrations of RuBP are essential for maintaining Rubisco activation (Woodrow &
Berry, 1988; Servaites & Geiger, 1995) and a high CO2 affinity. It is unlikely that the
replacements of the Rubisco – PCRC with a pathway having fewer low-Mr water-soluble P ester
intermediates would only decrease the content of the esters by 10% at most. Whether such
wholesale intervention as replacing Rubisco - PCRC is justified by the P saving needs further
investigation, since using the data in Table 1 with total plant P equal to 8.4 times the content of
low-Mr water-soluble ester P, decreasing the content of these P esters by 10% would decrease
the whole plant P content by not more than 1.2%.
A second possibility is to retain the existing pathways of metabolism and to alter one of more
enzymes in a way which, as an emergent property, involves lower concentrations of (a) low-Mr
water-soluble P ester(s). Whitney et al. (2011) survey achievements and opportunities in
replacing the native Form IB Rubisco in a C3 plant with a Rubisco (e.g. a Form ID Rubisco) with
different kinetic properties (Whitney et al., 2011). Whether this would economise on P through a
decreased number of Rubisco active sites binding RuBP for a given rate of CO2 assimilation
requires further investigation. It seems unlikely that this intervention could yield as large a
decrease in whole-plant P as the 1.2% for the first mechanism.
A third possibility is the addition of reactions to a pre-existing pathway which decreases the
overall use of P. The possibilities here include the addition of respiratory bypass enzymes to
eliminate Pi requiring steps (Plaxton & Podestá, 2006; Plaxton & Tran, 2011), a CCM based on
C4 metabolism (Kajala et al., 2011; Miyao et al., 2011) or of a cyanobacterial CCM (Price et al.,
2011) to C3 crop plants. Addition of CCMs would permit the Form IB Rubisco with high CO2
affinity, high CO2/O2 selectivity but low CO2-saturated specific reaction rate with a Rubisco with
lower CO2 affinity and low CO2/O2 selectivity but higher CO2-saturated specific reaction rate.
Such a Rubisco, supplied with saturating CO2 by the CCM, could be present as fewer copies
while retaining, or increasing, the photosynthetic rate. Such a replacement of Rubisco could
involve lower RuBP concentrations with less RuBP bound to Rubisco, consistent with data
showing higher phosphorus use efficiency in two C4 grasses than in a C3 grass (Ghannoum &
Conroy, 2007; Ghannoum et al., 2008). Further investigation is needed, especially, in the case of
a C4 CCM, of any trade-off in C4 plants between decreased RuBP requirement and a possible
increased phosphoenolpyruvate steady state concentration to serve the photosynthetic role of
phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase than is needed for
the glycolytic and anaplerotic involvement of phosphoenolpyruvate (Aubry et al., 2011). As with
the second mechanism, it is very unlikely that a whole-plant P saving could be achieved that is as
great as the maximum of 1.2% possible with the first mechanism discussed above,
To summarise, genetic modification of pathways could only yield small decreases in overall
plant P requirement by decreasing the concentration of low-Mr water-soluble P esters.
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